CN101886134B - Swine flu virus real-time fluorescent RT-PCR typing kit - Google Patents
Swine flu virus real-time fluorescent RT-PCR typing kit Download PDFInfo
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Abstract
The invention relates to a swine flu virus real-time fluorescent RT-PCR isotyping kit, in particular to a kit for performing isotyping of swine flu viruses by using a one-step method real-time reverse transcription-polymerase chain reaction (RT-PCR) technique. The kit has high sensitivity and specificity. Through the isotyping of the swine flu viruses in respiratory tract specimens and serum and lung tissue samples and other samples by using the kit of the invention, the early-period quick diagnosis of the swine flu viruses is realized, and the trend of the swine flu viruses can be monitored.
Description
Technical field
The present invention relates to a swine influenza virus parting kit, particularly relate to the test kit that carries out the swine influenza virus somatotype with one-step method real-time fluorescent reverse-transcription polymerase chain reaction (RT-PCR) technology.This test kit has very high sensitivity and specificity, through test kit of the present invention swine influenza virus in the samples such as respiratory tract sample, serum, lung tissue is carried out somatotype, thereby realizes the early stage quick diagnosis of swine influenza virus, monitors its fashion trend.
Background technology
Porcine influenza (SI) is a kind of respiratory diseases in pigs that causes because of swine influenza virus, and this disease all has generation all over the world, and harm is serious, and financial loss is huge, and human beings'health is constituted a threat to.Swine influenza virus (SIV) belongs to orthomyxoviridae family (Orthomyxoviridae), and influenza A virus belongs to (Influenza virus A).Typical case's virion is spherical, and diameter is 80nm～120nm, and cyst membrane is arranged.The projection gp that many radial arrangement are arranged on the cyst membrane is respectively hemagglutinin HA, neuraminidase NA and M2 albumen.Be nucleocapsid in the virion, shape is symmetrical in the shape of a spiral, and diameter is 10nm, it contain nucleoprotein (neucleoprotein, NP), 3 kinds of polymerase proteins (polymerase protein 1, PB1; Polymerase protein 2, PB2; Polymerase protein A) and viral single stranded RNA.Swine influenza virus is the sub-thread minus-stranded rna virus, and genome is about 13.6kb, is made up of 8 independent segments that differ in size.Swine influenza virus can be in cell and the chicken embryo propagation of multiple animal.Virus has hemagglutination activity, but the antigenicity of different strains does not have tangible differentiation.Because it is very big that virus receives the pressure of antibody, therefore the variation of virus is frequent.Although can form a variety of influenza virus serotypes between the different subtype, be separated to four kinds of main hypotype: H1N1, H1N2, H3N2 and H3N1 on one's body from pig, wherein there are three kinds of hypotypes to cause the people to infect, be respectively: H1N1, H1N2 and H3N2.
Swine influenza virus can be propagated the whole year in swinery, but majority breaks out latter stage in autumn and winter, has the characteristics of high incidence (almost 100%) and low case fatality rate (case fatality rate≤1%).Swine influenza virus infects ill pig production performance is descended, and postpones listing; Also can cause respiratory tract bacterium and viral secondary or polyinfection, make epidemic situation even more serious, cause pig only dead.Pig is the only common susceptible host of people source, fowl source and pig source influenza virus, so pig in the communication process, plays a part intermediate host and multiple host between the influenza kind of fowl-pig and human, and it can be propagated swine influenza virus to people or birds.The porcine influenza outburst once appearred in the U.S. in the soldier of New Jersey Fort Dix in 1976; Cause 200 many cases cases; Wherein at least 4 soldiers make progress into pneumonia, and 1 people is dead, this virus with at that time in the pig body isolating virus identical; Confirmed that first under field conditions (factors), swine influenza virus can be propagated to the people from pig.1988, the person-to-person sign of propagating of porcine influenza appearred in the U.S., has occurred slight influenza appearance disease among the medical personnel of contacted one routine porcine influenza case, and in serum, has detected porcine influenza antibody.During year February in December, 2005 to 2009, the U.S. has reported 12 routine people infected pigs influenza cases altogether, but all occurs dead.In October, 1999,1 the 10 monthly age girl baby in Hong Kong has infected swine influenza virus H3N2, returns to one's perfect health at present.In these years, the report of people infected pigs influenza virus different virus strain is arranged all over the world, but unpopular on a large scale.In March, 2009, people infected pigs influenza epidemic situation has been broken out in some areas such as the Mexico and the U.S., and promptly H 1 N 1 influenza A virus infection can the people infect the people, the potentially dangerous of global eruption and prevalence possibly occur.
The latent period of people infected pigs influenza is still indeterminate, with reference to being generally 1-3 days in latent period of influenza.Clinical symptom is similar with influenza, comprises heating, cough, pharyngalgia, body pain, headache, chilly and fatigue etc.Diarrhoea and vomiting also can appear in some people, even cause serious disease (pneumonia and respiratory insufficiency) and death.Behind the people infected pigs influenza virus, available data shows, infective stage is that premorbid 1 day is to falling ill back 7 days.If the case morbidity still had heating paresthesia after 7 days, expression still has infectivity.Children, especially child, in infective stage, possibly be longer than 7 days.
Pig is as influenza virus " mixing tank ", strides the kind obstacle and infect in new host's the process influenza virus to play an important role.Because the pig epithelial cell has sialyl 2; 6-galactoside and sialyl 2, the 3-galactoside, the human influenza virus can combine with the former; And bird flu virus combines with the latter; Therefore, the pig epithelial cell just can be by human influenza virus and avian influenza, and becomes the live vector of recombination between strain.Be separated to a strain H3N2 hypotype SIV from the North Carolina in 1998, its HA, NA and PB1 gene are human-like influenza virus gene, and M, NP and NS gene are pig type influenza virus gene, and PB2, PA gene are fowl type influenza virus gene.1978, the H1N2SIV that Japan breaks out was by H1N1 and H3N2 recombination and the new subtype that produces.Domestic from Shandong pig isolate the H9N2 influenza virus, analysis possibly be the result by the reorganization of chicken and duck virus.The H9N2 subtype influenza virus that continues was separated to from swinery in 1998 first, and research shows, finds pig source H9N2 hypotype and domestic isolating bird flu virus height homology through carrying out the partial sequence analysis.Between SI and the human influenza also cross infection and propagation can take place.1978, from a swinery in Taiwan, be separated to H3N2 hypotype SIV, find that through order-checking human influenza virus and the segmental reorganization of SI virogene have taken place this virus.The H1N1virus that the Mexico in 2009 and the U.S. occur is considered to the gene segment that this strain includes porcine influenza, bird flu and three kinds of influenza viruses of human influenza.
Therefore study the type of SIV and change, carry out corresponding epidemiology survey, can predict the development trend of human influenza, have far-reaching public hygienics meaning in different time and region.Because this disease is popular serious day by day with harm, therefore to SIV somatotype research having become one of focus of domestic and international animal doctor circle, medical circle and mikrobe educational circles.
Swine influenza virus genotyping detection method commonly used at present comprises:
1) serological method: comprise EUSA (ELISA), fluorescent immune method, hemagglutination-inhibition test etc.; These serological methods need be carried out chicken embryo or tissue culture in advance to sample; Can't realize quick diagnosis; And need the quite complete target antigen and the immune serum of type specificity, complex operation step.
2) nucleic acid detection method; Comprising the RT-PCR method, because round pcr has characteristics such as easy, quick, sensitive, high specificity, can be used for the swine influenza virus somatotype and detect with Molecule Epidemiology Investigation etc., is the recommend method of realization swine influenza virus fast typing detection.Method for gene chip, this method have fast, sensitivity and flux advantages of higher, but cost an arm and a leg, and are unfavorable for large-scale promotion.The method of sequencing and typing and genome sequencing has the accuracy height, and the characteristics of obtaining that contain much information can be carried out viral evolutionary analysis, but its requirement to instrument is high, and detection time is long than RT-PCR and gene chip.
Real-time fluorescence (Real-time) RT-PCR technology grows up in that RT-PCR is technical.The real-time fluorescence PCR technology is a kind of rapidly nucleic acid detection technique of development in recent years, uses a kind of pcr amplification appearance that has nuclear power coupling devices (CCD), reflects each round-robin level of amplification of PCR in real time through the dynamic change that detects fluorescent signal.CCD can periodically send the exciting light of specific wavelength according to certain procedure, collect to detect fluorescent signal, and is aggregated into workstation through software analysis and obtains amplification curve.One-step method real-time fluorescent RT-PCR technology is a kind of (Martell, M., the J.Gomez of real-time fluorescence PCR; J.Esteban; S.Sauleda, J.Quer, B.Cabot; R.Esteban, and J.Guardia.1999.High-throughput real-time reverse transcription-PCR quantitation of hepatitis Cvirus RNA.J.Clin.Microbiol.37:327-332; Lee CW, Suarez DL.2004.Application ofreal-time RT-PCR for the quantitation and competitive replication study of H5 andH7 subtype avian influenza virus.J Virol Methods.119 (2): 151-8.), it is the method for a kind of direct rapid detection RNA, compares with the real-time fluorescence PCR that detects DNA, difference is that the former has increased reversed transcriptive enzyme in reaction system, many simultaneously reverse transcription reaction steps; Something in common is that both all have the probe of a two ends difference mark fluorescent reporter group and quenching group in reaction system, and when probe structure was complete, the energy that the fluorescence report group sends fluorescence shifted to quenching group, presents quenching effect.If the existence of target sequence is arranged in the amplification procedure, the process middle probe molecule of amplification is hydrolyzed cut-out gradually, and fluorescence report group and quenching group dissociate each other, have blocked the two FRET effect, and the fluorescence report group sends fluorescent signal.Along with the carrying out of amplification, fluorescent signal presents linear the enhancing along with the segmental amplification of purpose.
Traditional RT-PCR is accomplished by two independent response procedures or step, and the first step is through the reversed transcriptive enzyme effect, and the RNA rt is become cDNA; Second step was under the effect of Taq enzyme, was that template is carried out pcr amplification with the cDNA that forms in the first step.Compare with traditional RT-PCR; One-step method real-time fluorescent RT-PCR technology has following advantage: two response procedures that 1, rt and two of pcr amplifications separately carried out in the differential responses pipe are merged into a response procedures; Stopped pipe is accomplished testing process fully in a PCR reaction tubes, has saved the reaction times greatly; 2, through analysis, abandon the regular-PCR method and receive multiple factor interferential end point analysis method, can carry out accurate quantitative analysis to test sample, thereby effectively monitor the effect of pharmacological agent the cycle threshold (Ct) of amplification curve and increased logarithmic phase; 3, be one with RNA rt, DNA cloning and detection three process fusion, can be in real time, the whole process of dynamic monitoring RNA amplification, saved PCR product postprocessing process, shortened analysis time as a result greatly, make that this method is more quick and easy; 4, owing to take a kind of detecting pattern of sealing, pollute and the false positive that causes thus thereby reduced aerosol; 5, since on common RT-PCR basis, increased by one can with the fluorescent probe of template complementary pairing, further improved the specificity that detects target polynucleotide.Therefore, this technology replaces traditional RT-PCR method gradually in the detection and analysis of RNA sample, obtain very using widely.
China FDA (SFDA) has ratified production and the clinical application such as the one-step method real-time fluorescent RT-PCR detection kit of pathogenic agent such as HIV-1, HCV.Therefore, developing a swine influenza virus parting kit through one-step method real-time fluorescent RT-PCR method is feasible technically, have on its methodology in addition easy fast, sensitivity and the high characteristics of specificity, can realize that early stage fast typing detects.But all still untapped at present go out to detect the swine influenza virus real-time fluorescent RT-PCR typing kit both at home and abroad, therefore developing this test kit becomes the task of top priority, and its application will greatly be satisfied the swine influenza virus fast typing and detect the needs with monitoring.
In using known one-step method real-time fluorescent RT-PCR technology for detection and quantitative analysis sample in the practice of certain particular target nucleic acid; In order to reduce and avoid the false negative or the false positive of detected result; Improve the accuracy of quantitative analysis, critical basic fundamental link is how based on known target polynucleotide sequences Design, screening and prepare suitable primer and oligonucleotide probe.The inventor has utilized one-step method real-time fluorescent RT-PCR technological invention on this basis a kind of test kit that carries out the swine influenza virus somatotype is applied to the swine influenza virus somatotype and detects and popular condition monitoring.
Summary of the invention
The present invention relates to a swine influenza virus parting kit, particularly relate to the test kit that carries out the swine influenza virus somatotype with one-step method real-time fluorescent reverse-transcription polymerase chain reaction (RT-PCR) technology.This test kit can carry out somatotype to the swine influenza virus in the samples such as respiratory tract sample, serum, lung tissue.
The purpose of this invention is to provide a kind of test kit that uses one-step method real-time fluorescent RT-PCR technology to carry out the swine influenza virus somatotype, particularly relate to application and the popular condition monitoring thereof of early infection in laboratory diagnosis of swine influenza virus.Its ultimate principle is to utilize the Auele Specific Primer of a pair of oligonucleotide and the specific probe of an oligonucleotide, at reversed transcriptive enzyme (RT enzyme), warm start Taq enzyme, RNA enzyme inhibitors (RNasin), high-quality deoxyribonucleoside triphosphate (dNTPs) and Mg
2+In the RT-PCR reaction buffer, realize the cyclic amplification of target polynucleotide through the fluorescent PCR amplification appearance that DA7600, ABI7500 etc. are commercially available, thereby reach purpose quick, real-time, the detection by quantitative target polynucleotide.
In order to realize the present invention, we have adopted following technical scheme:
1) use suitable foranalysis of nucleic acids software respectively the nucleotide sequence of known swine influenza virus H1 type, H3 type HA gene and N1 type, N2 type NA gene to be carried out homology relatively; Finding out on the basis of homology segment, further using suitable primer-design software (for example Primer Express 2) to select and design oligonucleotides primer and probe.Because institute's designed primer and probe all have the sequence that is complementary to the corresponding type of swine influenza virus; And there is not homology with the nucleotide sequence of other pathogenic agent; The situation that does not have cross reaction between other swine influenza virus of different shaped; Do not comprise the restriction enzyme site of any common endonuclease, so improved safety and accuracy that the swine influenza virus somatotype detects yet.
2) use the synthetic required Oligonucleolide primers of DNA synthesizer, separate processing with carrying out ammonia behind molecular sieve and fast protein liquid chromatography method (FPLC) purifying.Same synthetic required probe sequence, ammonia are separated after the processing respectively at the 6-FAM amidite of its 5 ' end mark as fluorescence generation group (reporter group), and its 3 ' end mark by active connecting arm coupling on as fluorescent quenching or suppress the TAMRA of group.With the fluorescently-labeled probe of FPLC purification by chromatography.Then, polyacrylamide gel (20%) electrophoretic method and spectrophotometry physical characterization institute synthetic primer and the probe under the use sex change condition.
3) be suitable for the reaction system that one-step method real-time fluorescent RT-PCR increases.Reaction system comprise reversed transcriptive enzyme, warm start Taq enzyme, 2 '-deoxynucleoside triphosphate, can with the forward primer of article one chain combination of double-stranded target polynucleotide, can with the reverse primer of the second chain combination of double-stranded target polynucleotide, can combine with the target polynucleotide and two ends be combined with respectively fluorescence generation group and fluorescent quenching group oligonucleotide probe, contain the damping fluid of mg ion.Owing to will confirm H antigen and the antigenic type of N respectively; Both combine the concrete hypotype that could judge swine influenza virus, so the reaction system of this test kit reagent will comprise four kinds: be respectively H1 hypotype reaction system, H3 hypotype reaction system, N1 hypotype reaction system and N2 hypotype reaction system.
4) from sample to be tested, extract RNA, add respectively in the aforesaid reaction system,, judge the type of swine influenza virus from the amplified fluorescence curve directly through single stage method RT-PCR amplification.
The test kit that swine influenza virus carries out somatotype in the sample provided by the present invention comprises: (1) is equipped with the packing box that these reagent bottles of packing or pipe are separated and concentrated to RNA extracting solution (being TRIZOL nucleic acid extraction agent), H1 hypotype reaction system, H3 hypotype reaction system, N1 hypotype reaction system and N2 hypotype reaction system, negative quality control product, H1 hypotype positive quality control product, H3 hypotype positive quality control product, N1 industry type positive quality control product and N2 hypotype positive quality control product and a plurality of reagent bottles that seal or pipe and (2) respectively.
According to another preferred embodiment of the present invention, wherein H1 hypotype reaction system comprises H1 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system; H3 hypotype reaction system comprises H3 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system; N1 hypotype reaction system comprises N1 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system; N2 hypotype reaction system comprises N2 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system.Wherein the component of RT-PCR reaction solution, RT-PCR enzyme system is all identical in H1 hypotype reaction system, H3 hypotype reaction system, N1 hypotype reaction system, the N2 hypotype reaction system.
According to another preferred embodiment of the present invention; Wherein H1 hypotype primer probe mixed solution is by (a) the H1 hypotype forward primer with article one chain combination of double-stranded target polynucleotide; (b) can with the H1 hypotype reverse primer of the second chain combination of double-stranded target polynucleotide; (c) and can combine with target polynucleotide and H1 hypotype oligonucleotide probe that two ends are combined with fluorescence report group and fluorescent quenching group is respectively formed; Above primer concentration and probe concentration is 5～15pmol/ reaction, and preferred primer concentration is that 5pmol/ reaction, concentration and probe concentration are the 5pmol/ reaction.
According to another preferred embodiment of the present invention; Wherein the sequence of H1 hypotype forward primer is in the H1 hypotype primer probe mixed solution: 5 '-TGGCACCAAGATATGCTTTTG-3 ' (SEQ ID NO:1); The sequence of H1 hypotype reverse primer is: 5 '-CAAGTCGCATTGCAGTCATG-3 ' (SEQID NO:2), forward and reverse primer can respectively extend 10 bases to 5 ' and 3 ' extreme direction.
According to another preferred embodiment of the present invention, wherein the sequence of H1 hypotype oligonucleotide probe is in the H1 hypotype primer probe mixed solution: 5 '-CGGGTATCATAACATCGGATGCACCA-3 ' (SEQ ID NO:3).
According to another preferred embodiment of the present invention; Wherein H3 hypotype primer probe mixed solution is by (a) the H3 hypotype forward primer with article one chain combination of double-stranded target polynucleotide; (b) can with the H3 hypotype reverse primer of the second chain combination of double-stranded target polynucleotide; (c) and can combine with target polynucleotide and H3 hypotype oligonucleotide probe that two ends are combined with fluorescence report group and fluorescent quenching group is respectively formed; Above primer concentration and probe concentration is 5～15pmol/ reaction, and preferred primer concentration is that 5pmol/ reaction, concentration and probe concentration are the 5pmol/ reaction.
According to another preferred embodiment of the present invention; Wherein the sequence of H3 hypotype forward primer is in the H3 hypotype primer probe mixed solution: 5 '-CGTCGTGAAAGCGTGAACAG-3 ' (SEQ ID NO:4); The sequence of H3 hypotype reverse primer is: 5 '-CGGCATGGTCACGTTCAG-3 ' (SEQ ID NO:5), forward and reverse primer can respectively extend 10 bases to 5 ' and 3 ' extreme direction.
According to another preferred embodiment of the present invention, wherein the sequence of H3 hypotype oligonucleotide probe is in the H3 hypotype primer probe mixed solution: 5 '-CCGTCTGAACTGGCTGCATAACCTGG-3 ' (SEQ ID NO:6).
According to another preferred embodiment of the present invention; Wherein N1 hypotype primer probe mixed solution is by (a) the N1 hypotype forward primer with article one chain combination of double-stranded target polynucleotide; (b) can with the N1 hypotype reverse primer of the second chain combination of double-stranded target polynucleotide; (c) and can combine with target polynucleotide and N1 hypotype oligonucleotide probe that two ends are combined with fluorescence report group and fluorescent quenching group is respectively formed; Above primer concentration and probe concentration is 5～15pmol/ reaction, and preferred primer concentration is that 5pmol/ reaction, concentration and probe concentration are the 5pmol/ reaction.
According to another preferred embodiment of the present invention; Wherein the sequence of N1 hypotype forward primer is in the N1 hypotype primer probe mixed solution: 5 '-GACCTTGCTTTTGGGTTGAACT-3 ' (SEQ ID NO:7); The sequence of N1 hypotype reverse primer is: 5 '-AAGGATATGCTGCTCCCACTAGTC-3 ' (SEQ ID NO:8), forward and reverse primer primer can respectively extend 10 bases to 5 ' and 3 ' extreme direction.
According to another preferred embodiment of the present invention, wherein the sequence of N1 hypotype oligonucleotide probe is in the N1 hypotype primer probe mixed solution: 5 '-CAGAGGGCGACCCAAAGAGAACACAATC-3 ' (SEQ ID NO:9).
According to another preferred embodiment of the present invention; Wherein N2 hypotype primer probe mixed solution is by (a) the N2 hypotype forward primer with article one chain combination of double-stranded target polynucleotide; (b) can with the N2 hypotype reverse primer of the second chain combination of double-stranded target polynucleotide; (c) and can combine with target polynucleotide and N2 hypotype oligonucleotide probe that two ends are combined with fluorescence report group and fluorescent quenching group is respectively formed; Above primer concentration and probe concentration is 5～15pmol/ reaction, and preferred primer concentration is that 5pmol/ reaction, concentration and probe concentration are the 5pmol/ reaction.
According to another preferred embodiment of the present invention; Wherein the sequence of N2 hypotype forward primer is in the N2 hypotype primer probe mixed solution: 5 '-GGGCACCACCCTGAACAA-3 ' (SEQ ID NO:10); The sequence of N2 hypotype reverse primer is: 5 '-CGTTCATCAGCAGGGTACGA-3 ' (SEQ ID NO:11), forward and reverse primer primer can respectively extend 10 bases to 5 ' and 3 ' extreme direction.
According to another preferred embodiment of the present invention, wherein the sequence of N2 hypotype oligonucleotide probe is in the N2 hypotype primer probe mixed solution: 5 '-CCATAGCAACGATACCGTGCATGATCG-3 ' (SEQ ID NO:12).
According to another preferred embodiment of the present invention; The RT-PCR reaction solution comprises single stage method RT-PCR reaction buffer and DEPC water, and wherein single stage method RT-PCR reaction buffer comprises 10mmol/L Tris-HCl (pH8.0), 150mmol/L KCl, 2.0mmol/L MgCl
2
According to another preferred embodiment of the present invention; RT-PCR enzyme system is that diluent is formed by reversed transcriptive enzyme (RT enzyme), warm start Taq enzyme, RNasin, dNTPs, enzyme; Wherein the RT enzyme dosage is that 1.5～3.5U/ reaction, warm start Taq enzyme dosage are that 3～7U/ reaction, RNasin consumption are that 15～25U/ reaction, dNTPs concentration are 0.20mmol/L, and enzyme is that diluent is that general commercially available enzyme is a diluent.
According to a preferred embodiment of the invention; Wherein negative quality control product is a deionized water; H1 hypotype positive quality control product is the recombinant plasmid that contains H1 hypotype HA gene order, and plasmid forms employed upstream and downstream primer sequence SEQ ID NO:1 and SEQ ID NO:2 by the PCR product of upstream and downstream primer amplification through commercially available carrier cloning structure; H3 hypotype positive quality control product is the recombinant plasmid that contains H3 hypotype HA gene order; Plasmid is formed through commercially available carrier cloning structure by the PCR product of upstream and downstream primer amplification, employed upstream and downstream primer sequence SEQ ID NO:4 and SEQ ID NO:5, and N1 hypotype positive quality control product is the recombinant plasmid that contains N1 hypotype NA gene order; Plasmid is formed through commercially available carrier cloning structure by the PCR product of upstream and downstream primer amplification; Employed upstream and downstream primer sequence SEQ ID NO:7 and SEQ ID NO:8, N2 hypotype positive quality control product is the recombinant plasmid that contains N2 hypotype NA gene order, plasmid is formed through commercially available carrier cloning structure by the PCR product of upstream and downstream primer amplification; Employed upstream and downstream primer sequence SEQ ID NO:10 and SEQ ID NO:11, above positive quality control product concentration is 1 * 10
6Copy/ml.
According to another preferred embodiment of the present invention, the optimal reaction temperature and the time that wherein are used for pcr amplification are: 50 ℃ of rt 15min, 1 circulation; 94 ℃ of 15min, 1 circulation; Last 95 ℃ of 15s, 55～60 ℃ of 45s, 45 circulations (collection fluorescent signal).
According to a preferred embodiment of the invention, wherein the sample to be checked of test kit is samples such as respiratory tract sample, serum, lung tissue.
The minimum concentration that one-step method real-time fluorescent RT-PCR test kit provided by the invention can detect the swine influenza virus different subtype is 1.0 * 10
2Copies/ml explains that this test kit has extraordinary sensitivity.
The present invention is respectively to swine influenza virus H1 hypotype, H3 hypotype HA gene and N1 hypotype, N2 hypotype NA gene design Auele Specific Primer and probe; Can detect the corresponding hypotype of swine influenza virus respectively; But can not detect porcine reproductive and respiratory syndrome virus, high-pathogenicity porcine reproductive and respiratory syndrome virus variant, Pseudorabies virus, pig parvoviral, porcine circovirus 2 type, foot and mouth disease virus positive sample, explain that this test kit has excellent specificity.
One-step method real-time fluorescent RT-PCR detection kit provided by the invention can detect the swine influenza virus different subtype in the samples such as respiratory tract sample, serum, lung tissue; Can be sensitivity, early diagnosis swine influenza virus infection fast, monitor its popular situation reliable experimental evidence is provided.
Description of drawings
Fig. 1 shows the detected result of H1 hypotype positive quality control product, H3 hypotype positive quality control product, N1 hypotype positive quality control product, N2 hypotype positive quality control product and negative quality control product.H1 hypotype positive quality control product, H3 hypotype positive quality control product, N1 hypotype positive quality control product, N2 hypotype positive quality control product amplification curve have the obvious Exponential growth phase, become the S type, can clearly be judged to be the positive.Negative quality control product amplification curve is straight or oblique do not have down and with baseline intersect, do not have the Ct value, can clearly be judged to be feminine gender.
Fig. 2 shows the detected result of specificity with reference to article.9 parts of specificitys with reference to the amplification curve of article straight or oblique do not have down and with baseline intersect, do not have the Ct value, can clearly be judged to be feminine gender.9 parts of specificitys are respectively each hypotype of bird flu virus H5/H7/H9, porcine reproductive and respiratory syndrome virus, high-pathogenicity porcine reproductive and respiratory syndrome virus variant, Pseudorabies virus, pig parvoviral, porcine circovirus 2 type, foot and mouth disease virus with reference to article.
Fig. 3 shows H1N1 type swine influenza virus pattern detection result.The amplification curve of H1 hypotype, N1 hypotype has the obvious Exponential growth phase to become the S type; Can clearly be judged to be the positive, the amplification curve amplification curve of all the other hypotypes is straight or oblique do not have down and with baseline intersect, do not have the Ct value; Feminine gender can be clearly be judged to be, therefore H1N1 type swine influenza virus can be clearly be judged to be.
Fig. 4 shows H3N2 type swine influenza virus pattern detection result.The amplification curve of H3 hypotype, N2 hypotype has the obvious Exponential growth phase to become the S type; Can clearly be judged to be the positive, the amplification curve amplification curve of all the other hypotypes is straight or oblique do not have down and with baseline intersect, do not have the Ct value; Feminine gender can be clearly be judged to be, therefore H3N2 type swine influenza virus can be clearly be judged to be.
Fig. 5 shows H1N2 type swine influenza virus pattern detection result.The amplification curve of H1 hypotype, N2 hypotype has the obvious Exponential growth phase to become the S type; Can clearly be judged to be the positive, the amplification curve amplification curve of all the other hypotypes is straight or oblique do not have down and with baseline intersect, do not have the Ct value; Feminine gender can be clearly be judged to be, therefore H1N2 type swine influenza virus can be clearly be judged to be.
Fig. 6 display sensitivity is with reference to the detected result of article.16 parts of sensitivity is with reference to being 1.0 * 10 except concentration in the article
14 parts of plasmid sample amplification curves of copies/ml are straight or oblique do not have down and with baseline intersect, do not have the Ct value, can clearly be judged to be feminine gender.All the other sample standard deviations can clearly be judged to be the positive.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: the development of swine influenza virus typing detection reagent
1, the design of primer and probe: through the nucleotide sequence of reporting swine influenza virus is carried out the sequence alignment analysis; Be the amplified target site with swine influenza virus H1 type, H3 type HA gene and N1 type, N2 type NA gene respectively; Select the section of no secondary structure and high conservative; According to the fundamental principle of primer probe design, utilize software many to primer and probe with artificial design.
2, the selection of clinical sample: show according to domestic and international pertinent literature report, can use samples such as respiratory tract sample, serum, lung tissue.
3, the foundation of reaction system and optimization
The preparation of sample: each purpose that contains to make up increases pulsating plasmid as positive quality control product, with each hypotype of bird flu virus H5/H7/H9, porcine reproductive and respiratory syndrome virus, high-pathogenicity porcine reproductive and respiratory syndrome virus variant, Pseudorabies virus, pig parvoviral, porcine circovirus 2 type, foot and mouth disease virus positive sample as negative reference article; As negative quality control product, extract above-mentioned positive quality control product and the specificity RNA with reference to article with TRIZOL with deionized water respectively, negative quality control product is for use.
The screening of primer probe: the many groups primer probe with design in above-mentioned 1 detects above-mentioned positive quality control product and negative RNA with reference to article respectively, through TE, filters out the best primer probe combinations of specificity, sensitivity and good reproducibility.(like SEQ ID NO:1 in the sequence table～SEQ ID NO:12).
The optimization of primer concentration and probe concentration: in reaction system under the constant situation of other components; Use respectively to react to react to the probe of 15pmol/ reaction density gradient and carry out the PCR reaction to the primer of 15pmol/ reaction gradient with from 2pmol/ from 2pmol/; Through revision test repeatedly find primer and concentration and probe concentration between 5～15pmol/ reaction all can, wherein best primer concentration is that 5pmol/ reaction, concentration and probe concentration are the 5pmol/ reaction.
The optimization of magnesium ion concentration: in reaction system, under the constant situation of other components, use respectively from the mg ion of 1mol/L to 2.5mmol/L concentration gradient and carry out the PCR reaction,, finally confirm that best magnesium ion concentration is 2.0mmol/L through revision test repeatedly.
The optimization of warm start Taq enzyme dosage: in 25 μ L reaction systems under the constant situation of other components; Use enzyme dosage/reaction to carry out the PCR reaction respectively from 1U (unit of enzyme) to the 8U concentration gradient; Warp is revision test repeatedly, and final definite best warm start Taq enzyme dosage is 3～7U/ reaction.
The optimization of RT enzyme dosage: in 25 μ L reaction systems under the constant situation of other components; Use enzyme dosage/reaction to carry out the PCR reaction respectively from 1U (unit of enzyme) to the 8U concentration gradient; Warp is revision test repeatedly, and final definite best RT enzyme dosage is 1.5～3.5U/ reaction.
The optimization of RNasin consumption: in 25 μ L reaction systems under the constant situation of other components; Use enzyme dosage/reaction to carry out the PCR reaction respectively from 5U (unit of enzyme) to the 40U concentration gradient; Warp is revision test repeatedly, and final definite best RNasin consumption is 15～25U/ reaction.
The optimization of dNTPs concentration: in reaction system under the constant situation of other components; Use respectively from the dNTPs of 0.1mmol/L to 0.25mmol/L concentration gradient and carry out the PCR reaction; Warp is revision test repeatedly, and final definite best dNTPs concentration is 0.20mmol/L.
The optimization of temperature of reaction: according to the activity of enzyme and the length of target polynucleotide, mainly annealing temperature and extension time are optimized, warp is revision test repeatedly, and final definite best temperature of reaction and time are: 50 ℃ of rt 15min, 1 circulation; 94 ℃ of 15min, 1 circulation; Last 95 ℃ of 15s, 55～60 ℃ of 45s, 45 circulations (collection fluorescent signal).
Embodiment 2: swine influenza virus parting detecting reagent and use thereof
1, preparation comprises the test kit of following moity: RNA extracting solution (50ml/ pipe) 1 pipe, H1 industry type primer probe mixed solution (50 μ l/ pipe) 1 pipe, H3 hypotype primer probe mixed solution (50 μ l/ pipe) 1 pipe,, N1 hypotype primer probe mixed solution (50 μ l/ pipe) 1 pipe, N2 hypotype primer probe mixed solution (50 μ l/ pipe) 1 pipe, RT-PCR reaction solution (1440 μ l/ pipe) 1 pipe, RT-PCR enzyme system (300 μ l/ pipe) 1 manage, negative quality control product (200 μ l/ pipe) 1 is managed, H1 hypotype positive quality control product (100 μ l/ pipe) 1 is managed, H3 hypotype positive quality control product (100 μ l/ pipe) 1 is managed, N1 hypotype positive quality control product (100 μ l/ pipe) 1 is managed, N2 hypotype positive quality control product (100 μ l/ pipe) 1 is managed.
2, collection of specimens, transport and preserve
Carry out collection of specimens according to practical situation, detectable sample comprises: samples such as respiratory tract sample, serum, lung tissue.Acquisition method is following: 1. respiratory tract sample: swab wiping bilateral pharyngeal tonsil and pharynx rear wall, immerse swab in the 4-5ml sample solution sealing censorship; 2. serum: the about 1ml of aseptic collection venous blood, leave standstill 2 hours and 1 hour respectively at room temperature and 4 ℃ after, centrifugal (8,000rpm, 5 minutes) are separated and are also collected serum specimen; 3. lung tissue: utilize bronchoscope that the suspicious focus of lung is carried out the living tissue sampling, and it is for use with tissue homogenizer tissue samples to be carried out homogenate.
Sample can be used for immediately the test, also can be stored in-70 ℃ to be measured, preservation period is 6 months.Sample transports and adopts 0 ℃ of curling stone
3, detect step
(1) RNA extracts
Get each 100 μ l (it is resuspended to add an amount of saline water during sample less than 100 μ l) of sample of the above-mentioned type, be positioned over 1.5ml sterilization centrifuge tube, add 200 μ l Trizol reagent and 100 μ l chloroforms, left standstill 3 minutes after 20 seconds with the powerful vibration of vibrator;
2～8 ℃ are descended 12, and the centrifugal 10min of 000rpm draws supernatant to another clean 1.5ml centrifuge tube;
Add the 0.2ml chloroform, cover tight lid, manually firmly put upside down and shake 15s (unavailable vibrator), incubated at room 2～3min;
Behind 2～8 ℃ of centrifugal 15min of following 12000rpm, the supernatant of getting the superiors is to another clean 1.5ml centrifuge tube;
The Virahol that adds the 0.5ml precooling, slowly put upside down abundant mixing after ,-20 ℃ leave standstill 15min, 2～8 ℃, the centrifugal 10min of 12000rpm carefully abandons most supernatant;
Add 70% ice-cold ethanol of 1ml precooling, manually put upside down washing precipitation for several times, 2～8 ℃, the centrifugal 5min of 8000rpm carefully abandons most supernatant;
Air or vacuum-drying 5～10min add 50 μ l DEPC H
2O is hatched 10min under 55～60 ℃, and light finger is flicked and beaten at the pipe end, and it is fully dissolved, and it is subsequent use directly to be used for experiment or-70 ℃ of preservations.
(2) PCR reaction and interpretation of result
Getting H1 hypotype primer probe mixed solution 2 μ l, RT-PCR reaction solution 15 μ l, RT-PCR enzyme respectively is that 3 μ l are mixed with H1 industry type reaction system; Getting H3 hypotype primer probe mixed solution 2 μ l, RT-PCR reaction solution 15 μ l, RT-PCR enzyme is that 3 μ l are mixed with H3 hypotype reaction system; Getting N1 hypotype primer probe mixed solution 2 μ l, RT-PCR reaction solution 15 μ l, RT-PCR enzyme is that 3 μ l are mixed with N1 hypotype reaction system; Getting N2 hypotype primer probe mixed solution 2 μ l, RT-PCR reaction solution 15 μ l, RT-PCR enzyme is that 3 μ l are mixed with N2 hypotype reaction system.
Get negative quality control product, each 5 μ l of sample; Add respectively in H1 hypotype reaction system, H3 hypotype reaction system, N1 hypotype reaction system, the N2 hypotype reaction system, get H1 hypotype positive quality control product 5 μ l in addition and add H1 hypotype reaction system, H3 hypotype positive quality control product 5 μ l and add that H3 hypotype reaction system, N1 hypotype positive quality control product 5 μ l add N1 hypotype reaction system, N2 hypotype positive quality control product 5 μ l add N2 hypotype reaction system and use commercially available quantitative real time PCR Instrument (like ABI7500) to carry out pcr amplification.The PCR cycling condition is: 50 ℃ of rt 15min, 1 circulation; 94 ℃ of 15min, 1 circulation; Last 95 ℃ of 15s, 55～60 ℃ of 45s, 45 circulations (collection fluorescent signal).
Reaction finishes the back and preserves the detection data file.According to the resulting curve of pcr amplification result, analyze experimental result.Like amplification curve there is the obvious Exponential growth phase, then is judged to be the corresponding hypotype positive, otherwise negative.
, the N1 hypotype positive positive like the H1 hypotype, all the other hypotypes are negative, then are H1N1 type swine influenza virus;
, the N2 hypotype positive positive like the H3 hypotype, all the other hypotypes are negative, then are H3N2 type swine influenza virus;
, the N2 hypotype positive positive like the H1 hypotype, all the other hypotypes are negative, then are H1N2 type swine influenza virus;
, the N1 hypotype positive positive like the H3 hypotype, all the other hypotypes are negative, then are H3N1 type swine influenza virus.
Embodiment 3: use swine influenza virus parting detecting reagent test sample
With the positive quality control product among the embodiment 1, negative quality control product, specificity with reference to article as sample to be checked; Method according to embodiment 2 detects these samples respectively; The positive quality control product detected result is positive, and negative quality control product and specificity detect result's all negative (referring to accompanying drawing 1,2) with reference to article.
Respectively with H1N1 type swine influenza virus, H3N2 type swine influenza virus, H1N2 type swine influenza virus sample as sample to be checked; Method according to embodiment 2 detects these samples respectively; The all accurate somatotype of H1N1 type swine influenza virus, H3N2 type swine influenza virus, H1N2 type swine influenza virus is (referring to accompanying drawing 3; 4,5).
Embodiment 4: swine influenza virus parting detecting reagent sensitivity experiment
Respectively with the plasmid of the plasmid of the plasmid of the plasmid of swine influenza virus H1 hypotype HA gene, H3 hypotype HA gene, N1 hypotype NA gene, N2 hypotype NA gene as sample to be tested, it is 1.0 * 10 that each plasmid concentration all dilutes
4Copies/ml, 1.0 * 10
3Copies/ml, 1.0 * 10
2Copies/ml, 1.0 * 10
1Copies/ml forms sensitivity with reference to article, according to the method for embodiment 2 detect respectively this 16 increment this, detected result shows that the sensitivity of this test kit is 1.0 * 10
2Copies/ml (seeing accompanying drawing 6).
Sequence table
< 110>Da
< 120>one swine influenza virus real-time fluorescent RT-PCR typing kits
<140>
<141>
<160>12
<210>1
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
tggcaccaagatatgcttttg
<210>2
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
caagtcgcattgcagtcatg
<210>3
<211>26
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>3
cgggtatcataacatcggatgcacca
<210>4
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>4
cgtcgtgaaagcgtgaacag
<210>5
<211>18
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>5
cggcatggtcacgttcag
<210>6
<211>26
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>6
ccgtctgaactggctgcataacctgg
<210>7
<211>22
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>7
gaccttgcttttgggttgaact
<210>8
<211>24
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>8
aaggatatgctgctcccactagtc
<210>9
<211>28
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>9
cagagggcgacccaaagagaacacaatc
<210>10
<211>18
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>10
gggcaccaccctgaacaa
<210>11
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>11
cgttcatcagcagggtacga
<210>12
<211>27
<212>DNA
< 213>artificial sequence
<220>
< 223>according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>12
ccatagcaacgataccgtgcatgatcg
Claims (3)
1. a swine influenza virus real-time fluorescent RT-PCR typing kit; Test kit comprises: the RNA extracting solution is housed respectively; H1 hypotype reaction system; H3 hypotype reaction system; N1 hypotype reaction system and N2 hypotype reaction system; Negative quality control product; H1 hypotype positive quality control product; H3 hypotype positive quality control product; N1 hypotype positive quality control product and N2 hypotype positive quality control product and a plurality of reagent bottles or the pipe that seal; (2) packing box of separation and concentrated these reagent bottles of packing or pipe; Wherein H1 hypotype reaction system comprises H1 hypotype primer probe mixed solution; The RT-PCR reaction solution; RT-PCR enzyme system; The sequence that it is characterized in that H1 hypotype forward primer in the H1 hypotype primer probe mixed solution is: 5 '-TGGCACCAAGATATGCTTTTG-3 '; The sequence of H1 hypotype reverse primer is: 5 '-CAAGTCGCATTGCAGTCATG-3 '
The sequence of H1 hypotype oligonucleotide probe is: 5 ' CGGGTATCATAACATCGGATGCACCA-3 '; Wherein H3 hypotype reaction system comprises H3 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system; Its characteristic is that also the sequence of H3 hypotype forward primer in the H3 hypotype primer probe mixed solution is: the sequence of 5 '-CGTCGTGAAAGCGTGAACAG-3 ', H3 hypotype reverse primer is: 5 '-CGGCATGGTCACGTTCAG-3 '
The sequence of H3 hypotype oligonucleotide probe is: 5 '-CCGTCTGAACTGGCTGCATAACCTGG-3 '; Wherein N1 hypotype reaction system comprises N1 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system; Its characteristic is that also the sequence of N1 hypotype forward primer in the N1 hypotype primer probe mixed solution is: the sequence of 5 '-GACCTTGCTTTTGGGTTGAACT-3 ', N1 hypotype reverse primer is: 5 '-AAGGATATGCTGCTCCCACTAGTC-3 '
The sequence of N1 hypotype oligonucleotide probe is: 5 '-CAGAGGGCGACCCAAAGAGAACACAATC-3 '; Wherein N2 hypotype reaction system comprises N2 hypotype primer probe mixed solution, RT-PCR reaction solution, RT-PCR enzyme system; Its characteristic is that also the sequence of N2 hypotype forward primer in the N2 hypotype primer probe mixed solution is: the sequence of 5 '-GGGCACCACCCTGAACAA-3 ', N2 hypotype reverse primer is: 5 '-CGTTCATCAGCAGGGTACGA-3 '
The sequence of N2 hypotype oligonucleotide probe is: 5 '-CCATAGCAACGATACCGTGCATGATCG-3 '.
2. according to the test kit of claim 1; Its characteristic also is the forward and reverse primer of H1 hypotype; The forward and reverse primer of H3 hypotype; The forward and reverse primer of N1 hypotype, the concentration of the forward and reverse primer of N2 hypotype are the 5pmol/ reaction, and the concentration of H1 hypotype oligonucleotide probe, H3 hypotype oligonucleotide probe, N1 hypotype oligonucleotide probe, N2 hypotype oligonucleotide probe is the 5pmol/ reaction.
3. according to the test kit of claim 1; RT-PCR enzyme system is that diluent is formed by reversed transcriptive enzyme, warm start Taq enzyme, RNasin, dNTPs, enzyme, and its characteristic is that also wherein the reversed transcriptive enzyme consumption is that 1.5～3.5U/ reaction, warm start Taq enzyme dosage are that 3～7U/ reaction, RNasin consumption are that 15～25U/ reaction, dNTPs concentration are 0.20mmol/L.
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| CN102586488B (en) * | 2012-03-16 | 2013-06-19 | 天津市畜牧兽医研究所 | Nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) kit for type A swine influenza viruses |
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| CN111850175A (en) * | 2020-08-12 | 2020-10-30 | 扬州大学 | Fluorescent quantitative RT-PCR primer, probe and kit for detecting and typing H1 and H3 subtype swine influenza viruses |
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| CN101163714A (en) * | 2005-02-24 | 2008-04-16 | 马萨诸塞大学 | Influenza nucleic acids, polypeptides, and uses thereof |
| CN101392298A (en) * | 2008-07-15 | 2009-03-25 | 江苏省疾病预防控制中心 | Method for detecting flu and H5N1 avian influenza virus by using liquid chip |
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| CN101392298A (en) * | 2008-07-15 | 2009-03-25 | 江苏省疾病预防控制中心 | Method for detecting flu and H5N1 avian influenza virus by using liquid chip |
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