CN105200049A - GeXP rapid detection kit capable of identifying H5 subtype avian influenza virus and four different NA subtypes thereof at the same time - Google Patents
GeXP rapid detection kit capable of identifying H5 subtype avian influenza virus and four different NA subtypes thereof at the same time Download PDFInfo
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Abstract
The invention belongs to the technical field of avian virus detection, and discloses a GeXP primer group and a kit capable of identifying an H5 subtype avian influenza virus and four different NA subtypes thereof at the same time. The inventor researches and designs six pairs of specific primers and one pair of universal primers based on a GeXP system, and builds the GeXP kit capable of identifying the H5 subtype avian influenza virus and four different NA subtypes thereof at the same time accordingly. Through adoption of the GeXP rapid detection kit, the H5 subtype avian influenza virus and the four different NA subtypes thereof can be identified at the same time, and the sensitivity is 102 copies per microliter. The GeXP rapid detection kit has the advantages of high flux, high specificity, high sensitivity, rapidness and the like, and has important significance to epidemiological investigation and differential diagnosis of the H5 subtype avian influenza virus.
Description
Technical field
The invention belongs to avian viral detection technique field, particularly relate to a kind of GeXP primer sets of discriminating H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof simultaneously, test kit and application thereof.
Background technology
Bird flu is a kind of bird acute infectious disease caused by avian influenza virus, and some subtype avian influenza virus also can infect the mankind simultaneously.Avian influenza virus, according to the antigenic relationship difference of its surface glycoprotein hemagglutinin (HA) and neuraminidase (NA), can be divided into 16 kinds of HA hypotypes and 9 kinds of NA hypotypes.Pathogenicly be divided into high pathogenic avian influenza and low pathogenicity bird flu according to it, high pathogenic avian influenza comprises H5 and H7 hypotype.Particularly the H5 subtype avian influenza epidemic situation of frequent outburst in recent years causes huge financial loss to the aviculture band of many countries and regions.Since 2003, H5N1 subtype avian influenza virus epidemic situation has progressively spread from Asia and has amounted to 56 countries and regions to Europe and Africa at interior.Since 2014, Chinese Heilungkiang, Jiangsu and Hunan etc. are economized and the epidemic situation that poultry infects H5N6 subtype avian influenza virus are successively occurred, in succession there is H5N8 bird flu epidemic situation in the areas such as Korea S, Japan, Holland, Britain, Germany, the U.S. and TaiWan, China, also have 2015 in the H5N2 subtype avian influenza epidemic situation of U.S.'s outburst, above-mentioned epidemic situation all causes the death of a large amount of poultry and is slaughtered.In addition, H5N1 hypotype and H5N6 hypotype AIV can direct infection people cause dead event prompt H5 hypotype AIV not only to cause tremendous economic to lose to aviculture, can also cross over host's species barrier direct infection people and then jeopardize human security.
At present, avian influenza virus laboratory detection technology mainly comprises the methods such as viral separation and cultivation, serological test, molecular biology experiment and immunofluorescence, and the separation and ientification of virus is diagnosis avian influenza " gold standard ".The variant numerous and new due to avian influenza subtypes constantly occurs, use above-mentioned detection method to carry out somatotype to it and diagnosis not only also exists working method complexity, and sense cycle is long and easily affect by factors such as antibody and make detected result can not accurately with timely.Although multiplex PCR has and can amplify multiple object because of fragment and then the quick diagnosis realizing multiple pathogens and reduce the advantage of inspection cost simultaneously, but common multiplex PCR is easily subject to the impact of the interaction between the characteristic of goal gene template, primer concentration and ratio and PCR reagent used etc. in the process of amplification, and then causes the inconsistent accuracy of detected result that makes of sample amplification efficiency greatly to reduce.Multiple reverse transcription polymerase chain reaction (the Multiplexreversetranscription-polymerasechainreaction of GeXP polygenic inheritance expression analysis system, mRT-PCR) be combined set up multiple reverse transcription polymerase chain reaction system by universal primer (upstream adds fluorescent mark) and specific chimeric primer (gene-specific primer 5' end connection universal primer sequence), the strategy adopting universal primer to cause, the problem that the amplification efficiency that can overcome the existence of common multiplex PCR differs.In addition, the method utilizes capillary electrophoresis to carry out the separation of product, substantially increases sensitivity and the reliability of detected result.At present, GeXP technology has applied to new H1N1virus and seasonal current Influenza Virus, hand foot mouth disease cause of disease, common respiratory tract disease cause of disease and HPV virus etc. infect detection and the somatotype of human pathogen, and this laboratory has also successfully established the GeXP method simultaneously detecting chicken nine kinds of respiratory tract disease pathogenic agent.
Outburst due to the H5AI of different N A hypotype all has that morbidity is anxious, velocity of propagation is fast, clinical symptom is similar and the common feature such as mortality ratio is high, existing diagnostic techniques is difficult to again make it judge accurately within a short period of time, is unfavorable for the formulation of its prevention and control and emergency schedule.At present, mostly the nucleic acid detection method detecting H5 hypotype AIV is for its HA gene, detects H5, N1, N2, N6 and N8 gene and the technology of carrying out somatotype there is not yet report simultaneously.
The information being disclosed in this background technology part is only intended to increase the understanding to general background of the present invention, and should not be regarded as admitting or imply in any form that this information structure has been prior art that persons skilled in the art are known.
Summary of the invention
In order to overcome the deficiencies in the prior art, the GeXP multiple PCR technique that the present invention sets up can detect and somatotype four kinds of different N A hypotypes of H5 subtype avian influenza virus simultaneously, reach the object of differentiating fast at present aviculture to be endangered to maximum H5N1, H5N2, H5N6 and H5N84 kind combination avian influenza virus, to epidemiology survey and the prevention and control thereof of H5 subtype avian influenza, ensure that the healthy and sustainable development of aviculture is significant.
The technical problem to be solved in the present invention be to provide a kind of differentiate H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof simultaneously GeXP primer sets, test kit and application thereof.
For solving the problems of the technologies described above, the present invention by the following technical solutions:
A kind of GeXP primer sets simultaneously differentiating H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof, comprise 6 pairs of Auele Specific Primers, be primer pair A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and D2, primer pair E1 and E1, primer pair F1 and F2 respectively, it has the sequence as shown in SEQIDNo.1 to SEQIDNo.12 respectively.
Differentiate a GeXP test kit for H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof simultaneously, comprise cDNA, 10 × PCRbuffer, MgCl
2, dNTP, JumpstartTaqpolymerase, 6 pairs of Auele Specific Primers, 1 pair of universal primer and RNA-free water, 6 pairs of described Auele Specific Primers, the 1 pair of universal primer have the sequence as shown in SEQIDNo.1 to SEQIDNo.14 respectively.
As preferably, the volumetric molar concentration that described 6 couples of Auele Specific Primer A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and D2, primer pair E1 and E2, primer pair F1 and F2 are corresponding in PCR reaction system is respectively 100nmol/L, 50nmol/L, 50nmol/L, 150nmol/L, 100nmol/L, 100nmol/L; Described universal primer Cy5-Tag-F, Tag-R volumetric molar concentration in PCR reaction system is 500nmol/L.
As preferably, described test kit cumulative volume is that 25 μ L/ manage, and wherein comprises: cDNA2.0 μ L, 10 × PCRbuffer2.5 μ L, MgCl
22.5 μ L, dNTP1 μ L, JumpstartTaqpolymerase1.2 μ L, 6 couples of Auele Specific Primer mixture 1.25 μ L, upstream and downstream universal primer mixture 1.25 μ L, RNA-free water complements to 25 μ L.
Present invention also offers described while differentiate H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof GeXP primer sets or described while differentiate that the GeXP test kit of H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof is differentiating the application in H5N1, H5N2, H5N6 and H5N8 subtype avian influenza virus.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention utilizes GenomeLab (tm) GeXP genetic analysis systems to establish a kind of avian influenza virus Multiplex RT-PCR (mRT-PCR) method that simultaneously can detect M, H5, N1, N2, N6 and N86 gene.First reaction conditions and multiple reaction system are optimized, then with the positive template verified, specificity verification are carried out to multiplex PCR system respectively.Multiple detection system can detect H5 subtype avian influenza virus 4 kinds of different N A hypotypes simultaneously, and sensitivity is 10
2copy/μ L.The method has high-throughput, high specificity, the highly sensitive and advantage such as quick, to the epidemiology survey of H5 subtype avian influenza virus and differential diagnosis significant.
2. the GeXP multiple PCR technique that the present invention sets up can detect and somatotype four kinds of different N A hypotypes of H5 subtype avian influenza virus simultaneously, reach the object of differentiating fast at present aviculture to be endangered to maximum H5N1, H5N2, H5N6 and H5N84 kind combination avian influenza virus, to epidemiology survey and the prevention and control thereof of H5 subtype avian influenza, ensure that the healthy and sustainable development of aviculture is significant.
Accompanying drawing explanation
Fig. 1 is the capillary electrophoresis analysis figure of GeXP multiplex PCR;
Fig. 2 is the detected result to clinical Simple infection H5N1 subtype avian influenza virus;
The explanation of appended with drawings mark:
The base number of X-coordinate-pcr amplification product; Ordinate zou-fluorescent signal value.
Embodiment
Below in conjunction with specific embodiment, elaboration detailed is further done to the present invention, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiments only for illustration of the present invention, but not for limiting the scope of the invention.In addition, after reading content of the present invention, those skilled in the art can do various amendment to the present invention, and these equivalent variations fall within appended claims limited range of the present invention equally.
The experimental technique used in following embodiment if no special instructions, is ordinary method.The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.The avian influenza strain used in embodiment and other avian viral Reference Strains are preserved by Veterinary Institute of Guangxi Zhuang Autonomous Region.
embodiment 1: avian influenza virus multiple RT-PCR design of primers
From GenBank database, avian influenza virus M is downloaded with reference to pertinent literature, H5, N1, N2, the sequence of N6 and N86 gene, DNAStar is utilized to carry out analysis and comparison to each gene nucleotide series respectively, find out the conservative region being applicable to design Auele Specific Primer, utilize GeXPexpressprofiler tool design for the Auele Specific Primer (see table 1) of avian influenza virus 6 genes, the primer designed adopts PrimerPremier5.0, NCBIPrimerBlast and Oligo7.0 carries out analyzing and screening, then one section of non-homology unique sequences is added respectively as universal primer (Uni-Primer) at the 5' end of whole forward primer and reverse primer, upstream universal primer 5' holds mark fluorescent dyestuff Cy5, i.e. Cy5-Tag-F, synthesized by Shanghai Invitrogen company, HPLC purifying.
Table 1 primer information
In table 1, annex base code R=A/G, D=A/G/T, Y=C/T, what underscore represented is upstream and downstream universal primer sequence; Fluorescence dye Cy5 marks upstream universal primer label, and downstream primer does not mark.The error of the different and GeXP system (as GenomeLabTMGeXPGeneticAnalysisSystem capillary electrophoresis apparatus) of the strain of the pathogenic agent detected according to reality, uses above-mentioned primer pair A-F and GeXP universal primer can to fluctuate up and down on the length estimating amplified production 3bp to detecting the actual amplified production length obtained.
embodiment2: the foundation of multiplex PCR detection system
The preparation of 2.1 templates and the mono-clonal plasmid standard containing target gene
According to TaKaRa company MiniBESTViralRNA/DNAExtractionKitVer.5.0 (catalog number (Cat.No.) DV819A) specification sheets from the nucleic acid extracting different subtype (H1-16 and N1-9) avian influenza virus and other avian viral, obtain the nucleic acid samples of 50 μ L, packing is placed in-80 DEG C of preservations.RT reaction system is carried out with reference to TaKaRa company reversed transcriptive enzyme (catalog number D2639A) specification sheets, the RNA sample of acquisition is carried out reverse transcription according to following reaction system and reaction conditions respectively, obtains cDNA; Using DEPC water as the contrast of total serum IgE.
Reaction system (25 μ L): 5 × ReverseTranscriptaseBuffer5 μ L, 50mmol/LRandomPrimer (9mer) 1 μ L, dNTPMixture (10mM/L) 2 μ L, 40URibonucleaseInhibitor0.5 μ L, 5U/ μ LMLVReverseTranscriptase0.5 μ L, template ribonucleic acid 1 μ g, RNA-free water complements to 25 μ L.
Reverse transcription temperature 42 DEG C of 1.5h, are placed in-20 DEG C of preservations.To increase respectively the fragment containing M, H5, N1, N2, N6 and N86 goal gene region with PCR, obtain after amplification PCR positive products be cloned in T-easy carrier and be built into plasmid, confirm that these 6 recombinant plasmids are the recombinant plasmid that T-easy carrier inserts a kind of said gene respectively through order-checking, and be respectively the target gene of above-mentioned 6 couples of primer pair A-F.
2.2 use substance RT-PCR method checking primer
2.2.1 substance Auele Specific Primer (SP-Primer) is diluted to working concentration 1 μm of ol/L, Cy5-Tag-F and Tag-R and is diluted to working concentration 10 μm of ol/L.
Reaction cumulative volume is 25 μ L, and system component is: cDNA2.0 μ L, 10 × PCRbuffer2.5 μ L, MgCl
2(25mM/L) 2.5 μ L, SingaJumpstartTaqpolymerase1.2 μ L (2.5U/ μ L) (Singa company, catalog number (Cat.No.): D4184), dNTP (10mM/L) 1 μ L (TaKaRa company, catalog number (Cat.No.): D4030RA), Auele Specific Primer mixture gets 1.25 μ L, and upstream and downstream universal primer mixture gets 1.25 μ L, finally supplies 25 μ L with RNA-free water.
Reaction conditions is: 94 DEG C of 5min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 10 circulations; 94 DEG C of 30s, 65 DEG C of 30s, 10 circulations; 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, 20 circulations; 72 DEG C extend 3min, are placed in 4 DEG C.
2.2.2 capillary electrophoresis
Use each PCR primer of GenomeLabGeXP genetic analysis systems to carry out capillary electrophoresis detection simultaneously, operation steps is as follows: be sample-loading buffer (Beckman Coulter Inc. of the U.S. with methane amide, catalog number 608082), DNAsizestandardKit-400BasePairs (Beckman Coulter Inc. of the U.S., catalog number 608098) and sample-loading buffer 1:(80-160 by volume) thoroughly mix, in sample panel, every hole adds the liquid that 39 μ L mix, carry out 10-100 by PCR primer doubly to dilute, get the product after dilution 1 μ L and add to sample panel, piping and druming mixing, the last dropstone wax oil that instills in every hole is closed, in order to avoid methane amide oxidation and sample evaporation.On damping fluid plate, every hole adds the damping fluid of 2/3, carries out capillary electrophoresis.The condition of capillary electrophoresis is as follows: kapillary heats up: temperature 50 C; Sex change: 90 DEG C, 120s; Inject sample: 2.0KV, 30s; Be separated: 6.0KV, 35min.GenomeLabGeXP genetic analysis systems is utilized to determine the actual detected magnitude of various specific primers amplify fragment.
2.2.3 the substance specific detection of primer pair A-F
To increase respectively the cDNA sample obtained in embodiment 1 with primer pair A-F: to increase H6N8 with primer pair A H5N1, primer pair B H5N1, primer pair C H5N1, primer pair D increase H6N6, the primer pair F of H9N2, primer pair E that increase that increase that increase that increase.The result display substance Auele Specific Primer of pcr amplification and capillary electrophoresis only has good amplification to target gene and there are no assorted peak.Magnitude range after different target fragment amplification is: AIVM, 210-213bp; AIV-H5,223-226bp; AIV-N1,160-163bp; AIV-N2,188-191bp; AIV-N6,240-243bp; AIV-N8,280-283bp.
The foundation of 2.3GeXP multiplex PCR system
Primer pair A, B, C, D, E and F are mixed into multiple mix primer (Mix-Primer) working fluid, by the optimization to primer concentration, the primer of target gene M, H5, N1, N2, N6 and N8 volumetric molar concentration corresponding in PCR reaction system is made to be respectively 100nmol/L, 50nmol/L, 50nmol/L, 150nmol/L, 100nmol/L, 100nmol/L; The volumetric molar concentration of universal primer Cy5-Tag-F and Tag-R in PCR reaction system is 500nmol/L.It is identical that all the other compositions and primer are verified.Using the single positive sample of many strains known viruse nucleic acid as the mixing cDNA sample of template or multiple cause of disease as template, carry out the reaction of many primer PCRs, it is identical that response procedures and electrophoresis and primer are verified.
2.4 result
Respectively single cDNA or mixing cDNA is detected after 6 couples of primer pair A-I mix.
As shown in Figure 1, result shows that many primers single mode version has detected the amplified production of 210bp, 223bp, 162bp, 188bp, 240bp, 280bp respectively, and negative control is without any amplified production; Many primers multi-template result shows that 6 target genes can be detected simultaneously, through the system anlysis of GeXPsystem Multiple detection, can detect with actual 6 object peaks conforming to and without other assorted peaks, carry out four different N A hypotypes of the H5 subtype avian influenza virus that district office is detected by product clip size simultaneously.Result also demonstrates the primer specificity in Multiple detection detection system.
embodiment3:GeXPsystem Multiple detection system specific detection
From allantoic fluid, HA (1-16) and NA (1-9) subtype avian influenza virus nucleic acid is extracted respectively according to MiniBESTViralRNA/DNAExtractionKitVer.5.0 specification sheets, the nucleic acid simultaneously extracting the common disease poultry and livestock poison such as IBV, NDV and ILTV joins in the GeXP Multiple detection system of embodiment 2 foundation respectively, detects the specificity of the method.After multiplex PCR completes, in PCR primer, machine carries out GeXP capillary electrophoresis analysis, and result shows each reaction and only occurs nonspecific signal, no cross reaction.HA gene is except H5 hypotype, and NA gene other NA hypotypes except N1, N2, N6 and N8 hypotype, and NDV, IBV, ILTV and all reactionless signal of blank, point out the method high specificity set up, with other detected object no cross reactions.
embodimentthe sensitivity test of 4:GeXPsystem Multiple detection system and the analysis of detection clinical sample ability
The sensitivity test of 4.1GeXPsystem Multiple detection system
Use SpeI and PvuII to carry out enzyme to the plasmid containing M, H5, N1, N2, N6 and N8 gene built in real-time example 2 with Promega company RiboMAXTMLargeScaleRNAProductionSystem-T7 test kit (catalog number (Cat.No.) P1300) respectively by its specification sheets to cut, carry out in-vitro transcription synthesis RNA by above-mentioned 6 containing different goal gene linearization plasmid.DU800UV/Vis spectrophotometer is utilized to carry out mensuration with quantitative to in-vitro transcription RNA concentration.The copy number of RNA is calculated according to nucleic acid concentration and molecular weight.10 times of serial dilutions to 10 are carried out after the in-vitro transcription RNA equal proportion of M, H5, N1, N2, N6 and N8 gene being mixed
6-10 copy/μ L.Take cut back as detected sample, the method set up with embodiment 2 carries out the examination and analysb of GeXP Multiple detection system sensitivity.Test and do not repeating 3 times on the same day.
Non-3 revision tests on the same day show, the method is for the minimum sample of nucleic acid that can detect to 100 copy/μ L of H5, M, N1, N2, N6 and N8 gene.
4.2GeXPsystem Multiple detection system detects the ability of clinical sample
From the cDNA of embodiment 1 Stochastic choice H5N1 subtype avian influenza virus, in embodiment 2, the method for step 2 detects.The detected result of H5N1 subtype avian influenza virus, as Fig. 2, can detect 162.46,210.60 and 224.86 3 object peaks simultaneously and without other assorted peaks, show the template only containing H5N1 subtype avian influenza virus in sample, conform to actual.
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (5)
1. differentiate the GeXP primer sets of H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof for one kind simultaneously, it is characterized in that: comprise 6 pairs of Auele Specific Primers, be primer pair A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and D2, primer pair E1 and E1, primer pair F1 and F2 respectively, it has the sequence as shown in SEQIDNo.1 to SEQIDNo.12 respectively.
2. differentiate a GeXP test kit for H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof simultaneously, comprise cDNA, 10 × PCRbuffer, MgCl
2, dNTP, JumpstartTaqpolymerase, 6 pairs of Auele Specific Primers, 1 pair of universal primer and RNA-free water, it is characterized in that: 6 pairs of described Auele Specific Primers, the 1 pair of universal primer have the sequence as shown in SEQIDNo.1 to SEQIDNo.14 respectively.
3. the GeXP test kit simultaneously differentiating H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof according to claim 2, is characterized in that: the volumetric molar concentration that described 6 couples of Auele Specific Primer A1 and A2, primer pair B1 and B2, primer pair C1 and C2, primer pair D1 and D2, primer pair E1 and E2, primer pair F1 and F2 are corresponding in PCR reaction system is respectively 100nmol/L, 50nmol/L, 50nmol/L, 150nmol/L, 100nmol/L, 100nmol/L; Described universal primer Cy5-Tag-F, Tag-R volumetric molar concentration in PCR reaction system is 500nmol/L.
4. the GeXP test kit simultaneously differentiating H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof according to claim 3, it is characterized in that: the cumulative volume of described test kit is that 25 μ L/ manage, wherein comprise: cDNA2.0 μ L, 10 × PCRbuffer2.5 μ L, MgCl
22.5 μ L, dNTP1 μ L, JumpstartTaqpolymerase1.2 μ L, 6 couples of Auele Specific Primer mixture 1.25 μ L, upstream and downstream universal primer mixture 1.25 μ L, RNA-free water complements to 25 μ L.
5. according to claim 1 differentiate H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof simultaneously GeXP primer sets or claim 2-4 arbitrary described while the test kit of H5 subtype avian influenza virus and four kinds of different N A hypotypes thereof differentiating the application in H5N1, H5N2, H5N6 and H5N8 subtype avian influenza virus.
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| CN109487011A (en) * | 2019-01-07 | 2019-03-19 | 广西壮族自治区兽医研究所 | For detecting the primer combination and its application of H5, H7 and H9 and 9 NA hypotype AIV |
| CN109628641A (en) * | 2019-01-07 | 2019-04-16 | 广西壮族自治区兽医研究所 | Primer for detecting H7 and 5 NA hypotype AIV combines and its application |
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| US10415103B2 (en) | 2015-11-27 | 2019-09-17 | Guangxi Veterinary Research Institute | GeXP rapid detection primer set for simultaneously identifying gene HA of eight different human-infected subtypes of avian influenza virus, kit and use thereof |
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| CN106191306A (en) * | 2016-07-14 | 2016-12-07 | 广西壮族自治区兽医研究所 | A kind of primer combination identifying H5 hypotype AIV and N6 hypotype AIV and application thereof |
| CN107312872A (en) * | 2017-06-22 | 2017-11-03 | 广州市华南农大生物药品有限公司 | Differentiate multiple PCR primer group, kit and the detection method of H5 subtype avian influenza virus and its NA hypotypes simultaneously |
| CN109487011A (en) * | 2019-01-07 | 2019-03-19 | 广西壮族自治区兽医研究所 | For detecting the primer combination and its application of H5, H7 and H9 and 9 NA hypotype AIV |
| CN109628641A (en) * | 2019-01-07 | 2019-04-16 | 广西壮族自治区兽医研究所 | Primer for detecting H7 and 5 NA hypotype AIV combines and its application |
| CN109628641B (en) * | 2019-01-07 | 2022-03-01 | 广西壮族自治区兽医研究所 | Primer combination for detecting H7 and 5 NA subtype AIV and application thereof |
| CN109487011B (en) * | 2019-01-07 | 2022-03-01 | 广西壮族自治区兽医研究所 | Primer combination for detecting H5, H7, H9 and 9 NA subtypes of AIV and application thereof |
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| CN105200049B (en) | 2018-11-09 |
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