CN101962691B

CN101962691B - Nucleotide sequences and kit for detecting H1N1 influenza A virus 2009 variants aiming at M genes

Info

Publication number: CN101962691B
Application number: CN 201010526991
Authority: CN
Inventors: 张鹤晓; 高志强; 乔彩霞; 蒲静; 谷强; 张伟; 刘环; 张利峰; 汪琳; 柏亚铎; 张向东
Original assignee: Beijing Entry Exit Inspection and Quarantine Bureau of Peoples Republic of China
Current assignee: Beijing Entry Exit Inspection and Quarantine Bureau of Peoples Republic of China
Priority date: 2010-11-01
Filing date: 2010-11-01
Publication date: 2011-12-07
Anticipated expiration: 2030-11-01
Also published as: CN101962691A

Abstract

The invention discloses a group of nucleotide sequences and a kit for detecting H1N1 influenza A virus 2009 variants aiming at M genes. The nucleotide sequences of the H1N1 influenza virus 2009 variants are expressed as sequence tables SEQ ID No: 1 to SEQ ID No: 3, wherein the sequences SEQ ID No: 1 and SEQ ID No: 2 are a positive primer and a negative primer for detecting the M genes of the H1N1influenza virus 2009 variants respectively, and the SEQ ID No: 3 is a fluorescent probe for detecting the LNA modification of the M genes of the H1N1 influenza virus 2009 variants. The invention further discloses the kit containing the primers and the probe for detecting the H1N1 influenza virus 2009 variants. The kit has the characteristics of quickness, sensitivity, specificity, good generality, good repeatability and difficult pollution.

Description

Nucleotide sequence and test kit at M gene test H1N1virus 2009 variants

Technical field

The present invention relates to nucleotide sequence and test kit at M gene test H1N1virus 2009 variants, especially refer to fast the short fluorescence probe RT-PCR that modifies based on LNA nucleotide sequence and test kit at M gene test H1N1virus (2009 variant), be not only applicable to the accurate detection of H1N1virus in the different animals tissue samples (2009 variant), also applicable to the detection of the H1N1virus (2009 variant) of different animals biopsy sample (being mainly the swab sample), can be used for the examination of animal H1N1virus (2009 variant), entry and exit quarantines etc. belong to inspection and quarantine field.

Background technology

Caused the whole world to be very popular by the Influenza A H1N1 epidemic situation that Mexico begins in April, 2009.This viral sequence evolutionary analysis is shown that this virus is a novel influenza A variant, and there are notable difference in seasonal H1N1 influenza virus of its gene order and people and classical H1N1 swine influenza virus.Sequential analysis shows that its 8 gene fragments have obvious singularity.But its HA fragment and American pig influenza virus HA fragment homology are the highest, are about 95%; NA and M are that Eurasian pig source viral fragment is the most close, and homology is respectively 92% and 97%.Therefore be in the diagnosis and detection of reality, when adopting nucleic acid amplification method to differentiate, must carry out careful sequence alignment analysis.

But study multiple Mammalss such as verified this strain natural infection pig and dog at present.On May 3rd, 2009, Canada reported, the pig that adds one pig farm, western Alberta detects this H1N1 influenza virus on one's body, and after this a lot of scholars detect this cause of disease again in animal bodies such as dog, cat.As from the foregoing should virus to stride the species transmission capacity stronger.

Novel nucleic acid amplification detection method development at present is quick, because it is simple and efficient, the result is accurate, and widespread use in clinical quarantine has obtained good effect, the existing at present trend that substitutes traditional detection method.Although after Influenza A H1N1 breaks out, the lot of domestic and international scholar's research has been set up fluorescent RT-PCR method for detecting.But all detection methods are all set up according to specific HA sequence.After we compare in a large number to H1N1virus (2009 variant) M gene and other H1N1 influenza virus M genes, find that all H1N1viruses (2009 variant) M gene 815-817 coded amino acid is a Threonine, and all the other H1N1 influenza virus coding leucines.But according to this difference designed probe and some H1N1 influenza virus corresponding position gene difference 2 Nucleotide only, we adopt LNA to modify the specificity that detecting probe method can further improve the probe recognition site for this reason, based on this, but we have designed the short probe specific recognition H1N1virus (2009 variant) that LNA modifies.LNA, have another name called lock nucleic acid, caused people's extensive concern in the biological study field as a kind of nucleotide derivative of novelty, it is a kind of special double-ring nucleotide derivative, contain in the structure one or more 2 '-O, 4 '-C-methylene radical-β-D-ribofuranose nucleic acid monomer, 2 of ribose '-O position and 4 '-the C position forms the oxygen methylene bridge by different shrink effect, sulphur methylene bridge or amine methylene bridge, and connect into annular, this annular bridge has locked the N configuration of furanose C3 '-Nei type, reduce the snappiness of ribose structure, increased the stability of phosphate backbone local structure.DNA, RNA there are good recognition capability and powerful avidity.LNA and DNA, RNA complementary two strands have very strong thermostability (Δ Tm=3～8 ℃), stability with anti-3 ' deoxynucleotide enzyme liberating, synthetic method is simple relatively, and partially or completely the few nucleic acid chains available amino end of the LNA that modifies phosphoric acid method is synthetic on automatic dna synthesizer.

This research is the specificity of method for guaranteeing, guarantee the matching of probe, we are on a large amount of sequential analysis bases, the short probe that designs the LNA modification at home first is used to detect H1N1virus (2009 variant), shorten the Nucleotide quantity of probe, and improved the specificity and the sensitivity of method.

Summary of the invention

First purpose of the present invention provides strong, the highly sensitive nucleotide sequence at M gene test H1N1virus (2009 variant) of a group-specific, comprises primer sequence and the short probe sequence of modifying based on LNA.

Another object of the present invention provide quick, accurate, easy to use based on LNA modify at M gene test H1N1virus (2009 variant) detection kit.

For achieving the above object, the present invention is by the following technical solutions:

The M gene nucleotide series of selection H1N1virus (2009 variant) and other A type influenza virus M gene coding region particular sequences on the basis of multiple sequence comparison, carry out primer and probe design as target region.Primer length is about 20 bases, and GC content is 50%-60%, no secondary structure and repeatability in the primer, and with the interior no complementary sequence of primer, the melting temperature (Tm) between primer (Tm value) differs less than 5 ℃ between primer.The length of probe adopts LNA to modify about 12～15 bases, and the mark fluorescent group.

One group at M gene test H1N1virus (2009 variant) nucleotide sequence, as follows:

1)5’-ATC?ATT?GGG?ATY?TTG?CAC-3′(SEQ?ID?NO：1)

2)5’-CGTAGAAGG?CCC?TCT?TTT?C-3’(SEQ?ID?NO：2)

3) 5 '-[FAM]-TA

[TAMRA or BHQ1]-3 ' (SEQ ID NO:3)

Y represents pyrimidine, is T or C herein;

Wherein sequence 1) and 2) be respectively the sense primer and the antisense primer that detect H1N1virus (2009 variant) M gene, sequence 3) the short probe of modifying for the LNA that detects H1N1virus (2009 variant) M gene of fluorescence, modify with LNA with the base that italic is represented, promptly the 3rd, 5,7,9,11,13 base is modified with LNA, 5 ' end mark report fluorophor FAM (6-carboxy-fluorescein) of probe, 3 ' end mark quenching group TAMRA or BHQ1.

We adopt TaqMan fluorescence RT-PCR detection technique to set up H1N1virus (2009 variant) M gene tester, the various conditions of reaction are optimized, comprising the screening of primer probe, Mg ²⁺The optimization of working concentration, the optimization of primer concentration and probe concentration obtains following test kit.

The LNA that detects H1N1virus (2009 variant) M gene modifies short probe in detecting test kit, and is composed of the following components:

1) lysate: available from INVITROGEN company, carry out packing, 2 bottle/boxes by the 15ml/ bottle.

2) RT-PCR reaction solution, table 1 is the reaction solution prescription.

Table 1RT-PCR reaction solution prescription

10 * PCR damping fluid, 25mM MgCl2 purchase the company in Promega; 5 * RT-damping fluid, 2.5mM dNTP is available from the precious biotinylated biomolecule in Dalian Engineering Co., Ltd, primer and probe all entrust Dalian precious biotinylated biomolecule Engineering Co., Ltd synthetic, consisting of of 10 * PCR damping fluid wherein: 500mmol/L KCl, 100mmol/L Tris-HCl (pH9.0,25 ℃), 1.0%Triton X-100.5 * RT-damping fluid consists of 375mmol/L KCl, 15mmol/L MgCl ₂, 50mmol/L DTT, 250mmol/L Tris-HCl (pH8.3,25 ℃).

3) RT-PCR enzyme granulate, available from AMERCIA company, 12/box.

4) Taq archaeal dna polymerase 5U/ μ L is available from Promega company.

5) DEPC water, with twice distillation of tap water, through Millipore MILLI-Q PF PLUS pure water instrument purifying, collect the water of resistivity 〉=18.0M Ω .cm, the Millipore-Q purified water add DEPC to final concentration be 0.1%, 37 ℃ of stir process 12hr, 15lbf/in2 (1.034 * 105Pa) high pressure steam sterilizations 15 minutes.

6) negative control: the negative tissue sample of influenza virus, make 20% suspension with 0.01mol/L pH7.2PBS buffer saline, 70 ℃ act on 1 hour.

7) positive control: be the non-infectious RNA fragment of in-vitro transcription.With H1N1virus A/California/04/2009 (H1N1) length of synthetic is that 982bp M gene clone is in pET-32a, transform the JM109 competent cell, alkaline lysis method of extracting plasmid DNA, cut through PCR and enzyme and to identify the positive recombinant plasmid of back acquisition, called after pET-32a-H1N1/2009-M.Plasmid with purifying is a template, after the linearization for enzyme restriction, carries out in-vitro transcription with the Ribo MAXTM Large Scale RNA Production System-T7 test kit of Promega company; The in-vitro transcription product is removed dna profiling wherein after after measuring after the TRIZOL extraction, promptly obtain preparing the required RNA positive reference substance mother liquor of H1N1virus (2009) M gene masculine reference substance with DNase.

H1N1virus A/California/04/2009 (H1N1) 982bp M purpose fragment gene has the nucleotide sequence shown in the sequence table SEQ ID NO:4.

Synthetic primer and probe are HPLC analyze, as the ratio that obtains single absorption peak collection of illustrative plates and measure OD260nm/OD280nm with ultraviolet spectrophotometer promptly is considered as qualified primer between 1.6-2.0.Respectively with after the above-mentioned in-vitro transcription 10 ⁵The RNA that extracts in the RNA of copy number and classical swine influenza virus A/Swine/2003 (H1N1) the chick embryo allantoic liquid culture is that template increases, the result shows that primer provided by the invention pair and probe are used, amplification A/California/04/2009 (H1N1) M gene RNA that can be special, but A/Swine/2003 (H1N1) RNA that can not increase.

The primer concentration that screening is good is that spacing increases progressively from 0.1 μ mol/L to 0.8 μ mol/L with 0.1 μ mol/L, and concentration and probe concentration increases progressively with 0.025 μ mol/L from 0.025 μ mol/L to 0.2 μ mol/L.Proportioning to primer and probe different concns compares, and from repeatedly finding the revision test: fluorescence amplification is relative higher when adopting the listed primer concentration of table 1 with concentration and probe concentration.

We are at Roche Light Cycler2.0, Roche480 and ABI 7900HT fluorescent PCR detector, at 42 ℃/30min, behind the reverse transcription of 94 ℃/3min, in this experiment denaturation temperature and time, annealing and elongating temperature and time having been carried out optimization experiment.During amplification, adopt cycle index to be respectively 40,45 and 50, compare the Ct value of amplification, and then determine that the cycle index of amplification is 40.Final definite employing expanding effect scheme preferably is the RT-PCR reaction parameter: reaction conditions is: 94 ℃/15s, and 53 ℃/5s, 60 ℃/35s, 40 circulations; When extending, each round-robin annealing collects fluorescence.

Studies show that Mg ²⁺Concentration has certain influence to the fluorescent PCR amplification, thus use the good primer probe of screening, with Mg ²⁺Concentration be that spacing increases progressively with 0.5mmol/L from 1.5mmol/L to 6.0mmol/L, to different Mg ²⁺Amplification under the concentration conditions compares.Mg after the optimization ²⁺Concentration is for seeing Table 1.

The Taq enzyme that we select Promega company to produce, the definition of a unit: 74 ℃ act on 30 minutes, the dNTPs of 10nM can be mixed needed enzyme amount in the acid soluble material.The active requirement: tool dna polymerase activity and 5 ' → 3 ' exonuclease activity, do not have 3 ' → 5 ' exonuclease activity and endonuclease activity; The tool thermostability, 94 ℃ still kept 50% activity after 1 hour.From repeatedly selecting 1.25U Taq enzyme as the Taq enzyme amount of using the revision test result.

The using method of the detection kit at M gene test H1N1virus 2009 variants of the present invention:

1) sample preparation: for tissue sample, in mortar, fully grind, add 5mL PBS mixing again, then tissue suspension is changed in the aseptic Eppendorf pipe, number standby with aseptic scissors and tweezers clip sample 1.0g to be checked.For liquid sample, directly draw to aseptic Eppendorf pipe with asepsis injector, number standby.The sample of gathering or handling is preserved under 2 ℃～8 ℃ conditions and should be no more than 24h; If need prolonged preservation, must place 70 ℃ of refrigerators, but should avoid multigelation (freeze thawing is 2 times at most).After the sample sealing of gathering, adopt thermo jug or insulated tank sealing on the rocks, be transported to the laboratory as early as possible.

2) extraction of RNA: carry out in the sample process district.

Get n 1.5ml sterilization centrifuge tube (n=sample number+1 pipe negative control+1 pipe positive control), perform mark.(lysate has very strong corrodibility at first to add 600 μ L lysates, be sure not to be stained with skin or clothing, otherwise immediately with a large amount of flushing with clean water and dry), add each 200 μ L (a sample is used a suction nozzle instead) of sample to be tested, negative control, positive control then respectively; Add 200 μ L chloroforms again, vibration mixing 5sec (should not be too strong, in order to avoid produce emulsion layer, also available hand is put upside down mixing) on the vortex mixer.

12, the centrifugal 15min of 000g (as have ready conditions, carry out centrifugal in 4 ℃).Get with the last step in the 1.5ml sterilization centrifuge tube of equal amts, add 400 μ L Virahols (20 ℃ of precoolings), perform mark.Draw supernatant liquor phase transition after centrifugal to corresponding pipe, put upside down mixing.

12, the centrifugal 15min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids (different samples should be stained with dried in the different places of thieving paper); Add 600 μ L, 75% ethanol (20 ℃ of precoolings), put upside down washing.

12, the centrifugal 10min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids as far as possible.4, the centrifugal 10sec of 000g is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted with micro sample adding appliance that (a sample is used a suction nozzle instead as far as possible, the attention suction nozzle is not run into the precipitation one side), drying at room temperature 3min (should not be too dry) in order to avoid RNA is insoluble.

Add 11 μ L DEPC water, mixing dissolves the RNA on the tube wall gently, and 2, the centrifugal 5sec of 000g preserves standby (note: the RNA of this moment is subject to the RNA enzyme liberating most, please carries out pcr amplification in 2 hours) on ice.

3) RT-PCR amplification: from test kit, take out RT-PCR reaction solution, Taq enzyme, after at room temperature melting, 2, the centrifugal 5sec of 000g.If required PCR pipe number is n (a n=sample number+1 pipe negative control+1 pipe positive control), each test reaction system needs 15 μ L RT-PCR reaction solutions and 0.25 μ L Taq enzyme.Calculate the usage quantity of good each reagent, add in the proper volume test tube, to wherein adding n/4 RT-PCR enzyme granulate, thorough mixing is even, and each packing 15 μ L in each PCR pipe are transferred to the sample process district.Each the 10 μ L of RNA solution that add preparation in the PCR of each setting pipe respectively, the tight pipe lid of lid is in the centrifugal 5sec of 800g.The PCR pipe sequenced put into the fluorescent PCR detector, the record sample is put order.

Reaction parameter is provided with: 94 ℃/15s, and 53 ℃/5s, 60 ℃/35s, 40 circulations; When extending, each round-robin annealing collects fluorescence.

4) result's judgement: the interpretation of result condition enactment, read detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.As negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is considered as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve shows (2009 variant) influenza virus that do not have H1N1 in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is H1N1 (2009 variant) influenza virus in the expression sample.

Advantage of the present invention is: the present invention selects H1N1 (2009 variant) influenza virus M gene coding region as target region, design degenerate primer and LNA modify short probe foundation and have optimized H1N1 (2009 variant) influenza virus fluorescent RT-PCR method for detecting, obtained excellent technique effect: 1) quick: this method is monitored in real time to the PCR product, and RT-PCR finishes to obtain the result.

2) more sensitive: owing to adopted specific LNA to modify fluorescent probe, higher than the sensitivity of general T aqMan probe, highly sensitive 100～1000 times than the PCR method of routine; In-vitro transcription RNA to the known copy number detects.The result shows that sensitivity all can reach 10 copy/reactions, and repeatability is better.

3) special: owing to not only adopted specific primer, and adopted specific probe, make the specificity of this method be higher than traditional RT-PCR method; But fluorescent RT-PCR method for detecting specific detection H1N1 (2009 variant) influenza virus of setting up, but can not detect classical pig H1N1 influenza virus and the seasonal H1N1 influenza virus of people.In addition, detecting collected other viruses and Avian pneumo-encephalitis virus, the result is negative, does not find cross reaction.

5) stable: the replica test result shows having good stability of institute's establishment method; CRNA to the known copy number of different concns carries out replica test, the results are shown in Table 2.

Table 2 replica test result

6) be difficult for polluting: totally-enclosed reaction need not the PCR aftertreatment, operational safety.

The invention will be further described below in conjunction with specification drawings and specific embodiments.

Description of drawings

The short fluorescence probe RT-PCR that Fig. 1 modifies for LNA is at M gene test H1N1virus (2009 variant) limit of detection measurement result.

The short fluorescence probe RT-PCR that Fig. 2 modifies for L LNA is at the specificity test-results of M gene test H1N1virus (2009 variant).

Embodiment

The viral nucleic acid of using among research of table 3 method and the embodiment

The preparation of embodiment 1, test kit and use

1, the preparation of test kit is formed, and sees Table 4.

The preparation of table 4 test kit is formed

Concrete material is formed as previously described.

2, the using method of test kit

2.1RNA extract:

2.2 amplifing reagent is prepared and preparation

From test kit, take out fluorescence RT-PCR reaction solution, Taq enzyme, after at room temperature melting, 2, the centrifugal 5sec of 000g.If required PCR pipe number is n (a n=sample number+1 pipe negative control+1 pipe positive control), each test reaction system needs 15 μ L RT-PCR reaction solutions and 0.25 μ L Taq enzyme.Calculate the usage quantity of good each reagent, add in the proper volume test tube, to wherein adding n/4 RT-PCR enzyme granulate, thorough mixing is even, and each packing 15 μ L in each PCR pipe are transferred to the sample process district.Each the 10 μ L of RNA solution that add preparation in the PCR of each setting pipe respectively, the tight pipe lid of lid is in the centrifugal 5sec of 400g.The PCR pipe sequenced put into the fluorescent PCR detector, the record sample is put order.

Reaction parameter is provided with: reaction conditions is: 94 ℃/15s, and 53 ℃/5s, 60 ℃/35s, 40 circulations; When extending, each round-robin annealing collects fluorescence.

2.3 the result judges

The interpretation of result condition enactment reads detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.As negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is considered as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve, showing does not have H1N1virus (2009 variant) in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is H1N1virus (2009 variant) in the expression sample.

The sensitivity test of embodiment 2, test kit

1, material:

The viral nucleic acid that is applied in the method research process is for entrusting the M gene of the Shanghai rising sun hat biotechnology synthetic H1N1virus A/California/04/2009 of Development Co., Ltd (H1N1) 982bp.

2, method

1) the M gene of synthetic H1N1virus A/California/04/2009 (H1N1) 982bp is carried out sequencing after, it is in full accord to show that the sequence that records and NCBI have delivered sequence, and it is cloned into the pET32a carrier.Transform the JM109 competent cell, alkaline lysis method of extracting plasmid DNA is cut through PCR and enzyme and to be identified the positive recombinant plasmid of back acquisition, called after pET32a-H1N1/2009-M.Plasmid with purifying is a template, after the linearization for enzyme restriction, carries out in-vitro transcription with the Ribo MAXTM Large Scale RNA Production System-T7 test kit of Promega company; The in-vitro transcription product after extracting, TRIZOL is removed foreign protein and various ion with the dna profiling that DNase removes wherein.RNA is precipitated in the aqua sterilisa of the no RNA enzyme that is dissolved in 1.0mL, packing, 20 μ L/ pipe, frozen under-80 ℃, promptly obtain the cRNA fragment for preparing.

2) the in-vitro transcription RNA that gets preparation does 200 times of dilutions respectively with the aqua sterilisa of no RNA enzyme, measures its A260 absorption value and calculates copy number.The cRNA standard substance of above-mentioned preparation are detected with the H1N1virus at the M gene (2009 variant) detection method of setting up after with 10 times of gradient dilutions, and with deionized water as negative standard.In detection, determine the minimum cRNA concentration that fluorescence quantitative RT-RCR can detect, and draw the limit of detection of this method thus different extent of dilution standard substance.

3, result

See shown in Figure 1ly, the sensitivity of method all can reach 10 copy/reactions.

The specificity test of embodiment 3, test kit

1, material

Listed as table 3.

2, method

With H1N1virus (2009 variant) detection method of the short probe of set up above-mentioned viral nucleic acid is detected, with the specificity of verification method at the M gene.

3, result

As shown in Figure 2, the result shows that the method for being set up can only detect H1N1virus (2009 variant) nucleic acid, but with other animal virus no cross reactions, specificity is good.

The laboratory report that embodiment 4, test kit detect clinical sample

1, material

592 parts of the known samples that China provides directly under Entry-Exit Inspection and Quarantine Bureau.

2, method

China is detected for 592 parts directly under the known sample that Entry-Exit Inspection and Quarantine Bureau provides.The susceptibility of verification method and specificity in actual sample.

3, result

Detected result sees Table 5.

The detected result of table 5 clinical sample

592 parts of known samples are detected.The result shows that the method for foundation can detect wherein all H1N1viruses (2009 variant) nucleic acid samples, and false positive results do not occur, the results are shown in Table 6.The method that shows foundation is reliable and practical.

Claims

1. one group of nucleotide primer and probe at M gene test H1N1virus 2009 variants, its nucleotide primer and probe such as sequence table SEQ ID NO:1 to SEQ ID NO:3, wherein sequence SEQ ID NO:1 and SEQ ID NO:2 are respectively sense primer and the antisense primer that detects H1N1virus 2009 variant M genes, the fluorescent probe that sequence SEQ ID NO:3 modifies for the LNA that detects H1N1virus 2009 variant M genes.

2. nucleotide primer and probe at M gene test H1N1virus 2009 variants according to claim 1, it is characterized in that: 5 ' the end mark report fluorophor FAM of described probe sequence SEQ ID NO:3,3 ' end mark cancellation fluorophor TAMRA or BHQ1, the 3rd, 5,7,9,11,13 base is modified with LNA.

3. test kit at M gene test H1N1virus 2009 variants, composed of the following components:

1) lysate;

2) RT-PCR reaction solution comprises: PCR damping fluid, RT damping fluid, MgCL ₂, dNTP, sense primer, antisense primer and fluorescent probe;

3) RT-PCR enzyme granulate;

4) TaqDNA polysaccharase;

5) DEPC water;

6) negative control, the negative tissue sample of influenza virus is made 20% suspension with 0.01mol/L pH7.2PBS buffer saline, and 70 ℃ act on 1 hour;

7) positive control: be the non-infectious RNA fragment of in-vitro transcription, its corresponding DNA sequences is the nucleotide sequence shown in the sequence table SEQ ID No.4;

Wherein, sense primer is the nucleotide sequence shown in the sequence table SEQ ID No.1, antisense primer is the nucleotide sequence shown in the sequence table SEQ ID No.2, fluorescent probe is the nucleotide sequence shown in the sequence table SEQ ID No.3, its 5 ' end mark report fluorophor FAM, 3 ' end mark cancellation fluorophor TAMRA or BHQ1, the 3rd, 5,7,9,11,13 base is modified with LNA.

4. the test kit at M gene test H1N1virus 2009 variants according to claim 3 is characterized in that, described RT-PCR reaction solution 0.5 * PCR damping fluid, 0.5 * RT damping fluid, 2.5mmol/LMgCL ₂, 0.2mmol/L dNTP, 0.3 μ mol/L sense primer, 0.3 μ mol/L antisense primer and 0.3 μ mol/L fluorescent probe.