CN107727865A

CN107727865A - The systemic detection method of tumor markers and its application

Info

Publication number: CN107727865A
Application number: CN201610657034.7A
Authority: CN
Inventors: 韩晓亮; 王建铭; 徐海; 马竣; 宋乐乐; 周光朋
Original assignee: Biochain Beijing Science and Technology Inc; Biochain Institute Inc
Current assignee: Biochain Beijing Science and Technology Inc
Priority date: 2016-08-11
Filing date: 2016-08-11
Publication date: 2018-02-23

Abstract

The invention provides a kind of systemic detection method of tumor markers, methods described includes：1) sample from check object is provided, the check object is behaved；2) sample described in step 1) is detected, it is described be detected as using high throughput nucleic acid sequencing measure include it is polygenic be mutated, it is polygenic methylate, multiple Micrornas etc. and measure multiple proteins tumor markers whether there is using protein-chip.Different tumor markerses is formed systematization detection, including DNA, RNA and protein etc. by the present invention, improves the sensitivity of tumour early detection.

Description

The systemic detection method of tumor markers and its application

Technical field

Mutually tied the present invention relates to the detection of disease marker, more particularly to by genetic marker thing and oncoprotein matter mark Share in the systemic detection method of the tumor markers of the external auxiliary diagnosis of disease.

Background technology

Cancer is one of main cause of death in the whole world：2004, global number of cancer deaths (accounted for owning up to 7,400,000 Death toll 13%), global number of cancer deaths will continue to increase, the year two thousand thirty, and death toll is estimated will be up to 12,000,000.Full generation Boundary has 84,000,000 people or so to die from cancer during 2005 to 2015, cause the major cancers species of cancer general mortality rate every year For：Lung cancer (1,300,000), stomach cancer (80.3 ten thousand), colon cancer (63.9 ten thousand), liver cancer (610,000) and breast cancer (51.9 ten thousand).U.S. in 2015 State shares 1,700,000 new cancer cases.It is about 429.16 ten thousand to estimate within 2015 Chinese annual new cancer cases, dead 281.42 ten thousand, account for new cases 65.6%.The major cancers species for estimating Chinese annual new cancer cases for 2015 is lung Cancer (73.33 ten thousand), stomach cancer (67.91 ten thousand), the cancer of the esophagus (47.79 ten thousand), liver cancer (46.61 ten thousand), colorectal cancer (37.63 ten thousand) and breast Cancer (27.24 ten thousand).Estimating within 2015 the Chinese annual major cancers species for causing cancer general mortality rate is：Lung cancer (61.02 ten thousand), Stomach cancer (49.80 ten thousand), liver cancer (42.2 ten thousand), the cancer of the esophagus (37.5 ten thousand), colorectal cancer (19.1 ten thousand), cancer of pancreas (7.94 ten thousand) and breast Cancer (7.07 ten thousand is dead).

Tumour is a kind of systemic disease, can almost occur all sites in body, tumour include malignant tumour and Benign tumour, most of in malignant tumour is cancer.The primary tumor for endangering the mankind at present has lung cancer, stomach cancer, liver cancer, Colon and rectum Cancer, breast cancer and cancer of the esophagus, other common tumours have cervix cancer, prostate cancer, carcinoma of urinary bladder, cancer of pancreas, thyroid cancer, leaching Bar knurl, carcinoma of uterine body, oophoroma and brain tumor etc..The most common cancer species in the whole world (by global death toll sort) be：Man Property：Lung cancer, stomach cancer, liver cancer, colorectal cancer, cancer of the esophagus and prostate cancer；Women：Breast cancer, lung cancer, stomach cancer, colorectal cancer And cervix cancer.The sequence of China is slightly different, the five kinds of most common tumours of Chinese male in 2015 lung cancer, stomach cancer, oesophagus successively Cancer, liver cancer and colorectal cancer, this five kinds of cancers account for the 2/3 of all cases of cancers.The most common tumour of women successively breast cancer, lung cancer, Stomach cancer, colorectal cancer and the cancer of the esophagus, account for the 60% of all cases of cancers.Total death toll sequence of Chinese major cancers in 2015 For lung cancer (21.68%), stomach cancer (17.7%), liver cancer 15.0%, cancer of the esophagus (13.33%), colorectal cancer (6.79%), pancreas Gland cancer (2.82)) and breast cancer (2.51%).

The mankind pass through and the arduous and very long game of tumour, it is understood that for the late tumor of III-IV phases, even if through Cross various complicated operative treatments, chemotherapy, radiotherapy, immunization therapy, gene therapy, targeted therapy etc., 5 annual survival rates Only 5-15%；And to the infantile tumour of I-II phases, 5 annual survival rates are up to 80-90%, precancerous lesion or 0 phase cancer or original position The therapeutic effect of cancer is more preferable.So the common recognition of industry is to want early detection and early intervention tumour in recent years, it is swollen that this is only solution The essential measure of knurl.Clinical research finds that most cancers initially form from focus, the process average of clinical symptoms occur to patient Time several years is needed, this is finds early-stage cancer, the diagnosis rate of raising early-stage cancer provides an effective window phase.Cause This, makes full use of this window phase, researches and develops detection and the examination technology of sensitive, accurate, noninvasive, convenient early-stage cancer, improves The discovery rate of early-stage cancer, it is to improve treatment of cancer effect, reduce the important channel of cancer mortality.

Regrettably in the inspection of tumour early detection or precancerous lesion, due to the main suit of not obvious person under inspection, Clinical symptoms and sign, it is difficult to targetedly select corresponding clinical auxiliary examination for example X- lines perspective, CT, MRI, ultrasonic wave or Hysteroscope inspection.These conventional methods are low to the sensitivity and specificity of early-stage cancer due to the limitation of detection technique, such as low dose It is high to measure the false positive rate of spiral CT detection lung cancer, improves the risk of over-treatment, and it is complex operation, invasive strong (such as：Intestines Mirror, gastroscope etc.).Testing cost is high (such as：CT, MRI need expensive equipment, and the technical staff of professional training) it is to be used for tumour The obvious obstacle of early detection, and only single cancer is detected, it is not suitable for the early detection of kinds cancer.Numerous Detection method in, people are difficult to select targetedly detection method and carry out the detection of early stage to go out a certain tumour, simultaneously The possibility of other tumours can be excluded.Because person under inspection is not have Symptomatic substantial amounts of normal (disease-free) crowd, to person under inspection For, lesion detection to sample conveniently, sample requirements are few, to person under inspection without pain and injury, price is appropriate, easily by by Inspection person receives.For the mechanism for implementing lesion detection, detection method is simple and quick, to detection device, place and personnel Do not require too, easily realize.This just needs one kind to have special, wide spectrum, sensitive and can be from blood from detection technique The method for detecting early-stage cancer, upper detection method that is simple, convenient and reducing person under inspection's burden is implemented from popularization.

Traditional protein tumor markers is widely used, but to kinds of tumors accurately early detection still people not to the utmost Meaning.Alpha-fetoprotein (α-fetoprotein, α FP or AFP) is the stronger protein tumor markers of a specificity, Ke Yi Raised in about 80% liver cancer patient blood serum, and as sb.'s illness took a turn for the worse, its content in serum can sharply increase, always It is considered the specific tumour mark of diagnosing primary liver cancer, has the function that to establish diagnosis, early diagnosis, antidiastole. But the detection can only detect primary carcinoma of liver, to other cancers, then Detection results are limited, and the stronger tumour of specificity is used alone Mark, such as alpha-fetoprotein detection, above-mentioned other cancers will be missed.

In order to not miss other tumours, infantile tumour can be detected using broad spectrum activity tumor markers.Carcinomebryonic antigen (carcino-embryonic antigen, CEA) is a broad spectrum activity tumor markers, except primary colon cancer, breast cancer and Beyond lung cancer, cancer of pancreas, cholangiocarcinoma, stomach cancer, the cancer of the esophagus, the tumer positive rate of myxoadenocarcinoma and urinary system also raise, its energy Reflect the presence of kinds of tumors to people, judgement the effect of to colorectal cancer, breast cancer and lung cancer, disease development, monitoring and Prognosis estimation is a preferable tumor markers, but industry, it is generally accepted that its specificity is not strong, sensitivity is not high, early to tumour Phase diagnostic effect unobvious.

In order to take into account broad spectrum activity, specificity, sensitivity and early-stage cancer these technical requirements can be detected from blood, compare For alpha-fetoprotein and carcinomebryonic antigen these traditional protein tumor markerses, detection nucleic acid tumor markers is acknowledged as according with Close and state technical requirements.The DNA that tumour discharges can be especially detected from blood, plays liquid biopsy (Liquid Biopsy effect).Conventional nucleic acid detection technique has solid phase chip, liquid-phase chip, real-time quantitative PCR (Polymerase Chain Reaction), digital pcr, Sanger DNA sequencings and high flux DNA sequencing (" Next-generation " Sequencing) etc..In these above-mentioned nucleic acid detection methods, best suited with high flux DNA sequencing take into account broad spectrum activity, specificity, Sensitivity and can detect early-stage cancer these technical requirements from blood, high throughput sequencing technologies are to tradition sequencing revolution Property change, sequencings are once carried out to hundreds of thousands to millions of DNA moleculars so as to the transcript profile and base of a tumour Because a group analysis for the careful overall picture of progress is possibly realized.

The content of the invention

The invention provides a kind of system, comprehensive, reliable and reduction person under inspection's burden to kinds of tumors mark Thing carries out the accurately method of systematicness detection and comprehensive analysis interpretation, and it is applied to examine the clinic of Diagnostic Value of Several Serum Tumor Markers Survey, people at highest risk's examination and the generaI investigation of general population.Wherein, the systematicness detection includes carrying out polygenes, more to person under inspection The high-throughout measure of characteristic, carry out the measure of Diagnostic Value of Several Serum Tumor Markers and carry out using the inspection of medicine equipment and equipment to person under inspection Look into.Wherein described comprehensive analysis interpretation method include the Clinical questionnaire of comprehensive person under inspection, main suit, symptom, sign, family history, Clinical main suit, clinical symptoms, clinical sign, the high-throughout measurement result of the more characteristics of polygenes, the measure of Diagnostic Value of Several Serum Tumor Markers As a result comprehensive analysis and using medicine equipment and equipment is carried out to the inspection result of person under inspection.By means of the invention it is also possible to Subject sample is once taken in realization, by systematicness detection and comprehensive analysis interpretation method, person under inspection is carried out a variety of swollen Tumor markers accurately determine.

The invention provides a kind of systemic detection method of tumor markers, methods described includes：

1) it is directed to and biological specimen or its extract is carried out using one or more nucleotide sequences with tumour-specific High flux detects；

2) biological specimen or its extract are detected for one or more protein tumor markerses；

3) testing result of the high flux testing result of step 1) and step 2) is combined, as systemic overall result；

Wherein, the biological specimen be selected from cell line, Histological section, organize the tissue of biopsy/FFPE, body fluid, Excrement, colonic effluent, urine, blood plasma, serum, whole blood, separation haemocyte, separated from blood cell, body fluid, sputum, Throat swab, or its combination；Be preferably chosen from colonic effluent, urine, blood plasma, serum, whole blood, separation haemocyte, by blood One or more in the cell of separation；

The nucleotide sequence with tumour-specific is selected from the mutator related to tumour generation, phase occurs with tumour The decorating state gene of pass, the recombinant DNA related to tumour generation, RNA, the copy number of the Fusion Strain related with tumour generation Amount changes the DNA related to tumour generation and RNA, related suppression tiny RNA (siRNA) occurs with tumour, phase occurs with tumour The one or more of the Microrna (microRNA) of pass or the non-transcribed RNA related to tumour generation nucleotide sequence；

The protein tumor markers is selected from the protein with tumour-specific, the albumen first related to tumour generation One kind in baseization modification or the Mechanisms of Histone Acetylation Modification related to tumour generation.

According in one embodiment of the invention, the mutator related to tumour generation be selected from KRAS, One or more in BRAF, EGFR；

Preferably, the decorating state gene related to tumour generation is that related methylated genes occur with tumour, Preferably described methylated genes be selected from GNG4, MIAT, DNM3, CHST2, HOXA9, C1orf70, NBLA00301, SIX6, OLIG2、SIM2、C9orf50、LONRF2、COL4A1、ADHFE1、ITGA4、SEPT9、CTSF、FAM159A、ZNF583、 EFHA2、ARHGAP22、CYP1B1、PPFIA4、SPAG6、RNF135、EFNB2、TRIL、LDHB、IGF1R、HOXD8、 HOXA11AS、HOXD9、SHOX2、CYBA、AOX1、AMOTL2、C2orf88、WFDC2、SIM1、GHSR、ZNF154、OXT、 WDR69、RNF180、C7orf51、ID3、VWCE、CRYGN、C10orf41、ARRDC2、AIM1、RAI1、GABRG3、PTGFR、 ZNF805、CCNA1、BCAN、RNLS、HOXD1、ELOVL5、JAKMIP1、CACNB2、PAX2、MCF2L、PDE4D、MAST4、 CHD3、PLIN1、PAK1、PROC、TFR2、PITPNM3、WNT7B、PTPRU、NDRG4、HOXXD10、NT5DC3、WNT3A、 One kind in UBXN10, CDH22, LYPLAL1, F11R, TMEM101, PYY, TERC, PTGER4, FOXL2, BNC1, ADAMTS1 It is or a variety of；

Preferably, the Microrna related to tumour generation is selected from hsa-let-7c, hsa-miR-122, hsa-miR- 182、hsa-miR-193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、hsa-let-7b、 hsa-miR-411、hsa-miR-450b、hsa-miR-485、hsa-miR-519a、hsa-miR-642、hsa-miR-517b、 hsa-miR-520f、hsa-miR-206、hsa-miR-566、hsa-miR-661、hsa-miR-340、hsa-miR-1243、 One or more in hsa-miR-720, hsa-miR-543 and hsa-miR-1267.

It is further preferred that the nucleotide sequence with tumour-specific be selected from KRAS, BRAF, EGFR, RNF180, SHOX2、PTGER4、FOXL2、GNG4、C1orf70、C9orf50、CTSF、CYP1B1、RNF135、HOXD8、CYBA、SIM1、 C7orf51, ARRDC2, GABRG3, RNLS, PAX2, PROC, PITPNM3, HOXXD10, LYPLAL1, hsa-miR-122 and One or more in hsa-let-7b；The assay method of the preferably described nucleotide sequence with tumour-specific be selected from PCR, Real-time quantitative PCR, digital pcr, liquid chip, solid-state chip, in situ hybridization, common sequencing and one kind in high-flux sequence or It is a variety of；

Protein tumor markers described in step 2) is carcinomebryonic antigen (carcino-embryonic antigen CEA), IDH1, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, CA125, CA50, PSA, FPSA/TPSA、NSE、SCC、proGRP、PG、PG I/PG II、TPA、TPS、Beta-HCG、SF、H3K4me1、H3K4me3、 H4K8ac, Auto-ab p53, Auto-Ab MMP-7, Auto-Ab Hsp70, Auto-Ab Prx VI, Auto-Ab Bmi-1 and β-HCG；The assay method of preferably described protein tumor markers is selected from liquid chip, solid-state chip, enzyme linked immunological, radiation One or more in immune, chemiluminescence immunoassay, mass spectrum, high performance liquid chroma- tography, the Western markings and sequencing.

According in one embodiment of the invention, step 1) is to determine the sample extraction thing by high-flux sequence In the mutator related to tumour generation, the preferentially one kind or more of the mutator in KRAS, BRAF and EGFR Kind；

Protein tumor markers described in step 2) is carcinomebryonic antigen (carcino-embryonic antigen CEA), IDH1, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, CA125, CA50, PSA, FPSA/TPSA、NSE、SCC、proGRP、PG、PG I/PG II、TPA、TPS、Beta-HCG、SF、H3K4me1、H3K4me3、 H4K8ac, Auto-ab p53 and β-HCG；The assay method of preferably described protein tumor markers is selected from liquid chip, consolidated In state chip, enzyme linked immunological, radio-immunity, chemiluminescence immunoassay, mass spectrum, high performance liquid chroma- tography, the Western markings and sequencing It is one or more.

According in one embodiment of the invention, the gene methylation with tumour-specific is examined described in step 1) The nucleotide sequence of survey be selected from GNG4, MIAT, DNM3, CHST2, HOXA9, C1orf70, NBLA00301, SIX6, OLIG2, SIM2, C9orf50、LONRF2、COL4A1、ADHFE1、ITGA4、SEPT9、CTSF、FAM159A、ZNF583、EFHA2、ARHGAP22、 CYP1B1、PPFIA4、SPAG6、RNF135、EFNB2、TRIL、LDHB、IGF1R、HOXD8、HOXA11AS、HOXD9、SHOX2、 CYBA、AOX1、AMOTL2、C2orf88、WFDC2、SIM1、GHSR、ZNF154、OXT、WDR69、RNF180、C7orf51、ID3、 VWCE、CRYGN、C10orf41、ARRDC2、AIM1、RAI1、GABRG3、PTGFR、ZNF805、CCNA1、BCAN、RNLS、 HOXD1、ELOVL5、JAKMIP1、CACNB2、PAX2、MCF2L、PDE4D、MAST4、CHD3、PLIN1、PAK1、PROC、TFR2、 PITPNM3、WNT7B、PTPRU、NDRG4、HOXXD10、NT5DC3、WNT3A、UBXN10、CDH22、LYPLAL1、F11R、 One or more in TMEM101, PYY, TERC, PTGER4, FOXL2, BNC1, ADAMTS1；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to breast cancer tissue：1~5；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to Bladder Cancer：6~10；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to intestinal cancer tissue：11~16；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to esophageal cancer tissue：17~21；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to renal carcinoma tissue：22~24；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to liver cancer tissue：25~29；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to cancerous lung tissue：30~34；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to prostate cancer tissue：35~40；

Preferably, nucleotide sequence corresponding to the cancerous tissue of uterus is SEQ ID NO：41~45；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to stomach organization：46；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to breast tissue：47~51；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to bladder body：52~56；

Preferably, nucleotide sequence corresponding to its midgut tissue is SEQ ID NO：57~61；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to esophageal tissue：62~66；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to nephridial tissue：67~71；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to hepatic tissue：72~76；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to lung tissue：77~80；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to prostata tissue：81~85；

Preferably, nucleotide sequence is SEQ ID NO wherein corresponding to uterine tissue：86~90.

The assay method of the preferably described nucleotide sequence for being used for gene methylation detection with tumour-specific is selected from One in PCR, real-time quantitative PCR, digital pcr, liquid chip, solid-state chip, in situ hybridization, common sequencing and high-flux sequence Kind is a variety of；

According in one embodiment of the invention, step 1) is by described in liquid chip or high-flux sequence measure The Microrna related to tumour generation in sample extraction thing, the Microrna be selected from hsa-let-7c, hsa-miR-122, hsa-miR-182、hsa-miR-193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、 hsa-let-7b、hsa-miR-411、hsa-miR-450b、hsa-miR-485、hsa-miR-519a、hsa-miR-642、hsa- miR-517b、hsa-miR-520f、hsa-miR-206、hsa-miR-566、hsa-miR-661、hsa-miR-340、hsa- One or more in miR-1243, hsa-miR-720, hsa-miR-543 and hsa-miR-1267；Albumen described in step 2) Matter tumor markers is carcinomebryonic antigen (carcino-embryonic antigen CEA), IDH1, CA724, alpha-fetoprotein (AFP)、CA242、CA199、Cyfra21-1、CA153、CA125、CA50、PSA、FPSA/TPSA、NSE、SCC、proGRP、PG、 PG I/PG II, TPA, TPS, Beta-HCG, SF, H3K4me1, H3K4me3, H4K8ac, Auto-ab p53 and β-HCG；It is preferred that The assay method of the ground protein tumor markers is selected from liquid chip, solid-state chip, enzyme linked immunological, radio-immunity, chemistry One or more in electrochemiluminescent immunoassay, mass spectrum, high performance liquid chroma- tography, the Western markings and sequencing.

According in one embodiment of the invention, the nucleotide sequence with tumour-specific is selected from described in step 1) KRAS、BRAF、EGFR、RNF180、SHOX2、PTGER4、FOXL2、GNG4、C1orf70、C9orf50、CTSF、CYP1B1、 RNF135、HOXD8、CYBA、SIM1、C7orf51、ARRDC2、GABRG3、RNLS、PAX2、PROC、PITPNM3、HOXXD10、 One or more in LYPLAL1, hsa-miR-122, hsa-let-7b；The preferably described nucleic acid sequence with tumour-specific The assay method of row be selected from PCR, real-time quantitative PCR, digital pcr, liquid chip, solid-state chip, in situ hybridization, common sequencing and One or more in high-flux sequence；

Step 2) is that the carcinomebryonic antigen (carcino- in the sample extraction thing is determined by enzyme linked immunoassay embryonic antigen CEA)。

Step 2) determines the protein tumor markers in the sample extraction thing, institute by liquid chip or solid-state chip State protein tumor markers and be selected from carcinomebryonic antigen (carcino-embryonicantigen CEA), CA724, alpha-fetoprotein (AFP), the one or more in CA242, CA199, Cyfra21-1, CA153, CA125, PSA and Beta-HCG.

Step 2) passes through sample extraction thing described in liquid chip or solid-state chip enzyme linked immunological or chemiluminescence immunoassay In protein tumor markers, the protein tumor markers be selected from Auto-Ab p53, Auto-Ab MMP-7, Auto- One or more in Ab Hsp70, Auto-Ab Prx VI and Auto-Ab Bmi-1.

Step 2) passes through the protein tumor markers in sample extraction thing described in liquid chip or mass spectroscopy, the egg One or more of the white matter tumor markers in H3K4me1, H3K4me3 and H4K8ac.

One or more of protein tumor markers described in step 2) in CEA, CA199, CA242 and CA724； Preferably, described CEA, CA199, CA242 and/or CA724 pass through ELISA reaction assays；It is highly preferred that step 2) also includes blood The detection of Lactoferrin, it is further preferred that the hemoglobin is determined by immunochromatography FIT method；

According in one embodiment of the invention, the nucleotide sequence with tumour-specific is selected from described in step 1) KRAS、BRAF、EGFR、RNF180、SHOX2、PTGER4、FOXL2、GNG4、C1orf70、C9orf50、CTSF、CYP1B1、 RNF135、HOXD8、CYBA、SIM1、C7orf51、ARRDC2、GABRG3、RNLS、PAX2、PROC、PITPNM3、HOXXD10、 LYPLAL1、hsa-miR-122、hsa-let-7bhsa-let-7c、hsa-miR-122、hsa-miR-182、hsa-miR- 193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、hsa-let-7b、hsa-miR-411、 One or more in hsa-miR-450b, hsa-miR-485, hsa-miR-519a and hsa-miR-642；Preferably described tool The assay method for having the nucleotide sequence of tumour-specific is high-flux sequence；

Swollen in step 2) by the protein in sample extraction thing described in liquid chip, solid-state chip or enzyme-linked immunoassay Tumor markers, one or more of the protein tumor markers in Cyfra 21-a, NSE, SCC and IDH1.

On the other hand, the invention provides the kit for the above method, the kit includes measure tomour specific Property nucleotide sequence required for primer, probe and/or reagent, and measure protein tumor markers required for reagent；

Wherein, the nucleotide sequence with tumour-specific is selected from the mutator and tumour related to tumour generation The RNA of the Fusion Strain of the related decorating state gene of generation, the recombinant DNA related to tumour generation and tumour generation correlation, Number of copies changes the DNA related to tumour generation and RNA, related suppression tiny RNA (siRNA) and tumour occurs with tumour The one or more of related Microrna (microRNA) or the non-transcribed RNA related to tumour generation nucleotide sequence occur；

Preferably, one or more of the mutator related to tumour generation in KRAS, BRAF, EGFR；

Preferably, the decorating state gene related to tumour generation is that related methylated genes occur with tumour, Preferably described methylated genes be selected from GNG4, MIAT, DNM3, CHST2, HOXA9, C1orf70, NRLA00301, SIX6, OLIG2、SIM2、C9orf50、LONRF2、COL4A1、ADHFE1、ITGA4、SEPT9、CTSF、FAM159A、ZNF583、 EFHA2、ARHGAP22、CYP1B1、PPFIA4、SPAG6、RNF135、EFNB2、TRIL、LDHB、IGF1R、HOXD8、 HOXA11AS、HOXD9、SHOX2、CYBA、AOX1、AMOTL2、C2orf88、WFDC2、SIM1、GHSR、ZNF154、OXT、 WDR69、RNF180、C7orf51、ID3、VWCE、CRYGN、C10orf41、ARRDC2、AIM1、RAI1、GABRG3、PTGFR、 ZNF805、CCNA1、BCAN、RNLS、HOXD1、ELOVL5、JAKMIP1、CACNB2、PAX2、MCF2L、PDE4D、MAST4、 CHD3、PLIN1、PAK1、PROC、TFR2、PITPNM3、WNT7B、PTPRU、NDRG4、HOXXD10、NT5DC3、WNT3A、 One kind in UBXN10, CDH22, LYPLAL1, F11R, TMEM101, PYY, TERC, PTGER4, FOXL2, BNC1, ADAMTS1 It is or a variety of；

Preferably, the Microrna related to tumour generation is selected from hsa-let-7c, hsa-miR-122, hsa-miR- 182、hsa-miR-193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、hsa-let-7b、 hsa-miR-411、hsa-miR-450b、hsa-miR-485、hsa-miR-519a、hsa-miR-642、hsa-miR-517b、 hsa-miR-520f、hsa-miR-206、hsa-miR-566、hsa-miR-661、hsa-miR-340、hsa-miR-1243、 One or more in hsa-miR-720, hsa-miR-543 and hsa-miR-1267；

It is highly preferred that the nucleotide sequence with tumour-specific be selected from KRAS, BRAF, EGFR, RNF180, SHOX2, PTGER4、FOXL2、GNG4、C1orf70、C9orf50、CTSF、CYP1B1、RNF135、HOXD8、CYBA、SIM1、C7orf51、 ARRDC2、GABRG3、RNLS、PAX2、PROC、PITPNM3、HOXXD10、LYPLAL1、hsa-miR-122、hsa-let- 7bhsa-let-7c、hsa-miR-122、hsa-miR-182、hsa-miR-193a、hsa-miR-200c、hsa-miR-203、 hsa-miR-218、hsa-miR-155、hsa-let-7b、hsa-miR-411、hsa-miR-450b、hsa-miR-485、hsa- One or more in miR-519a and hsa-miR-642；

Preferably, the protein tumor markers is selected from carcinomebryonic antigen (carcino-embryonic antigen CEA), IDH1, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, CA125, CA50, PSA, FPSA/TPSA、NSE、SCC、proGRP、PG、PG I/PG II、TPA、TPS、Beta-HCG、SF、H3K4me1、H3K4me3、 H4K8ac, Auto-ab p53Auto-Ab MMP-7, Auto-Ab Hsp70, Auto-Ab Prx VI, Auto-Ab Bmi-1 and One or more in β-HCG；

It is highly preferred that the kit includes determining the egg in the sample extraction thing by liquid chip or solid-state chip White matter tumor markers carcinomebryonic antigen, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, CA125, The reagent required for one or more in PSA or Beta-HCG；Or

The kit is included by sample described in liquid chip or solid-state chip enzyme linked immunological or chemiluminescence immunoassay Protein tumor markers Auto-Ab p53, Auto-Ab MMP-7, Auto-Ab Hsp70, Auto-Ab in product extract The reagent required for one or more in Prx VI and Auto-Ab Bmi-1；Or

The kit is included by the protein tumor-marker in sample extraction thing described in liquid chip or mass spectroscopy The reagent required for one or more in thing H3K4me1, H3K4me3 and H4K8ac；Or

The kit includes passing through ELISA reaction assay protein tumor markerses CEA, CA199, CA242 and CA724 In one or more required for reagent；

It is further preferred that the kit also includes the reagent needed for measure hemoglobin；It is further preferred that institute It is that the reagent needed for hemoglobin is determined by immunochromatography FIT method to state reagent.

The invention has the advantages that：

The invention provides a kind of system it is comprehensively reliable and and reduce person under inspection's burden to kinds of tumors accurately system Property detection and comprehensive analysis interpretation method, applied to the clinical detection to kinds of tumors, people at highest risk's examination and general population GeneraI investigation.Wherein described systemic detection includes carrying out person under inspection the high-throughout measure of the more characteristics of polygenes, carries out a variety of swell The measure of tumor markers and carry out using the inspection of medicine equipment and equipment to person under inspection.Wherein described comprehensive analysis interpretation Method includes Clinical questionnaire, main suit, symptom, sign, family history, clinical main suit, clinical symptoms, the clinical body of comprehensive person under inspection Sign, the high-throughout measurement result of the more characteristics of polygenes, the measurement result of Diagnostic Value of Several Serum Tumor Markers and use medicine equipment and equipment Comprehensive analysis is carried out to the inspection result of person under inspection.Subject sample is once taken by means of the invention it is also possible to realize, is led to The more characteristic high flux detections of polygenes and comprehensive analysis interpretation method are crossed, kinds of tumors is carried out to person under inspection and accurately determined.

Another aspect of the present invention, there is provided a kind of comprehensive analysis interpretation method and application.Because the present invention is pair Infantile tumour is detected, and person under inspection is Silent cerebral infarction mostly, collection and the clinical information with reference to person under inspection, including is asked Volume, main suit, symptom, sign, age, sex, body weight, smoking history, family history, infecting medical history, diabetic history etc. can be to group Conjunction property testing result provides guidance quality clue, there is provided to comprehensive analysis interpretation method and application.

The present invention is needed to gather and refer to other clinical adjunct test results, and especially associativity testing result prompting is asked In the case that topic is, it is necessary to further examine, other clinical adjunct tests are used.Other clinical adjunct test methods include image Learn the inspection with hysteroscope.Iconography detection method includes X-ray examination, CT, MRI and ultrasonic wave etc., and hysteroscope inspection includes gastroscope, intestines Mirror, laparoscope, ERCP, bronchoscope, thoracoscope, cystoscope, hysteroscope etc..

, be according to the result detected in the present invention to the associativity of kinds of tumors, with reference to being examined when analyzing testing result The clinical information of person and other clinical adjunct test results, comprehensive analysis and interpretation are carried out to lesion detection result.These are just Constitute the present invention to kinds of tumors accurately associativity detection and comprehensive analysis interpretation method and application.

The present invention also to gather and the clinical information with reference to person under inspection, including questionnaire, main suit, symptom, sign, age, property Not, body weight, smoking history, family history, medical history, diabetic history etc. is infected.Also to gather and with reference to other clinical adjunct test knots Fruit, such as the inspection of iconography and hysteroscope.When analyzing testing result, the systematicness of kinds of tumors is detected according in the present invention Result, with reference to person under inspection clinical information and other clinical adjunct test results, comprehensive point is carried out to lesion detection result Analysis and interpretation.These just constitute the present invention to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and Using.

Brief description of the drawings

Fig. 1 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- overall project；

Fig. 2 are to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- liquid biopsy；

Fig. 3 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- gene mutation group Close；

Fig. 4 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- gene methylation Combination；

Fig. 5 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- Microrna group Close；

Fig. 6 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- Septin9+；

Fig. 7 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- CEA+；

Fig. 8 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- ten big tumour marks Will thing combines；

Accurately systematicness detection and comprehensive analysis interpretation method and application --- tumour itself is anti-to kinds of tumors by Fig. 9 Body combines

To kinds of tumors, accurately systematicness is detected Figure 10 and comprehensive analysis interpretation method and application --- histone is repaiied Decorations

Figure 11 to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- colorectal cancer Joint inspection；

Figure 12 are to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- lung cancer early stage Diagnosis, medication guide, monitoring recurrence.

Embodiment

The present invention detects DNA, RNA, Microrna and protein etc. simultaneously on same sample with high-throughout method The inspection of system property.Apply and check tumour in system medicine and systematicness, reach the purpose of tumour early screening.It is representational specific Experimental method be provided with 46 methylated genes do high-flux sequence and/or provide 21 Micrornas do high-flux sequence, simultaneously Tens tumours are detected using protein-chip high flux.Business can be used in high-flux sequence and high throughput protein chip product Industry product, with reference to its corresponding product specification.

Protein tumor markers used in the present invention has enhancing sensitive with corresponding nucleic acids marker combine detection The evidence of the synergy of the technique effects such as degree, specificity is shown in Examples 1 and 2.Embodiment 1 is to detect nucleic acid and detection egg simultaneously The tumor markers of white matter, the overall detection sensitivity of tumour improves 17%, and the detection sensitivity of infantile tumour improves 18%. Embodiment 2 is to detect different nucleic acid tumor markerses simultaneously, the overall detection sensitivity raising 18% of tumour, and infantile tumour Detection sensitivity improve 10%.Above-mentioned testing result is obtained according to clinical large sample, to confirm the reliability of the present invention.Fig. 1 To 12 new invention exactly proposed on the basis of the embodiment above.

The present invention is further illustrated with reference to embodiment, it will be appreciated that embodiment is only used for further illustrating and explained The present invention, it is not intended to limit the present invention.

Unless specifically stated otherwise, reagent and material used in the present invention can be obtained by commercial sources.

Unless otherwise indicated, the implementation of the application is by using conventional molecular biology (including recombinant technique), microorganism , cell biology, biochemistry and genetics technology, it is in the range of the conventional technical means of this area.In the literature Such technology is described in detail such as Molecular Cloning：A Laboratory Manual, the second edition (Sambrook etc., 1989)；Oligonucleotide Synthesis (M.J.Gait, 1984 editions)；Animal Cell Culture (R.I.Freshney, 1987 editions)；Methods in Enzymology book series (the limited public affairs in academic press of the U.S. Department)；Current Protocols in Molecular Biology (F.M.Ausubel etc., 1987 editions, and regularly update)； PCR：The Polymerase Chain Reaction (Mullis etc., 1994 editions).Primer used herein, probe and Kit can use standard technique well known in the art to prepare.

Unless otherwise defined, technology used in this application and scientific terminology and ordinary skill people of the art Member's is generally understood that with identical implication.Singleton etc., Dictionary of Microbiology and Molecular Biology, the second edition, J.Wiley＆Sons (New York, N.Y.1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, fourth edition, John Wiley＆Sons (New York, N.Y.1992), provided for multiple terms used herein for those skilled in the art general Instruct.

Definition

" cancer " of the application represents and including any malignant tumour (malignancy) or malignant cell division or pernicious Tumour (malignant tumour), or any morbid state with uncontrolled or unsuitable cell propagation, and Include but is not limited to any disease for being characterized as uncontrolled or unsuitable cell propagation.

" lung cancer (the 1ung cancer) " of the application, is a kind of malignant tumour of lung, is characterized as thin in lung tissue Born of the same parents uncontrollably grow.Lung cancer is broadly divided into ED-SCLC (small-cell lungcarcinoma, SCLC) and non-small Cell lung cancer (non-small-cell lung carcinoma, NSCLC).

" lung carcinoma cell " of the application represents the cell with lung cancer feature, and including pre-cancerous cells.

" pre-cancer " of the application represents in the early stage for being converted into cancer cell or tends to be converted into cancer cell Cell.Such cell can show one or more phenotypic characters with cancer cell feature.

" biomarker " in the application refers to a kind of material of such as gene, the variable related to disease measure, Can be as the indicator or predictive factor of that disease.The presence of disease or risk can be from this parameters of biomarker It is inferred to, it is not necessary to determine disease in itself.

" nucleic acid ", " nucleotide sequence " in the application etc., refer to polynucleotide, can be gDNA, cDNA or RNA, Can also be single-stranded or double-stranded.Term also includes peptide nucleic acid (PNA), or any chemical DNA classes or RNA class materials. " cDNA " refers to the DNA replicated from the mRNA naturally occurred." gDNA " refers to genomic DNA.These materials can also be made Combination (be partly gDNA and part be cDNA recombinant nucleic acid).

" DNA methylation " of the application refers to that methyl is added to 5 of cytimidine (C), this generally (but necessarily) be In the case of CpG (being guanine after cytimidine) dinucleotides." increased methylation " used herein or " significant Methylation " refers to a C nucleotide to methylate in DNA sequence dna at least be present, and wherein normal reference sample is (such as from non- The DNA sample or the DNA sample that is handled of methylating to DNA residues of cancer cell or tissue sample extraction) in corresponding C be Non- to methylate, in certain embodiments, at least 2,3,4,5,6,7,8,9,10 or more C can methylate, its The C of these positions in middle comparison DNA sample is non-methylates.

In embodiments, a variety of different methods can be used for detection DNA methylation to change.Detect the side of DNA methylation Method includes, for example, the restriction endonuclease for the methyl-sensitive analyzed using southern or polymerase chain reaction (PCR) (MSRE) the mononucleotide primer extension of the PCR (MS-PCR) of measure, methylation-specific or methyl-sensitive, methyl-sensitive (Ms-SnuPE), high-resolution melts (HRM) analysis, bisulfite sequencing, pyrosequencing, the single-stranded structure of methylation-specific Picture analysis (MS-SSCA), combination bisulfite restriction analysis (COBRA), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE), methylation-specific melting curve analysis (MS-MCA), methylation-specific denaturing high-performance chromatography (MS- DHPLC), methylation-specific microarray (MSO).These measure can be PCR analysis, using fluorescence labeling quantitative analysis or The southern markings are analyzed.

" methylation assay " of the application, which refers to, determines that one or more CpG dinucleotides sequences methylate in DNA sequence dna Any measure of state.

" biological sample " or " sample " of the application includes the histotomy of such as biopsy and autopsy samples and in order to organize Form after the processing of the freezing microtome section or these any samples learning purpose and obtain.Biological sample includes blood and blood constitutent Or product (such as serum, blood plasma, blood platelet, red blood cell etc.), sputum or saliva, lymph and tongue tissue, such as primary culture, The cell of the culture of explant and the cell of conversion, excrement, urine, gastric biopsy etc..Biological sample is generally obtained from eucaryon and given birth to Thing, the eucaryote can be mammals, can be primate and can be human individuals.

" biopsy " of the application refers to the process of, in order to diagnose or prognosis evaluation takes out tissue sample, and also refer to tissue sample Sheet itself.Any Biopsy known in the art can be used for diagnosis and the method for prognosis of the present invention.Biopsy skill used Art depends on organization type to be assessed (such as tongue, colon, prostate, kidney, bladder, lymph node, liver, marrow, haemocyte, stomach Tissue etc.) etc. factor.Representational Biopsy includes but is not limited to Biopsy, cuts biopsy, needle biopsy, open surgical biopsy And bone marrow biopsy, and colonoscopy can be included.A variety of Biopsies are that well known to a person skilled in the art they need Carrying out seldom experiment can be to select and use from these technologies.

" separation " nucleic acid molecules of the application are represented from other nucleic acid generally associated with the nucleic acid molecules that this is separated The nucleic acid molecules isolated in molecule.Therefore, " separation " nucleic acid molecules include but is not limited to such nucleic acid molecules：It does not have Have nucleic acid source in separation in organism genome in natively flank connect one or two end of the nucleic acid Sequence (for example, by PCR or restriction endonuclease digestion and caused cDNA or genomic DNA fragment).Generally by this The nucleic acid molecules of the separation of sample introduce carrier (for example, cloning vector or expression vector), in order to manipulate or produce integrative nucleic acid Molecule.In addition, separation nucleic acid molecules can include engineering nucleic acid molecules, such as restructuring or synthesis nucleic acid molecules. It is present in such as nucleic acid library (such as cDNA or genomic library) or the gel (example of the genomic DNA containing restrictive digestion Such as, agarose or polyacrylamide) a part in hundreds of to millions of other nucleic acid molecules in nucleic acid molecules do not recognized For be separation nucleic acid.

" cell " of the application can be separation, can be contained in cell colony, can be in culture, or can It to be comprised in individual living, and can be mammalian cell, and can be the cell of people.Equally, " tissue " can be with Including any number of cell, and it can be contained in individual living or can be separated therefrom.

" detection " of the application represents mark in observation biological sample or mark changes that (such as mark methylates The change of state or the expression of nucleic acid or protein sequence) any process, no matter actually whether detect mark or Mark changes.In other words, it is " detection " to detect the behavior that the mark of sample or mark change, even if mark is measured To be not present or less than level of sensitivity.Detection can be quantitative, sxemiquantitative or non-quantitation observation, and can be based on and one Or the comparison of multiple control samples.It should be appreciated that detecting lung cancer disclosed herein includes detection pre-cancerous cells, the cancer Progenitor cells start to develop into lung carcinoma cell or will develop into lung carcinoma cell, or develop into inclining for lung carcinoma cell with increased To.Detect the possible prognosis that lung cancer can also include detecting possible probability of death or disease conditions.

Technology as " polymerase chain reaction " or " PCR " expression of the application：Denaturation and the annealing of primer and and DNA The circulation of the extension of polymerase be used to the copy number of target DNA sequence being expanded to about 10⁶Times or more.For amplification of nucleic acid Pcr process can be found in U.S. Patent No. No. 4,683,195 and No. 4,683,202.

" sensitivity " expression of the application detects the ratio of cancer from certain cancer sample, and its calculation formula is：Spirit Sensitivity=(cancer detected/all cancer), and " specificity " represents to detect normally to compare in certain normal person's sample Example, its calculation formula are specificity=(undetected negative/total feminine gender).

A variety of distinct methods detection nucleic acid molecules can be used.Nucleic acid detection method includes, for example, PCR and nucleic acid hybridization (for example, Southern traces, Northern traces or in situ hybridization).Specifically, the oligonucleotides of target nucleic acid can be expanded (for example, Oligonucleolide primers) can be used for PCR reactions.PCR method generally includes following steps：Obtain sample, from the sample Product seperated nuclear acid (for example, DNA, RNA or both) and the nucleic acid is set to be contacted with one or more Oligonucleolide primers, it is described to draw Thing specifically hybridizes under conditions of template nucleic acid amplification can occur with template nucleic acid.In the presence of template nucleic acid, production Raw amplified production.Nucleic acid amplification and the condition of amplified production detection are well known by persons skilled in the art.It is a variety of right to have developed In the improvement of basic round pcr, include but is not limited to, anchor PCR, RACE PCR, RT-PCR and ligase chain reaction (LCR).Primer pair in amplified reaction must the relative chain annealing with template nucleic acid, and should keep each other suitably away from From so that polymerase can effectively be polymerize across region and allow to for example easily detect amplification production using electrophoresis Thing.It is, for example, possible to use such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) meter Calculation machine program carrys out design oligonucleotides primer, to help primer of the design with similar melting temperature.Generally, Oligonucleolide primers Length be 10-30 or 40 or 50 nucleotides (for example, length be 10,11,12,13,14,15,16,17,18,19,20,21, 22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、 47th, 48,49 or 50 nucleotides), but Oligonucleolide primers can be with longer or shorter, as long as using suitable amplification condition.

It is one group of highly conserved GTP knot that " the SEPT9 genes " of the application, which is located at chromosome 17q25.3, septin families, Hop protein, it is widely present in human cell.They provide structural support in fission process.In the mankind, a total of 13 Septin genes, 15 polypeptides of corresponding encoded.All septin can form different poly- compound and participate in the composition of higher structure, Such as filament, the formation of ring shape structure and cage structure.These unique structures be control cell processes necessary to. It is in actin dynamics, angiogenesis, cell movement, cell propagation, cell shape, cytokinesis, micro-pipe regulation, capsule Bubble targeting in terms of exocytosis with having important physiological action.

The application " carcinomebryonic antigen, CEA " they are a kind of glycoprotein caused by Colorectal Carcinoma, can cause trouble as antigen The immune response of person.Such a antigen is referred to as carcinomebryonic antigen (carcino-embryonic antigen CEA), can be widely present in The Alimentary System of endoderm origin, exists in the digestion tubing of normal fetus, can also have in normal human serum micro- Amount is present.Carcinomebryonic antigen is a broad spectrum activity tumor markers, and it can reflect the presence of kinds of tumors to people, to Colon and rectum The judgement of the effect of cancer, breast cancer and lung cancer, disease development, monitoring and prognosis estimation are a preferable tumor markerses, but its Specificity is not strong, and sensitivity is not high, and unobvious are acted on to early diagnosis of tumor.

The application " CA19-9 " and be a kind of carbohydrate protein tumor mark of mucin type, be cell membrane on glycolipid Matter, molecular weight are more than 1000kD.It is hitherto reported to cancer of pancreas sensitiveness highest mark.It is glued with saliva in serum Protein form is present, and is distributed in normal fetus pancreas, gall-bladder, liver, intestines and normal adult human pancreas, epithelial duct etc..It is to deposit It is sanguimotor gastroenteric tumor related antigen.

" CA242 " of the application is a kind of sialylated sugar antigen, can be by colon cancer cell line through hybridoma technology A series of one of obtained monoclonal antibodies CA242 identified, it is in mucoprotein that it, which is that one kind is present in multiple organ malignant tumour, The glycoprotein of type can not can not react CanAg with LewisA type antigen-reactives with sialylated galactoside. Immunochemistry research has shown that it is different from other known tumor-associated mucin such as：CA199、CA50、CA125、CA153 Deng content is relatively low in Healthy People and benign disease serum.CA242 is to be applied to a kind of clinical newer tumor-marker in recent years Thing, cancer of pancreas and the preferable tumor markers of colon cancer.

" CA72-4 " of the application is a kind of mucinoid HMW sugar by two plants of monoclonal antibody identifications of cc49 and B72.3 Albumen, molecular weight 220-400KD, content ＜ 6U/mL in normal human serum, abnormal rise is in various tumor in digestive tract, ovary Cancer can produce.Detection specificity for stomach cancer is higher, using ＞ 6U/mL as critical value.Benign stomach disease only ＜ is raised, And stomach cancer rise person ratio such as detects, positive rate is up to 56% simultaneously up to 42.6% with CA19-9.

The application " alpha-fetoprotein, AFP " they are a kind of glycoprotein, and under normal circumstances, this albumen is essentially from embryo's Liver cell, about two weeks alpha-fetoproteins disappear from blood after fetal birth, thus in normal human serum alpha-fetoprotein content still Less than 20 micrograms per litres.

The application " ferritin, SF " they are a kind of solvable histone for storing iron in body, are contained in normal human serum A small amount of ferritin, but different detection methods has different normal values, normally average male about 80-130ug/L (80- 130ng/ml) women about 35-55ug/L (35-55ng/ml), serum iron levels reduce in the gestational period and acute anemia, anxious slow Property liver damage and raise during liver cancer, country's report liver cancer patient positive rate is up to 90%.

" prostate specific antigen (Prostate the Specific Antigen, PSA) " of the application is by prostatic acini With a kind of single chain glycoprotein of the epithelial cells of conduit, functionally belong to a kind of serine stretch protein of class kallikrein Enzyme, the liquefaction process of seminal fluid is participated in, be that routine clinical to be used for prostate benign with malignant disease Diagnosis and differential diaggnosis and forefront The important indicator of adenocarcinoma patients' Follow-up After.

" β2-microglobulin (the β 2-microglobulin, p2-m) " of the application is expressed on most of karyocyte surfaces. Clinically it is used for diagnosing lymphoproliferative disease, such as leukaemia, lymthoma and Huppert's disease.Its horizontal and tumour cell Quantity, growth rate, prognosis and Disease Activity are relevant.In addition, patients with malignant myeloma can be additionally used in by stages according to this level.Serum β 2-MG be able to can increase in renal failure, inflammation and a variety of diseases.Therefore it should exclude due to some diseases associated with inflammation or kidney Serumβ 2-MG caused by bead filtering function lowers increases.

The application " cancer antigen 50, also known as CA50, sugar chain antigen 50, be one kind with saliva acid esters and sialic acid sugar egg Bai Weizhu glycoprotein, and a kind of tumour antigen.Cancer antigen 50 is a kind of nonspecific broad-spectrum tumor mark, is resisted with cancer Former 19-9 has certain cross antigenicity, is mainly used in cancer of pancreas, the colon/carcinoma of the rectum, the wherein auxiliary diagnosis of stomach cancer, cancer of pancreas Patient increases most obvious.Increase：See cancer of pancreas (positive rate is up to 87%), the colon/carcinoma of the rectum, stomach cancer, lung cancer.Liver cancer.Ovum The malignant tumours such as nest cancer, breast cancer；Ulcerative colitis, hepatic sclerosis, melanoma, lymthoma, autoimmune disease etc. Increase.

The application " tissue polypeptide antigen, TPA " they are to be present in placenta and most of tumor tissue cell's film and cytoplasm In a kind of single chain polypeptide.Recall rate in sera of patients with malignant tumors may be up to more than 70%, but it increase and tumour Happening part and organization type non-correlation.But then there is higher sensitiveness in observation curative effect.Sera of patients with malignant tumors TPA levels can be raised significantly.After treatment takes a turn for the better, TPA is horizontal to be reduced；If TPA increases again, prompt have tumor recurrence.TPA with CEA is detected simultaneously can be advantageous to the pernicious antidiastole with non-malignant breast disease.TPA is raised in 80% ovarian cancer patients blood. In addition, the TPA such as lung cancer, oxyhepatitis, pancreatitis, pneumonia levels can also increase.

The application " tissue polypeptide specific antigen, TPS) be tumor cell secretion a kind of polypeptide antigen.It is TPA in blood In specific part .1, TPS rise phenomenon is generally may occur in which in serum of ovarian cancer patients, other are such as lung cancer, patient with breast cancer Also it may occur in which that its serum levels raises；2nd, auxiliary of the TPS with other marks together as diagnosis, tracking of knub and change of illness state Index.

" CA15-3 " of the application is the most important Specific marker of breast cancer.30%-50% patient with breast cancer CA15-3 it is significantly raised, the change of its content is closely related with therapeutic effect, be patient with breast cancer diagnosis and monitor postoperative multiple The optimal parameter of hair, observation curative effect.CA15-3 dynamic measure contributes to after II phases and III primary breast cancer Case treatments the morning recurred Phase finds；When CA15-3 is more than 100U/ml, it is believed that have metastatic lesion.

" CA125 " of the application is that detected by Bast etc. from epithelial ovarian cancer antigen nineteen eighty-three can be by monoclonal antibody A kind of glycoprotein that OC125 is combined, from embryonic development period coelomic epithelium, is not present in normal ovarian tissue, therefore most It is common in the serum of epithelial ovarian tumor (serous tumor) patient, the sensitiveness that it is diagnosed is higher, but specificity is poor. It is not present in mucus ovarian neoplasm.80% patients with epithelial ovarian tumor Serum tumor marker CA125 rise, but the early stage disease of nearly half Example does not raise, therefore is not individually used for the early diagnosis of epithelial ovarian cancer.90% patients serum CA125 has with course advancement Close, therefore be used for state of an illness detection and curative effect evaluation.95% healthy adult women CA125 level≤40U/ml, if being increased to More than 2 times of normal value should draw attention.During other CA125 checks also seen in the serum of tubercular peritonitis patient, and CA125 is horizontal to be raised in decades of times, preoperative in oophoroma it should be understood that exclusion tubercular peritonitis, pelvic infecton may.

" human chorionic gonadotrophin, the β-HCG " of the application is by a kind of sugared egg of trophocyte's secretion of placenta In vain, it is made up of the glycoprotein of α and β dimers.β-HCG rises also have following several possibility：Normal pregnancy, twins, Portugal Grape tire or some diseases or tumour.Such as in endocrine system disease, such as pituitary disease, hyperthyroidism, gynecological disease such as The HCG such as ovarian cyst, uterine cancer can also increase.It was found that for example tacit tire knurl of malignant tumour, cancer of pancreas, stomach cancer, liver cancer, breast cancer, In the blood such as lung cancer therefore HCG can be also raised in oncology, regard HCG one of as carcinoma markers.

" cyfra21-1 " of the application is the soluble fragments of Cyfra21-1, is mainly used as tumor markers at present, Diagnosis to lung cancer has greater significance.Cytokeratin (cytokeratins) is formed in one of structural proteins of epithelial cell Between silk subunit.According to the difference of isoelectric point in its molecular weight and dielectrophoresis, 20 kinds of different types can be divided into.Their quilts It is divided into two subgroups：I classes (acidic protein), II classes (basic protein).Cytokeratin is made up of I classes and II keratin like protein Different condensate.Cyfra21-1 (CYK-19) is a molecular weight about 40,000Da I keratin like protein (acidic protein), is angle egg Minimum member in white family.CYK-19 is widely distributed in normal structure surface, in stratiform or scaly epithelium.In Malignant Epithelium In cell, the protease of activation accelerates the degraded of cell so that a large amount of cytokeratin fragments are released into blood, its soluble piece Two plants of monoclonal antibody KS19.1 and BM19.21 specific bindings of Duan Keyu, therefore referred to as CYFRA21-1.CYFRA21-1 molecular weight About 30,000Da.In pernicious cancerous lung tissue, CYFRA21-1 rich contents, especially there is high expression in lung squamous cancer.

" neuronspecific enolase (the neuron-specific enolase, NSE) " of the application is to participate in sugared ferment One kind in the enolase of solution approach, is present in nerve fiber and neuroendocrine tissue.Work of the NSE in brain tissue cell The activity level of property highest, peripheral nerve and neurosecretion tissue is placed in the middle, and minimum sees non-nervous tissue, serum and spinal cord Liquid.It is found in the tumour relevant with neuroendocrine tissue origin, is had excessive NSE expression in particularly SCLC, is led Cause NSE in serum significantly raised.

The application " squamouse cell carcinoma antigen SCC " is the preferred tumor markers of cervical squamous cancer.Its Cutoff values typically exist 2.5 μ g/L, SCC participate in normally with protein breakdown during canceration regulating and controlling, significantly raised during cervical squamous cancer.But because not yet sending out at present Existing 100% specificity and the tumor markers of 100% sensitivity, are not one-to-one relationship between tumour and tumor markers, They are correlation.Therefore, it is cervical squamous cancer that only can not just be made a definite diagnosis from index rise merely.Need to combine closely and face Bed and other detection methods comprehensive descisions.In addition, part lung cancer, stomach cancer, the ovarian cancer patients index can also raise.

The application " propepsin (PGI/II) be by stomach secretion participation digest pepsin precursor, lead to Often about 1% PG can enter blood circulation by stomach lining, can be divided into two kinds of hypotypes of PGI and PGII, Serum Pepsinogen can Relatively accurately to show the state of stomach lining and function.PG is superficial gastritis, erosive gastritis, gastric ulcer, duodenum The initial screening index of the disease of stomach such as ulcer, atrophic gastritis, stomach cancer and the monitor control index for the treatment of.In routine physical examination everyone It is unpractical to make gastroscope, can be detected by Noninvasive serum PG by superficial gastritis, erosive gastritis, gastric ulcer, 12 The stomach trouble people at highest risk such as Duodenalulcer, stomach cancer, which checks, to come, then it is a realistic plan to carry out gastrocopy.Research hair It is existing, the serum PG horizontal abnormality of about 15% or so people in routine physical examination, and gastrocopy is further carried out, wherein More than 90% patient has different degrees of superficial gastritis, erosive gastritis, gastric ulcer, duodenal ulcer, atrophic stomach The disease of stomach such as inflammation, stomach cancer.The stomach cancer incidence for the people at highest risk that PGI and PGI/PGII ratios substantially reduce typically compares normal person The high decades of times of group.

General introduction

The detection that tumor-specific genes methylate is to detect the dissociative DNA in blood plasma, and detection uses 10ml EDTA vacuum Heparin tube (purple cap) gathers Venous Blood, and whole blood sample amount is at least the pipe of the pipe of 10ml × 1 or 5ml × 2, and centrifugation is equipped with blood sample Heparin tube 12 minutes, 1350 ± 150rcf of centrifugal force.Heparin tube is taken out from centrifuge, it is disposable with a clean 15cm Pipette is transferred to blood plasma in the 15ml centrifuge tubes of polypropylene material, conical bottom.

Centrifuge blood plasma 12 minutes, 1350 ± 150rcf of centrifugal force.Will with new disposable pipette or serum pipette 3.5ml blood plasma is moved into the centrifuge tube of the conical bottom marked.By 3.5ml plasma samples, positive reference substance, negative controls point It is not transferred in the 15ml centrifuge tubes marked in advance；Sequentially add 3.5ml cracking adsorption liquids.Centrifugation lid is covered tightly, is vortexed mixed It is even.Placed 10 ± 1 minutes in centrifuge tube room temperature (15-30 DEG C).

Following reagent is added in 15ml centrifuge tubes successively：90 μ l magnetic beads (fresh suspension)；2.5ml absolute ethyl alcohols (point Sub- biology rank, purity >=99.5%).Centrifugation lid is covered tightly, overturns and mixes 5-6 times.It is mixed that centrifuge tube is placed in colyliform rotation On even device, room temperature middling speed (about 10-20rpm) rotates 45 ± 5 minutes, about 35~45 ° of the anglec of rotation.

15ml centrifuge tubes are positioned over DynaMag^TM5-10 minutes are adsorbed on -15 magnetic rack for test tubes.It is careful to outwell supernatant (note Meaning not outwell magnetic bead, keep 15ml centrifuge tubes to be positioned over DynaMag when outwelling supernatant^TMOn -15 magnetic rack for test tubes).Add 1.5ml washing lotion A, vortex mixing ensure that magnetic bead is thoroughly resuspended.Bead suspension is moved to what is marked with disposable pipette In 2.0ml centrifuge tubes.Bead suspension is remained with pipette, extract again, is transferred them in 2.0ml centrifuge tubes.

2.0ml centrifuge tubes are positioned over DynaMag^TM2-6 minutes are adsorbed on -2 magnetic rack for test tubes.Use up with disposable pipette Amount removes liquid, is careful not to draw magnetic bead.Of short duration centrifugation 2.0ml centrifuge tubes.2.0ml centrifuge tubes are placed in DynaMag^TM- 2 magnetic Property rack for test tube on adsorb 2-6 minutes again, with 10-100 μ l pipettors as far as possible remove residual liquid.

Centrifuge tube is moved on nonmagnetic rack for test tube.It is vortexed and mixes eluent, adds 100 μ l eluents to each centrifuge tube In, centrifuge tube is covered tightly, is vortexed and mixes, magnetic bead is resuspended.Centrifuge tube is positioned in constant temperature oscillator, rotating speed is set to 1000 ± 100rpm, temperature are set to 80 DEG C, shake 10 ± 1 minutes.Of short duration centrifugation centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 magnetic are tried 2-6 minutes on pipe support.Whole eluents are moved in new 2.0ml centrifuge tubes (about 100 μ l DNA eluents).

150 μ l sulfite solutions, 25 μ l protection liquid are once added in the 2.0ml centrifuge tubes containing DNA eluents.Lid After tight centrifuge tube, be vortexed the reaction solution mixed in centrifuge tube.Of short duration centrifugation centrifuge tube.Centrifuge tube is placed in constant temperature oscillator, 80 DEG C of constant-temperature incubations 45 ± 5 minutes, not vibrate.After 45 ± 5 minutes, centrifuge tube is taken out immediately.

The of short duration above-mentioned reacted 2.0ml centrifuge tubes of centrifugation.Sequentially added in centrifuge tube：1000 μ l washing lotions A；20 μ l magnetic Pearl (fresh suspension).It is vortexed and mixes.Centrifuge tube being placed in 23 DEG C of constant temperature oscillator, adjustment rotating speed is 1000 ± 100rpm, It is incubated 45 ± 5 minutes.Of short duration centrifugation centrifuge tube, DynaMag is placed in by centrifuge tube^TM- 2 magnetic rack for test tube 2-6 minutes.With disposable Pipette removes liquid as far as possible.

Wash for the first time, centrifuge tube is removed from magnetic frame, add 800 μ l washing lotions A.It is vortexed to mix and magnetic bead is resuspended, it is short Temporarily centrifugation centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 magnetic rack for test tube 2-6 minutes.Liquid is removed as far as possible with disposable pipette Body.

Second of washing, centrifuge tube is removed from magnetic frame, adds 800 μ l washing lotions B.It is vortexed to mix and magnetic bead is resuspended.It is short Temporarily centrifugation centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 magnetic rack for test tube 2-6 minutes.Liquid is removed as far as possible with disposable pipette Body.

Third time is washed, and centrifuge tube is removed from magnetic frame, adds 400 μ l washing lotions B.It is vortexed to mix and magnetic bead is resuspended.It is short Temporarily centrifugation centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 magnetic rack for test tube 2-6 minutes.Liquid is removed as far as possible with disposable pipette Body.Of short duration centrifugation centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 magnetic rack for test tube 2-6 minutes, use up with 10-100ul pipettors Amount removes residual liquid.Of short duration centrifugation centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 magnetic rack for test tube 2-6 minutes, use 10- 100ul pipettors remove residual liquid as far as possible.

Dry and elute, open centrifuge tube lid.Centrifuge tube is placed in constant temperature oscillator, 23 ± 2 DEG C stand 10 minutes Drying to be precipitated, it not shake.Centrifuge tube is moved on nonmagnetic rack for test tube, adds 60 μ l eluents.Centrifuge tube is covered, is vortexed Mix and magnetic bead is resuspended.Centrifuge tube is put into constant temperature oscillator, 23 ± 2 DEG C, 1000 ± 100rpm is incubated 10 minutes.Of short duration centrifugation Centrifuge tube.Centrifuge tube is placed in DynaMag^TM- 2 Magnetic rack 2-6 minutes.Eluent (about 60 μ l) is turned with 10-100 μ l pipettors Move in 96 orifice plates, and 96 orifice plates are sealed using adhesive membrane and sealer device.

PCR is set, and each PCR reactions need 32 μ l PCR reaction solutions and 1.6 μ l polymerases.In proportion by respective volume PCR reaction solutions and polymerase are added in 2.0ml centrifuge tube.It is vortexed and mixes PCR pre-reaction liquid, of short duration centrifugation centrifuge tube will Tube wall drop is from getting off.30 μ l PCR pre-reaction liquid are added in selected 96 orifice bores.30 μ l BisDNA is added to PCR plate In corresponding hole.WithOpticalAdhesive Film glued membranes are sealed, and 1000 ± 100rcf is centrifuged 1 minute. PCR plate after sealing can place at 2-8 DEG C to be no more than 4 hours.

PCR reacts, and PCR processes use Applied Biosystems 7500PCR instrument, instrument SDS v2.0.5 softwares Had been verified by with SDS v1.4 softwares.ROX or other dyestuffs are not included in PCR pre-reaction liquid.Therefore, Passive Reference sets and is necessary for " none ", and FAM fluorescence channels are chosen in the detection of tumor-specific genes, and ACTB chooses JOE and led to Road, response procedures are：In the stage 1, one circulates 94 DEG C, 20 minutes；2,45 circulations of stage, 62 DEG C, 5 seconds, 55.5 DEG C, 35 seconds, 93 DEG C, 30 seconds；3,40 DEG C, 5 seconds of stage.

Analysis condition sets (1) SDS v2.0.5 versions, sets 10-22 circulation of baseline, sets Septin9 threshold values 30000, ACTB threshold values 14000.With the validity of reference substance checking PCR reactions：Positive control Septin9 Ct value≤41.1, ACTB Ct value≤29.8；Negative control Septin9 Ct value nothings, ACTB Ct value≤37.2；Any one condition is not met, Then this experimental result engineering noise.(2) SDS V1.4 version softwares are set, and are set 10-22 circulation of baseline, are set Septin9 Threshold value 100000, ACTB threshold values 60000.With the validity of reference substance checking PCR reactions：Positive control Septin9 Ct values≤ 41.1, ACTB Ct value≤29.8；Negative control Septin9 Ct value nothings, ACTB Ct value≤37.2；Any one condition is not Meet, then this experimental result engineering noise.

The explanation of sample PCR reaction results, single sample result are explained：If ACTB Ct value≤32.1, tomour specific Property gene Ct value≤41, define the sample for the positive；If ACTB Ct value≤32.1, the Ct values ＞ of tumor-specific genes 41, the sample is defined as feminine gender；If ACTB Ct values ＞ 32.1, PCR reaction engineering noises are defined.

For example, primer, probe and blocking agent (Blocker) they are oligonucleotides used in Septin9 genetic tests, wherein The sequence of primer, probe and Blocker is：Sense primer, SEQ ID NO：91GTAGTAGTTAGTTTAGTATTTATTTT；Under Swim primer, SEQ ID NO：92CCCACCAACCATCATAT；Blocking agent (Blocker), SEQ ID NO： 93CATCATATCAAACCCCACAATCAACACACAAC；Probe 1, SEQ ID NO： 94GTTCGAAATGATTTTATTTAGTTGC；Probe 2, SEQ ID NO：95CGTTGATCGCGGGGTTC.Primer, probe, Blocker position is located at the CpG islands of 1 λ extrons of Septin9 gene v2 transcription products, and Blocker covers 5 CpG first Base site, probe 1 cover 3 CpG methylation sites, and probe 2 covers 1 CpG methylation sites.

ACTB primers, detecting probe information：ACTB primer, probe design alternative housekeeping gene ACTB conserved sequence area Domain.Sequence information is：Sense primer, SEQ ID NO：96GTGATGGAGGAGGTTTAGTAAGTT；Anti-sense primer, SEQ ID NO：97CCAATAAAACCTACTCCTCCCTTAA；Probe, SEQ ID NO：98ACCACCACCCAACACACAATAACAAACAC A。

Protein tumor-marker analyte detection is the chemoluminescence method conventional reagent and protein-chip using hospital laboratory Method conventional reagent.The present invention use it is representative with chemoluminescence method come to detect protein tumor markers be Shenzhen The instrument and reagent system of NPD projects biomedical engineering limited company, can detect AFP, CEA, PSA, F-PSA, CA125、CA153、CA199、FER、NSE、CA50、SCC、CYFRA211、CA724、CA242、HE4、S-100、PG-I、PG-II、 HCG and HGH etc..It is representative with protein-chip method come to detect protein tumor markers be Shanghai number health biology section The instrument and reagent system of skill Co., Ltd, can detect AFP, CEA, PSA, F-PSA, CA125, CA153, CA199, FER, NSE, CA242, HCG and HGH etc.

Fecal Immunochemical detection (Fecal Immunochemical Test, FIT) is the routine using hospital laboratory Reagent.

The assay method of the nucleotide sequence of with tumour-specific and gene methylation detection is determined selected from PCR, in real time Measure the one or more in PCR, digital pcr, liquid chip, solid-state chip, in situ hybridization, common sequencing and high-flux sequence. If number gene to be determined is few, method or the method that is commonly sequenced using PCR, if number gene to be determined is more, The methods of liquid chip, solid-state chip and high-flux sequence are then included using high-throughout detection method.Embodiments of the invention In using high-flux sequence method.

High throughput sequencing technologies (High-throughput sequencing) are also known as " next generation " sequencing technologies (" Next- Generation " sequencing technology), so that sequence once can be carried out to millions of DNA moleculars to hundreds of thousands parallel Row measure and the long shorter grade of general reading are mark.The new-generation sequencing instrument Hiseq 2000 and Hiseq 2500 of Illumina companies With high accuracy, the outstanding advantage such as high flux, high sensitivity, and low operating cost, traditional genomics can be completed simultaneously Research (sequencing and annotation) and functional genomics (gene expression and regulation, gene function, albumen/nucleic acid interaction) are ground Study carefully.Hiseq be it is a kind of based on unimolecule cluster while synthesis while sequencing technologies, based on proprietary reversible termination chemical principle. The random fragment of genomic DNA is attached to optically transparent glass surface (i.e. Flow cell), these DNA fragmentations during sequencing Through extension and bridge amplification after, form hundreds of millions of Cluster on Flow cell, each Cluster be have it is thousands of The unimolecule cluster of part same template.Then four kinds of special deoxyribonucleotides with fluorophor are utilized, it is whole by invertibity SBS only technology (is sequenced) in synthesis template DNA to be measured is sequenced.Main operating procedure has the preparation of 1. samples (sample fragmentation)；2. library construction (library preparation)；3. sequencing reaction (sequencing reaction)；4. data analysis (data analysis).

The detection of high flux gene methylation is to detect the dissociative DNA in blood plasma, and preparation of samples step includes blood sample Prepared by product collection, blood plasma, extraction dissociative DNA, sulphite conversion DNA, and the dissociative DNA after purifying conversion is used for library construction Second step.Blood sample collection is using 10ml EDTA vacuum blood collection tubes (purple cap) collection Venous Blood, and whole blood sample amount is extremely Managed less for 10ml × 1 or 5ml × 2 are managed, heparin tube of the centrifugation equipped with blood sample 12 minutes, 1350 ± 150rcf of centrifugal force.From from Heparin tube is taken out in scheming, blood plasma is transferred to polypropylene material, conical bottom with clean 15cm disposable pipette In 15ml centrifuge tubes.

Library construction, sequencing reaction and the data analysis of high-flux sequence are professional and technical very single-minded technologies Field, the invention provides the target gene to methylate needed with high-flux sequence, and the core of the present invention, follow-up skill Art operates, and is completed by Illumina company techniques service department.

Embodiment 1Combination gene methylates and protein tumor markers

Gene methylation and protein tumor markers, which is applied in combination, can increase the sensitivity of detection, improve tumour early stage The success rate of diagnosis.In the Septin9 gene methylations kit detection colorectal cancer clinical test of 1031, hospital inspection Section detects the gene methylation content in self serum with Septin9 gene methylations kit, while uses hospital laboratory Fecal Immunochemical detection (Fecal Immunochemical Test, FIT) kit detection person under inspection stool in it is blood red Albumen, or the content with CEA in CEA detection person under inspection's blood of hospital laboratory.

As shown in table 1, when detecting three indexs respectively, the sensitivity of Septin9 detection tumours is 77%, and specificity is 96%, the sensitivity for detecting infantile tumour is 68%, all respectively than FIT (70%, 95%, 58%) and CEA (52%, 85%, 31%) it is good, and by Septin9 result and FIT result comprehensive analysis, the sensitivity of combine detection tumour is 94%, than combination The sensitivity that preceding Septin9 is individually detected 77% improves 17%, and specificity declines 4%.If by Septin9 result and CEA result comprehensive analysis, the sensitivity of combine detection tumour is 88%, the sensitivity individually detected than Septin9 before combination 77% improves 11%, and specificity declines 6%.If by Septin9 result and FIT and CEA result comprehensive analysis, combination Detect the sensitivity of tumour and improve 20% for 97%, but specificity also accordingly decline it is more, less than 90% (not shown in table) With caution from excessive and unsuitable combination.

It is that can increase the detection of infantile tumour that gene methylation and protein tumor markers sharpest edges, which is applied in combination, Sensitivity, Septin9 detections and FIT detection combinations in the present invention, can improve 18% by the detection sensitivity of infantile tumour, by The sensitivity that Septin9 individually detects infantile tumour brings up to 86% for 68%.Its main cause is Septin9 gene methylations Occur in tumorigenic very early stage, be detection even in precancerous stage, detection Septin9 gene methylations The early changes of tumour, this is the place less than protein tumor markers, although protein tumor markers is swollen to early stage The detection sensitivity of knurl also has certain contribution.Tumor tissues are completely in the change of nucleic acid level and the change of protein level Two different biology aspects, the change of nucleic acid level tends in early days and wide spectrum, the change of protein level tend to middle and advanced stage Certain tissue specificity with formation.Combination can form good cooperative effect.

Table 1

Septin9 gene methylations detection method is with reference to the sincere companies of Bo Er《Septin9 gene methylation detection kits (PCR fluorescence probe methods) specification》, Septin9 gene methylations detection kit includes two steps：

Step 1, the dissociative DNA in blood plasma is extracted using plasma treatment kit, is not then occurred with sulphite conversion The cytimidine to methylate, uracil sulphonate is produced by desamination reaction, the cytimidine to methylate then will not be by sulfurous Hydrochlorate converts；

Step 2, the DNA (BisDNA) that sulphite converts is done into double PCR amplification, blocking agent and spy in PCR reactions Pin can be distinguished to methylate and preferentially expanded with non-methylated DNA fragments, methylated DNA fragments, with the Septin9 gene orders spy that methylates The fluorescein probe that the opposite sex combines can exclusively detect methylated DNA fragments in PCR reactions.Internal reference ACTB (β-actin) Whether gene is used to assess amount of DNA in detection enough.Positive and negative control is provided in kit, is all needed in detecting each time Positive and negative control is detected simultaneously.

Embodiment 2Two gene methylation tumor markerses are applied in combination

Different gene methylation tumor markerses, which is applied in combination, can increase the sensitivity of detection, improve tumour early stage and examine Disconnected success rate.In the Septin9+RNF180 gene methylations kit detection stomach cancer clinical test of 202, hospital inspection Section detects the gene methylation content in self serum with Septin9+RNF180 gene methylations kit.As shown in table 2, When analyzing two indexs respectively, the sensitivity of Septin9 and RNF180 detection tumours is respectively 43% and 48%, all 50% Hereinafter, and by Septin9 result and RNF180 result comprehensive analysis, the sensitivity of combine detection tumour is 66%, is improved 18%, specificity is constant.As a result prompting combination uses inspection of the different gene methylation tumor markerses to the overall tumour of raising Survey sensitivity (18%) and combination gene methylate close with protein tumor-marker object detecting method (17%).

The excellent of detection sensitivity of the different gene methylation tumor markerses also with increase infantile tumour is applied in combination Gesture, the sensitivity of Septin9 and RNF180 detection infantile tumours is respectively 28% and 38% in the present invention, and Septin9 is detected With RNF180 detection combinations, the detection sensitivity of infantile tumour can be improved 10%, infantile tumour is detected from independent RNF180 38% sensitivity brings up to 48%.As a result prompting combination is swollen to improving early stage using different gene methylation tumor markerses The detection sensitivity (10%) and combination gene of knurl methylate and had significantly with protein tumor-marker object detecting method (18%) Gap.Its possible cause is that Septin9 gene methylations and RNF180 gene methylations were in tumorigenic identical morning In stage phase, even in precancerous stage, it is all that detection is swollen to detect Septin9 gene methylations and RNF180 gene methylations The early changes of knurl, both are repeatedly more on Biological Mechanism, there is normal synergy, are 1+1 effects.And detect Septin9 gene methylations and detection protein tumor-marker analyte detection are different materials, both mechanism in biology Upper difference is very big, there is extraordinary synergy, is the effect that 1+1 is more than 2.And this phenomenon occurs over just detection infantile tumour Stage, if each phase tumour merged, effect then unobvious of this 1+1 more than 2.Because combining on the whole The detection sensitivity (18%) of overall stomach cancer, its cooperative effect are improved using different gene methylation tumor-marker analyte detections Methylated with combination gene and the detection sensitivity (17%) of overall colorectal cancer is improved with protein tumor-marker object detecting method It is close.The extraordinary association that combine detection Septin9 gene methylations and detection protein tumor markers diagnose to infantile tumour Same-action, i.e. 1+1 are more than 2 effect, not yet meet report.Main cause is the sensitivity for detecting overall tumour necessarily than detection The sensitivity height of infantile tumour is the unquestionable natural law, thus people it is typically natural think that combine detection improves The sensitivity of overall lesion detection also should just be higher than the sensitivity to improving infantile tumour detection.And the combine detection of the present invention Effects of the 1+1 more than 2 of Septin9 gene methylations and detection protein tumor markers method is not apparent.

Table 2

Embodiment 3Systematicness detection implements and combination

Different according to the purpose of detection, systematicness detection as shown in table 3, can form different combination sides with motor-driven combination Case, including 1) detection method by the more characteristic high flux detections of polygenes and various protein tumor markerses combines shape Into overall project；2) detection method by the more characteristic high flux detections of polygenes and various protein tumor markerses is combined one Rise and form liquid biopsy scheme；3) the more characteristic high fluxs of polygenes are detected into the mutation of emphasis gene and various protein tumour marks The detection method of will thing is grouped together into gene mutation assembled scheme；4) the more characteristic high fluxs of polygenes are detected into emphasis base Methylating for cause is grouped together into gene methylation assembled scheme with the detection method of various protein tumor markerses；5) The detection method of the more characteristic high flux detection emphasis Micrornas of polygenes and various protein tumor markerses is combined Form Microrna assembled scheme；6) by the gene methylation for determining person under inspection and the detection side of various protein tumor markerses Method, which is combined, forms the assembled scheme that single-gene methylates；7) by the Septin9 gene methylations of measure person under inspection and respectively The detection method of kind protein tumor markers combines the assembled scheme for forming Septin9+, and it is big to form digestive system five The combination inspection of cancer, including stomach cancer, liver cancer, colorectal cancer, cancer of the esophagus and cancer of pancreas, include the early detection of these tumours, Further form the combination inspection of ten big cancers, including lung cancer, stomach cancer, liver cancer, colorectal cancer, cancer of the esophagus, breast cancer, pancreas Cancer, oophoroma, prostate cancer and uterine cancer, include the early detection of these tumours；8) the more characteristic high fluxs of polygenes are detected With the Combination of Methods for detecting single protein tumor markers together with form the assembled scheme of single protein tumor markers； 9) it will form CEA+'s together with the detection of polygenes more characteristic high fluxs and detection protein tumor markers CEA Combination of Methods Assembled scheme；10) detection method by the more characteristic high flux detections of polygenes and emphasis protein tumor markers is combined one Act the assembled scheme for forming tumor markers；11) by the more characteristic high flux detections of polygenes and the detection side of tumour autoantibody Method is grouped together into the assembled scheme of tumour autoantibody；12) the more characteristic high flux detections of polygenes and histone are repaiied Decorations include methylating or the detection method of acetylation is grouped together into the assembled scheme of histone modification；13) by polygenes More characteristic high flux detections and fecal hemoglobin and blood CEA detection method are grouped together into colorectal cancer joint inspection Assembled scheme；14) it is the more characteristic high flux detections of polygenes are related to lung cancer early diagnosis, medication guide, monitoring recurrence The change of gene mutation, gene methylation and Microrna is related to lung cancer early diagnosis, medication guide, monitoring recurrence to detecting The detection method of emphasis tumor markers be grouped together into lung cancer early diagnosis, the combination of medication guide, monitoring recurrence Scheme.

Embodiment 4：To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- it is comprehensively square Case

As shown in figure 1, the more characteristic high fluxs of polygenes are detected to the detection method group with various protein tumor markerses It is combined to form overall project, by more to detect to person under inspection's implementation liquid biopsy and the detection of whole body other biological sample Number tumour and infantile tumour.It is comprehensively reliable accurately systemic to kinds of tumors with reduction person under inspection's burden to establish a kind of system Detection and comprehensive analysis interpretation method, applied to the clinical detection to kinds of tumors, people at highest risk's examination and general population GeneraI investigation.Methods described includes 1) systemic detection including more to the high-throughout measure of person under inspection's progress more characteristics of polygenes, progress Plant the measure of tumor markers and carry out using the inspection of medicine equipment and equipment to person under inspection；With 2) comprehensive analysis interpretation Method includes Clinical questionnaire, main suit, symptom, sign, family history, clinical main suit, clinical symptoms, the clinical body of comprehensive person under inspection Sign, the high-throughout measurement result of the more characteristics of polygenes, the measurement result of Diagnostic Value of Several Serum Tumor Markers and use medicine equipment and equipment Comprehensive analysis is carried out to the inspection result of person under inspection.

The kinds of tumors detected includes but is not limited to lung cancer, stomach cancer, liver cancer, colorectal cancer, the cancer of the esophagus, breast cancer, brain Cancer, cervical carcinoma, cancer of pancreas, thyroid cancer, lymph cancer, carcinoma of urinary bladder, leukaemia, kidney, uterine cancer, prostate cancer, oophoroma and Other various tumours.

The high-throughout assay method of the more characteristics of polygenes includes but is not limited to high-throughout method such as high-flux sequence, liquid phase Chip and solid phase chip etc. determine state of multiple different genes in different qualities, include but is not limited to, and the sequence of gene becomes Different, gene mutation and missing, the decorating state of gene, DNA restructuring and RNA Fusion Strains, the change of DNA and RNA number of copies Change, Microrna (microRNA) and the change, non-transcribed RNA change etc. for suppressing tiny RNA (siRNA).

The assay method of Diagnostic Value of Several Serum Tumor Markers includes but is not limited to using liquid chip, solid-state chip, enzyme linked immunological, put Penetrate immune, chemiluminescence immunoassay, mass spectrum, high performance liquid chroma- tography, the Western markings, sequence analysis and other various assay methods.

Used medicine equipment and equipment include but is not limited to use iconography detection method to the inspection method of person under inspection, Including but not limited to X-ray examination, CT, MRI and ultrasonic wave etc., hysteroscope inspection include gastroscope, colonoscopy, laparoscope, ERCP, Bronchoscope, thoracoscope, cystoscope, hysteroscope etc..

Systematicness detection includes but is not limited to the more characteristic high throughput methods of polygenes for determining person under inspection and various albumen The Combination of Methods of matter tumor markers is together.

The clinical information outside the systematicness inspection for offer comprehensive analysis interpretation method is also provided, including it is but unlimited In the Clinical questionnaire of person under inspection, main suit, symptom, sign, family history, clinical main suit, clinical symptoms, clinical sign reviewing party Method, used for comprehensive analysis interpretation, so as to provide accurately to the testing result of kinds of tumors.

Comprehensive analysis interpretation method include but is not limited to combine systemic testing result, Clinical questionnaire, main suit, symptom, Sign, family history, clinical main suit, clinical symptoms, clinical sign, the inspection result of iconography detection method and hysteroscope, there is provided essence The accurate testing result to kinds of tumors.

Overall project is by combining systemic testing result, Clinical questionnaire, main suit, symptom, sign, family history, clinic Main suit, clinical symptoms, clinical sign, the inspection result of iconography testing result and hysteroscope, there is provided accurately to kinds of tumors Testing result.Applied to the method to kinds of tumors accurately clinical detection, people at highest risk's examination and the generaI investigation of general population.

Overall project is a kind of method that system is comprehensively reliable and reduces person under inspection's burden, uses the more characteristic high passes of polygenes The tumor markers of the wide spectrum of amount and special tumor markers, avoid repeating the biological sample for taking person under inspection, avoid complexity Complementary medicine include the detection of iconography and hysteroscope, make person under inspection acceptant, the detection method that testing agency easily realizes.

The biological sample for the person under inspection for being used to detect in overall project is selected from cell line, Histological section, tissue work The tissue of inspection/FFPE, body fluid, excrement, urine, blood plasma, serum, whole blood, separation haemocyte, separated from blood it is thin Born of the same parents, body fluid, sputum, throat swab, or its combination.The biological sample of person under inspection used in liquid biopsy is blood plasma or blood Clearly.

Real whole example 5：To kinds of tumors, accurately systematicness detection and comprehensive analysis interpretation method and application --- liquid is lived Procuratorial organ's case

This programme main contents are with embodiment 4, as shown in Fig. 2 the biological sample of the person under inspection used in liquid biopsy is main For for blood plasma or serum.By the way that the detection of liquid biopsy is carried out person under inspection detect most tumors and infantile tumour.

Embodiment 6：To kinds of tumors, accurately systematicness is detected and comprehensive analysis interpretation method and application --- gene is dashed forward Become combination

This programme main contents are with embodiment 4, as shown in figure 3, the more characteristic high passes of polygenes in gene mutation assembled scheme Amount detection be emphasis gene gene mutation, such as the gene such as KRAS, BRAF and EGFR.Lived by carrying out liquid to person under inspection The detection of inspection detects most tumors and infantile tumour.

Embodiment 7To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- gene first Baseization combines

The same example IV of this programme main contents, as shown in figure 4, the more characteristic high passes of polygenes in gene mutation assembled scheme What amount detected is the change of the gene methylation of emphasis gene.The emphasis assortment of genes of detection gene methylation includes primary tumor In have the gene of the change that methylates, the title of the assortment of genes is as shown in table 4：

Table 4

Embodiment 8To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- Microrna Combination

This programme main contents are with embodiment 4, as shown in figure 5, the more characteristic high fluxs of polygenes in Microrna assembled scheme What is detected is the change of Microrna.The title of the emphasis Microrna combination related to lung cancer detected is as shown in table 5：

Table 5

hsa-let-7c
	hsa-miR-122
hsa-miR-182
	hsa-miR-193a
hsa-miR-200c
	hsa-miR-203
hsa-miR-218
	hsa-miR-155
hsa-let-7b
	hsa-miR-411
hsa-miR-450b
	hsa-miR-485
hsa-miR-519a
	hsa-miR-642
hsa-miR-517b
	hsa-miR-520f
hsa-miR-206
	hsa-miR-566
hsa-miR-661
	hsa-miR-340
hsa-miR-1243
	hsa-miR-720
hsa-miR-543
	hsa-miR-1267

Embodiment 9：To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- Septin9+

This programme main contents are with embodiment 4, as shown in fig. 6, the more characteristic high passes of polygenes in Septin9+ assembled schemes What amount detected is the assembled scheme that single-gene methylates, and determines the change of Septin9 gene methylations.Operated to simplify, detection Method can use real-time quantitative PCR.

According to the discovery of Examples 1 and 2, present invention design and develop to kinds of tumors accurately systematicness detection and comprehensive Conjunction property analysis interpretation method and application --- Septin9+.As shown in fig. 6, the present invention is combination S eptin9 gene methylations and egg White matter tumor-marker object detecting method, the detection method of Septin9 gene methylations use the approach of liquid biopsy, are determined with real-time Measure the Septin9 genes that methylate to dissociate in PCR method measure detection self serum.Protein tumor-marker analyte detection side Method can take the method measure that ELISA or chemistry are given out light immune, can also use the side of liquid phase protein chip or Solid protein chip Method determines.These assay methods can be operated by product description.Detection Septin9 gene methylations are to detect early stage Tumour, and using its broad spectrum activity the characteristics of, kinds of tumors is detected, detection multiple proteins tumor markers be in order to Tissue specificity positioning is carried out to different tumours.Combination determines, and can improve the detection sensitivity of infantile tumour 20%, improve the overall sensitivity 17% of each phase tumour.

Described kinds of tumors include lung cancer, stomach cancer, liver cancer, colorectal cancer, the cancer of the esophagus, breast cancer, the cancer of the brain, cervical carcinoma, Cancer of pancreas, thyroid cancer, lymph cancer, carcinoma of urinary bladder, leukaemia, kidney, uterine cancer, prostate cancer, oophoroma etc..

Another aspect of the present invention, there is provided a kind of comprehensive analysis interpretation method and application.Because the present invention is pair Infantile tumour is detected, and person under inspection is Silent cerebral infarction mostly, collection and the clinical information with reference to person under inspection, including is asked Volume, main suit, symptom, sign, age, sex, body weight, smoking history, family history, infecting medical history, diabetic history etc. can be to being System property testing result provides guidance quality clue, there is provided to comprehensive analysis interpretation method and application.

The present invention will also be gathered and asked with reference to other clinical adjunct test results, especially systemic testing result prompting In the case that topic is, it is necessary to further examine, other clinical adjunct tests are used.Other clinical adjunct test methods include image Learn the inspection with hysteroscope.Iconography detection method includes X-ray examination, CT, MRI and ultrasonic wave etc., and hysteroscope inspection includes gastroscope, intestines Mirror, laparoscope, ERCP, bronchoscope, thoracoscope, cystoscope, hysteroscope etc..

, be according to the result detected in the present invention to the systematicness of kinds of tumors, with reference to being examined when analyzing testing result The clinical information of person and other clinical adjunct test results, comprehensive analysis and interpretation are carried out to lesion detection result.These are just Constitute the present invention to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application.

Embodiment 10：Digestive system tumor joint inspection is combined

The Septin9 genes to methylate elevated ratio highest in the blood plasma of colorectal cancer patients, according to 1031 Clinical test results, up to 77%.Elevated ratio is up to 50% in the blood plasma of patients with gastric cancer.Cancer of the esophagus, cancer of pancreas and There is different degrees of elevated ratio in the blood plasma of the patient of liver cancer.It is crucial that Septin9 gene methylations can detect Infantile tumour.Protein tumor markers has certain tissue specificity, and the detection of Septin9 gene methylations has well mutually Benefit property and concertedness.According to the design and framework of embodiment three, the present invention derives digestive system tumor joint inspection combination, in detail It is shown in Table 6.

Protein the tumor markers FIT and CEA of colorectal cancer synergy are described in detail in embodiment one.To stomach For cancer, CA724 is a preferred tumor markers, and PGI/II is also taken seriously in recent years, and PG is superficial gastritis, erosion The initial screening index of disease of stomach and the monitoring for the treatment of such as property gastritis, gastric ulcer, duodenal ulcer, atrophic gastritis, stomach cancer Index, add combination advantageously.Detections of the AFP and CEA to liver cancer in clinical practice for many years, AFP is to primary carcinoma of liver There is good specificity.CA242 has preferable specificity to cancer of the esophagus, mixes TPA, TPS and CEA combination, and effect is more preferable. CA199 has preferable specificity to cancer of pancreas, is hitherto reported to cancer of pancreas sensitiveness highest mark.CA50 is in pancreas The positive rate of gland cancer is up to 87%.

Once blood drawing can complete the sample collection for the combine detection, Septin9 real-time quantitative PCR inspection Survey, a performance liquid chip detection can be completed to detect all protein tumor markers.Medical inspection mechanism automates Electrochemiluminescent immunoassay or ELISA can also complete the detection of protein tumor markers.

Table 6

Embodiment 11：Ten tumour joint inspection combinations

The Septin9 genes to methylate elevated ratio in the blood plasma of patients with lung cancer there are about more than 40%, breast cancer, ovum Also there is certain increased proportion in the blood plasma of nest cancer, uterine cancer and patients with prostate cancer.According to the design and framework of embodiment 3, The present invention derives ten tumour joint inspection combinations, wherein the joint inspection combination of five primary tumors refers to table 7, plus table The tumour of five digestive systems in 6, it is ten tumour joint inspection combinations altogether.

Cyfra21-1 is the soluble fragments of Cyfra21-1, is mainly used as tumor markers at present, and lung cancer is examined It is disconnected to have greater significance.In pernicious cancerous lung tissue, CYFRA21-1 rich contents, especially there is high expression in lung squamous cancer. CA15-3 is the most important Specific marker of breast cancer.The CA15-3 of 30%-50% patient with breast cancer is significantly raised, its The change of content is closely related with therapeutic effect, is patient with breast cancer's diagnosis and monitoring postoperative recurrence, the optimal finger for observing curative effect Mark.80% patients with epithelial ovarian tumor Serum tumor marker CA125 rise, but the early-stage cases of nearly half do not raise, thus it is not independent Early diagnosis for epithelial ovarian cancer.Prostate specific antigen (Prostate Specific Antigen, human chorionic Promoting sexual gland hormone is by a kind of glycoprotein of trophocyte's secretion of placenta, there is good specificity, but lung cancer to uterine cancer It is that can also raise.PSA) it is a kind of single chain glycoprotein by the epithelial cells of prostatic acini and conduit, is routine clinical For the benign important indicator with malignant disease Diagnosis and differential diaggnosis and patients with prostate cancer Follow-up After of prostate.

Table 7

Embodiment 12：To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- CEA+

This programme main contents are with embodiment 4, as shown in fig. 7, by the more characteristic high fluxs of polygenes in CEA+ assembled schemes Detect with the Combination of Methods for detecting single protein tumor markers CEA together with form the group of single protein tumor markers Conjunction scheme.By the way that the detection of liquid biopsy is carried out person under inspection detect most tumors and infantile tumour.

Embodiment 13：To kinds of tumors, accurately systematicness detects and comprehensive analysis interpretation method and application --- ten is big Tumor markers combines

This programme main contents are with embodiment 4, as shown in figure 8, in ten big tumor markers assembled schemes that polygenes is more Characteristic high flux detect with the Combination of Methods of detection multiple proteins tumor markers together with form a variety of emphasis protein and swell The assembled scheme of tumor markers.This programme apparatus representational protein tumor markers detects ten kinds of main tumours, Lung cancer (Cyfra 21-1), stomach cancer (CA72-4), colorectal cancer (CEA), liver cancer (AFP), cancer of the esophagus (CA242), breast cancer (CA153), prostate cancer (PSA), cancer of pancreas (Ca199), oophoroma (CA125) and uterine cancer (Beta-HCG).By to by The detection that inspection person carries out liquid biopsy detects most tumors and infantile tumour.

Embodiment 14：To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- tumour Autoantibody combines

This programme main contents are with embodiment 4, as shown in figure 9, in tumour autoantibody assembled scheme that polygenes is how special Property high flux detection and the Combination of Methods of detection kinds of tumors autoantibody together with form the assembled scheme of tumour autoantibody. This programme detection tumour autoantibody Auto-Ab p53, Auto-Ab MMP-7, Auto-Ab Hsp70, Auto-Ab Prx VI With Auto-Ab Bmi-1.By the way that the detection of liquid biopsy is carried out person under inspection detect most tumors and infantile tumour.

Embodiment 15：To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- group egg White modification

This programme main contents are with embodiment 4, as shown in Figure 10, by the more characteristics of polygenes in histone modification assembled scheme High flux detect with the Combination of Methods of detection histone modification together with form the assembled scheme of histone modification.This programme detects Histone modification, for example, histone methylate (H3K4mel and H3K4me3) and the acetylation (H4K8ac) of histone etc..It is logical The detection to person under inspection's implementation liquid biopsy is crossed to detect most tumors and infantile tumour.

Embodiment 16：To kinds of tumors, accurately systematicness detection and comprehensive analysis interpretation method and application --- knot is straight Intestinal cancer joint inspection

How special this programme main contents are as shown in figure 11, by polygenes in colorectal cancer joint inspection assembled scheme with embodiment 4 Property high flux detection form colorectal cancer joint inspection together with hemoglobin and the Combination of Methods of CEA in blood with detecting in excrement Assembled scheme.This programme detects hemoglobin in excrement with FIT methods, detects CEA, CA199, CA242 and CA724 in blood Deng, with reference to the more characteristic high flux testing results of polygenes, carry out person under inspection the detection of liquid biopsy detect colorectal cancer and Early stage colorectal cancer.

The methylating of several genes, gene mutation and Microrna can be detected in colorectal cancer joint inspection combination Change, can also only detection Septin9 gene methylations, plus detection blood in Diagnostic Value of Several Serum Tumor Markers such as CEA, CA199, CA242 and CA724 etc., along with FIT detect excrement.These assay methods can be operated by product description.Detection Septin9 gene methylations be in order to detect infantile tumour, detection multiple proteins tumor markers be in order to increase sensitivity, Guiding treatment and Prognosis scoveillance etc..

Embodiment 17：To kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method and application --- lung cancer Early diagnosis, medication guide, monitoring recurrence

This programme main contents are the same as embodiment 4, as shown in figure 12, lung cancer early diagnosis, medication guide, the group of monitoring recurrence The more characteristic high fluxs of polygenes are detected into the gene related to lung cancer early diagnosis, medication guide, monitoring recurrence in conjunction scheme to dash forward Become, the change of gene methylation and Microrna, the emphasis related to lung cancer early diagnosis, medication guide, monitoring recurrence to detecting The detection method of tumor markers is grouped together into lung cancer early diagnosis, medication guide, the assembled scheme of monitoring recurrence, leads to The detection to person under inspection's implementation liquid biopsy is crossed to realize lung cancer early diagnosis, medication guide and monitoring recurrence.

Embodiment 18：Analysis and the implementation steps of interpretation

The implementation steps of analysis and interpretation are divided into three major parts, and Part I is data acquisition, including 1. collection bases Because of methylate tumor markers and protein tumor markers testing result, the clinical information of 2. collection persons under inspection, including questionnaire, Main suit, symptom, sign, age, sex, body weight, smoking history, family history, infect medical history, diabetic history；3. collection and other Clinical adjunct test result includes the inspection of iconography and hysteroscope.Iconography detection method includes X-ray examination, CT, MRI and ultrasound Ripple etc., hysteroscope inspection include gastroscope, colonoscopy, laparoscope, ERCP, bronchoscope, thoracoscope, cystoscope, hysteroscope etc. Result.

Part II is the result of the three sets of heterogeneitys and reflection person under inspection's different conditions of analyzing Part I collection.Often One result is defined as a factor, because being multiplicity, weight, distribution are distributed each factor during analysis The principle of weight is according to the comprehensive analysis to above-mentioned three sets of factors.First set factor is analysis nucleic acid tumor markers and albumen Matter tumor markers testing result, 1) positive analysis method can be that any one positive is then analyzed in several tumor markerses Then analyzed as the positive for the positive, or any two, three, four or multiple positives.2) Broad Spectrum Analysis of Infinitesimal of infantile tumour：Nucleic acid The tumor markers of high-flux sequence is good at the cancer of detection early stage and wide spectrum, using nucleic acid tumor markers as high weight 3) tumor tissue specificity analysis is weighed using protein tumor markers, as in table 6 and table 7 " tumour-specific is relatively good " Cited protein tumor markers more has the tissue specificity that relatively respective a line corresponds to tumour, such as FIT in one row Colorectal cancer weight is aggravated, AFP aggravates liver cancer weight, and Cyfra21-1 aggravates lung cancer weight, and PSA aggravates prostate cancer weight etc. The weight to tissue specificity can be added Deng, these protein tumor markerses.

Part II will also analyze second set of factor, i.e. clinical information, including questionnaire, main suit, symptom, sign, age, property Not, body weight, smoking history, family history, medical history, diabetic history etc. is infected.The main Early manifestation for being to provide tumour and tumour Tissue specificity.For example, abdominal discomfort, appetite, which die-off, aggravates the weight of stomach cancer, complexion jaundice aggravates liver cancer weight, stool type shape Change and aggravate colorectal cancer weight, dysphagia aggravates the weight of the cancer of the esophagus, and fast weight, which declines, aggravates cancer of pancreas weight, smokes History aggravates lung cancer weight, and family blood relationship relatives someone, which suffers from breast cancer, aggravates knot breast cancer weight.Part II will also analyze Three sets of factors, other clinical adjunct test results include the inspection of iconography and hysteroscope.It is saturating that iconography detection method includes x-ray Depending on, CT, MRI and ultrasonic wave etc., hysteroscope inspection include gastroscope, colonoscopy, laparoscope, ERCP, bronchoscope, thoracoscope, The result of cystoscope, hysteroscope etc..For example, when X-ray examination found gastric ulcer, the weight of stomach cancer is aggravated, ultrasonic wave was found There is occupying lesion in liver, aggravate the weight of liver cancer, CT had found there is pulmonary nodule, aggravated the weight of lung cancer, and gastroscope is found Atrophic gastritis or intestinal metaplasia are crossed, aggravates the weight of stomach cancer, colonoscopy found polyposis, aggravated the weight of colorectal cancer, X Line perspective found breast tubercle, aggravated weight of breast cancer etc..

Part III is comprehensive interpretation, the result of three sets of heterogeneitys of summary and reflection person under inspection's different conditions or because Element, the weight of comprehensive each factor come whether interpretation person under inspection suffers from infantile tumour, and various factors is from several to tens kinds or up to a hundred Kind.The few available simple analysis of factor, needing more than factor carry out aggregation of data analysis interpretation with computer, or further use " big data " processing mode carries out comprehensive analysis interpretation.Formation algorithm formula carries out interpretation after Integrated Analysis of Multi-Factors Involved.Lift Under such as, example 1, the tumor markers of person under inspection's high throughput sequencing of nucleic acids is positive, and protein tumor markers CA724 positive, Prompting person under inspection has the possibility of early carcinoma of stomach.Example 2, the tumor markers Septin9 gene first of person under inspection's high throughput sequencing of nucleic acids Baseization is positive, and other protein tumor markerses are feminine gender, because Septin9 gene methylations mostly occur in colorectal cancer, It is colorectal cancer that person under inspection, which should be first determined whether, but person under inspection has abdominal discomfort, appetite to die-off, and more prompts person under inspection to have early carcinoma of stomach Possibility.Example 3, the tumor markers of person under inspection's high throughput sequencing of nucleic acids is negative, protein tumor markers CA724 and MG7 bis- Person is positive, and person under inspection had the history that x-ray stomach radiography found gastric ulcer, should be judged as stomach cancer；Example 4, person under inspection The tumor markers Septin9 gene methylations of high throughput sequencing of nucleic acids are positive, protein tumor markers CEA and Cyfra21- 1 is positive, has lineal blood relationship relatives to suffer from colorectal cancer.Due to having the Septin9 gene methylations positive, protein tumor markers CEA is positive plus there is lineal blood relationship relatives to suffer from colorectal cancer these and the great factor of colorectal cancer correlative weight, is judged as that knot is straight The possibility of intestinal cancer is big.But pass through it is further check, find person under inspection also have cough, chest CT find nodosity these and The great factor of lung cancer correlative weight, person under inspection are judged as that the possibility of lung cancer is bigger, because the Cyfra21-1 positives are and lung cancer The very big factor of associated weight, CEA are also larger with the associated weight of lung cancer.So weight by comprehensive analysis various factors Size, can have and more accurately judge that person under inspection is probably to suffer from lung cancer.

Above-mentioned example 4 involves how to assign the weight size of various factors this key link, and this is one and is highly desirable to The integration of disease and medical knowledge, and the process of comprehensive logic judgment is carried out, the weight of each factor is not unalterable , such as this factor of coughing, if chest CT does not find tubercle, the weight of cough in tumour interpretation before just very It is low, but after chest CT finds tubercle, the weight of cough is just very high in tumour interpretation, and the weight of other factors, such as Lineal blood relationship relatives suffer from the colorectal cancer many that just become low, and it is lung cancer that person under inspection, which is more likely to interpretation,.The medical history of person under inspection because Element is also critically important, and for example 4, the new factor in medical history can change the weight of other factors again.For example, looking back person under inspection Medical history in did the record of Sigmoidoscope before 7 years, and cut off the villous adenoma of 2.2 centimetres of sizes, this it is new because Element, can change the weight of other factors again again, and the weight that lineal blood relationship relatives suffer from colorectal cancer this factor just becomes high Many, it is colorectal cancer that person under inspection is more likely to interpretation again, because the possibility of more than 2 centimetres of villous adenoma canceration is very Greatly, person under inspection is probably the canceration of villous adenoma after performing the operation 7 years, and Septin9 gene methylations are positive, protein tumour mark The will thing CEA positives still support the interpretation of colorectal cancer.Now, the weight for this factor of coughing does not lower, it is possible to by The colorectal cancer of inspection person is transferred into lung, so as to cause cough and lung CT to find tubercle.

It is not unalterable from the weight described above that can see each factor, is changed by the change of other factors Become.So the present invention gathers the result or factor of three sets of heterogeneitys and reflection person under inspection's different conditions, including 1. detection of nucleic acids As a result include questionnaire with the testing result of protein tumor markers, 2. clinical information, main suit, symptom, sign, the age, sex, Body weight, smoking history, family history, infect medical history, diabetic history etc., and 3. other clinical adjunct test results include iconography and The inspection of hysteroscope.Iconography detection method includes X-ray examination, CT, MRI and ultrasonic wave etc., hysteroscope inspection include gastroscope, colonoscopy, The result of laparoscope, ERCP, bronchoscope, thoracoscope, cystoscope, hysteroscope etc. carries out comprehensive analysis, obtains to swollen Knurl includes the accurate interpretation of infantile tumour.Because the factor that the present invention gathers is more, these above-mentioned comprehensive analysis interpretations can lead to The computing for crossing computer precisely judges that tumour includes infantile tumour to find the maximum combination of composite factor weight.

In embodiment above, almost each embodiment can be separately formed the product of practicality, apply a variety of Early diagnosis, guiding treatment and Prognosis scoveillance of tumour etc..In each embodiment can with system comprehensively reliably check and Comprehensive analysis, reach the purpose for needing to can be only achieved with a variety of and multiple detections presently, there are, complex and expensive.To being examined For person, hospital's blood drawing and this number of sampling and the financial burden of detection are reduced to, so the invention provides a kind of system It is comprehensively reliable and reduce person under inspection's burden to kinds of tumors accurately systematicness detection and comprehensive analysis interpretation method.

Sensitivity and specificity of the tumor markers in lesion detection in the present invention, tried from inventor in clinic The data of measure and accumulation in testing, referring also to current existing data.The portion of inventor is merely illustrative in embodiment Subsystem detects, and also has some systematicness detections not embody in this manual.Table 8 is in tumour to Partial tumors mark Sensitivity and specific Macro or mass analysis in detection, different combinations is entered to the tumor markers for screening different, there is provided Foundation, veteran can also make different systematicness according to the present invention and detect.

Although present invention has been a certain degree of description, it will be apparent that, do not departing from the spirit and scope of the present invention Under the conditions of, the appropriate change of each condition can be carried out.It is appreciated that the invention is not restricted to the embodiment, and it is attributed to right It is required that scope, it includes the equivalent substitution of each factor.

Claims

1. the systemic detection method of a kind of tumor markers, it is characterised in that methods described includes：

1) high flux inspection is carried out to biological specimen or its extract for one or more nucleotide sequences with tumour-specific Survey；

Wherein, the biological specimen be selected from cell line, Histological section, organize the tissue of biopsy/FFPE, body fluid, excrement, Colonic effluent, urine, blood plasma, serum, whole blood, the haemocyte of separation；The cell that is separated from blood, body fluid, sputum, pharynx are wiped Son, or its combination；Be preferably chosen from colonic effluent, urine, blood plasma, serum, whole blood, separation haemocyte, by being separated in blood Cell in one or more；

The nucleotide sequence with tumour-specific is selected from the mutator related to tumour generation, correlation occurs with tumour Decorating state gene, the recombinant DNA related to tumour generation, the RNA of the Fusion Strain related with tumour generation, copy number quantitative change Change the DNA related to tumour generation and RNA and tumour occurs related suppression tiny RNA (siRNA), correlation occurs with tumour The one or more of Microrna (microRNA) or the non-transcribed RNA related to tumour generation nucleotide sequence；

The protein tumor markers is selected from protein, the albumen related to tumour generation with tumour-specific and methylated One or more in modification or the Mechanisms of Histone Acetylation Modification related to tumour generation.

2. the method as described in claim 1, it is characterised in that the mutator related to tumour generation be selected from KRAS, One or more in BRAF, EGFR；

Preferably, the decorating state gene related to tumour generation is that related methylated genes occur with tumour, preferably The ground methylated genes be selected from GNG4, MIAT, DNM3, CHST2, HOXA9, C1orf70, NBLA00301, SIX6, OLIG2, SIM2、C9orf50、LONRF2、COL4A1、ADHFE1、ITGA4、SEPT9、CTSF、FAM159A、ZNF583、EFHA2、 ARHGAP22、CYP1B1、PPFIA4、SPAG6、RNF135、EFNB2、TRIL、LDHB、IGF1R、HOXD8、HOXA11AS、 HOXD9、SHOX2、CYBA、AOX1、AMOTL2、C2orf88、WFDC2、SIM1、GHSR、ZNF154、OXT、WDR69、RNF180、 C7orf51、ID3、VWCE、CRYGN、C10orf41、ARRDC2、AIM1、RAI1、GABRG3、PTGFR、ZNF805、CCNA1、 BCAN、RNLS、HOXD1、ELOVL5、JAKMIP1、CACNB2、PAX2、MCF2L、PDE4D、MAST4、CHD3、PLIN1、PAK1、 PROC、TFR2、PITPNM3、WNT7B、PTPRU、NDRG4、HOXXD10、NT5DC3、WNT3A、UBXN10、CDH22、 One or more in LYPLAL1, F11R, TMEM101, PYY, TERC, PTGER4, FOXL2, BNC1, ADAMTS1；

Preferably, the Microrna related to tumour generation be selected from hsa-let-7c, hsa-miR-122, hsa-miR-182, hsa-miR-193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、hsa-let-7b、hsa- miR-411、hsa-miR-450b、hsa-miR-485、hsa-miR-519a、hsa-miR-642、hsa-miR-517b、hsa- miR-520f、hsa-miR-206、hsa-miR-566、hsa-miR-661、hsa-miR-340、hsa-miR-1243、hsa- One or more in miR-720, hsa-miR-543 and hsa-miR-1267.

3. method as claimed in claim 1 or 2, it is characterised in that the measure of the nucleotide sequence with tumour-specific Method is selected from PCR, real-time quantitative PCR, digital pcr, liquid chip, solid-state chip, in situ hybridization, common sequencing and high pass and measured One or more in sequence.

4. such as the method any one of claim 1-3, it is characterised in that the protein tumor markers is selected from cancer embryo Antigen (carcino-embryonic antigen CEA), IDH1, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1、CA153、CA125、CA50、PSA、FPSA/TPSA、NSE、SCC、proGRP、PG、PGⅠ/PGⅡ、TPA、TPS、 Beta-HCG、SF、H3K4me1、H3K4me3、H4K8ac、Auto-ab p53Auto-AbMMP-7、Auto-Ab Hsp70、 One or more in Auto-Ab Prx VI, Auto-Ab Bmi-1 and β-HCG.

5. such as the method any one of claim 1-4, it is characterised in that the measure side of the protein tumor markers Method be selected from liquid chip, solid-state chip, enzyme linked immunological, radio-immunity, chemiluminescence immunoassay, mass spectrum, high performance liquid chroma- tography, One or more in the Western markings and sequencing.

6. such as the method any one of claim 1-5, it is characterised in that the nucleotide sequence with tumour-specific Selected from KRAS, BRAF, EGFR, RNF180, SHOX2, PTGER4, FOXL2, GNG4, C1orf70, C9orf50, CTSF, CYP1B1、RNF135、HOXD8、CYBA、SIM1、C7orf51、ARRDC2、GABRG3、RNLS、PAX2、PROC、PITPNM3、 One or more in HOXXD10, LYPLAL1, hsa-miR-122, hsa-let-7b.

7. method as claimed in claim 6, it is characterised in that step 2) carries to determine the sample by enzyme linked immunoassay Take the carcinomebryonic antigen in thing；Or

Step 2) is that the protein tumor markers in the sample extraction thing is determined by liquid chip or solid-state chip, described Protein tumor markers be selected from carcinomebryonic antigen, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, One or more in CA125, PSA and Beta-HCG；Or

Step 2) is by sample extraction thing described in liquid chip or solid-state chip enzyme linked immunological or chemiluminescence immunoassay Protein tumor markers, the protein tumor markers be selected from Auto-Ab p53, Auto-Ab MMP-7, Auto-Ab One or more in Hsp70, Auto-Ab Prx VI and Auto-Ab Bmi-1；Or

Step 2) is to pass through the protein tumor markers in sample extraction thing described in liquid chip or mass spectroscopy, the albumen One or more of the matter tumor markers in H3K4me1, H3K4me3 and H4K8ac.

8. method as claimed in claim 6, it is characterised in that protein tumor markers described in step 2) be selected from CEA, One or more in CA199, CA242 and CA724；Preferably, described CEA, CA199, CA242 and/or CA724 pass through ELISA reaction assays；It is highly preferred that step 2) also includes the detection of hemoglobin, it is further preferred that the hemoglobin leads to Cross immunochromatography FIT method measure.

9. method as claimed in claim 1 or 2, it is characterised in that there is the nucleic acid sequence of tumour-specific described in step 1) Column selection from KRAS, BRAF, EGFR, RNF180, SHOX2, PTGER4, FOXL2, GNG4, C1orf70, C9orf50, CTSF, CYP1B1、RNF135、HOXD8、CYBA、SIM1、C7orf51、ARRDC2、GABRG3、RNLS、PAX2、PROC、PITPNM3、 HOXXD10、LYPLAL1、hsa-miR-122、hsa-let-7bhsa-let-7c、hsa-miR-122、hsa-miR-182、hsa- miR-193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、hsa-let-7b、hsa-miR- 411st, the one or more in hsa-miR-450b, hsa-miR-485, hsa-miR-519a and hsa-miR-642；Preferably institute The assay method for stating the nucleotide sequence with tumour-specific is high-flux sequence；

Pass through the protein tumour mark in sample extraction thing described in liquid chip, solid-state chip or enzyme-linked immunoassay in step 3) Will thing, one or more of the protein tumor markers in Cyfra 21-a, NSE, SCC and IDH1.

A kind of 10. kit being used for such as method according to any one of claims 1 to 9, it is characterised in that the kit Primer, probe and/or reagent required for nucleotide sequence comprising measure tumour-specific, and measure protein tumor markers Required reagent；

Wherein, the nucleotide sequence with tumour-specific is selected from the mutator related to tumour generation, occurred with tumour The RNA of related Fusion Strain, copy occur for related decorating state gene, the recombinant DNA related to tumour generation and tumour The number change DNA related to tumour generation and RNA, related suppression tiny RNA (siRNA) and tumour generation occur with tumour Related Microrna (microRNA) or the one or more of the non-transcribed RNA related to tumour generation nucleotide sequence；

Preferably, the Microrna related to tumour generation be selected from hsa-let-7c, hsa-miR-122, hsa-miR-182, hsa-miR-193a、hsa-miR-200c、hsa-miR-203、hsa-miR-218、hsa-miR-155、hsa-let-7b、hsa- miR-411、hsa-miR-450b、hsa-miR-485、hsa-miR-519a、hsa-miR-642、hsa-miR-517b、hsa- miR-520f、hsa-miR-206、hsa-miR-566、hsa-miR-661、hsa-miR-340、hsa-miR-1243、hsa- One or more in miR-720, hsa-miR-543 and hsa-miR-1267；

Preferably, the protein tumor markers be selected from carcinomebryonic antigen (carcino-embryonic antigen CEA), IDH1, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, CA125, CA50, PSA, FPSA/ TPSA、NSE、SCC、proGRP、PG、PGⅠ/PGⅡ、TPA、TPS、Beta-HCG、SF、H3K4me1、H3K4me3、H4K8ac、 In Auto-ab p53Auto-Ab MMP-7, Auto-Ab Hsp70, Auto-Ab Prx VI, Auto-Ab Bmi-1 and β-HCG It is one or more；

It is highly preferred that the kit includes determining the protein in the sample extraction thing by liquid chip or solid-state chip Tumor markers carcinomebryonic antigen, CA724, alpha-fetoprotein (AFP), CA242, CA199, Cyfra21-1, CA153, CA125, PSA Or the reagent required for the one or more in Beta-HCG；Or

The kit includes carrying by sample described in liquid chip or solid-state chip enzyme linked immunological or chemiluminescence immunoassay Take protein tumor markers Auto-Ab p53, Auto-Ab MMP-7, Auto-Ab Hsp70, the Auto-Ab Prx VI in thing With the reagent required for the one or more in Auto-Ab Bmi-1；Or

The kit is included by the protein tumor markers in sample extraction thing described in liquid chip or mass spectroscopy The reagent required for one or more in H3K4me1, H3K4me3 and H4K8ac；Or

The kit includes passing through in ELISA reaction assay protein tumor markerses CEA, CA199, CA242 and CA724 Reagent required for one or more；

It is further preferred that the kit also includes the reagent needed for measure hemoglobin；It is further preferred that the examination Agent is that the reagent needed for hemoglobin is determined by immunochromatography FIT method.