CN107385037B - MiRNA indirect real-time fluorescence quantitative PCR detection method - Google Patents

MiRNA indirect real-time fluorescence quantitative PCR detection method Download PDF

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CN107385037B
CN107385037B CN201710605544.4A CN201710605544A CN107385037B CN 107385037 B CN107385037 B CN 107385037B CN 201710605544 A CN201710605544 A CN 201710605544A CN 107385037 B CN107385037 B CN 107385037B
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CN107385037A (en
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张晓玮
张新
邓可基
彭春梅
李家导
张嘉
乐小炎
李海茵
罗园香
杨芬
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Dongguan Microprecision Testing Research Institute Co ltd
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Abstract

The invention discloses a miRNA indirect real-time fluorescence quantitative PCR detection method, which combines single-stranded DNA and miRNA, then uses DSN enzyme to digest the paired single-stranded DNA, and finally quantifies the residual single-stranded DNA by a Taqman method, thereby indirectly detecting the content of target miRNA. The invention avoids the problem of non-specific amplification in the amplification of the traditional method, still uses mature Taqman technology, and has extremely convenient and stable application. Based on the advantages, the method has wide application prospect in the field of tumor detection.

Description

MiRNA indirect real-time fluorescence quantitative PCR detection method
Technical Field
The invention belongs to the technical field of molecular biology, and discloses an indirect real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for miRNA.
Background
miRNA is a non-coding micro RNA with the length of about 22 nt and has the following structural characteristics: mirnas are ubiquitous in eukaryotes, do not encode proteins, and do not have open reading frames per se; mature miRNA is not a simple RNA degradation fragment, its 5' end and 3' end are linked with phosphate group and hydroxyl group respectively, and the first base pair of 5' end has strong tendency to uracil (U) and some resistance to hey mountain (G). miRNA gene sequences are not randomly arranged, some are clustered (cluster), and clustered genes are often expressed synergistically; most mirnas are produced by processing on one arm of a Precursor miRNA (Pre miRNA), and few mirnas are produced simultaneously on both arms of a Pre-miRNA. The expression of miRNA has obvious tissue specificity and cell specificity, certain time sequence and high conservative property of its sequence.
Research shows that miRNA regulates the gene expression of more than 30% of human beings, thereby participating in various life processes. There is a lot of evidence that abnormal miRNA expression levels are in Mount link with human tumorigenesis. It is currently believed that down-regulation or up-regulation of mirnas may lead to activation of proto-oncogenes or inhibition of cancer suppressor genes. miR-21 is up-regulated in expression level in various tumor diseases such as gastric cancer, lung cancer, prostatic cancer, colon cancer, breast cancer and the like, and is positively correlated with malignancy degree of tumors such as gastric cancer and the like. most target mRNAs of miR-21 are transcription products of cancer suppressor genes, such as PDCD4, PTEN and the like. The mature sequence of miR-21 is hsa-miR-21-5p MIMAT 0000076: UAGCUUAUCAGACUGAUUGAUUGA (SEQ ID NO: 5).
The mainstream method for detecting miRNA is based on Taqman fluorescence quantitative PCR, and comprises a PolyA tail method and a stem-loop method.
The reverse transcription primer for the tailed approach is essentially the universal oligo (dT) primer used for mRNA processing. One step prior to reverse transcription is to add a PolyA tail to the RNA sample, followed by reverse transcription. Therefore, its reverse transcription primer comprises a universal sequence, a poly-T and a single base anchor. The concept of single base anchoring stems from the Differential display (Differential display) technique in the mRNA analysis technique. The principle is to classify a large number of mRNAs by means of their polyA tail. This degenerate reverse transcription primer of A or C or G followed by poly-T classifies all mRNAs into 3 types, which are single base anchored.
"Stem loop" refers to a reverse transcription primer. The stem-loop structure not only can effectively prolong the length of miRNA, but also can avoid the combination with other homologous genes due to the self-complementary conformation, thereby reducing the probability of nonspecific amplification. The whole primer consists of 2 parts, a universal stem-loop structure and 5-8 bases which are reversely complementary with the 3' end of the target miRNA. The general stem-loop structure is designed by Chen and the like, and the sequence is 5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC-3' (SEQ ID NO: 6). Wherein the underlined part is the stem-loop self-complementary part. Then 5-8 bases are added to the 3 'end of the sequence, and the sequence is reversely complementary with the 3' end of the target MicroRNA.
Reverse transcription is required by both the PolyA tail method and the stem-loop method, but the reverse transcription process has the problem of insufficient primer specificity, so the specificity of the two methods cannot meet the requirement of a diagnostic reagent product.
Double-strand specific nuclease (DSN) is a specific nuclease from red-gold crab, has thermal stability, maximum activity at 60-65 deg.C, and 25% activity even after incubation at 70 deg.C for 20 min. DSN enzymes cleave double-stranded DNA, as well as DNA in DNA/RNA hybrid strands, and are routinely used for cDNA homogenization. Since 2013, some technologies such as a DSN enzyme combined graphene oxide sensor, specific magnetic beads, nanogold, molecular beacons and the like are used for miRNA detection. However, these methods are complicated in design and high in cost, and are not suitable for industrialization.
Disclosure of Invention
The invention aims to provide a miRNA indirect real-time fluorescent quantitative PCR detection method.
The invention also aims to provide a miRNA indirect real-time fluorescent quantitative PCR detection kit.
The technical scheme adopted by the invention is as follows:
an indirect real-time fluorescence quantitative PCR detection method for miRNA comprises the following steps:
1) synthesizing single-stranded DNA, wherein the single-stranded DNA comprises a segment 1 which can be subjected to base complementation with a target miRNA, and the 5 'end and the 3' end of the segment 1 are respectively connected with a segment 2 and a segment 3, the base numbers of which are not less than 20 bp;
2) and (3) hybridization digestion: hybridizing the synthesized single-stranded DNA with a sample to be detected, and digesting the DNA in a hybrid by using DSN enzyme;
3) carrying out real-time fluorescent quantitative PCR on the sample to be detected after hybridization digestion, and detecting the Ct value of the single-stranded DNA left after DSN enzyme digestion; and determining the concentration of the target miRNA corresponding to the Ct value according to a standard curve established by the positive quality control product of the target miRNA, thereby obtaining the concentration of the target miRNA in the sample to be detected.
Further, in the step 1), the base sequences of the segment 2 and the segment 3 have no homology with the genome of the species to which the target miRNA belongs.
Further, in the step 2), the reaction process of the hybridization digestion is 30-60 min at 55-65 ℃ and 10-20 min at 92-97 ℃.
Further, in step 2), the amount of the single-stranded DNA added to the reaction system for the hybridization digestion is such that the final concentration thereof is 103～108copies/mL。
Further, in step 3), the fragment amplified by the real-time fluorescence quantitative PCR contains the fragment 1, or contains the base sequence of more than 6bp of the fragment 1.
Further, the target miRNA is miR-21, and the single-stranded DNA is shown in SEQ ID NO: as shown in figure 1, the first and second main bodies,
further, the sequences of the primers and the fluorescent probes used for real-time fluorescent quantitative PCR are as follows:
an upstream primer F: 5'-TGGATTGATGTGATATCTCCACTGA-3' (SEQ ID NO: 2),
a downstream primer R: 5'-CCTTATATAGAGGAAGGGTCTTGAGTTG-3' (SEQ ID NO: 3),
and (3) a fluorescent probe P: 5 '-Fam-TAAGGGATATCGAATAGTCTG-3' MGB (SEQ ID NO: 4).
An indirect real-time fluorescence quantitative PCR kit of miRNA, the kit contains single-stranded DNA, primer and fluorescent probe of real-time fluorescence quantitative PCR;
the single-stranded DNA contains a segment 1 which can be subjected to base complementation with a target miRNA, and the 5 'end and the 3' end of the segment 1 are respectively connected with a segment 2 and a segment 3, the base numbers of which are not less than 20 bp;
the primer can enable the fragment amplified by the real-time fluorescent quantitative PCR to contain the fragment 1 or contain the base sequence of more than 6bp of the fragment 1;
the fluorescent probe can detect the amplification product of real-time fluorescent quantitative PCR.
Further, the base sequences of the segment 2 and the segment 3 have no homology with the genome of the species to which the target miRNA belongs.
An indirect real-time fluorescence quantitative PCR detection kit of miR-21, the kit contains SEQ ID NO: 1, SEQ ID NO: 2-3, SEQ ID NO: 4, and (b) a fluorescent probe.
The invention has the beneficial effects that:
the invention provides an indirect real-time fluorescence quantitative PCR detection method of miRNA, which comprises the steps of combining the miRNA with artificially synthesized single-stranded DNA (deoxyribonucleic acid) which has a complementary sequence with a target miRNA, digesting the paired single-stranded DNA by using DSN (deoxyribose nucleic acid) enzyme, and combining the released target miRNA with the complementary single-stranded DNA to realize signal amplification. And finally, quantifying the residual single-stranded DNA by a Taqman method, thereby indirectly detecting the content of the target miRNA.
The method does not need reverse transcription of the target miRNA, and avoids the problem of non-specific amplification in the traditional method.
The method still uses mature Taqman technology, is extremely convenient to apply, and does not need to use technologies such as a graphene oxide sensor, a specific magnetic bead, nanogold, a molecular beacon and the like.
The principle of the method is universal, and the method can be used for detecting the miR-21 mentioned herein and can be applied to the detection of other miRNA after the sequence is modified.
All enzymes used in the method have certain thermal stability, so that the product based on the method has stronger stability.
Based on the advantages, the method has wide application prospect in the field of tumor detection.
Drawings
FIG. 1 shows an experiment for detecting miR-21 by using the indirect real-time fluorescent quantitative PCR detection method of the invention, wherein A is a standard curve obtained after detection of a miRNA standard substance diluted in a gradient manner, the slope of the standard curve is 3.30165, the intercept is-6.37831, the correlation coefficient is 0.99982, and the amplification efficiency is-0.50212; b is 1 multiplied by 10 from left to right4、1×105、1.236×105、1×106、1×107copy/mL miR-21 amplification result, wherein the concentration is 1.236 x 105copying/mL of a sample to be detected, and amplifying the rest samples which are miR-21 standard samples;
FIG. 2 is a specific experiment for detecting miR-21 by applying the indirect real-time fluorescent quantitative PCR detection method of the invention, and the specific experiment is carried out by using normal saline because miR-21 also exists in normal human serum.
Detailed Description
An indirect real-time fluorescence quantitative PCR detection method for miRNA comprises the following steps:
1) synthesizing single-stranded DNA, wherein the single-stranded DNA comprises a segment 1 which can be subjected to base complementation with a target miRNA, and the 5 'end and the 3' end of the segment 1 are respectively connected with a segment 2 and a segment 3, the base numbers of which are not less than 20 bp;
2) and (3) hybridization digestion: hybridizing the synthesized single-stranded DNA with a sample to be detected, and digesting the DNA in a hybrid by using DSN enzyme;
3) carrying out real-time fluorescent quantitative PCR on the sample to be detected after hybridization digestion, and detecting the Ct value of the single-stranded DNA left after DSN enzyme digestion; and determining the concentration of the target miRNA corresponding to the Ct value according to a standard curve established by the positive quality control product of the target miRNA, thereby obtaining the concentration of the target miRNA in the sample to be detected.
Preferably, in step 1), the base sequences of the fragments 2 and 3 have no homology with the genome of the species to which the target miRNA belongs.
Preferably, in the step 2), the reaction process of the hybridization digestion is 30-60 min at 55-65 ℃ and 10-20 min at 92-97 ℃.
Preferably, in step 2), the reaction system for the hybridization digestion contains DSN enzyme, Tris-HCl and MgCl2KCl and DTT.
Preferably, the reaction system for hybridization digestion contains 5-10U DSN enzyme, 45-55 mM Tris-HCl pH 7.2-7.8, 4-6 mM MgCl2、18～22mM KCl、0.8～1.2mM DTT。
Preferably, in step 2), the single-stranded DNA is added to the reaction system of the hybridization digestion in such an amount that the final concentration thereof is 103～108copies/mL。
Preferably, in step 3), the fragment amplified by real-time fluorescent quantitative PCR contains fragment 1, or contains the base sequence of fragment 1 of more than 6 bp.
Preferably, in step 3), the fluorescent probe used in the real-time fluorescent quantitative PCR is capable of detecting an amplification product of the real-time fluorescent quantitative PCR.
Preferably, when the detection result has no Ct, the sample is positive and exceeds the upper limit of quantification, and detection is carried out after dilution;
when the Ct is more than or equal to 6 and less than or equal to 40 in the detection result, the test result is judged to be positive;
when the detection result Ct is less than 6, the retest is recommended, and if the retest result Ct is more than or equal to 4, the sample can be judged to be positive by the target miRNA; if the Ct of the rechecking result is less than 4, the target miRNA is judged to be negative;
when the Ct of the detection result is less than 4, the miRNA is judged to be negative.
Preferably, the sample to be detected is an miRNA extraction sample.
Preferably, the target miRNA is miR-21, and the single-stranded DNA is represented by SEQ ID NO: as shown in figure 1, the first and second main bodies,
preferably, the sequences of the primers and the fluorescent probes used for real-time fluorescent quantitative PCR are as follows:
an upstream primer F: 5'-TGGATTGATGTGATATCTCCACTGA-3' (SEQ ID NO: 2),
a downstream primer R: 5'-CCTTATATAGAGGAAGGGTCTTGAGTTG-3' (SEQ ID NO: 3),
and (3) a fluorescent probe P: 5 '-Fam-TAAGGGATATCGAATAGTCTG-3' MGB (SEQ ID NO: 4).
An indirect real-time fluorescence quantitative PCR kit of miRNA, the kit contains single-stranded DNA, primer and fluorescent probe of real-time fluorescence quantitative PCR;
the single-stranded DNA contains a segment 1 which can be subjected to base complementation with a target miRNA, and the 5 'end and the 3' end of the segment 1 are respectively connected with a segment 2 and a segment 3, the base numbers of which are not less than 20 bp;
the primer can enable the fragment amplified by the real-time fluorescent quantitative PCR to contain the fragment 1 or contain the base sequence of more than 6bp of the fragment 1;
the fluorescent probe can detect the amplification product of real-time fluorescent quantitative PCR.
Preferably, the base sequences of the fragment 2 and the fragment 3 have no homology with the genome of the species to which the target miRNA belongs.
Preferably, the kit further comprises DSN enzyme, Tris-HCl, MgCl2、KCl、DTT。
An indirect real-time fluorescence quantitative PCR detection kit of miR-21, the kit contains SEQ ID NO: 1, SEQ ID NO: 2-3, SEQ ID NO: 4, and (b) a fluorescent probe.
Preferably, the kit further comprises a DSN enzyme.
The present invention will be further described with reference to the following examples.
Example 1 MiR-21 indirect real-time fluorescence quantitative PCR detection method
Firstly, designing a single-stranded DNA and a primer probe sequence:
artificially designing, and synthesizing a segment of nucleotide sequence shown as SEQ ID NO: 1, wherein the underlined part is complementary to the sequence of miR-21 in the reverse direction, the remainder is Camv35S sequence having no homology with the human genome, and the synthetic company is shanghai jiere biotechnology limited.
Single-stranded DNA:
5’-AGTGGATTGATGTGATATCTCCACTGACGTAAGGGATATCGAATAGTCTGACTACAACTCAAGACCCTTCCTCTATATAAGGAAG-3’(SEQ ID NO：1)
the matched real-time fluorescent quantitative PCR primers and fluorescent probe sequences are designed as follows:
an upstream primer F: 5'-TGGATTGATGTGATATCTCCACTGA-3' (SEQ ID NO: 2),
a downstream primer R: 5'-CCTTATATAGAGGAAGGGTCTTGAGTTG-3' (SEQ ID NO: 3),
and (3) a fluorescent probe P: 5 '-Fam-TAAGGGATATCGAATAGTCTG-3' MGB (SEQ ID NO: 4).
The synthetic company is Shanghai Yiwei Jie based (Shanghai) trade company Limited.
Secondly, sample collection and pretreatment
Exosome separation kit produced by Guangzhou Hailite Biotechnology limited company is used for separating exosomes from serum samples according to the requirements of instructions, and the serum samples are collected from Zhongshan three hospitals in Guangzhou city.
The exosome miRNA extraction kit produced by Guangzhou Hailite Biotechnology limited company is used for extracting miRNA according to the instruction requirements to obtain a sample to be detected.
Preparation of positive quality control material
Artificially synthesizing a miR-21 sequence: 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO: 5), and the concentration was determined. The synthetic company is Shanghai Jima pharmaceutical technology, Inc.
Four, cross digestion
And (3) carrying out hybridization and DSN enzyme digestion on the sample to be detected and the positive quality control product which are subjected to exosome separation and miRNA extraction respectively.
The hybridization digestion system contained 50mM Tris-HCl pH 7.5, 5mM MgCl2，20mM KCl，1mM DTT，108Single-stranded DNA of copies, 5-10U DSN enzyme, 5-20 mu L of sample to be detected, and the balance of RNAase-Free water.
The hybridization digestion reaction process is as follows: 45min at 60 ℃ and 15min at 95 ℃.
Fifth, real-time fluorescent quantitative PCR amplification
Reaction system of real-time fluorescence quantitative PCR: amplification was performed using a 55. mu.L system containing 400nM forward primer F, 400nM downstream primer R, 200nM fluorescent probe P, 25. mu.l PCR Mix, 25. mu.L of the sample to be tested, and the balance water. A positive control group (artificially synthesized miR-21) and a negative control group (water) were set according to the same system.
The PCR Mix component is 65mM Tris pH 8.8, MgCl2 10mM，(NH4)2SO4 30mM，KCl 20mM，Taq 5U/25μL，dNTP 700mM。
Placing each reaction tube into a reaction tank of a quantitative PCR instrument, setting the name and the type of each detected fluorescent group (setting a report group as FAM and a quenching group as MGB), and setting the cycle conditions:
amplifying by using a macro time SLAN 96P fluorescence PCR instrument, wherein the circulating condition is 95 ℃ for 2 minutes; 15 seconds at 95 ℃, 30 seconds at 60 ℃ and 40 cycles; fluorescence was collected at 60 ℃.
Sixthly, analyzing and judging results
1. Setting of conditions for analysis of results
Setting baseline (baseline): the default settings of software of the SLAN 96P fluorescence PCR instrument when the macro is used can be properly adjusted according to specific conditions.
Setting a threshold (threshold): macro time SLAN 96P fluorescence PCR instrument software defaults.
2. Result judgment
And when the detection result has no Ct, the sample is positive and exceeds the upper limit of quantification, and the detection is carried out after dilution.
When the Ct is more than or equal to 6 and less than or equal to 40 in the detection result, judging the test result to be positive, and taking the quantitative value as the detection result;
when the detection result Ct is less than 6, a retest is recommended, if the retest result Ct is more than or equal to 4, the sample is determined to be miRNA-21 positive, but the sample concentration is lower than the reagent quantitative limit, and the quantitative value is only used for reference; if the Ct of the rechecking result is less than 4, the miRNA-21 is judged to be negative;
when the Ct of the detection result is less than 4, the miRNA-21 is judged to be negative.
3. Concentration of miRNA
The miRNA standard substance with gradient dilution is detected, a standard curve (shown in figure 1-A) is obtained by software calculation of a macro-time SLAN 96P fluorescence PCR instrument, and figure 1-B shows an experiment for detecting positive miR-21 with different concentrations by applying the indirect real-time fluorescence quantitative PCR detection method of the invention, wherein the experiment is sequentially 1 multiplied by 10 from left to right4、1×105、1.236×105、1×106、1×107copy/mL miR-21 amplification result, wherein the concentration is 1.236 x 105And copying/mL of the sample to be detected, and amplifying the other samples which are miR-21 standard products.
The embodiment is calculated by the self-contained software of the fluorescence quantitative PCR instrument according to the standard curveThe concentration of the miRNA-21 in the sample to be detected is 1.236 multiplied by 105copy/mL.
FIG. 2 shows the result of the amplification of negative control (physiological saline) detected by the indirect real-time fluorescent quantitative PCR detection method of the present invention, where Ct is 2.89.
Seventh, specific detection
And carrying out specificity detection on the miR-21 indirect real-time fluorescence quantitative PCR detection method, wherein a physiological saline is used for carrying out specificity experiments due to the fact that miR-21 also exists in serum of normal human. The results of the detection are shown in FIG. 2.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
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Claims (5)

1. An indirect real-time fluorescence quantitative PCR detection method of miRNA for non-disease diagnosis application is characterized by comprising the following steps:
1) synthesizing single-stranded DNA, wherein the single-stranded DNA comprises a segment 1 which can be subjected to base complementation with a target miRNA, and the 5 'end and the 3' end of the segment 1 are respectively connected with a segment 2 and a segment 3, the base numbers of which are not less than 20 bp;
2) and (3) hybridization digestion: hybridizing the synthesized single-stranded DNA with a sample to be detected, and digesting the DNA in a hybrid by using DSN enzyme;
3) carrying out real-time fluorescent quantitative PCR on the sample to be detected after hybridization digestion, and detecting the Ct value of the single-stranded DNA left after DSN enzyme digestion; determining the concentration of the target miRNA corresponding to the Ct value according to a standard curve established by the positive quality control product of the target miRNA, thereby obtaining the concentration of the target miRNA in the sample to be detected;
the target miRNA is miR-21, and the single-stranded DNA is shown in SEQ ID NO: 1 is shown in the specification;
the sequences of the primers and the fluorescent probes used in the real-time fluorescent quantitative PCR are as follows:
an upstream primer F: 5'-TGGATTGATGTGATATCTCCACTGA-3' (SEQ ID NO: 2),
a downstream primer R: 5'-CCTTATATAGAGGAAGGGTCTTGAGTTG-3' (SEQ ID NO: 3),
and (3) a fluorescent probe P: 5'-TAAGGGATATCGAATAGTCTG-3' (SEQ ID NO: 4),
the 5 'end of the fluorescent probe P is marked with Fam, and the 3' end is marked with MGB.
2. The method as claimed in claim 1, wherein in the step 2), the reaction process of the hybridization digestion is 30-60 min at 55-65 ℃ and 10-20 min at 92-97 ℃.
3. The method according to claim 1, wherein in step 2), the amount of the single-stranded DNA added to the reaction system for the hybridization digestion is such that the final concentration thereof is 103～108copies/mL。
4. The method according to claim 1, wherein the fragment amplified by real-time fluorescent quantitative PCR in step 3) comprises fragment 1, or comprises a base sequence of more than 6bp of fragment 1.
5. An indirect real-time fluorescence quantitative PCR detection kit for miR-21 is characterized by comprising the following components in parts by weight of SEQ ID NO: 1, SEQ ID NO: 2-3, SEQ ID NO: 4, DSN enzyme.
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CN109234413A (en) * 2018-11-27 2019-01-18 中国农业大学 A kind of visualization Cascaded amplification functional nucleic acid sensor for pig derived component quantitative detection
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102618664A (en) * 2012-05-03 2012-08-01 武汉大学 MiRNA (Micro Ribonucleic Acid) detection probe and method for visually detecting miRNA
CN103834719A (en) * 2012-11-28 2014-06-04 深圳先进技术研究院 miRNA detection probe and miRNA amplification detection method
CN103882132A (en) * 2014-03-27 2014-06-25 中国人民解放军第三军医大学第一附属医院 Real-time dynamic detection method of trace RNA (Ribose Nucleic Acid)
CN104342486A (en) * 2013-07-30 2015-02-11 中国科学院上海生命科学研究院 MiRNA detection method and its application
CN105132577A (en) * 2015-09-30 2015-12-09 基因科技（上海）有限公司 Method for conducting multiplex quantitative detection on miRNA

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102618664A (en) * 2012-05-03 2012-08-01 武汉大学 MiRNA (Micro Ribonucleic Acid) detection probe and method for visually detecting miRNA
CN103834719A (en) * 2012-11-28 2014-06-04 深圳先进技术研究院 miRNA detection probe and miRNA amplification detection method
CN104342486A (en) * 2013-07-30 2015-02-11 中国科学院上海生命科学研究院 MiRNA detection method and its application
CN103882132A (en) * 2014-03-27 2014-06-25 中国人民解放军第三军医大学第一附属医院 Real-time dynamic detection method of trace RNA (Ribose Nucleic Acid)
CN105132577A (en) * 2015-09-30 2015-12-09 基因科技（上海）有限公司 Method for conducting multiplex quantitative detection on miRNA

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
One-Step, Multiplexed Fluorescence Detection of microRNAs Based on Duplex-Specific Nuclease Signal Amplification;Yin, Bin-Cheng等;《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》;20120321;第134卷(第11期);第5064-5067页 *
谢振华.相关肿瘤标志性microRNA新型检测体系的构建及其应用.《中国优秀硕士学位论文全文数据库中国优秀硕士学位论文全文数据库》.2017,摘要，第3-4章. *

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