CN101875933A - Kit for identifying subtypes of influenza A virus - Google Patents
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Abstract
The invention discloses a kit for identifying subtypes of influenza A virus. The kit comprises the following primer pairs: one primer of which the sequence is shown in a sequence 5 in a sequence table and the other primer of which the sequence is shown in a sequence 6 in the sequence table; and the subtypes of influenza A virus are subtypes of new influenza A H1N1 virus, influenza H1N1 virus, influenza A H5N1 virus or influenza A H3N2 virus. Experiments prove that the method has the advantages of reliable detection results, high sensitivity (capable of detecting 103 copy number of the new influenza A H1N1 virus to the lowest), and good specificity; and particularly, the method can also separate the subtypes of the new influenza A H1N1 virus from the subtypes of common seasonal H1N1 influenza virus, which cannot be realized by the conventional technology. Besides, the method has the advantages of no need of precious equipment, simple and quick operation, and capacity of realizing high-flux quick detection. The kit and the PCR reagent have the advantages of high sensitivity, good specificity, wide sources and low cost. The invention provides a simple, feasible and effective method for early diagnosis of the infections of human influenza viruses.
Description
Technical field
The present invention relates to a kind of test kit that is used to differentiate subtypes of influenza A virus.
Background technology
Influenza infection is the public health hidden danger that the mankind are difficult to capture always.The novel swine influenza virus swine H1N1 that spread out of by Mexico in March, 2009 is wide-scale distribution worldwide, is explosive popular, has brought great loss to the mankind.
Influenza virus is mainly with the first type and the B-mode infection mankind, and influenza A virus wherein is with H1N1, and H3N2, H5N1 are common pathogenic hypotype.The Highly Pathogenic Avian Influenza Virus (HPAIV) that find recent years, it can be crossed over the population obstacle mankind are caused infection.The influenza virus of different subtype has different virulence and propagation characteristic, has brought very big difficulty for the Clinics and Practices of infectious diseases.Quick diagnosis for virus subtype is to improve the key of patient's curative effect and the diffusion of control virus, needs to seek effective fast discrimination method.Useful microchip, flow cytometry method detect in the prior art, but big multiprogram complexity, the cost costliness.
Summary of the invention
An object of the present invention is to provide a kind of primer sets compound.
Primer sets compound provided by the present invention is following 1) or 2) shown in:
1) by following primer to a), b), c) and d) form;
2) by following primer to a), b), c) and d) at least a the composition;
A) primer sequence is shown in sequence in the sequence table 1, and another primer sequence is shown in sequence in the sequence table 2; (promptly differentiate whether be the new H1N1 hypotype of influenza A virus primer to);
B) primer sequence is shown in sequence in the sequence table 3, another primer sequence shown in sequence in the sequence table 4 (promptly differentiate whether be influenza A virus H1N1 hypotype primer to);
C) primer sequence is shown in sequence in the sequence table 5, another primer sequence shown in sequence in the sequence table 6 (promptly differentiate whether be influenza A virus first type H5N1 hypotype primer to);
D) primer sequence is shown in sequence in the sequence table 7, another primer sequence shown in sequence in the sequence table 8 (promptly differentiate whether be influenza A virus first type H3N2 hypotype primer to).
Another object of the present invention provides a kind of test kit that is used to differentiate subtypes of influenza A virus.
The test kit that is used to differentiate subtypes of influenza A virus provided by the present invention is above-mentioned primer sets compound;
Described subtypes of influenza A virus is new H1N1 hypotype, H1N1 hypotype, first type H5N1 hypotype or first type H3N2 hypotype.
Another object of the present invention provides a kind of PCR reagent that is used to differentiate subtypes of influenza A virus.
Provided by the present inventionly be used to differentiate that the PCR reagent of subtypes of influenza A virus is made up of above-mentioned arbitrary described primer sets compound, PCR damping fluid, dNTPs and ExTaq enzyme.
In the above-mentioned PCR reagent, described a) shown in the concentration of two primers in described PCR reagent of primer centering be 0.2mM; Described b) concentration of two of primer centering primers shown in described PCR reagent is 0.1mM; Described c) concentration of two of primer centering primers shown in described PCR reagent is 0.1mM; Described d) concentration of two of primer centering primers shown in described PCR reagent is 0.2mM.
In the above-mentioned PCR reagent, described dNTPs be dATP, dTTP, dGTP and dCTP etc. molar mixture, described dATP, dTTP, dGTP or the dCTP concentration in described PCR reagent is 0.8mM.
Above-mentioned arbitrary described primer sets compound is used for differentiating that in preparation the application of the test kit of subtypes of influenza A virus also belongs to protection scope of the present invention.
Above-mentioned arbitrary described primer sets compound is used for differentiating that in preparation the application of the PCR reagent of subtypes of influenza A virus also belongs to protection scope of the present invention.
Last purpose of the present invention provides a kind of method of differentiating subtypes of influenza A virus.
Kind provided by the present invention is differentiated the method for subtypes of influenza A virus, comprise the steps: the cDNA of testing sample to be carried out pcr amplification with above-mentioned arbitrary described PCR reagent, detect the size of PCR product, determine subtypes of influenza A virus in the described testing sample;
The method of subtypes of influenza A virus is following 1 in described definite described testing sample) or 2) shown in:
1) sequence of the described PCR product of detection,
If contain the dna fragmentation that sequence size is 495bp in the described PCR product, the candidate is contained the new H1N1 subtype virus of influenza A virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 363bp in the described PCR product, the candidate is contained influenza A virus H1N1 subtype virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 113bp in the described PCR product, the candidate is contained influenza A virus first type H3N2 subtype virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 188bp in the described PCR product, the candidate is contained influenza A virus first type H5N1 subtype virus in the then described testing sample;
2) detect described PCR product with agarose gel electrophoresis,
If described PCR product shows the band of 480-510bp on sepharose, the candidate is contained the new H1N1 subtype virus of influenza A virus in the then described testing sample;
If described PCR product shows the band of 350-370bp on sepharose, the candidate is contained influenza A virus H1N1 subtype virus in the then described testing sample;
If described PCR product shows the band of 100-130bp on sepharose, the candidate is contained influenza A virus first type H3N2 subtype virus in the then described testing sample;
If described PCR product shows the band of 170-200bp on sepharose, the candidate is contained influenza A virus first type H5N1 subtype virus in the then described testing sample;
The method of described discriminating subtypes of influenza A virus does not comprise treatment of diseases and diagnostic method.
In the aforesaid method, the annealing temperature of described pcr amplification is 54 ℃.
The method of above-mentioned detection virus not only is used for the clinical infection determination of pathogens, can also be used for breadboard pathogen detection research.
The present invention has set up and can detect human influenza A virus H1N1 simultaneously, H3N2, and the multiplex RT-PCR method of H5N1 hypotype, this method is at first carried out pcr amplification, just can observe the result intuitively by gel electrophoresis then.
The present invention has carried out optimizing exploration to the reaction conditions of multiplex PCR, at first, primer concentration in the hybrid reaction is optimized, detect by primer (concentration 0.4mM-0.05mM) and to find best primer concentration twice dilution, the result shows as H3N2, newly the H1N1 primer concentration is 0.2mM, H5N1, when seasonal influenza H1N1 primer concentration is 0.1mM, the susceptibility of experiment is best, when Type B influenza virus primer concentration is 0.1mM, when A type influenza virus primer concentration was 0.05mM, the susceptibility of experiment was best.Secondly, by reaction conditions is optimized in the test of 52 ℃ to 58 ℃ of the annealing temperatures of multiplex PCR, when the annealing temperature of finally finding each reaction was 54 ℃, the PCR effect was best.
Experiment showed, that the inventive method detected result is reliable, highly sensitively (minimumly detect 10
3The new first type H1 gene of copy number), specificity is good; Especially the inventive method can also distinguish new H1N1 subtype influenza virus and common seasonal influenza virus H1N1 hypotype, and this is that prior art never realizes; In addition, the inventive method need not expensive equipment, and is simple to operate, quick, rapid detection when can realize multiple pathogenic agent.Test kit of the present invention and PCR reagent sensitivity height, good, the wide material sources, with low cost of specificity.The present invention provides simple, efficient ways for the early diagnosis that human influenza virus infects.
Description of drawings
Fig. 1 is the PCR detected result to virus.
Fig. 2 is the specific detection result of method.
Fig. 3 is the susceptibility detected result of method.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The different subtype of embodiment 1, influenza A virus is identified
The concrete virus strain that this experiment is used is as follows:
The virus strain of the new H1N1 hypotype of influenza A virus is that A/Beijing/501/2009 (H1N1) (swine influenza virus strain) is at document (Liu, B., Qin, C., Jiang, T., Hu, Y., Li, X., Zhang, X., Kang, X., Deng, Y., Zhang, Y., Li, Y., Liu, H., Jia, N., Chang, G, Zhao, H., Tong, Y., Qin, E., Zhu, Q.and Cao, W.Direct Submission Submitted (30-MAY-2009) Department of Virology, Beijing Institute of Microbiology and Epidemiology, 20DongdaStreet, Beijing, Beijing 100071, disclose in Chin); (providing) by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL;
The virus strain of influenza A virus H1N1 hypotype (being seasonal H1N1 hypotype) is that A/PR/8/34 (H1N1) (seasonal influenza virus) is at document (Ryan Vander Veen, Kurt Kamrud, Mark Mogler, Alan T.Loynachan, Jerry McVicker, Peter Berglund, Gary Owens, Sarah Timberlake, Whitney Lewis, Jonathan Smith, and DL Hank Harris, Rapid Development of an Efficacious Swine Vaccine forNovel H1N1 PLoS Curr Influenza.2009 October 29:RRN1123) (being provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL) disclosed in;
The virus strain of influenza A virus first type H3N2 hypotype is that A/Wisconsin/67/2005 (H3N2) is at document (Deyde, V.M., Xu, X., Bright, R.A., Shaw, M., Smith, C.B., Zhang, Y., Shu, Y., Gubareva, L.V., Cox, N.J.andKlimov, A.I.Surveillance of resistance to adamantanes among influenza A (H3N2) and A (H1N1) virusesisolated worldwide, J.Infect.Dis.196 (2), 249-257 (2007)) (being provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL) disclosed in;
The virus strain of influenza A virus first type H5N1 hypotype is that A/Beijing/01/2003 (H5N1) (avian influenza virus) is at document (4Zhu, Q.Y., Qin, E.D., Wang, W., Yu, J., Liu, B.H., Hu, Y., Hu, J.F.and Cao, W.C Fatalinfection with influenza A (H5N1) virus in China.N.Engl.J.Med.354 (25), (being provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL) disclosed 2731-2732 (2006)).
Den4 is at document (Dengue virus 4 strain 341750) Kelly for virus, E.P., Puri, B., Sun, W.and Falgout, B.Identification of mutations in a candidate dengue 4 vaccinestrain 341750 PDK20 and construction of a full-length cDNA clone of the PDK20vaccine candidateJ.Vaccine 28 (17), 3030-3037 disclosed in (2010), (being provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL).
Mdck cell is numbered ATCC No.CRL-2935 available from ATCC.
QIAamp Viral RNA test kit is available from Qiagen company, and catalog number is 52904.
The random hexamer primer is available from Promega company.
DNTP is available from Promega company.
The EXtaq enzyme is available from TaKaLa Bio InC, Dalian, China.
One, testing sample is a virus
1, virus culture: cultivate the virus strain of above-mentioned each hypotype respectively with mdck cell, obtain the cell culture supernatant of each subtype virus strain respectively.
2, viral supernatant extracts RNA: use QIAamp Viral RNA test kit respectively the cell culture supernatant of each subtype virus strain to be carried out the RNA extraction, extraction step carries out according to the product service manual, obtains the RNA of each subtype virus strain.
3, the reverse transcription of RNA:
Reverse transcription system: get virus strain RNA 5ul, random hexamer primer 0.5ul, 1ul dNTP (10mM), the ThermoScript II 1ul of above-mentioned H3N2 hypotype, H5N1 hypotype, seasonal H1N1 hypotype, new H1N1 hypotype respectively, add ddH
2O replenishes volume to 20ul.
Reverse transcription reaction condition: 42 ℃ of reaction 60min.With the RNA reverse transcription is cDNA.
The RNA of each subtype virus strain carries out reverse transcription respectively.
4, the PCR of cDNA
The primer that is used to differentiate subtypes of influenza A virus all designs on the HA gene of each hypotype of influenza A virus.The theoretical amplification length of concrete primer sequence and each primer is as shown in table 1.
Under primer sequence guides in table 1, the extension increasing sequence of new H1N1 hypotype as among the Genebank accessionnumber GQ223408 from shown in the Nucleotide of 5 ' terminal 408-902 position, the extension increasing sequence of H1N1 hypotype as among the Genebank accession number EF467821 from 5 ' terminal 265-627 position Nucleotide shown in, the extension increasing sequence of first type H3N2 hypotype as among the Genebank accession numberEU103823 from 5 ' terminal 902-1014 position Nucleotide shown in, the extension increasing sequence of first type H5N1 hypotype as among the Genebank accession numberEF587277 from shown in the Nucleotide of 5 ' end 914-1101 position.
The PCR reaction system of each group is as follows:
Combined group (mix primer): template is the cDNA mixture (cDNA of four subtype virus is 3 μ l) of four subtype virus, primer is that (concentration of two primers of four kinds of primer centerings in reaction system is: new H1N1 0.2mM for the mixture of four pairs of primers in the table 2, H1N1 0.1mM, first type H3N2 0.2mM, first type H5N1 0.1mM), 5 μ l, 10 * PCR buffer, 0.8mM dNTP (being dATP0.8mM, dTTP0.8mM, dGTP0.8mM, dCTP0.8mM), EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
Below be independent Auele Specific Primer:
New H1N1 group: new H1N1 cDNA 5 μ l, new H1N1 primer 1mM, 5 μ l, 10 * PCRbuffer, 0.8mM dNTP (being dATP0.8mM, dTTP0.8mM, dGTP0.8mM, dCTP0.8mM), EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
H1N1 group: H1N1 cDNA 5 μ l, H1N1 primer 1mM, 5 μ l, 10 * PCR buffer, 0.8mM dNTP (being dATP0.8mM, dTTP0.8mM, dGTP0.8mM, dCTP0.8mM), EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
First type H3N2 group: first type H3N2 cDNA 5 μ l, first type H3N2 primer 1mM, 5 μ l, 10 * PCR buffer, 0.8mM dNTP (being dATP0.8mM, dTTP0.8mM, dGTP0.8mM, dCTP0.8mM), EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
First type H5N1 group: first type H5N1 cDNA 5 μ l, first type H5N1 primer 1mM, 5 μ l, 10 * PCR buffer, 0.8mM dNTP (being dATP0.8mM, dTTP0.8mM, dGTP0.8mM, dCTP0.8mM), EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
Negative control group: template is irrelevant viral Den4 cDNA (template volume 5ul), 5 μ l, 10 * PCR buffer, and 0.8mM dNTP, EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.Primer is consistent with above-mentioned combined group.
PCR reaction conditions of above-mentioned each group is: 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, 30 circulations, last circulation was extended 10 minutes for 72 ℃.
Table 2, be used to differentiate that the primer of subtypes of influenza A virus is right
5, the detection of PCR product
(1) the above-mentioned PCR product of respectively organizing is carried out 2% agarose gel electrophoresis analysis respectively; PCR product and 0.5 μ g/ml ethidium bromide aqueous solution are carried out electrophoresis.The band of the marker that uses is respectively: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp.
The result as shown in Figure 1.Among the figure, H1 represents the H1N1 group, and H3 represents first type H3N2 group, and H5 represents first type H5N1 group, and sH1 represents new H1N1 group, and Mix represents combined group, and N represents negative control group.
The H1N1 group, the size of pcr amplification band is shown as 350-370bp on gel;
First type H3N2 group, the size of pcr amplification band is shown as 100-130bp on gel;
First type H5N1 group, the size of pcr amplification band is shown as 170-200bp on gel;
New H1N1 group, the size of pcr amplification band is shown as 480-510bp on gel;
Mix represents combined group, and the pcr amplification band is shown as 4 band: 350-370bp, 100-130bp, 170-200bp, the 480-510bp of following size on gel;
Negative control group does not have any amplified band.
The gel result shows that the independent experimental group of each hypotype, combined group all detect the purpose band, and purpose stripe size and theoretical band are approaching and in the limit of error of gel electrophoresis, and negative control group does not increase and obtains any band; It is clear that each organizes the PCR band, special nothing but background.
(2) the above-mentioned PCR product of respectively organizing is checked order respectively
The result:
Combined group: record four fragments of 363bp, 113bp, 188bp and 495bp, size is consistent with theoretical amplification size, and base sequence is correct;
The H1N1 group: record the fragment of 363bp, size is consistent with theoretical amplification size, and base sequence is correct;
First type H3N2 group: record the fragment of 113bp, size is consistent with theoretical amplification size, and base sequence is correct;
First type H5N1 group: record the fragment of 188bp, size is consistent with theoretical amplification size, and base sequence is correct;
New H1N1 group: record the fragment of 495bp, size is consistent with theoretical amplification size, and base sequence is correct;
To sum up the result shows, under above-mentioned system and reaction conditions, the purpose fragment of primer amplification is correct.The specificity of primer of the present invention and amplification system and response procedures is good, can detect four hypotypes of influenza A virus accurately.
Two, testing sample is clinical pharynx formula sample
Clinical pharynx formula sample is taken from 39 parts of doubtful new H1N1 patients, all passes through patient's agreement, picks up from different infectious hospitals respectively.
1, extracts the RNA method of pharynx formula sample: the throat swab sample is placed the 3mlDMED nutrient solution, pathogenic agent in the sample is entered in the nutrient solution, get the 200ulDMED nutrient solution then, utilize QIAamp Viral RNA test kit that the throat swab sample is carried out RNA and extract, extraction step carries out according to the product service manual.
2, the RNA reverse transcription becomes the method for cDNA: with consistent described in the experiment one.
3, the PCR of cDNA
The PCR reaction system of every part of pharynx formula sample is as follows:
The cDNA 3 μ l of pharynx formula sample, primer is that (concentration of two primers in reaction system of four kinds of primer centerings is: new H1N1 0.2mM for the mixture of four pairs of primers in the table 2, H1N1 0.1mM, first type H3N2 0.2mM, first type H5N1 0.1mM), 5 μ l, 10 * PCR buffer, 0.8mM dNTP (being dATP0.8mM, dTTP0.8mM, dGTP0.8mM, dCTP0.8mM), EX Taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
The PCR reaction conditions is: 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, 30 circulations, last circulation was extended 10 minutes for 72 ℃.
4, judge the influenza A virus that contains which kind of hypotype in the pharynx formula sample to be measured according to the PCR product, determination methods is shown in following (1) or (2):
(1) above-mentioned each PCR product is carried out 2% agarose gel electrophoresis analysis respectively; PCR product and 0.5 μ g/ml ethidium bromide aqueous solution are carried out electrophoresis.The band of the marker that uses is respectively: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp.
If described PCR product shows the band of 480-510bp on sepharose, contain the new H1N1 subtype virus of influenza A virus in the then described testing sample;
If described PCR product shows the band of 350-370bp on sepharose, contain influenza A virus H1N1 subtype virus in the then described testing sample;
If described PCR product shows the band of 100-130bp on sepharose, contain influenza A virus first type H3N2 subtype virus in the then described testing sample;
If described PCR product shows the band of 170-200bp on sepharose, contain influenza A virus first type H5N1 subtype virus in the then described testing sample.
As a result, positive 4 parts (10.2%) of new H1N1, positive 2 parts (5.1%) of seasonal influenza H1N1 hypotype, positive 12 parts (30.7%) of seasonal influenza H3N2 hypotype.
(2) sequence of the described PCR product of detection, judge that according to sequencing result decision method is as follows:
If contain the dna fragmentation that sequence size is 495bp in the described PCR product, contain the new H1N1 subtype virus of influenza A virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 363bp in the described PCR product, contain influenza A virus H1N1 subtype virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 113bp in the described PCR product, contain influenza A virus first type H3N2 subtype virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 188bp in the described PCR product, contain in the then described testing sample; Influenza A virus first type H5N1 subtype virus;
As a result, positive 4 parts (10.2%) of new H1N1, positive 2 parts (5.1%) of seasonal influenza H1N1 hypotype, positive 12 parts (30.7%) of seasonal influenza H3N2 hypotype.
The real-time PCR method of recommending to use with WHO detects above-mentioned 39 parts of pharynx formula samples respectively again, detection method such as World Health Organization.CDC protocol of real-time RT-PCR for swine influenza A (H1N1) .http: //www.who.int/csr/resources/publications/swineflu/realtime ptpcr/en/index.html (Accessed June 12,2009). described.The result is with identical with above-mentioned the inventive method detected result.Proof the invention described above method accurately and reliably.
Three, the susceptibility of method detects
1, the H1 gene of the new H1N1 subtype virus of synthetic strain A/California/04/2009.(the synthetic sequence as among the Genebank accession number FJ966082 from 5 ' terminal 408-902 position Nucleotide shown in).New H1N1 subtype virus strain A/California/04/2009 is at document (Ryan Vander Veen, Kurt Kamrud, Mark Mogler, Alan T.Loynachan, Jerry McVicker, Peter Berglund, Gary Owens, Sarah Timberlake, Whitney Lewis, Jonathan Smith, and DL Hank Harris, Rapid Development ofan Efficacious Swine Vaccine for Novel H1N1 PLoS Curr Influenza.2009 October 29:RRN1123.) in disclosed.
2, with the H1 gene of synthetic with
Plasmid connects, and will connect the product transformed into escherichia coli, resistance screening, picking mono-clonal; The liquid culture mono-clonal extracts plasmid, carries out sequence verification, and the result shows that the structure of recombinant plasmid of structure is correct.
3, in-vitro transcription: working method is according to the Riboprobe system-T7 test kit process specifications of Promega company, products catalogue: 2710556.
4, survey the expression amount that OD260 detected and calculated RNA in the in-vitro transcription product by the NanoDrop1000 instrument.
5, with distilled water doubling dilution in-vitro transcription product, obtain the solution of different RNA copy number, viral RNA concentration is 1x10 in the solution
8Copies/ μ l-lcopies/ μ l.
6, the reverse transcription of RNA and PCR:
Reverse transcription system: get viral RNA 5ul, random hexamer primer 0.5ul, 1ul dNTP (10mM), ThermoScript II 1ul respectively, add ddH
2O replenishes volume to 20ul.
Reverse transcription reaction condition: 42 ℃ of reaction 60min.With the RNA reverse transcription is cDNA.
The PCR system: primer is that (concentration of four kinds of each bar primers of primer centering in reaction system is: new H1N1 0.2mM for the mixture of four pairs of primers in the table 2, H1N1 0.1mM, first type H3N2 0.2mM, first type H5N1 0.1mM), 5 μ l, 10 * PCR buffer, 0.8mM dNTP, EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
The PCR reaction conditions is: 95 ℃ of pre-sex change 5 minutes, and 94 ℃ of sex change 30 seconds, 54 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, 30 circulations, last circulation was extended 10 minutes for 72 ℃.
Above-mentioned each PCR product is carried out 2% agarose gel electrophoresis analysis respectively.
The result as shown in Figure 3.The result shows that the sensitivity of the inventive method is 10 for the RNA copy number
3Copies/ul." N " negative control group does not promptly add template cDNA group.
Four, the specific detection of method
Detect the specificity of the inventive method with Respirovirus.The virus strain of using is as follows:
Human airway syncytial virus A C-type virus C strain RSB5857[Human respiratory syncytial virus A (strainrsb5857) .] at document (Timsit E, Maingourd C, Le Dr é an E, Belloc C, Seegers H, DouartA, Assi é S.Evaluation of a commercial real-time reverse transcriptionpolymerase chain reaction kit for the diagnosis of Bovine respiratory syncytialvirus infection.J Vet Diagn Invest, 2010 Mar; 22 (2): disclosed 238-41), and provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.
Human airway syncytial virus Type B virus strain 18537[Human respiratory syncytial virus B (strain 18537)] at document [Lim CS, Kumarasinghe G, Chow VT.Sequence and phylogenetic analysis ofSH, G, and F genes and proteins of Human respiratory syncytial virus isolatesfrom Singapore.j Acta Virol.2003; 47 (2): 97-104] disclosed in, provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.
Human rhinovirus 3 C-type virus C strain QCE[Human rhinovirus C strain QCE] at document [Arden KE, Faux CE, O ' Neill NT, McErlean P, Nitsche A, Lambert SB, Nissen MD, Sloots TP, MackayIM.b Molecular characterization and distinguishing features of a novelhuman rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough andwheeze during 2003.J Clin Virol.2010 Mar; 47 (3): 219-23.Epub 2010 Jan 27.] disclosed in, provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.
Human parainfluenza virus 1 C-type virus C strain Washington/1964[Human parainfluenza virus 1strain Washington/1964] at document [Newman JT, Surman SR, Riggs JM, Hansen CT, Collins PL, Murphy BR, Skiadopoulos MH.Sequence analysis of theWashington/1964 strain of human parainfluenza virus type 1 (HPIV1) and recoveryand characterization of wild-type recombinant HPIV1 produced by reversegenetics.J Virus Genes.2002; 24 (1): 77-92.] disclosed in, provided by Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.
Each respiratory tract disease poison strain is carried out following test experience respectively.
Virus culture, viral supernatant extract the reverse transcription of RNA, RNA all with consistent described in the experiment one.
The PCR reaction system of cDNA: template is the cDNA 3 μ l of each Respirovirus, primer is that (concentration of two primers in reaction system of four kinds of primer centerings is: new H1N1 0.2mM for the mixture of four pairs of primers in the table 2, H1N1 0.1mM, first type H3N2 0.2mM, first type H5N1 0.1mM), 5 μ l, 10 * PCR buffer, 0.8mMdNTP, EX taq enzyme 2.5U adds DEPC water at last and replenishes volume to the 50ul system.
The PCR response procedures of cDNA: with consistent described in the experiment one.
The result is (RSA represents human airway syncytial virus A type, and RSB represents human airway syncytial virus Type B, and RHV represents human rhinovirus's 3 types, and PAIF represents human parainfluenza virus's 1 type) as shown in Figure 2.Show: when being template, all do not amplify any band, show that the specificity of the inventive method is good with the cDNA of each Respirovirus.
Sequence table
＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL
＜120〉a kind of test kit that is used to differentiate subtypes of influenza A virus
<160>8
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gacaagttca?tggcccaatc 20
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
cccttgggtg?tttgacaagt 20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
cttaggaaac?ccagaatgcg 20
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
gcgggtgatg?aacacccca 19
<210>5
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
actccaatgg?gggcgataaa?c 21
<210>6
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
ccctctataa?aacctgctat?ag 22
<210>7
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
atggaagcat?tcccaatgca?a 21
<210>8
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)，(4)，(10)
＜223〉r is a or g, and y is c or t
<400>8
rttycgcaty?cctgttgcca 20
Claims (9)
1. a primer sets compound is following 1) or 2) shown in:
1) by following primer to a), b), c) and d) form;
2) by following primer to a), b), c) and d) at least a the composition;
A) primer sequence is shown in sequence in the sequence table 1, and another primer sequence is shown in sequence in the sequence table 2;
B) primer sequence is shown in sequence in the sequence table 3, and another primer sequence is shown in sequence in the sequence table 4;
C) primer sequence is shown in sequence in the sequence table 5, and another primer sequence is shown in sequence in the sequence table 6;
D) primer sequence is shown in sequence in the sequence table 7, and another primer sequence is shown in sequence in the sequence table 8.
2. a test kit that is used to differentiate subtypes of influenza A virus is the compound of primer sets described in the claim 1;
Described subtypes of influenza A virus is new H1N1 hypotype, H1N1 hypotype, first type H5N1 hypotype or first type H3N2 hypotype.
3. a PCR reagent that is used to differentiate subtypes of influenza A virus is made up of the compound of primer sets described in the claim 1, PCR damping fluid, dNTPs and ExTaq enzyme.
4. PCR reagent according to claim 3 is characterized in that: described a) shown in the concentration of two primers in described PCR reagent of primer centering be 0.2mM; Described b) concentration of two of primer centering primers shown in described PCR reagent is 0.1mM; Described c) concentration of two of primer centering primers shown in described PCR reagent is 0.1mM; Described d) concentration of two of primer centering primers shown in described PCR reagent is 0.2mM.
5. according to claim 3 or 4 described PCR reagent, it is characterized in that: described dNTPs be dATP, dTTP, dGTP and dCTP etc. molar mixture, described dATP, dTTP, dGTP or the dCTP concentration in described PCR reagent is 0.8mM.
6. the compound of primer sets described in the claim 1 is used for differentiating the application of the test kit of subtypes of influenza A virus in preparation.
7. the compound of primer sets described in the claim 1 is used for differentiating the application of the PCR reagent of subtypes of influenza A virus in preparation.
8. method of differentiating subtypes of influenza A virus comprises the steps: with arbitrary described PCR reagent among the claim 3-5 cDNA of testing sample to be carried out pcr amplification, detects the size of PCR product, determines subtypes of influenza A virus in the described testing sample;
The method of subtypes of influenza A virus is following 1 in described definite described testing sample) or 2) shown in:
1) sequence of the described PCR product of detection,
If contain the dna fragmentation that sequence size is 495bp in the described PCR product, the candidate is contained the new H1N1 subtype virus of influenza A virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 363bp in the described PCR product, the candidate is contained influenza A virus H1N1 subtype virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 113bp in the described PCR product, the candidate is contained influenza A virus first type H3N2 subtype virus in the then described testing sample;
If contain the dna fragmentation that sequence size is 188bp in the described PCR product, the candidate is contained influenza A virus first type H5N1 subtype virus in the then described testing sample;
2) detect described PCR product with agarose gel electrophoresis,
If described PCR product shows the band of 480-510bp on sepharose, the candidate is contained the new H1N1 subtype virus of influenza A virus in the then described testing sample;
If described PCR product shows the band of 350-370bp on sepharose, the candidate is contained influenza A virus H1N1 subtype virus in the then described testing sample;
If described PCR product shows the band of 100-130bp on sepharose, the candidate is contained influenza A virus first type H3N2 subtype virus in the then described testing sample;
If described PCR product shows the band of 170-200bp on sepharose, the candidate is contained influenza A virus first type H5N1 subtype virus in the then described testing sample;
The method of described discriminating subtypes of influenza A virus does not comprise treatment of diseases and diagnostic method.
9. method according to claim 8 is characterized in that: the annealing temperature of described pcr amplification is 54 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111004869A (en) * | 2020-02-08 | 2020-04-14 | 吉林大学 | Method for genetic evolution pedigree identification of H1N1 subtype influenza virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101376912A (en) * | 2008-06-25 | 2009-03-04 | 浙江省疾病预防控制中心 | Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method |
| CN101649356A (en) * | 2009-07-24 | 2010-02-17 | 浙江省疾病预防控制中心 | Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101376912A (en) * | 2008-06-25 | 2009-03-04 | 浙江省疾病预防控制中心 | Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method |
| CN101649356A (en) * | 2009-07-24 | 2010-02-17 | 浙江省疾病预防控制中心 | Fluorescent quantitative detection kit of H1N1 influenza virus A and detection method thereof |
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| 《Arch Virol》 20091210 Arch Virol Subtype identification of the novel A H1N1 and other human influenza A viruses using an oligonucleotide microarray 摘要、第59页表2第1、2行 1-7 第155卷, 2 * |
| 《军事医学科学院院刊》 20100825 林方 等 人类流感病毒多重PCR分型方法的建立 331-344 1-9 第34卷, 第4期 2 * |
| 《检验医学》 20090831 龙凤兴 等 流感病毒快速检测方法特异性和敏感性研究 598-601 1-9 第24卷, 第8期 2 * |
| 《生物技术同学》 20100330 尹慧琼 等 甲型H1N1流感病毒核酸荧光定量RT-PCR检测技术 33-35 1-9 , 第2期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111004869A (en) * | 2020-02-08 | 2020-04-14 | 吉林大学 | Method for genetic evolution pedigree identification of H1N1 subtype influenza virus |
| CN111004869B (en) * | 2020-02-08 | 2023-05-23 | 吉林大学 | Fluorescent quantitative PCR (polymerase chain reaction) primer and reference standard for identifying genetic evolutionary lineages of H1N1 subtype influenza viruses |
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| CN101875933B (en) | 2012-07-18 |
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