CN103773878A

CN103773878A - Plasma microRNA (Ribonucleic Acid) detection kit and detection method based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction)

Info

Publication number: CN103773878A
Application number: CN201410041147.5A
Authority: CN
Inventors: 张忠英; 唐晶
Original assignee: Zhongshan Hospital Xiamen University
Current assignee: Zhongshan Hospital Xiamen University
Priority date: 2014-01-28
Filing date: 2014-01-28
Publication date: 2014-05-07
Anticipated expiration: 2034-01-28
Also published as: CN103773878B

Abstract

The invention discloses a plasma microRNA (Ribonucleic Acid) detection kit and detection method based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction), relating to microRNA. The kit is provided with a kit body, a partition plate, a miRNA extraction reagent bottle, a stem-loop reverse transcription reagent bottle and a real-time fluorescent quantitative PCR reagent bottle. The detecting method comprises the steps: after sufficiently cracking 200mu L of plasma, adding the plasma with the working concentration of 5n mol as a microRNA exogenous reference, immediately carrying out vortex vibration for 15s, and carrying out the rest steps according to the conventional method, wherein the plasma with the working concentration of 5n is contained in the kit; carrying out reverse transcription on miRNA by using a stem-loop reverse transcription reagent provided by the kit to form cDNA; carrying out real-time fluorescent quantitative PCR amplification on cDNA by using a real-time fluorescent quantitative PCR reagent, wherein the real-time fluorescent quantitative PCR reagent is provided by the kit; and comprehensively analyzing all data provided by instruments, setting reasonable threshold values and base lines and finally carrying out analysis. The detection kit is high in specificity and sensibility and capable of rapidly and accurately detecting the content of miRNA in a sample.

Description

Blood plasma microRNA detection kit and detection method thereof based on AllGlo fluorescence probe quantitative PCR

Technical field

The present invention relates to microRNA, be specifically related to a kind of blood plasma microRNA detection kit and detection method thereof based on AllGlo fluorescence probe quantitative PCR.

Background technology

MicroRNA (miRNA) is that a class is extensively present in the non-coding RNA molecule that is about 22 Nucleotide in eukaryotic cell, many pathological processes in organism are participated in, express by regulatory gene, at cell proliferation, apoptosis, grow, play a significant role in the process such as cytodifferentiation, metabolism.Be embodied in miRNA process in nucleus and form initial primary transcribe pri-miRNA to pre-miRNA, after transport out nucleus, form ripe miRNA by shearing, by forming with Ago albumen etc. the silencing complex that RISC(RNA induces), part suppresses or degraded said target mrna sequence, participate in gene expression regulation (Bartel DP.MicroRNAs:genomics, biogenesis, mechanism, and function[J] .Cell, 2004,116 (2): 281-297.).

Because miRNA is playing the part of key player under generation, development, prognosis judgement and many other diseases states of tumour, accurately detect its expression and be significant for the effect of research miRNA, the method that detects at present miRNA mainly contains northern blot technology, Real-Time Fluorescent Quantitative PCR Technique, hybridization in situ technique, micro-array chip technology etc.Wherein northern blot technology is one of early stage means of RNA detection, but its specificity and susceptibility are low, if sample rna content is low or have degraded, may can't detect; Hybridization in situ technique, for detection of the distribution situation of tissue and cell levels miRNA, carries out half-quantitative detection to miRNA simultaneously, and its weak point is accurately to detect the miRNA of low expression; Micro-array chip technology is a kind of high-throughout detection technique, can detect a large amount of miRNA of one or more samples simultaneously, but have that chip manufacturing is time-consuming, effort, mark cost are high, detection sensitivity and the problem such as specificity is low.

Real time fluorescent quantitative technology (RT-qPCR) is one of technology the most generally detecting for genetic expression at present, have easy fast, the feature such as the high and specificity of susceptibility is good.The quantitative fluorescent PCR two portions that comprise at present RNA reverse transcription and downstream for detection of the real time fluorescence quantifying PCR method of miRNA, wherein reverse transcription is divided into two kinds according to the difference of reverse transcription primer: homopolymeric tailing method and stem ring reverse transcription method; The detection of quantitative fluorescent PCR is divided into dye method and probe method, and the probe that wherein detects at present miRNA mostly is Taqman-MGB probe (a kind of fluorescent probe of the short-movie section that is suitable for increasing).Due to ripe miRNA, to have fragment short and small, special detection, and probe method more has superiority, and in susceptibility and specificity, is better than dye method.Reverse transcriptase primer energy specific recognition based on loop-stem structure, the ripe miRNA sequence that increases, and the precursor of miRNA is not amplified, the shortcoming of Taqman-MGB probe in detecting miRNA is synthetic price, is unfavorable for promoting.

AllGlo probe has been successfully applied to and has detected simplexvirus, hepatitis B virogene sudden change (Feng, Z.L.Yu, X.Y.Lu at present, Z.M.Geng, D.Y.Zhang, L.Chen, S.J.Rapid detection of the hepatitis B virus YMDD mutant using AllGlo ^tMprobes[J] .Clin Chim Acta, 2011,412 (11-12): 1018-1021), invasive aspergillosis (Wu, D.S.Shen, J.Z.Zhou, X.Q.Shen, S.F.Wu, X.M.The establishment and evaluation of diagnostic accuracy of AllGlo (TM) probe-based techniques for invasive aspergillosis[J] .Zhong hua Nei Ke Za Zhi, 2010,49 (2): 142-145), the detection such as K-ras transgenation, ankylosing spondylitis.

Summary of the invention

For the above-mentioned defect of the test kit using in existing detection blood plasma miRNA method and detection method existence, first object of the present invention is to provide the special primer that detects three kinds of blood plasma miRNA.

Second order of the present invention is to provide the detection kit of the blood plasma miRNA based on AllGlo fluorescence probe quantitative PCR.

The 3rd object of the present invention is to provide a kind of three kinds of blood plasma miRNA detection methods based on AllGlo fluorescence probe quantitative PCR.

Described three kinds of blood plasma miRNA refer to respectively hsa-miR-504, hsa-miR-133b, cel-miR-39; Wherein hsa-miR-504 and hsa-miR-133b are humanized miRNA, cel-miR-39 is the miRNA in nematode source, exogenous with reference to miRNA as one, extract and start to add blood plasma from blood plasma, effect is to start to monitor from extracting miRNA the whole process of whole real-time fluorescence quantitative PCR.

The special primer of three kinds of blood plasma miRNA of described detection comprises three kinds of blood plasma miRNA specificity reverse transcriptase primers and three kinds of blood plasma miRNA specificity forward primers;

Described three kinds of blood plasma miRNA specificity reverse transcriptase primers are made up of specificity reverse transcriptase primer, the specificity reverse transcriptase primer of hsa-miR-133b and the specificity reverse transcriptase primer of cel-miR-39 of hsa-miR-504;

The nucleotides sequence of the specificity reverse transcriptase primer of described hsa-miR-504 is classified SEQ ID NO.1 5 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGATAGAGT-3 ＇ as;

The nucleotides sequence of the specificity reverse transcriptase primer of described hsa-miR-133b is classified SEQ ID NO.2 5 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTAGCTGGT-3 ＇ as;

The nucleotides sequence of the specificity reverse transcriptase primer of described cel-miR-39 is classified SEQ ID NO.3 5 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCAAGCTGA-3 ＇ as.

Described three kinds of blood plasma miRNA specificity forward primers are made up of specificity forward primer, the specificity forward primer of hsa-miR-133b and the specificity forward primer of cel-miR-39 of hsa-miR-504;

The nucleotides sequence of the specificity forward primer of described hsa-miR-504 is classified SEQ ID NO.4 5 ＇-GCTGGTTAAGACCCTGGT-3 ＇ as;

The specificity forward primer nucleotides sequence of described hsa-miR-133b is classified SEQ ID NO.5 5 ＇-TAGCGTCTTTGGTCCCCT-3 ＇ as;

The nucleotides sequence of the specificity forward primer of described cel-miR-39 is classified SEQ ID NO.6 5 ＇-CAGAGTCACCGGGTGTAAAT-3 ＇ as.

Described three kinds of blood plasma miRNA specificity AllGlo probes are made up of specificity AllGlo probe, the specificity AllGlo probe of hsa-miR-133b and the specificity AllGlo probe of cel-miR-39 of hsa-miR-504;

The nucleotides sequence of the specificity AllGlo probe of described hsa-miR-504 is classified SEQ ID NO.7MAR-CTGGATACGACGATAGAGTGC-MAR as;

The nucleotides sequence of the specificity AllGlo probe of described hsa-miR-133b is classified SEQ ID NO.8JUP-CTGGATACGACTAGCTGGTTGA-JUP as;

The nucleotides sequence of the specificity AllGlo probe of described cel-miR-39 is classified SEQ ID NO.9NEP-TGCACTGGATACGACCAAGCT-NEP as.

The nucleotides sequence of the universal primer sequence of described three kinds of blood plasma miRNA is classified SEQ ID NO.105 ＇-CAGTGCGTGTCGTGGAGT-3 ＇ as.

The described blood plasma microRNA detection kit based on AllGlo fluorescence probe quantitative PCR is provided with box body, dividing plate, miRNA extraction reagent bottle, stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, miRNA extraction reagent bottle, stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, in miRNA extraction reagent bottle, miRNA is housed and extracts reagent, stem ring reverse transcription reagent is housed in stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent is housed in real-time fluorescence quantitative PCR reagent bottle.

Described miRNA extracts reagent and comprises the exogenous reference of blood plasma miRNA, the exogenous reference of described blood plasma miRNA refers to the stand-in of the cel-miR-39 of synthetic, and (stand-in of the cel-miR-39 of synthetic are as a kind of exogenous reference adding, its working concentration is 5n mol, and effect is to monitor from extracting microRNA to the reaction efficiency of real-time fluorescence quantitative PCR whole process below).

Described stem ring reverse transcription reagent comprises following component: (5 × RT damping fluid is by 250m mol Tris-HCl, 375m mol KCl, 15m mol MgCl for the MMLV enzyme of 200 units/μ L, 10m mol dNTP mixed solution, 5 × RT damping fluid ₂, 50m molDTT composition), the RNA enzyme inhibitors of 40 units/μ L, the MgCl of 10m mol ₂, 1 μ mol reverse transcriptase primer, the miRNA standard substance (the miRNA standard substance here refer to the miRNA of synthetic, and concentration is 100nmol) of special miRNA.

Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100mmolTris-HCl, 500m mol KCl), 10m mol ₂, 5 units/μ L Taq polysaccharase, 10mMdNTP mixed solution, 100ml nuclease free water, the special forward primer of 10 μ mol, the general reverse primer of 10 μ mol and AllGlo probe.

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

1. it is the exogenous reference of blood plasma miRNA of 5n mol that the working concentration providing in the described blood plasma miRNA detection kit based on AllGlo fluorescence probe quantitative PCR is provided after the abundant cracking of 200 μ L blood plasma, carry out immediately whirlpool concussion 15s, the same ordinary method of all the other steps;

2. the stem ring reverse transcription reagent that the described blood plasma miRNA detection kit based on AllGlo fluorescence probe quantitative PCR of application provides is cDNA by miRNA reverse transcription;

3. cDNA is carried out real-time fluorescence quantitative PCR amplification by the real-time fluorescence quantitative PCR reagent that the described blood plasma miRNA detection kit based on AllGlo fluorescence probe quantitative PCR of application provides;

4. every data that composite analyser provides, set rational threshold value (Threshold) and baseline (Baseline), carry out interpretation of result.

Described AllGlo probe can adopt the fluorescent quantitation probe of A1leLogic Biosciences Corporation company of the U.S., it has general T aqman, the all advantages of Taqman-MGB and molecular beacon (molecular beacon) probe, remove the maximum drawback of current these several probes, the restriction that it has broken reporter group one end, traditional Taqman one end quenching group, utilize the special fluorescence dye of the several frequently seen wavelength of A1leLogic Biosciences Corporation company of U.S. development, be marked at above oligonucleotide each other reporter group and the essence group that goes out, and contain the special chemical group that can improve Tm value (annealing temperature) above, improve probe specificity, hybridization specificity improves greatly, after hybridization hydrolysis, the dyestuff of two ends mark all becomes again reporter group, improve fluorescence increment.

Described AllGlo probe has following advantage: 1. improve Tm value (can reach more than 10 ℃), probe is shorter can reach 15～16 bases, can be suitable for A, the sequences Design probe that T content is higher; 2. strengthened the selection of multiple fluorescence quantitative, because every kind of dyestuff is that reporter group is again quenching group, the Taqman probe that breaks traditions, because of wavelength reason Marker selection difficulty, is not limited by quenching group wavelength; 3. greatly improve signal to noise ratio, without background signal, space length is near, better cancellation effect; 4. cost is lower, and price is only equivalent to the half of Taqman-MGB probe, has MGB probe and has superiority.

The present invention has adopted AllGlo probe technique, three kinds of special forward primers and specific probe and general reverse primer are designed, three kinds of special fluorophors of mark (MAR, JUP, NEP) only need be distinguished in three kinds of probe two ends, can detect three kinds of miRNA simultaneously, and this technology reaction conditions is optimized, set up a kind of method that simultaneously detects three kinds of blood plasma miRNA based on AllGlo probe technique associating Real-Time Fluorescent Quantitative PCR Technique, had a extensive future.

The present invention has adopted AllGlo probe technique, has set up the method that can detect the real-time fluorescence quantitative PCR of miRNA, and compared with Taqman-MGB probe method, linearity range all can reach 7 orders of magnitude.The minimum 10 copies/μ L that reaches of real-time fluorescence quantitative PCR sensitivity that the present invention sets up, reaches as high as 10 ⁷copy/μ L.

Beneficial effect of the present invention is as follows:

The invention provides detection method and the test kit of a kind of miRNA of detection based on AllGlo probe RT-qPCR, its specificity and susceptibility are high, can quick and precisely detect the content of miRNA in sample, compared with prior art have following advantage:

1. with northern blot, miRNA chip is compared, and it is high that the specificity of AllGlo probe in detecting and susceptibility are all wanted, and it is cheap that cost ratio microRNA chip is wanted;

2. compared with taqman-MGB probe, AllGlo probe adopts identical fluorophor, reporter group and quenching group each other, and the fluorescent signal discharging is on the one hand stronger, and background signal is lower on the other hand; And synthesis program is simple, price is only equivalent to the half of taqman-MGB.

3. the RT-qPCR that the present invention has set up based on AllGlo probe detects miRNA method, be suitable for as cDNA microarray miRNA later stage clinical sample checking, and research miRNA is at the differential expression of different field etc., has promoted the further investigation of miRNA at relative disease.

Accompanying drawing explanation

Fig. 1 is the structure composition schematic diagram of the blood plasma microRNA detection kit of the embodiment of the present invention based on AllGlo fluorescence probe quantitative PCR.

Fig. 2 is hsa-miR-133b(humanized miR-133b in three kinds of standard substance mixed solutions of AllGlo probe for real-time fluorescence quantitative PCR detection) amplification curve diagram.

Fig. 3 is hsa-miR-133b(humanized miR-133b in three kinds of standard substance mixed solutions of AllGlo probe for real-time fluorescence quantitative PCR detection) canonical plotting.

Embodiment

Embodiment 1

Referring to Fig. 1, the blood plasma microRNA detection kit embodiment based on AllGlo fluorescence probe quantitative PCR of the present invention is provided with box body 1, dividing plate 2, miRNA extraction reagent bottle 3, stem ring reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent bottle 5; Dividing plate 2 is located in box body 1, miRNA extraction reagent bottle 3, stem ring reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent bottle 5 are inserted on dividing plate 2, in miRNA extraction reagent bottle 3, miRNA is housed and extracts reagent, stem ring reverse transcription reagent is housed in stem ring reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent is housed in real-time fluorescence quantitative PCR reagent bottle 5.

Embodiment 2

Concrete operation step of the present invention and reaction system are as follows:

1. extract miRNA

Adopt the miRNA of the production of Tian Gen bio tech ltd to extract test kit, carrying out sample (blood plasma) microRNA according to process specifications extracts, wherein 200 μ L blood plasma add exogenous with reference to cel-mir395 μ L (working concentration is 5n mol) after adding equal-volume lysate to leave standstill 5min, after to specifications operation carry out, finally use the stoning enzyme water dissolution RNA of 30 μ L, on nucleic acid spectrophotometric instrument Nano Drop2000, measure respectively concentration and purity, get the miRNA having extracted of 2～20ng for the reverse transcription in downstream, remaining miRNA is all frozen in-80 ℃.

The reverse transcription of 2.miRNA:

2.1 dissolve required reverse transcription composition under room temperature, and concussion mixes and is placed on ice;

2.2 preparation reverse transcription reaction mixed solutions, prepare by table 1:

Table 1

Component	Volume/pipe (μ L)
		MMLV reversed transcriptive enzyme (200 units/μ L) (promega company)	0.2
MMLV5 times of reversed transcriptive enzyme damping fluid (promega company)	4
		Deoxynucleoside mixed solution (10m mol)	0.8
RNA enzyme inhibitors (40 units/μ L)	0.2
		Specificity stem ring reverse transcriptase primer (1 μ mol)	1.2
Nuclease free water	11.6
		Cumulative volume	18

2.3 every pipe packing in the independent PCR pipe of 0.2 μ L

The 18 μ L mixed solutions that prepare are sub-packed in the PCR pipe without RNA enzyme, after add the 2 μ L miRNA solution of Extraction and enrichment, with fully mix (generation that bubble is avoided in this operation) without RNA enzyme rifle head as far as possible, to be placed on regular-PCR instrument (production of Biorad company) upper to managing the end for of short duration centrifugal drying, and reverse transcription reaction condition is as table 2.

Table 2

Step	Condition
			1	25℃5min
2	42℃60min
		3	70℃15min

Reaction finishes to be placed on 4 ℃.

The fluorescence quantitative PCR detection of 3.miRNA

Each component of quantitative fluorescent PCR is placed in room temperature by 3.1 dissolves, and mixes and is placed on ice.

3.2 preparation PCR reaction mixtures are as table 3.

Table 3

Component	Every pipe volume (μ L)
		DNTP mixed solution (10m mol)	2
MgCl2(25m?mol)	2
		10 × Taq damping fluid is (without Mg ²⁺)	2
RTaq (5 units/μ L) (takara company)	0.2
		Forward primer (10 μ mol)	0.4
Reverse primer (10 μ mol)	0.4
		Probe (10 μ mol)	0.2
Stoning enzyme water	10.8
		Cumulative volume	18

3.3 in 8 unions packing prepare PCR reaction mixture, every pipe adds the cDNA of 2 μ L, fully mixes, whole process is carried out on ice, avoids the generation of bubble.

3.4 quantitative fluorescent PCR reaction response conditions are as table 4.

Table 4

Detect on real-time fluorescence quantitative PCR instrument, carry out, can use ABI7300, Applied Biosystems company of the 7500(U.S.) etc. multiple instrument.

4. data analysis and standardization: application SPSS13.0 and Excel2007 carry out data analysis, target miRNA and the exogenous CT value with reference to cel-mir-39 in the sample blood plasma that can survey with aforesaid method, try to achieve the relative content of object miRNA in blood plasma according to the CT value level of exogenous reference, with 2 of relative quantification in qPCR ^{-Δ Ct}mode represents the level (Δ Ct is Ct value poor of object microRNA and exogenous reference) of object miRNA in blood plasma.

Embodiment 3

The quantitative fluorescent PCR reaction system of 3 kinds of miRNA mixing and the foundation of typical curve are as table 5.

Table 5

Composition	Volume/every pipe (μ L)
		DNTP mixed solution (10m mol)	4
MgCl2(25m?mol)	4
		10 × Taq damping fluid (without Mg2+)	4
RTaq (5 units/μ L) (takara company)	0.4
		3 align, reverse primer mixed solution (10 μ mol)	4.8
3 kinds of probe mixed solutions (10 μ mol)	1.2
		Stoning enzyme water	19.6
Cumulative volume	38

In advance 3 kinds of microRNA standard substance are diluted to 5 × 10 ⁷the miRNA initial concentration of copy/μ L, after mixing, carries out continuous 10 times of dilutions, after carry out RT-qPCR detection, take hsa-miR-133b as example, its amplification curve and typical curve are as Fig. 2 and 3.

Embodiment 4

Clinical sample detects:

Choose each 5 examples of patient's plasma sample, application AllGlo probe method detects hsa-miR-133b, compares with the method for the microRNA Taqman MGB of the detection hsa-miR-133b of the life technologies company of the U.S., does respectively 2 multiple holes.

Three kinds of methods detect respectively the result of hsa-miR-133b in 5 routine Colorectal Carcinoma samples relatively as table 6.

Table 6

Hsa-miR-133b/CT value relatively	Substance AllGlo probe method	Taqman-MGB method	Triple AllGlo probe methods
				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

Illustrate: detect same sample, the lower explanation susceptibility of CT value is higher, data presentation from upper table: detect 5 different samples from Taqman-MGB probe method and compare, substance AllGlo probe method and triple AllGlo probe method will be lower than Taqman-MGB probe methods in the CT value that detects plasma sample has-miR-133b, illustrate that both susceptibility will be apparently higher than Taqman-MGB probe method.

Claims

1. the special primer that detects three kinds of blood plasma miRNA, is characterized in that described three kinds of blood plasma miRNA refer to respectively hsa-miR-504, hsa-miR-133b, cel-miR-39; Wherein hsa-miR-504 and hsa-miR-133b are humanized miRNA, cel-miR-39 is the miRNA in nematode source, exogenous with reference to miRNA as one, extract and start to add blood plasma from blood plasma, effect is to start to monitor from extracting miRNA the whole process of whole real-time fluorescence quantitative PCR;

The nucleotides sequence of the specificity reverse transcriptase primer of described cel-miR-39 is classified SEQ ID NO.3 5 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCAAGCTGA-3 ＇ as;

2. three kinds of blood plasma miRNA specificity AllGlo probes, is characterized in that described three kinds of blood plasma miRNA refer to respectively hsa-miR-504, hsa-miR-133b, cel-miR-39; Wherein hsa-miR-504 and hsa-miR-133b are humanized miRNA, cel-miR-39 is the miRNA in nematode source, exogenous with reference to miRNA as one, extract and start to add blood plasma from blood plasma, effect is to start to monitor from extracting miRNA the whole process of whole real-time fluorescence quantitative PCR;

The nucleotides sequence of the specificity AllGlo probe of described cel-miR-39 is classified SEQ ID NO.9NEP-TGCACTGGATACGACCAAGCT-NEP as;

The nucleotides sequence of the universal primer sequence of described three kinds of blood plasma miRNA is classified SEQ ID NO.10 5 ＇-CAGTGCGTGTCGTGGAGT-3 ＇ as.

3. the blood plasma microRNA detection kit based on AllGlo fluorescence probe quantitative PCR, is characterized in that being provided with box body, dividing plate, miRNA extraction reagent bottle, stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, miRNA extraction reagent bottle, stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, in miRNA extraction reagent bottle, miRNA is housed and extracts reagent, stem ring reverse transcription reagent is housed in stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent is housed in real-time fluorescence quantitative PCR reagent bottle.

4. the blood plasma microRNA detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 3, it is characterized in that described miRNA extracts reagent and comprises the exogenous reference of blood plasma miRNA, the exogenous reference of described blood plasma miRNA refers to the stand-in of the cel-miR-39 of synthetic, the stand-in of the cel-miR-39 of synthetic are as a kind of exogenous reference adding, its working concentration is 5n mol, and effect is to monitor from extracting microRNA to the reaction efficiency of real-time fluorescence quantitative PCR whole process below.

5. the blood plasma microRNA detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 3, is characterized in that described stem ring reverse transcription reagent comprises following component: the MMLV enzyme of 200 units/μ L, 10m mol dNTP mixed solution, 5 × RT damping fluid, the RNA enzyme inhibitors of 40 units/μ L, the MgCl of 10m mol ₂, 1 μ mol reverse transcriptase primer, the miRNA standard substance of special miRNA, concentration is 100n mol; Described 5 × RT damping fluid is by 250m molTris-HCl, 375m mol KCl, 15m mol MgCl ₂, 50m mol DTT composition.

6. the blood plasma microRNA detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 3, is characterized in that described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid, 10m mol ₂, 5 units/μ L Taq polysaccharase, 10mMdNTP mixed solution, 100ml nuclease free water, the special forward primer of 10 μ mol, the general reverse primer of 10 μ mol and AllGlo probe; Described 10 × Taq damping fluid comprises 100m molTris-HCl, 500mmol KCl.

7. the blood plasma microRNA detection method based on AllGlo fluorescence probe quantitative PCR, is characterized in that comprising the following steps:

1) after the abundant cracking of 200 μ L blood plasma, adding the working concentration providing in the described blood plasma miRNA detection kit based on AllGlo fluorescence probe quantitative PCR is the exogenous reference of blood plasma miRNA of 5n mol, carry out immediately whirlpool concussion 15s, the same ordinary method of all the other steps;

2) the stem ring reverse transcription reagent that the described blood plasma miRNA detection kit based on AllGlo fluorescence probe quantitative PCR of application provides is cDNA by miRNA reverse transcription;

3) cDNA is carried out real-time fluorescence quantitative PCR amplification by the real-time fluorescence quantitative PCR reagent that the described blood plasma miRNA detection kit based on AllGlo fluorescence probe quantitative PCR of application provides;

4) every data that composite analyser provides, set rational threshold value and baseline, carry out interpretation of result.

8. the blood plasma microRNA detection method based on AllGlo fluorescence probe quantitative PCR as claimed in claim 7, is characterized in that described AllGlo probe adopts the fluorescent quantitation probe of A1leLogic Biosciences Corporation company of the U.S..