CN103740846B

CN103740846B - Based on hsa-miR-1321 detection kit and the detection method thereof of AllGlo fluorescence probe quantitative PCR

Info

Publication number: CN103740846B
Application number: CN201410041061.2A
Authority: CN
Inventors: 方赞熙; 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: 2015-10-28
Anticipated expiration: 2034-01-28
Also published as: CN103740846A

Abstract

Based on hsa-miR-1321 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, exogenous reference bottle, stem ring reverse transcription reagents bottle, real-time fluorescence quantitative PCR reagent bottle.Extract miRNA in sample, if serum/plasma or other body fluid samples, then the concentration that adding test kit after the abundant cracking of sample provides is the exogenous with reference to cel-miR-395 μ L of 5nmol, and whirlpool shakes; If cell or tissue sample, then exogenous with reference to cel-miR-39 without the need to adding; The stem ring reverse transcription reagents reverse transcription miRNA adopting test kit to provide is cDNA; CDNA is carried out real time PCR amplification by the real-time fluorescence quantitative PCR reagent adopting test kit to provide; Every data that composite analyser provides, set rational threshold value and baseline, carry out interpretation of result.

Description

Based on hsa-miR-1321 detection kit and the detection method thereof of AllGlo fluorescence probe quantitative PCR

Technical field

The present invention relates to MicroRNA, especially relate to a kind of hsa-miR-1321 detection kit based on AllGlo fluorescence probe quantitative PCR and detection method thereof.

Background technology

MicroRNA (miRNA) is the non-coding RNA molecule being about 22 Nucleotide that a class is extensively present in eukaryotic cell, take part in many pathological processes in organism, expressed by regulatory gene, in cell proliferation, apoptosis, grow, cytodifferentiation, play a significant role in the processes such as metabolism, be embodied in miRNA in nucleus through forming initial primary transcribe pri-miRNA to pre-miRNA, after transport out nucleus, ripe miRNA is formed 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 plays key player under the generation of tumour, development, Index for diagnosis and many other diseases states, accurately detect its expression and the effect of research miRNA is significant.At present, the method detecting miRNA mainly contains northern blot technology, Real-Time Fluorescent Quantitative PCR Technique, hybridization in situ technique, microarray chip technology etc.Wherein, northern blot technology is one of early stage means that RNA detects, but its specificity and susceptibility low, if sample rna content is low or there is degraded, may can't detect; Hybridization in situ technique, for detecting the distribution situation of tissue and cell levels miRNA, carries out half-quantitative detection to miRNA simultaneously, and it is not enough, and place is the miRNA that low expression accurately can not be detected; Microarray chip technology is a kind of high-throughout detection technique, can detect a large amount of miRNA of one or more samples, but it is time-consuming to there is chip manufacturing simultaneously, effort and mark cost high, detection sensitivity and the problem such as specificity is low.

Real time fluorescent quantitative technology (RT-qPCR) is commonly used to most at present one of technology that genetic expression detects, have easy fast, the feature such as the high and specificity of susceptibility is good.At present, the real time fluorescence quantifying PCR method for detecting miRNA comprises the quantitative fluorescent PCR two portions in miRNA reverse transcription and downstream, and 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, the probe wherein detecting miRNA mostly at present is Taqman-MGB probe (a kind of fluorescent probe being suitable for amplification short-movie section), miRNA due to maturation has the short and small feature of fragment, want special detection, probe method advantageously, Sensitivity and Specificity is better than dye method.Based on the reverse transcriptase primer energy specific recognition of loop-stem structure, the miRNA sequence that amplification is ripe, and the precursor of miRNA is not amplified, Taqman-MGB probe in detecting miRNA shortcoming is to synthesize price, is unfavorable for promoting.

At present, AllGlo probe has been successfully applied to and has detected simplexvirus, hepatitis B virogene sudden change (Feng, Z.L.Yu, X.Y.Lu, Z.M.Geng, D.Y.Zhang, L.Chen, S.J.Rapid detection of the hepatitis B virus YMDDmutant 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 ofdiagnostic accuracy of AllGlo (TM) probe-based techniques for invasive aspergillosis [J] .Zhong huaNei Ke Za Zhi, 2010,49 (2): 142-145) detections such as, K-ras transgenation, ankylosing spondylitis.

Recent research is reported, uses cerebro-cardiac apoplexy knurl and the pairing tissue thereof of microRNA microarray chip analysis children's, finds that hsa-miR-1321 expresses in cancerous tissue and significantly rise.By the analysis of gene database and signal path, confirm that hsa-miR-1321 take part in the biological processes that children's's cerebro-cardiac apoplexy knurl occurs and the signal path (Liu be correlated with, F., Y.Xiong, et al. (2013) .Identification of aberrant microRNA expression pattern in pediatric gliomas bymicroarray. diagn Pathol8 (158): 1746-1596), has-miR-1321 is a novel children's's brain tumor Biomarkers having application prospect.

Summary of the invention

An object of the present invention is the above-mentioned defect existed for the test kit used in existing detection miRNA method and detection method, provides the hsa-miR-1321 detection kit based on AllGlo fluorescence probe quantitative PCR.

Two of object of the present invention is the detection method of the hsa-miR-1321 provided based on AllGlo fluorescence probe quantitative PCR.

Described hsa-miR-1321 is a kind of humanized miRNA, and its nucleotides sequence is classified as: SEQ ID NO.15 ＇-CAGGGAGGUGAAUGUGAU-3 ＇.

The described hsa-miR-1321 detection kit based on AllGlo fluorescence probe quantitative PCR, is provided with box body, dividing plate, exogenous reference bottle, stem ring reverse transcription reagents bottle, real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, exogenous reference bottle, stem ring reverse transcription reagents bottle, real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, exogenous reference bottle is built with exogenous reference, stem ring reverse transcription reagents bottle is built with stem ring reverse transcription reagents, and real-time fluorescence quantitative PCR reagent bottle is built with real-time fluorescence quantitative PCR reagent.

Described exogenous reference can adopt cel-miR-39 etc., and described cel-miR-39 is a kind of threadworms miRNA, and its nucleotides sequence is classified as: SEQ ID NO.25 ＇-UCACCGGGUGUAAAUCAGCUUG-3 ＇; Its working concentration can be 5nmol.

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

The special reverse transcriptase primer of described miRNA is made up of the special reverse transcriptase primer of hsa-miR-1321, the exogenous special reverse transcriptase primer with reference to cel-miR-39 and the endogenous special reverse transcriptase primer with reference to U6:

The nucleotides sequence of the special reverse transcriptase primer of described hsa-miR-1321 is classified as: SEQ ID NO.35 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACATCACATT-3 ＇;

The nucleotides sequence of the described exogenous special reverse transcriptase primer with reference to cel-miR-39 is classified as: SEQ ID NO.45 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCTGTTCCT-3 ＇;

Described endogenous is classified as with reference to the nucleotides sequence of the special reverse transcriptase primer of U6: SEQ ID NO.55 ＇-AACGCTTCACGAATTTGCGT-3 ＇.

Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100mmol Tris-HCl, 500m mol KCl), 10m mol ₂, the Taq polysaccharase of 5 units/μ L, 10m moldNTP mixed solution, 100mL nuclease free water, 10 μm of ol specific forward primer, 10 μm of general reverse primers of ol and AllGlo probes.

Described specific forward primer is made up of the special primer of hsa-miR-1321, the exogenous special primer with reference to cel-miR-39 and the endogenous special primer with reference to U6:

The nucleotides sequence of the special primer of described hsa-miR-1321 is classified as:

SEQ ID NO.65＇-GTTTGGGCAGGGAGGTG-3＇；

The nucleotides sequence of the described exogenous special primer with reference to cel-miR-39 is classified as:

SEQ ID NO.75＇-CAGAGTCACCGGGTGTAAAT-3＇；

Described endogenous is classified as with reference to the nucleotides sequence of the special primer of U6:

SEQ ID NO.85＇-CTCGCTTCGGCAGCACA-3＇。

The nucleotides sequence of described general reverse primer is classified as:

SEQ ID NO.95＇-CAGTGCGTGTCGTGGAGT-3＇。

Described AllGlo probe is made up of the specific probe of hsa-miR-1321, the specific probe of endogenous reference U6 and the exogenous specific probe with reference to cel-miR-39;

The nucleotides sequence of the specific probe of described hsa-miR-1321 is classified as:

SEQ ID NO.10MAR-TTGCACTGGATACGACATCACATTCA-MAR；

Described endogenous is classified as with reference to the nucleotides sequence of the specific probe of U6:

SEQ ID NO.11JUP-CGATACAGAGAAGATTAGCATGGCCCC-JUP；

The nucleotides sequence of the described exogenous specific probe with reference to cel-miR-39 is classified as:

SEQ ID NO.12JUP-TGCACTGGATACGACCAAGCT-JUP。

The detection method of the described hsa-miR-1321 based on AllGlo fluorescence probe quantitative PCR, its step is as follows:

1) ordinary method is adopted to extract miRNA in sample, if serum/plasma or other body fluid samples, after the abundant cracking of sample, then add the described concentration provided based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR is the exogenous with reference to cel-miR-395 μ L of 5n mol, carries out whirlpool concussion 15s immediately; If cell or tissue sample, then exogenous with reference to cel-miR-39 without the need to adding;

2) the described stem ring reverse transcription reagents reverse transcription miRNA provided based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR is adopted to be cDNA;

3) adopt the described real-time fluorescence quantitative PCR reagent provided based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR that cDNA is carried out real time PCR amplification;

4) every data of providing of composite analyser, 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, Taqman-MGB and all advantages of molecular beacon (molecular beacon) probe, overcome the maximum drawback of this several probe at present, it has broken the restriction of reporter group one end, traditional Taqman one end quenching group, make use of the special fluorescence dye of the several frequently seen wavelength of A1leLogic Biosciences Corporation company of U.S. development, to be marked at above oligonucleotide reporter group and essence each other to go out group, and above containing the special chemical group that can improve Tm value (annealing temperature), improve probe specificity, hybrid specificities improves greatly, after hybridization hydrolysis, the dyestuff of two ends mark all becomes reporter group again, improve fluorescence increment.

Described AllGlo probe has following advantage: 1. improve Tm value (can reach more than 10 DEG C), probe is shorter can reach 15 ~ 16 bases, can be suitable for A, the sequences Design probe that T comparision contents is high; 2. increase the selection of multiple fluorescence quantitative, because not only often kind of dyestuff is reporter group but also be quenching group, the Taqman probe that breaks traditions, because of wavelength reason Marker selection difficulty, does not limit by quenching group wavelength; 3. greatly improve signal to noise ratio, without background signal, space length is near, better quenching effects; 4. cost is lower, and price is only equivalent to the half of Taqman-MGB probe, has MGB probe and had superiority.

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

Present invention employs AllGlo probe technique, devise Auele Specific Primer and corresponding AllGlo probe, probe marks 2 kinds of special fluorophors (MAR, JUP) respectively, and this technology reaction conditions is optimized, establish a kind of method of AllGlo fluorescence probe quantitative PCR technology for detection hsa-miR-1321, have a extensive future.

Beneficial effect of the present invention is as follows:

The invention provides a kind of hsa-miR-1321 detection kit based on AllGlo probe RT-qPCR and detection method, its specificity and susceptibility high, quick and precisely can detect the content of miRNA in sample, compared with prior art there is following advantage:

1. compare with northern blot, miRNA chip, the specificity of AllGlo probe in detecting and susceptibility all want high, and cost ratio miRNA chip wants cheap;

2. compared with taqman-MGB probe, AllGlo probe adopts identical fluorophor, each other reporter group and quenching group, and the fluorescent signal of release 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. compare generally adopt at present detect miRNA method based on SYBGreen dyestuff, the reaction times based on AllGlo probe in detecting miRNA shortens greatly, and sensitivity and specificity will apparently higher than the method detecting miRNA based on SYBGreen;

4. the present invention establishes the method for the RT-qPCR detection hsa-miR-1321 based on AllGlo probe, and be suitable for the clinical sample checking as screening miRNA, the effect of playing the part of in relative disease for studying miRNA further plays a role in promoting.

Accompanying drawing explanation

Fig. 1 is the structure composition schematic diagram of the hsa-miR-1321 detection kit embodiment that the present invention is based on AllGlo fluorescence probe quantitative PCR.

Embodiment

Embodiment 1

See Fig. 1, the hsa-miR-1321 detection kit embodiment based on AllGlo fluorescence probe quantitative PCR of the present invention, is provided with box body 1, dividing plate 2, exogenous reference bottle 3, stem ring reverse transcription reagents bottle 4, real-time fluorescence quantitative PCR reagent bottle 5; Dividing plate 2 is located in box body 1, exogenous reference bottle 3, stem ring reverse transcription reagents bottle 4, real-time fluorescence quantitative PCR reagent bottle 5 are inserted on dividing plate 2, exogenous reference bottle 3 is built with exogenous reference, stem ring reverse transcription reagents bottle 4 is built with stem ring reverse transcription reagents, and real-time fluorescence quantitative PCR reagent bottle 5 is built with real-time fluorescence quantitative PCR reagent.

1. described exogenous reference can adopt cel-miR-39 etc., and described cel-miR-39 is a kind of threadworms miRNA, and its working concentration can be 5n mol, its role is to monitor the reaction efficiency extracting follow-up real-time fluorescence quantitative PCR whole process from miRNA;

2. stem ring reverse transcription reagents 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 ₂50m mol DTT forms), the RNA enzyme inhibitors of 40 units/μ L, the MgCl2 of 10m mol, the reverse transcriptase primer of the special miRNA of 1 μm of ol, miRNA standard substance (miRNA standard substance here refer to the miRNA of synthetic, and concentration is 100n mol);

3. real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100m molTris-HCl, 500m mol KCl), 10m mol ₂, the Taq polysaccharase of 5 units/μ L, 10m moldNTP mixed solution, 100mL nuclease free water, 10 μm of ol specific forward primer, 10 μm of general reverse primers of ol and AllGlo probes.

Embodiment 2

The detection of serum or blood plasma hsa-miR-1321, comprises the following steps:

1. collect blood plasma or serum:

Extract fresh blood sample 2mL and be loaded on EDTA anticoagulant tube or without in antithrombotics pipe, immediately above-mentioned test tube is put upside down mixing 6 ~ 8 times, then above-mentioned test tube is placed in the centrifugal 10min of whizzer 3000r, taking out after terminating is placed on test-tube stand, careful absorption supernatant is placed in the centrifuge tube without RNA enzyme of new 1.5mL, the centrifuge tube of the 1.5mL that supernatant is housed is placed in the centrifugal 10min of whizzer 13000r, after terminating, by upper plasma or serum transfers, to the centrifuge tube of new 1.5mL without RNA enzyme, (this step notes the cell precipitation not being drawn to lower floor.), often pipe packing 400 ~ 500 μ L, gets 200 μ L and extracts for next step, all the other blood plasma or serum frozen in-80 DEG C.

2. extract microRNA

The miRNA of the production of Tian Gen bio tech ltd is adopted to extract test kit (DP501), carry out sample (blood plasma or serum) miRNA according to process specifications to extract, wherein 200 μ L blood plasma or serum leave standstill 5min after adding the lysate concuss 30s of equal volume, then add exogenous with reference to cel-mir-39 (its working concentration is 5n mol) 5 μ L, after to specifications operation carry out, finally use the stoning enzyme water dissolution miRNA of 30 μ L, concentration and purity is measured respectively on nucleic acid spectrophotometric instrument Nano Drop2000, get purity A260/A280 ratio between 1.6 ~ 2.2, get the reverse transcription of the miRNA extracted for downstream of concentration 2 ~ 20ng/ μ L, remaining miRNA is all frozen in-80 DEG C.

The reverse transcription of 3.miRNA:

Composition needed for reverse transcription dissolves by 3.1 under room temperature, and concussion mixing is placed on ice; Please prepare reverse transcription reaction liquid by table 1.

Table 1

Component	Add volume (μ L)
		MMLV reversed transcriptive enzyme (200 units/μ L) (promega company)	0.4
MMLV5 times of reverse transcriptase buffer (promega company)	8
		Deoxynucleoside mixed solution (10mL)	1.6
RNA enzyme inhibitors (40 units/μ L)	0.4
		Specificity stem ring reverse transcriptase primer (1 μ L)	Each 1.2, totally 2.4
Nuclease free water	25.2
		Cumulative volume	38

Note: in table 1, each 1.2 μ L of specific reverse transcriptase primer refer to the special reverse transcriptase primer of object hsa-miR-1321 and the exogenous special primer with reference to cel-miR-39 respectively.

The 38 μ L mixed solutions prepared are sub-packed in the PCR pipe without RNA enzyme by 3.2, after add the miRNA solution of 2 μ L Extraction and enrichment, with fully mixing without RNA enzyme rifle head (generation that bubble is avoided in this operation) as far as possible, be placed at the bottom of of short duration centrifugal drying to pipe on regular-PCR instrument (production of Biorad company), reverse transcription reaction condition is as table 2.

Table 2

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

Reaction end is placed on 4 DEG C or on ice.

4. real-time fluorescence quantitative PCR detects hsa-miR-1321

Each component of quantitative fluorescent PCR is placed in room-temperature dissolution by 4.1, and mixing is placed on ice.

4.2 preparation PCR reaction mixtures are as table 3.

Table 3

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

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

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

Table 4

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

5. data analysis and standardization: apply ABI instrument and carry software and Excel carries out data analysis, the CT value of hsa-miR-1321 and exogenous contrast cel-mir-39 in the sample (serum or blood plasma) can surveyed with aforesaid method, CT value level according to reference tries to achieve the relative content of hsa-miR-1321 in serum or blood plasma, with 2 of relative quantification in qPCR ^{-Δ Ct}mode represents the level (Δ Ct is the difference of the Ct value of hsa-miR-1321 and outer source reference) of object hsa-miR-1321 in serum or blood plasma.

Embodiment 3. is organized or cell miRNA detects

1. sample preparation:

A. organize: will be organized in liquid nitrogen and grind.Every 30 ~ 50mg animal tissues or 100mg plant tissue add 1mL lysate MZ(Tian Gen bio tech ltd and produce), carry out homogenized with Syrup-homogenizing instrument.Sample volume should not exceed 10% of lysate MZ volume.

B. single-layer culturing cell: directly add lysate MZ lysing cell in culture plate, every 10cm ²area adds 1mLMZ.Lash several times with sampler.

C. cell suspension: centrifugal 800r5min gets cell, abandons supernatant.Add 1mL lysate MZ, oscillator vibrates or pipettor are inhaled and are played mixing for several times.Washed cell is not wanted, in order to avoid degradation of rna before adding lysate MZ.

2. organize, cell miRNA extracts and enrichment

The miRNA of the production of Tian Gen bio tech ltd is adopted to extract test kit (DP501), carry out organizing according to process specifications or cell miRNA extracts, finally use the stoning enzyme water dissolution RNA of 50 μ L, concentration and purity is measured respectively on nucleic acid spectrophotometric instrument NanoDrop2000

3. organize, reverse transcription and the enforcement fluorescent quantitation of cell miRNA detect.

Subsequent step with reference to step 2 ~ 4 of embodiment 2, but does following change:

1. in embodiment 2 step 2, the special primer of the mixed solution exogenous reference cel-miR-39 of reverse transcription reaction liquid will change the special primer of U6 into, the concentration of primer and constancy of volume;

2. the PCR mixed solution exogenous of follow-up real time fluorescent quantitative will change special primer and the probe of U6 into reference to the special primer of cel-miR-39 and probe.

Can range of application:

The detection of cell hsa-miR-1321 expression level in human tissue, hemocyte and body fluid is can be applicable to, above display and describe ultimate principle of the present invention, principal character and advantage of the present invention based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR and detection method thereof.

Claims

1. based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR, it is characterized in that described hsa-miR-1321 is a kind of humanized miRNA, its nucleotides sequence is classified as: SEQ ID NO.15 ＇-CAGGGAGGUGAAUGUGAU-3 ＇;

The described hsa-miR-1321 detection kit based on AllGlo fluorescence probe quantitative PCR, is provided with box body, dividing plate, exogenous reference bottle, stem ring reverse transcription reagents bottle, real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, exogenous reference bottle, stem ring reverse transcription reagents bottle, real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, exogenous reference bottle is built with exogenous reference, stem ring reverse transcription reagents bottle is built with stem ring reverse transcription reagents, and real-time fluorescence quantitative PCR reagent bottle is built with real-time fluorescence quantitative PCR reagent;

Describedly exogenously adopt by reference cel-miR-39, described cel-miR-39 is a kind of threadworms miRNA, and its nucleotides sequence is classified as: SEQ ID NO.25 ＇-UCACCGGGUGUAAAUCAGCUUG-3 ＇; Its working concentration is 5nmol;

Described stem ring reverse transcription reagents 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 μm of ol reverse transcriptase primer, the miRNA standard substance of special miRNA, concentration is 100n mol; Described 5 × RT damping fluid is by 250m mol Tris-HCl, 375mmol KCl, 15m mol MgCl ₂, 50m mol DTT forms;

Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid, 10m mol ₂, the Taq polysaccharase of 5 units/μ L, 10m mol dNTP mixed solution, 100mL nuclease free water, 10 μm of ol specific forward primer, 10 μm of general reverse primers of ol and AllGlo probes; Described 10 × Taq damping fluid comprises 100m mol Tris-HCl, 500m mol KCl;

SEQ ID NO.10MAR-TTGCACTGGATACGACATCACATTCA-MAR；

SEQ ID NO.11JUP-CGATACAGAGAAGATTAGCATGGCCCC-JUP；

SEQ ID NO.12JUP-TGCACTGGATACGACCAAGCT-JUP。

2., as claimed in claim 1 based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR, it is characterized in that the special reverse transcriptase primer of reverse transcriptase primer by hsa-miR-1321 of described special miRNA, the exogenous special reverse transcriptase primer with reference to cel-miR-39 and endogenous form with reference to the special reverse transcriptase primer of U6;

The nucleotides sequence of the special reverse transcriptase primer of described hsa-miR-1321 is classified as: SEQ ID NO.3 5 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACATCACATT-3 ＇;

The nucleotides sequence of the described exogenous special reverse transcriptase primer with reference to cel-miR-39 is classified as: SEQ ID NO.4 5 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCTGTTCCT-3 ＇;

Described endogenous is classified as with reference to the nucleotides sequence of the special reverse transcriptase primer of U6: SEQ ID NO.5 5 ＇-AACGCTTCACGAATTTGCGT-3 ＇.

3., as claimed in claim 1 based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR, it is characterized in that described specific forward primer is made up of the special primer of hsa-miR-1321, the exogenous special primer with reference to cel-miR-39 and the endogenous special primer with reference to U6;

SEQ ID NO.6 5＇-GTTTGGGCAGGGAGGTG-3＇；

SEQ ID NO.7 5＇-CAGAGTCACCGGGTGTAAAT-3＇；

SEQ ID NO.8 5＇-CTCGCTTCGGCAGCACA-3＇。

4., as claimed in claim 1 based on the hsa-miR-1321 detection kit of AllGlo fluorescence probe quantitative PCR, it is characterized in that the nucleotides sequence of described general reverse primer is classified as:

SEQ ID NO.9 5＇-CAGTGCGTGTCGTGGAGT-3＇。