CN103305600B - Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof - Google Patents
Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof Download PDFInfo
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Abstract
The invention discloses a kit for synchronously detecting related gene expression level of 14 antitumor drugs by using a paraffin embedding biopsy sample, and a detection method thereof. The kit comprises DEPC (diethylpyrocarbonate) water, 5*RT buffer solution, a reverse transcription primer, a reverse transcription enzyme, an X solution, 10*PCR buffer solution, a PCR primer, 25mM magnesium chloride solution, DNA (deoxyribonucleic acid) polymerase and a positive reference substance; the reverse transcription primer comprises related genes of the 14 antitumor drugs, and RT amplification primers of an RNA (ribonucleic acid) internal reference; gene sequences are shown in SEQ ID NO.1 to NO.18; the PCR primer includes related genes of the 14 antitumor drugs, and positive and negative PCR amplification primers of a DNA internal reference; the gene sequences are shown in SEQ ID NO.19 to NO.38. The kit has the advantages of strong specificity, high sensitivity, high flux, strong reliability, low cost and absence of false-negative result.
Description
Technical field
The present invention relates to test kit and the detection method thereof of antitumor medication related gene expression level, especially relate to a kind of test kit and detection method thereof of using 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection based on GeXP multiple gene expression genetic analysis systems.
Background technology
Recent years, pharmacogenetics/pharmacogenomics has obtained breakthrough in the research of the aspects such as antitumor drug mechanism of action, finds that some antitumor drug is to the lethal effect of tumour cell and specifically expression and/or the polymorphism significant correlation of a kind of (or one group) gene.By the detection of genes involved, the curative effect of prediction chemotherapy and targeted drug, selects suitable medicine to carry out Individual Chemotherapy, the choose reasonable that become and improved curative effect, reduces futile treatment.
Individualized treatment is the genetics feature according to patient, the method that adopts special and best pharmaceutical admixtures to treat.A large amount of clinical datas show, in tumor tissues, the target gene mrna expression level such as ERCC1/RRM1/TYMS/TUBB3 can be predicted respectively the reaction of patient to platinum class/gemcitabine/fluorouracil/Common Chemotherapy medicines such as anti-microtubule class.According to the detected result of mrna expression level in specimens, work out individualized treatment scheme, contribute to select to be applicable to patient's chemotherapeutics, improve the specific aim for the treatment of.By target detection, select chemotherapeutics can avoid invalid or harmful chemotherapy, save valuable treatment time and medical expense, improve patients ' life quality.
At present, the method for traditional gene expression detection (mRNA level) has following multiple:
(1) PCR detection method: at present often application have real-time fluorescence quantitative PCR, immuno-PCR, a reverse transcription PCR etc., be all that the specific target gene for pathogenic agent detects.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of the method is that susceptibility is high, and can accurate quantification; The method also has its shortcoming, and flux is low: the expression level that once can only detect a gene; Cost is relatively high: because once detecting a gene, when a sample needs to detect a plurality of genetic expression simultaneously, must detect one by one, cost increases relatively.
(2) molecular hybridization---Northern hybridization and RNA in situ hybridization: the process that complementary nucleotide sequence forms stable heteroduplex molecular dna molecule by Walson-Crick base pairing is called hybridization.Crossover process is high degree of specificity, can to known array, carry out specific target sequence detection according to used probe.Molecular hybridization has the specificity of very high sensitivity and height, thereby this technology in biology field, is used in widely the screening of clone gene, the qualitative and quantitative analysis of specific gene sequence and the aspects such as diagnosis of disease in the making of restriction enzyme mapping, genome.But its complex operation step, and technical requirements is higher; This technology flux is low simultaneously, once can only detect a goal gene.
(3) gene chips: gene chip is to pass through micro-processing technology, by ten hundreds of, and even the DNA fragmentation of 1,000,000 particular sequence (gene probe), arrange and be fixed on the upholders such as silicon chip, slide regularly, the two-dimentional DNA probe array forming, utilize the biological sample of this class chip and mark to hybridize, can carry out fast qualitative and quantitative analysis to the gene expression profile bioinformation of sample.The advantage of gene chips is: 1) high-flux parallel detects: when a sample needs to detect a plurality of gene expression dose simultaneously, once experiment can draw whole results; 2) easy and simple to handle quick: whole detection only needs 4-8 hour substantially can go out result.But also there is following shortcoming: 1) cost is higher: each sample needs a chip, and cost great Yu $1000/ sample, is unfavorable for large-scale promotion; Synthetic and the fixing more complicated of probe, particularly makes highdensity probe array, is main rate-limiting step; 2) can not accurate quantification, poor repeatability; 3) sensitivity is lower and need nucleic acid amount larger, in addition because the kind of chip is more, is difficult to formulate a unified quality control standard.
(4) transcribe group order-checking: transcribe group (transcriptome) and broadly refer under a certain physiological condition, in cell, the set of all transcription products, comprises messenger RNA(mRNA), ribosome-RNA(rRNA), transfer RNA and non-coding RNA; Refer to narrowly the set of all mRNA.Transcribing group order-checking is generally that ripe mRNA and the ncRNA that transcribes generation to carry out the rna plymerase ii of affinity purification with poly thymus pyrimidine (oligo-dT) carries out high-flux sequence.Obtain rapidly a certain species certain organs or be organized in the nearly all transcript under a certain state comprehensively, reflecting their expression level.With respect to traditional chip hybridization platform, transcribe group order-checking without in advance for known array designing probe, can detect the integral body activity of transcribing of any species, more exact figure signal is provided, higher detection flux and widely sensing range are the powerful tool that group complicacy is transcribed in further investigation at present.Along with the continuous progress of sequencing technologies, sequencing throughput is increasing, but its cost is always high, and common patient cannot bear, and the complicated operation of order-checking, and the cycle is longer, wouldn't be applicable to clinical treatment at present.
GenomeLab
tMcapillary electrophoresis separation technology and the highly sensitive laser Induced Fluorescence Technology research and development of GeXP multiple gene expression genetic analysis systems based on Beckman company maturation form, the kapillary display and design in a branch of 8 roads takes full advantage of the alignment characteristics of 96 orifice plates, has reduced cost and the complicacy of using larger display to bring.Adopt multiple PCR method, by Beckman Coulter dye marker, in same EP pipe, analyze the expression of a plurality of genes simultaneously, can fast and effeciently detect the expression situation of gene, overcome the defect that above-mentioned fluorescence quantitative PCR method exists, had the following advantages:
1, high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 30-40 site, can do simultaneously 192 reactions (as 192 patient's samples, 30 kinds of diarrhea viruses of each sample detection, 30 sites), within one day, go out result; For co-infected patients, present method can disposablely provide accurate report, avoids undetected.
2, accuracy is strong: GeXP adopts capillary electrophoresis to carry out separation detection to PCR product, can non-specific amplification product, primer dimer and specific amplification products is separated, at utmost reduce false positive;
3, susceptibility is high, and result is reproducible: GeXP system has overcome the deviation that the unequal amplification of normal PCR amplification method causes, and has improved a set of goal gene is carried out to quantitative speed and susceptibility, adopts laser induced fluorescence(LIF)-PMT, has hypersensitivity;
4, method is easy, uses economical: GeXP provides from a complete set of experimental programs such as reagent, multiple PCR primer design, result and quantitative expression spectrum analysis; The testing cost Shao Yu $50 of each sample, is beneficial to large-scale promotion;
5, accurate quantification, handiness are strong: can accurate quantification pathogen gene copy number, can adjust according to demand at any time the target gene of detection.
6, easily be automated: with regard to sample preparation, Biomek series automated fluid processing instrument can mate completely with GeXP analyser and Ampligrid amplification instrument, and integrated bar code reader has guaranteed that sample is followed the trail of and report the test accurately.
At present, both at home and abroad also not about the test kit of 14 kinds of antitumor medication related gene expression levels of the synchronous detection based on GeXP multiple gene expression genetic analysis systems and the correlative study of detection method report thereof.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of high specificity, highly sensitive, flux is high, reliability is strong, cost is low, without test kit and the detection method thereof of the 14 kinds of antitumor medication related gene expression levels of use specimens paraffin embedding slices sample synchronous detection based on GeXP multiple gene expression genetic analysis systems of false negative result.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection, comprise DEPC water, 5 * RT damping fluid, reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, PCR primer, 25mM magnesium chloride solution, archaeal dna polymerase and positive reference substance, described reverse transcription primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds in following table 1 and the RT amplimer of RNA internal reference, described PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds in following table 1, forward and reverse pcr amplification primer of the pcr amplification primer of RNA internal reference and DNA internal reference, gene order is as shown in table 1 below:
Table 1
Described X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
Described positive reference substance is the RNA mixture extracting from tumour cell, and described RNA mixture comprises 14 kinds of described antitumor medication related gene mRNAs.
A method of utilizing the antitumor medication related gene expression of the detection level of the test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection, specifically comprises the following steps:
(1) collecting sample extract nucleic acid
The separation and Culture thing that gathers specimens specimens paraffin embedding slices sample extracts nucleic acid from separation and Culture thing;
(2) take patient's nucleic acid carries out RT reaction as template
Get the nucleic acid samples RNA5 μ L of 5-20ng/ul, DEPC water 8 μ L, 5 * RT damping fluid, 4 μ L, RT primer solution 2 μ L, join in 96 hole sample panel and carry out reverse transcription, reaction conditions after RT enzyme 1 μ L mixes: 48 ° of C1 minute; 42 ° of C60 minute; 95 ° of C5 minute; 4 ° of C are until collect RT product; Wherein in RT primer solution, each RT primer concentration is 500nM, and described RT primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds and the RT amplimer of RNA internal reference, and gene order is as shown in SEQ ID NO.1～NO.18;
(3) take reverse transcription product carries out PCR reaction as template
Get RT product 8.6 μ L, 10 * PCR damping fluid, 2 μ L, the magnesium chloride 4 μ L of 25mM, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L, join the enterprising performing PCR reaction of 96 hole sample panel, reaction conditions: 95 ° of C10 minute after X solution 2 μ L mix; In 94 ° of C30 seconds, in 55 ° of C30 seconds, 70 ° of C1 minute, circulate 35 times; 70 ° of C1 minute; 4 ° of C are until collect PCR product; Wherein said X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark, in PCR primer solution, each PCR primer concentration is 200nM, PCR primer comprises forward and reverse pcr amplification primer of the pcr amplification primer of 14 kinds of antitumor medication genes involveds, the pcr amplification primer of RNA internal reference and DNA internal reference, and gene order is as shown in SEQ ID NO.19～NO.38;
(4) GeXP genetic analyzer electrocapillary phoresis sample separation
Get PCR product 0.1-1 μ L, the sample-loading buffer 38.75 μ L that GeXP genetic analyzer is supporting, DNAMarker0.5 μ L, after one, mineral oil mixes, join on 96 hole parting liquid plates and carry out electrocapillary phoresis sample separation, the collection of illustrative plates that the software of GeXP genetic analyzer is obtained and standard diagram contrast, and obtain the expression level of antitumor medication genes involved.
Compared with prior art, the invention has the advantages that: a kind of test kit and detection method thereof of using 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection of the present invention, because this test kit has been introduced for 14 genes that antitumor medication is relevant, the designed specificity amplification primers such as 4 reference genes, according to the expression level of these genes, instruct the nearly medicining condition of 24 kinds of antitumor drugs, within one day, can complete the detection of 192 patient's samples, both production cost and testing cost had been saved, improved again detection efficiency and shortened the time, the use of four reference genes can be used for monitoring the efficiency of whole reactive system, RT-PCR reaction and the quality of assessment RNA template, avoids false negative, the use of reaction internal reference can be used for monitoring the efficiency of whole reactive system, PCR reaction, avoids false negative, makes to detect to have better sensitivity and specificity, thereby has avoided the not high problem of other detection method specificitys.
In sum, the present invention is based on test kit and the detection method thereof of 14 kinds of antitumor medication related gene expression levels of use specimens paraffin embedding slices sample synchronous detection of GeXP multiple gene expression genetic analysis systems, can for 14 antitumor medication genes involveds, detect simultaneously, detection sensitivity is high, specificity is good, reduce the false positive rate of conventional pcr amplification, can also effectively solve the easy pollution problem of conventional PCR; There is Noncompetitive internal comparison system, reliability is strong, without false negative result, utilizes that GeXP genetic analysis systems is sensitive, accurate quantitative analysis, quick, high-throughout technical superiority, for clinical, provide a kind of superior molecular diagnosis method cheaply, contribute to chemotherapeutics to use safely and effectively.
Accompanying drawing explanation
Fig. 1 is the electrocapillary phoresis sample separation result standard collection of illustrative plates of GeXP genetic analyzer.
Embodiment
Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.
A kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection of the present invention, this test kit comprises following reagent:
1) RT primer (reverse transcription primer RT primer Mix)
2) PCR primer (PCR Primer Mix)
3) 25mM magnesium chloride (25mM MgCl2)
4) reversed transcriptive enzyme (Reversetranscripatase)
5) archaeal dna polymerase (Taq DNA Polymerase)
6) X solution (Solution X)
7) 10 * PCR damping fluid (10 * PCR Buffer)
8) 5 * RT damping fluid (5 * RT buffer)
9) positive control (Positive Control)
11) without RNA enzyme/DNA enzyme ultrapure water (Dnase/Rnase Free ddH2O)
12) positive reference substance
Above-mentioned reverse transcription primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds in following table 1 and the RT amplimer of RNA internal reference, described PCR primer comprises forward and reverse pcr amplification primer of the pcr amplification primer of 14 kinds of antitumor medication genes involveds in following table 1, the pcr amplification primer of RNA internal reference and DNA internal reference, and gene order is as shown in table 1:
The multiple gene test oligonucleotide sequence of the antitumor medication genes involved of table 1
Above-mentioned X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
Above-mentioned positive reference substance is the RNA mixture extracting from tumour cell, comprises above-mentioned 14 kinds of antitumor medication related gene mRNAs.
According to the expression level of above-mentioned 14 kinds of antitumor medication genes involveds, instruct the nearly medicining condition of 24 kinds of antitumor drugs, specific as follows:
(1) BRCA1: expression level and patient are proportionate to the susceptibility of platinum class and anti-microtubule class medicine;
(2) DPYD: expression level and 5-FU(5-Fluracil) susceptibility is negative correlation;
(3) EGFR: expression level and Cetuximab resistance are proportionate;
(4) PTEN: down-regulated expression is the potential sign of mammary cancer differentiation and transfer;
(5) ERCC1: expression level and therapeutic effectiveness of platinum medicaments are negative correlation;
(6) STMN1: it is better that low expression level is accepted taxanes/cis-platinum (drug combination that refers to taxol and cis-platinum) result for the treatment of; High expression level patient tumors shifts risk;
(7) TUBB3: low expression level accepts taxanes/cis-platinum or vinca result for the treatment of is better; High expression level patient tumors shifts risk;
(8) RRM1: expression level and gemcitabine resistance are proportionate;
(9) VEGFR: expression level and rhuMAb-VEGF curative effect are proportionate;
(10) HER2: expression level and Trastuzumab curative effect are proportionate;
(11) TYMP: expression level and capecitabine curative effect are proportionate; The low patient of expression level has good prognosis;
(12) TYMS: expression level and 5-FU curative effect are proportionate;
(13) PDGFR: expression level variation prediction Xarelto curative effect;
(14) TOP2A: expression level and patient are proportionate to the susceptibility of Etoposide.
Specific embodiment two
A kind of detection method of using 14 kinds of antitumor medication genes involveds of specimens paraffin embedding slices sample synchronous detection of the present invention, gather specimens and extract nucleic acid, the patient's nucleic acid of take carries out reverse transcription and PCR reaction as template, finally uses electrocapillary phoresis method sample separation, and concrete steps are as follows:
1, the test kit of producing the 14 kinds of antitumor medication genes involveds of use specimens paraffin embedding slices sample synchronous detection based on GeXP multiple gene expression genetic analysis systems, the component that test kit comprises is with above-described embodiment 1;
2, collecting sample extract nucleic acid
The separation and Culture thing that gathers specimens specimens paraffin embedding slices sample extracts nucleic acid from separation and Culture thing;
3, take patient's nucleic acid carries out reverse transcription (RT) reaction as template
1) in following ratio, in 96 hole sample panel, add reagent and sample (RT plate is in Table 2):
Table 2RT reaction reagent and sample mix ratio
| RT reaction reagent | Amount/hole |
| DEPC water (without RNA enzyme/DNA enzyme ultrapure water Dnase/Rnase Free) | 8μL |
| 5 * RT damping fluid | 4μL |
| RT primer solution (each RT primer concentration is 500nM) | 2μL |
| RT enzyme | 1μL |
| Sample RNA (5-20ng/ul) | 5μL |
| Total | 20μL |
Note: add in RT reaction, positive reference substance is the RNA mixture extracting from tumour cell, comprises 14 kinds of described antitumor medication related gene mRNAs: consumption is 5 μ L/ reactions
2) by following temperature, hatch (in Table 3) after mixing:
Table 3RT reaction conditions
4, take reverse transcription product carries out PCR reaction as template
1) in following ratio, in 96 hole sample panel, add reagent and sample (PCR plate is in Table 4):
Table 4PCR reaction reagent and sample mix ratio
| PCR reaction reagent | Amount/hole |
| 10 * PCR damping fluid | 4μL |
| 25mM?MgCl2 | 4μL |
| PCR primer | 2μL |
| Archaeal dna polymerase | 1.4μL |
| X solution | 2μL |
| RT product | 8.6μL |
| Total | 20μL |
Note: X solution comprises triphosphate deoxy-nucleotide (dNTPs) and universal primer, universal primer forward amplimer sequence is AGGTGACACTATAGAATA, reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, universal primer forward amplimer band fluorescent mark.
2) by following temperature, carry out thermal cycle reaction (in Table 5) after mixing:
Table 5PCR reaction conditions
| Step | Temperature | Time |
| 1 | 95°C | 10 minutes |
| 2 | 94°C | 30 seconds |
| 3 | 55°C | 30 seconds |
| 4 | 70°C | 1 minute |
| 5 | N/A | Repeat 2-4 step 34 time (totally 35 times) |
| 6 | 70°C | 1 minute |
| 7 | 4°C | Continue: until collect PCR product |
5, GeXP genetic analyzer electrocapillary phoresis sample separation
1) prepare GeXP sample (in Table 6):
Table 6GeXP sample mix ratio
2) electrocapillary phoresis sample separation
GeXP sample is added in the hole of proper number on 96 hole capillary electrophoresis separation plates and carry out capillary electrophoresis separation; Capillary electrophoresis separation is that a class be take the Novel liquid-phase isolation technique that kapillary is motivating force as split tunnel, the high-voltage dc of take, and specific procedure is 90 ℃ of sex change 120 seconds, sample introduction voltage 2kv, 30 seconds, separation voltage 6kv, 35 minutes.
6, interpretation of result (seeing GenomeLabGeXP genetic analyzer specification sheets)
According to the parameter of giving tacit consent on the own software of GeXP genetic analyzer, result is carried out to clip size analysis, its X-coordinate represents clip size, and ordinate zou is that signal is strong and weak.The peak collection of illustrative plates obtaining according to the software of GeXP genetic analyzer, the calculated by peak area at each gene peak obtains relative expression's level of antitumor medication genes involved, according to relative expression's level of these genes, instructs the nearly medicining condition of 24 kinds of antitumor drugs.As shown in Figure 1, its result can accurately detect the expression level of 14 kinds of antitumor medication genes involveds to standard diagram, and each target fragment size interval is moderate, and signal is unlikely to supersaturation, and between each target, signal is relatively fair, and there is no broad peak, the phenomenon such as bimodal.
Specific embodiment three
Detection kit sensitivity, specificity analyses
Sensitivity analysis: by after certain ng value doubling dilution, until can't detect signal, this ng value is lowest detection line, namely the sensitivity of test kit through pcr amplification and capillary electrophoresis detection by positive reference substance.The sensitivity of this test kit is 1.0ng.
Specificity analyses: it is the unimodal of target fragment size that substance pcr amplification detects through capillary electrophoresis.
Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (2)
1. a test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection, comprise DEPC water, 5 * RT damping fluid, reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, PCR primer, 25 mM magnesium chloride solutions, archaeal dna polymerase and positive reference substance, it is characterized in that described reverse transcription primer comprises the RT amplimer of 14 kinds of antitumor medication genes involveds in following table and the RT amplimer of RNA internal reference, described PCR primer comprises the pcr amplification primer of 14 kinds of antitumor medication genes involveds in following table, forward and reverse pcr amplification primer of the pcr amplification primer of RNA internal reference and DNA internal reference, gene order is as shown in the table:
Wherein said X solution comprises triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Reverse primer extension increasing sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
2. a kind of test kit that uses 14 kinds of antitumor medication related gene expression levels of specimens paraffin embedding slices sample synchronous detection according to claim 1, it is characterized in that: described positive reference substance is the RNA mixture for extracting from tumour cell, comprises 14 kinds of described antitumor medication related gene mRNAs.
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| CN102251036A (en) * | 2011-07-07 | 2011-11-23 | 中国人民解放军总医院 | Method for simultaneously detecting 17 genes |
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