CN103773878B - Based on blood plasma microRNA detection kit and the detection method thereof of AllGlo fluorescence probe quantitative PCR - Google Patents

Abstract

Based on blood plasma microRNA detection kit and the detection method thereof of AllGlo fluorescence probe quantitative PCR, relate to microRNA.Test kit is provided with box body, dividing plate, miRNA extract reagent bottle, stem ring reverse transcription reagents bottle, real-time fluorescence quantitative PCR reagent bottle.Does is after the abundant cracking of 200 μ L blood plasma, add the working concentration provided in described test kit 5n? the exogenous reference of blood plasma miRNA of mol, carries out whirlpool concussion 15s, the same ordinary method of all the other steps immediately; MiRNA reverse transcription is cDNA by the stem ring reverse transcription reagents that applying described test kit provides; CDNA is carried out real-time fluorescence quantitative PCR amplification by the real-time fluorescence quantitative PCR reagent that applying described test kit provides; Every data that composite analyser provides, set rational threshold value and baseline, analyze.Its specificity and susceptibility high, quick and precisely can detect the content of miRNA in sample.

Description

Plasma microRNA detection kit and detection method based on AllGlo probe fluorescence quantitative PCR

technical field

The invention relates to microRNA, in particular to a plasma microRNA detection kit based on AllGlo probe fluorescent quantitative PCR and a detection method thereof.

Background technique

MicroRNA (miRNA) is a kind of non-coding RNA molecule of about 22 nucleotides widely present in eukaryotic cells, which participates in many physiological and pathological processes in organisms, by regulating gene expression, in cell proliferation, apoptosis , growth and development, cell differentiation, metabolism and other processes play an important role. The specific performance is that miRNA forms the initial primary transcript pri-miRNA to pre-miRNA in the nucleus, and then transports out of the nucleus, forms mature miRNA by shearing, and forms RISC (RNA-induced silencing complex) with Ago protein, etc. Partially inhibit or degrade the target mRNA sequence and participate in the regulation of gene expression (BartelDP. MicroRNAs: genomics, biogenesis, mechanism, and function [J]. Cell, 2004, 116(2): 281-297.).

Since miRNA plays an important role in tumor occurrence, development, prognosis judgment and many other disease states, accurate detection of its expression is of great significance for studying the role of miRNA. At present, the methods for detecting miRNA mainly include northern blot technology and real-time fluorescent quantitative PCR. technology, in situ hybridization technology, microarray chip technology, etc. Among them, northern blot technology is one of the early means of RNA detection, but its specificity and sensitivity are low. If the sample RNA content is low or there is degradation, it may not be detected; in situ hybridization technology is used to detect the distribution of miRNA at the tissue and cell level , while performing semi-quantitative detection of miRNA, its disadvantage is that it cannot accurately detect low-expression miRNA; microarray chip technology is a high-throughput detection technology that can simultaneously detect a large number of miRNAs in one or more samples , but there are problems such as time-consuming and labor-intensive chip fabrication, high labeling costs, and low detection sensitivity and specificity.

Real-time fluorescence quantitative technique (RT-qPCR) is one of the most commonly used techniques for gene expression detection, which is characterized by simplicity, rapidity, high sensitivity and good specificity. The real-time fluorescent quantitative PCR method currently used to detect miRNA includes two parts: RNA reverse transcription and downstream fluorescent quantitative PCR. The reverse transcription is divided into two types according to the different reverse transcription primers: homopolymer tailing method and stem-loop reverse transcription. The detection method of fluorescent quantitative PCR is divided into dye method and probe method, among which the probe for detecting miRNA is mostly Taqman-MGB probe (a fluorescent probe suitable for amplifying short fragments). Since the mature miRNA has the characteristics of short fragments, the probe method has more advantages for specific detection, and is superior to the dye method in terms of sensitivity and specificity. The reverse transcription primer based on the stem-loop structure can specifically recognize and amplify the mature miRNA sequence, but the precursor of miRNA has not been amplified. The disadvantage of Taqman-MGB probe detection of miRNA is that the synthesis is expensive, which is not conducive to popularization.

At present, AllGlo probes have been successfully applied to detect gene mutations of herpes virus and hepatitis B virus (Feng, ZLYu, XYLu, ZMGeng, DYZhang, L. Chen, SJ Rapid detection of the hepatitis B virus YMDD mutation using AllGlo ^TM probes [J]. ClinChimActa, 2011, 412 (11-12) : 1018-1021), invasive aspergillosis (Wu, DSShen, JZZhou, XQShen, SFWu, XM The establishment and evaluation of diagnostic accuracy of All Glo (TM) probe-based techniques for invasive aspergillosis [J]. ZhonghuaNeiKeZaZhi, 2010, 49 (2): 142K-145 Detection of gene mutation, ankylosing spondylitis, etc.

Contents of the invention

In view of the above-mentioned defects in the kits and detection methods used in the existing plasma miRNA detection methods, the first purpose of the present invention is to provide specific primers for detection of three plasma miRNAs.

The second object of the present invention is to provide a plasma miRNA detection kit based on AllGlo probe fluorescence quantitative PCR.

The third object of the present invention is to provide a method for detecting three plasma miRNAs based on AllGlo probe fluorescent quantitative PCR.

The three plasma miRNAs refer to hsa-miR-504, hsa-miR-133b, and cel-miR-39 respectively; wherein hsa-miR-504 and hsa-miR-133b are human-derived miRNAs, and cel-miR-39 is The nematode-derived miRNA, as an exogenous reference miRNA, is added to the plasma from plasma extraction, and its function is to be able to monitor the whole process of real-time fluorescent quantitative PCR from the extraction of miRNA.

The specific primers for detecting three plasma miRNAs include three plasma miRNA specific reverse transcription primers and three plasma miRNA specific forward primers;

The three plasma miRNA specific reverse transcription primers are composed of hsa-miR-504 specific reverse transcription primers, hsa-miR-133b specific reverse transcription primers and cel-miR-39 specific reverse transcription primers;

The nucleotide sequence of the hsa-miR-504 specific reverse transcription primer is SEQ ID NO.15'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGATAGAGT-3';

The nucleotide sequence of the specific reverse transcription primer of hsa-miR-133b is SEQ ID NO.25'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTAGCTGGT-3';

The nucleotide sequence of the specific reverse transcription primer of cel-miR-39 is SEQ ID NO. 35'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCAAGCTGA-3'.

The three plasma miRNA-specific forward primers are composed of hsa-miR-504 specific forward primers, hsa-miR-133b specific forward primers and cel-miR-39 specific forward primers;

The nucleotide sequence of the hsa-miR-504 specific forward primer is SEQ ID NO.45'-GCTGGTTAAGACCCTGGT-3';

The specific forward primer nucleotide sequence of hsa-miR-133b is SEQ ID NO.55'-TAGCGTCTTTGGTCCCCT-3';

The nucleotide sequence of the specific forward primer of cel-miR-39 is SEQ ID NO.65'-CAGAGTCACCGGGTGTAAAT-3'.

The three plasma miRNA specific AllGlo probes are composed of hsa-miR-504 specific AllGlo probes, hsa-miR-133b specific AllGlo probes and cel-miR-39 specific AllGlo probes;

The nucleotide sequence of the hsa-miR-504-specific AllGlo probe is SEQ ID NO. 7MAR-CTGGATACGACGATAGAGTGC-MAR;

The nucleotide sequence of the specific AllGlo probe of hsa-miR-133b is SEQ ID NO.8JUP-CTGGATACGACTAGCTGGTTGA-JUP;

The nucleotide sequence of the specific AllGlo probe of cel-miR-39 is SEQ ID NO.9NEP-TGCACTGGATACGACCAAGCT-NEP.

The nucleotide sequence of the universal primer sequence of the three plasma miRNAs is SEQ ID NO. 105'-CAGTGCGTGTCGTGGAGT-3'.

The plasma microRNA detection kit based on AllGlo probe fluorescent quantitative PCR is provided with a box body, a partition, a miRNA extraction reagent bottle, a stem-loop reverse transcription reagent bottle, and a real-time fluorescent quantitative PCR reagent bottle; the partition is arranged in the box body, and the miRNA Extraction reagent bottles, stem-loop reverse transcription reagent bottles, and real-time fluorescence quantitative PCR reagent bottles are inserted on the partition plate, miRNA extraction reagent bottles are filled with miRNA extraction reagents, and stem-loop reverse transcription reagent bottles are filled with stem-loop reverse transcription reagents for real-time fluorescence quantitative The PCR reagent bottle is equipped with real-time fluorescent quantitative PCR reagents.

The miRNA extraction reagent includes plasma miRNA exogenous reference, and the plasma miRNA exogenous reference refers to the artificially synthesized cel-miR-39 mimic (the artificially synthesized cel-miR-39 mimic serves as an exogenous The working concentration of the original added reference is 5nmol, which is used to monitor the reaction efficiency of the whole process from microRNA extraction to real-time fluorescent quantitative PCR).

The stem-loop reverse transcription reagent includes the following components: 200 units/μL of MMLV enzyme, 10mmoldNTP mixture, 5×RT buffer (5×RT buffer consists of 250mmol Tris-HCl, 375mmol KCl, 15mmolMgCl ₂ , 50mmol DTT), 40 Unit/μL of RNase inhibitor, 10 mmol of MgCl ₂ , 1 μmol of specific miRNA reverse transcription primer, and miRNA standard (the miRNA standard here refers to artificially synthesized miRNA with a concentration of 100 nmol).

The real-time fluorescent quantitative PCR reagent includes the following components: 10×Taq buffer (10×Taq buffer includes 100mmol Tris-HCl, 500mmol KCl), 10mmol of MgCl ₂ , 5 units/μL of Taq polymerase, 10mMdNTP mixture, 100ml Nuclease-free water, 10 μmol specific forward primer, 10 μmol universal reverse primer and AllGlo probe.

The plasma microRNA detection method based on AllGlo probe fluorescence quantitative PCR, comprises the following steps:

1. After 200 μL of plasma is fully lysed, add the plasma miRNA exogenous reference with a working concentration of 5 nmol provided in the plasma miRNA detection kit based on AllGlo probe fluorescent quantitative PCR, and immediately vortex for 15 seconds, and the rest of the steps are the same as the conventional method ;

2. Using the stem-loop reverse transcription reagent provided by the plasma miRNA detection kit based on AllGlo probe fluorescence quantitative PCR, the miRNA is reverse-transcribed into cDNA;

3. Apply the real-time fluorescent quantitative PCR reagent provided by the plasma miRNA detection kit based on AllGlo probe fluorescent quantitative PCR to carry out real-time fluorescent quantitative PCR amplification of cDNA;

4. Comprehensively analyze the various data given by the instrument, set a reasonable threshold (Threshold) and baseline (Baseline), and analyze the results.

The AllGlo probe can adopt the fluorescent quantitative probe of AlleLogic Biosciences Corporation of the United States, which has all the advantages of common Taqman, Taqman-MGB and molecular beacon (molecular beacon) probes, and removes the biggest drawback of these several probes at present. To overcome the limitation of the traditional Taqman reporter group at one end and the quencher group at the other end, the special fluorescent dyes with several common wavelengths developed by AleLogic Biosciences Corporation of the United States are used to mark each other on the oligonucleotide as the reporter group and the quencher group, and It contains a special chemical group that can increase the Tm value (annealing temperature), improve the specificity of the probe, and greatly improve the hybridization specificity. After the hybridization hydrolysis, all the dyes labeled at both ends become reporter groups, increasing the fluorescence increment. .

The AllGlo probe has the following advantages: ① Increase the Tm value (up to 10°C or more), the shorter probe can reach 15-16 bases, and it can be used to design probes for sequences with relatively high A and T content; ② Add Increase the choice of multiple fluorescence quantification, because each dye is both a reporter group and a quencher group, which breaks the difficulty of label selection due to the wavelength of the traditional Taqman probe, and is not limited by the wavelength of the quencher group; ③ greatly improves the signal-to-noise ratio, no background signal, close spatial distance, and better quenching effect; ④ lower cost, the price is only half of that of Taqman-MGB probes, and has all the advantages of MGB probes.

The present invention adopts the AllGlo probe technology, and designs three kinds of specific forward primers, specific probes and universal reverse primers. The two ends of the three probes only need to be labeled with three special fluorescent groups (MAR, JUP, NEP). , can simultaneously detect three miRNAs, and optimize the reaction conditions of this technology, and establish a method based on AllGlo probe technology combined with real-time fluorescent quantitative PCR technology to simultaneously detect three plasma miRNAs, which has broad application prospects.

The present invention adopts the AllGlo probe technology and establishes a real-time fluorescent quantitative PCR method capable of detecting miRNA. Compared with the Taqman-MGB probe method, the linear range can reach 7 orders of magnitude. The sensitivity of the real-time fluorescence quantitative PCR established by the present invention can be as low as 10 copies/μL and as high as 10 ⁷ copies/μL.

The beneficial effects of the present invention are as follows:

The present invention provides a detection method and kit based on AllGlo probe RT-qPCR for detecting miRNA, which has high specificity and sensitivity, can quickly and accurately detect the content of miRNA in a sample, and has the following advantages compared with the prior art:

1. Compared with northern blot and miRNA chips, the specificity and sensitivity of AllGlo probe detection are higher, and the price is cheaper than microRNA chips;

2. Compared with the taqman-MGB probe, the AllGlo probe uses the same fluorescent group, which acts as a reporter group and a quencher group for each other. On the one hand, the released fluorescent signal is stronger, and on the other hand, the background signal is lower; Moreover, the synthesis procedure is simple, and the price is only equivalent to half of taqman-MGB.

3. The present invention establishes an RT-qPCR method for detecting miRNA based on AllGlo probes, which is suitable for later stage clinical sample verification of microarray screening miRNA, as well as research on differential expression of miRNA in different fields, etc., which promotes miRNA in related diseases in-depth research.

Description of drawings

FIG. 1 is a schematic diagram of the structure and composition of a plasma microRNA detection kit based on AllGlo probe fluorescent quantitative PCR according to an embodiment of the present invention.

Figure 2 is the amplification curve of hsa-miR-133b (human-derived miR-133b) detected in three standard mixtures by AllGlo probe real-time fluorescent quantitative PCR.

Fig. 3 is a standard curve diagram of hsa-miR-133b (human miR-133b) detected in three standard mixtures by AllGlo probe real-time fluorescent quantitative PCR.

detailed description

Example 1

Referring to Fig. 1, the plasma microRNA detection kit embodiment based on AllGlo probe fluorescence quantitative PCR of the present invention is provided with box body 1, partition 2, miRNA extraction reagent bottle 3, stem-loop reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent bottle 5; Partition plate 2 is located in box body 1, miRNA extraction reagent bottle 3, stem-loop reverse transcription reagent bottle 4, real-time fluorescent quantitative PCR reagent bottle 5 are inserted on the partition plate 2, and miRNA extraction reagent bottle 3 is equipped with The miRNA extraction reagent, the stem-loop reverse transcription reagent bottle 4 is equipped with a stem-loop reverse transcription reagent, and the real-time fluorescent quantitative PCR reagent bottle 5 is equipped with a real-time fluorescent quantitative PCR reagent.

The miRNA extraction reagent includes plasma miRNA exogenous reference, and the plasma miRNA exogenous reference refers to the artificially synthesized cel-miR-39 mimic (the artificially synthesized cel-miR-39 mimic is used as a The working concentration of exogenously added reference is 5nmol, which is used to monitor the reaction efficiency of the whole process from microRNA extraction to real-time fluorescent quantitative PCR).

The stem-loop reverse transcription reagent includes the following components: 200 units/μL of MMLV enzyme, 10mmoldNTP mixture, 5×RT buffer (5×RT buffer consists of 250mmol Tris-HCl, 375mmol KCl, 15mmolMgCl ₂ , 50mmol DTT), 40 Unit/μL of RNase inhibitor, 10 mmol of MgCl ₂ , 1 μmol of specific miRNA reverse transcription primer, and miRNA standard (the miRNA standard here refers to artificially synthesized miRNA with a concentration of 100 nmol).

The real-time fluorescent quantitative PCR reagent includes the following components: 10×Taq buffer (10×Taq buffer includes 100mmol Tris-HCl, 500mmol KCl), 10mmol of MgCl ₂ , 5 units/μL of Taq polymerase, 10mMdNTP mixture, 100ml Nuclease-free water, 10 μmol specific forward primer, 10 μmol universal reverse primer and AllGlo probe.

Example 2

Concrete operating steps of the present invention and reaction system are as follows:

1. Extraction of miRNA

The miRNA extraction kit produced by Tiangen Biotechnology Co., Ltd. was used to extract the microRNA from the sample (plasma) according to the operating instructions. Among them, 200 μL of plasma was added to an equal volume of lysate and allowed to stand for 5 minutes, and then 395 μL of exogenous reference cel-mir (working concentration) was added. 5nmol), then follow the instructions, and finally dissolve the RNA with 30 μL of denuclease water, measure the concentration and purity on the nucleic acid spectrophotometer NanoDrop2000, and take 2-20ng of the extracted miRNA for downstream reverse transcription. All other miRNAs were frozen at -80°C.

2. Reverse transcription of miRNA:

2.1 Dissolve the components required for reverse transcription at room temperature, shake and mix well, and place on ice;

2.2 Prepare the reverse transcription reaction mixture according to Table 1:

Table 1

components Volume/tube (μL) MMLV reverse transcriptase (200 units/μL) (promega company) 0.2 MMLV 5x reverse transcriptase buffer (Promega) 4 Deoxynucleoside mixture (10mmol) 0.8 RNase inhibitor (40 units/μL) 0.2 Specific stem-loop reverse transcription primer (1 μmol) 1.2 Nuclease-free water 11.6 total capacity 18

2.3 Aliquot each tube in separate 0.2 μL PCR tubes

Dispense 18 μL of the prepared mixture into RNase-free PCR tubes, then add 2 μL of the extracted and enriched miRNA solution, mix thoroughly with RNase-free pipette tips (try to avoid the generation of air bubbles during this operation), and centrifuge briefly Throw it to the bottom of the tube and place it on an ordinary PCR instrument (produced by Biorad Company). The reverse transcription reaction conditions are shown in Table 2.

Table 2

step condition 1 25℃5min 2 42℃60min 3 70℃15min

Place at 4°C after the reaction is complete.

3. Fluorescent quantitative PCR detection of miRNA

3.1 Dissolve the components of fluorescent quantitative PCR at room temperature, mix well and place on ice.

3.2 Prepare the PCR reaction mixture as shown in Table 3.

table 3

components Volume per tube (μL) dNTP mixture (10mmol) 2 MgCl2 (25mmol) 2 10×Taq buffer (Mg ²⁺ free) 2 rTaq (5 units/μL) (takara) 0.2 Forward primer (10 μmol) 0.4 Reverse primer (10μmol) 0.4 Probe (10μmol) 0.2 nuclease water 10.8 total capacity 18

3.3 Dispense and prepare the PCR reaction mixture in 8 tubes, add 2 μL of cDNA to each tube, and mix well. The whole process is carried out on ice to avoid the generation of air bubbles.

3.4 Fluorescent quantitative PCR reaction The reaction conditions are shown in Table 4.

Table 4

The detection is carried out on a real-time fluorescent quantitative PCR instrument, and various instruments such as ABI7300 and 7500 (Applied Biosystems, USA) can be used.

4. Data analysis and standardization processing: SPSS13.0 and Excel2007 were used for data analysis, and the CT values of the target miRNA in the sample plasma and the exogenous reference cel-mir-39 measured by the above method, according to the CT value of the exogenous reference The relative content of the target miRNA in the plasma was obtained at the value level, and the level of the target miRNA in the plasma was expressed in the form of relative quantitative 2 ^-ΔCt in qPCR (ΔCt is the difference between the Ct value of the target microRNA and the exogenous reference).

Example 3

The fluorescent quantitative PCR reaction system and the establishment of the standard curve of the three miRNA mixtures are shown in Table 5.

table 5

Element Volume/tube (μL) dNTP mixture (10mmol) 4 MgCl2 (25mmol) 4 10×Taq buffer (Mg2+-free) 4 rTaq (5 units/μL) (takara) 0.4 3 pairs of forward and reverse primer mixture (10μmol) 4.8 3 kinds of probe mixture (10μmol) 1.2 nuclease water 19.6 total capacity 38

Dilute the three microRNA standards in advance to the initial miRNA concentration of 5×10 ⁷ copies/μL. After mixing, perform serial 10-fold dilutions, and then perform RT-qPCR detection. Taking hsa-miR-133b as an example, its amplification The increasing curve and standard curve are shown in Figures 2 and 3.

Example 4

Clinical sample testing:

Select 5 cases of plasma samples from each patient, and use the AllGlo probe method to detect hsa-miR-133b, and compare it with the microRNATaqmanMGB method of hsa-miR-133b detected by American lifetechnologies company, and make 2 duplicate holes.

Table 6 compares the results of three methods for detecting hsa-miR-133b in 5 cases of colorectal cancer tissue samples.

Table 6

Hsa-miR-133b/CT value comparison Single-plex AllGlo probe method Taqman-MGB method Triple AllGlo probe method sample 1 30.703±0.61 33.107±0.27 30.729±0.738 sample 2 30.487±0.49 33.053±0.13 28.946±0.537 sample 3 31.073±0.403 32.43±0.51 30.4±0.129 Sample 4 30.183±0.084 34.085±0.106 32.431±0.134 Sample 5 31.862±0.234 32.81±0.27 29.88±0.455

Explanation: When detecting the same sample, the lower the CT value, the higher the sensitivity. The data in the above table shows that compared with the detection of 5 different samples by the Taqman-MGB probe method, the single AllGlo probe method and the triple AllGlo probe method The CT value of has-miR-133b in plasma samples detected by the needle method is lower than that of the Taqman-MGB probe method, indicating that the sensitivity of the two methods is significantly higher than that of the Taqman-MGB probe method.

Claims (3)

1. detect the specific primer of three kinds of plasma miRNAs, it is characterized in that described three kinds of plasma miRNAs refer to hsa-miR-504, hsa-miR-133b, cel-miR-39 respectively; Wherein hsa-miR-504 and hsa- miR-133b is a human-derived miRNA, and cel-miR-39 is a nematode-derived miRNA. As an exogenous reference miRNA, it is added to the plasma from the plasma extraction, and the role is to monitor the progress of the real-time fluorescent quantitative PCR from the extraction of miRNA. The whole process; The specific primers for detecting three kinds of plasma miRNAs include three kinds of plasma miRNA specific reverse transcription primers, three kinds of plasma miRNA specific forward primers and three kinds of plasma miRNA general reverse primers; The three plasma miRNA specific reverse transcription primers are composed of hsa-miR-504 specific reverse transcription primers, hsa-miR-133b specific reverse transcription primers and cel-miR-39 specific reverse transcription primers; The nucleotide sequence of the hsa-miR-504 specific reverse transcription primer is SEQ ID NO.15'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGATAGAGT-3'; The nucleotide sequence of the specific reverse transcription primer of hsa-miR-133b is SEQ ID NO.25'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTAGCTGGT-3'; The nucleotide sequence of the specific reverse transcription primer of cel-miR-39 is SEQ ID NO.35'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCAAGCTGA-3'; The three plasma miRNA-specific forward primers are composed of hsa-miR-504 specific forward primers, hsa-miR-133b specific forward primers and cel-miR-39 specific forward primers; The nucleotide sequence of the hsa-miR-504 specific forward primer is SEQ ID NO.45'-GCTGGTTAAGACCCTGGT-3'; The nucleotide sequence of the hsa-miR-133b specific forward primer is SEQ ID NO.55'-TAGCGTCTTTGGTCCCCT-3'; The nucleotide sequence of the specific forward primer of cel-miR-39 is SEQ ID NO.65'-CAGAGTCACCGGGTGTAAAT-3' The nucleotide sequence of the three plasma miRNA universal reverse primers is SEQ ID NO. 105'-CAGTGCGTGTCGTGGAGT-3'. 2. Three kinds of plasma miRNA-specific AllGlo probes, characterized in that the three plasma miRNAs refer to hsa-miR-504, hsa-miR-133b, cel-miR-39 respectively; wherein hsa-miR-504 and hsa -miR-133b is a human-derived miRNA, and cel-miR-39 is a nematode-derived miRNA. As an exogenous reference miRNA, it is added to the plasma from the plasma extraction, and the role is to monitor the entire real-time fluorescent quantitative PCR from the extraction of miRNA the whole process; The three plasma miRNA specific AllGlo probes are composed of hsa-miR-504 specific AllGlo probes, hsa-miR-133b specific AllGlo probes and cel-miR-39 specific AllGlo probes; The nucleotide sequence of the hsa-miR-504-specific AllGlo probe is SEQ ID NO. 7MAR-CTGGATACGACGATAGAGTGC-MAR; The nucleotide sequence of the specific AllGlo probe of hsa-miR-133b is SEQ ID NO.8JUP-CTGGATACGACTAGCTGGTTGA-JUP; The nucleotide sequence of the specific AllGlo probe of cel-miR-39 is SEQ ID NO.9NEP-TGCACTGGATACGACCAAGCT-NEP. 3. The plasma microRNA detection kit based on AllGlo probe fluorescent quantitative PCR is characterized in that it is provided with a box body, a partition, a miRNA extraction reagent bottle, a stem-loop reverse transcription reagent bottle, and a real-time fluorescent quantitative PCR reagent bottle; the partition is located at Inside the box, miRNA extraction reagent bottles, stem-loop reverse transcription reagent bottles, and real-time fluorescent quantitative PCR reagent bottles are inserted into the partition plate, miRNA extraction reagent bottles are filled with miRNA extraction reagents, and stem-loop reverse transcription reagent bottles are filled with stem-loop reverse transcription reagents , the real-time fluorescent quantitative PCR reagent bottle is equipped with real-time fluorescent quantitative PCR reagent; The miRNA extraction reagent includes plasma miRNA exogenous reference, the plasma miRNA exogenous reference refers to artificially synthesized cel-miR-39, and artificially synthesized cel-miR-39 is used as an exogenously added reference, Its working concentration is 5nmol/L, and its function is to monitor the reaction efficiency of the whole process from microRNA extraction to real-time fluorescent quantitative PCR; The stem-loop reverse transcription reagent includes the following components: 200 units/μL of MMLV enzyme, 10 mmol/LdNTP mixture, 5×RT buffer, 40 units/μL of RNase inhibitor, 10 mmol/L of MgCl ₂ , 1 μmol The reverse transcription primer and miRNA standard substance of specific miRNA/L, the concentration is 100nmol/L; The 5×RT buffer solution is made up of 250mmol/LTris-HCl, 375mmol/LKCl, 15mmol/LMgCl ₂ , 50mmol/LDTT; The reverse transcription primer of the specific miRNA is three kinds of plasma miRNA specific reverse transcription primers described in claim 1; The real-time fluorescence quantitative PCR reagent includes the following components: 10×Taq buffer, 10mmol/L MgCl ₂ , 5 units/μL Taq polymerase, 10mMdNTP mixture, 100ml nuclease-free water, 10μmol/L specific positive Primer, 10 μmol/L universal reverse primer and AllGlo probe; Described 10×Taq damping fluid comprises 100mmol/LTris-HCl, 500mmol/LKCl; Described specific forward primer is three kinds of plasma miRNA specificity described in claim 1 forward primer, the universal reverse primer is the three plasma miRNA universal reverse primers described in claim 1, and the AllGlo probe is the three plasma miRNA-specific AllGlo probes described in claim 2.