CN102329891A - RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primer group for detecting H9 subtype avian influenza virus as well as detection method and application thereof - Google Patents
RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primer group for detecting H9 subtype avian influenza virus as well as detection method and application thereof Download PDFInfo
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Abstract
The invention discloses an RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primer group for detecting H9 subtype avian influenza virus as well as a detection method and an application thereof, and the primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers, wherein the sequences of the outer primers are as shown in SEQ ID No. 1, 2 (sequence identity numbers 1 and 2) respectively, the sequences of the inner primers are as shown in SEQ ID No. 3, 4 respectively and the sequences of the loop primers are as shown in SEQ ID No. 5, 6 respectively. The primer group can be used for preparing a kit for detecting the H9 subtype avian influenza virus, and the kit comprises Reaction Mix, Enzyme Mix, Distilled Water, a fluorescence test reagent and the primer group provided by the invention. The test kit has the characteristics of less samples, strong specificity, high sensitivity, fastness, accuracy and the like, thereby providing a simple and effective early rapid diagnosis way for a prevention, control and early warning mechanism of H9 subtype avian influenza.
Description
Technical field
The present invention relates to the detection method of bird flu virus, relate in particular to primer sets, and detection method and application, belong to the detection range of bird flu virus based on the detection H9 subtype avian influenza virus of RT-LAMP design.
Background technology
Bird flu virus (Avian influenza virus AIV) is the sub-thread strand RNA, by 8 independently the RNA sections form, one of significant biological property is that hypotype is numerous, variation is frequent.The different subtype strain is to host's virulence significant difference, and the H9N2 hypotype is middle low pathogenicity strain, is characterized in distributing extensively, can cause host's immunosuppression, and can cause the bird morbidity through synergy with other pathogenic micro-organism.
Though H9N2 is medium low pathogenicity strain, it can not be ignored the harm of aviculture equally, and in the period of the 1996-2000, the sickness rate of China H9N2 subtype avian influenza accounts for 93.89% of total bird flu sickness rate, becomes the main AIV hypotype that influences China's aviculture.From 1994 since China's Guangdong chicken house is separated to the H9N2 subtype avian influenza virus first, the H9N2 subtype avian influenza is in rising trend always in China, also extensively takes place in the international coverage.Chinese scholar successively has been separated to the bird flu virus of identical hypotype from different places in the host from difference.The particularly repeatedly generation of H9N2 subtype avian influenza virus infected person incident since 1999; Serious threat human health, livestock industry production and livestock product international trade; Especially public health meaning has caused global extensive concern, and the research of this subtype virus has also been obtained progress faster.
Because the slight respiratory symptom that the H9 subtype avian influenza virus causes, degradation is very similar with poultry diseases such as newcastle disease, infectious laryngotracheitis, infectious bronchitiss under the laying hen egg productivity, difficult clinically the differentiation.After occurring in epidemic situation, can react in time fast, the detection method of setting up a kind of conveniently H9 subtype avian influenza virus has great importance.The detection method of bird flu has virus separation, blood clotting (HA) and blood clotting to suppress (HI) test, IFA, ELISA, RT-PCR, Real-time, PCR, NASBA (Nucleic acid sequence-based amplification) and micro-array chip (Microarray) etc., and the RT-PCR technology is one of focus of research.
Quick diagnosis is the first step and an important ring of bird flu prevention and control with monitoring in time.At present; The detection method of relevant bird flu is more; Virus is separated and biological Characteristics Study is the most feasible and illustrative problem in the classical influenza virus etiological diagnosis method, but the isolation identification of virus need need the time (several days to a week) of length with pathological material of disease inoculated into chick embryo or tissue culture; Can not satisfy the anti-system of the urgent epidemic situation of acute deadly infectious disease requirements of one's work; Infect mammiferous potentially dangerous because HPAIV exists simultaneously, the manipulation require of virus carries out in three grades of laboratories of Biosafety, and general sanitary authority does not possess this test conditions.Fast, sensitive, special molecular Biological Detection technology, like RT-PCR, RT-PCR-ELISA, real-time RT-PCR have brought into play important effect (Shan, 2003 in avian flu virus detection; Collins, 2002; Wang, 2005; Spackman, 2003; Poddar, 2002).But all need complicated detecting instrument (like PCR appearance, quantitative real time PCR Instrument etc.), when the laboratory operation of some basic units, be restricted.(Loop-mediated Isothermal Amplification Method, LAMP) technology is a kind of molecular biology method (Notomi, 2000) of new amplification in vitro DNA fragment specific to ring mediated isothermal amplification.This method does not need extra reverse transcription step; Only need simple water-bath; A large amount of nucleic acid can increase in 30-90min; Need not come detected result, only need to precipitate observations (if can more be prone to observe after adding optical dye) through the magnesium pyrophosphate of its by product-white through the method for nucleic acid electrophoresis.Simple, quick, efficient, practical characteristics that LAMP had be highly suitable for the basic unit laboratory, even quick diagnosis are carried out at the scene.This method successfully has been applied to the detection of H9 and H9 hypotype AIV.The prerequisite of setting up the RT-LAMP detection method of a kind of H9 hypotype AI V that can be sensitive, special is to need to obtain Auele Specific Primer to H9HA gene conserved regions design.
Summary of the invention
Main purpose of the present invention provides the primer sets of a kind of detection H9 subtype avian influenza virus RT-LAMP that can be sensitive, special;
Above-mentioned purpose of the present invention realizes through following technical scheme:
A kind of RT-LAMP primer sets that is used for the detection of H9 subtype avian influenza virus is characterized in that described RT-LAMP primer sets by a pair of outer primer, and inner primers and a pair of ring primer are formed; Said outer primer is respectively shown in SEQ ID No.1 and the SEQ ID No.2 sequence, and said inner primer is respectively shown in SEQ ID No.3 and the SEQ ID No.4 sequence, and said ring primer is respectively shown in SEQ ID No.5 and the SEQ ID No.6 sequence.
The present invention also provides a kind of method of the H9 of detection subtype avian influenza virus, it is characterized in that: use described primer sets that testing sample is carried out the RT-LAMP amplification.
Described RT-LAMP primer sets of the present invention can be used for preparing the biological reagent that detects or diagnose the H9 subtype avian influenza virus.
The invention still further relates to the test kit that a kind of H9 of being used for subtype avian influenza virus RT-LAMP detects, comprise Reaction Mix, Enzyme Mix and Distilled Water, it is characterized in that also comprising luciferase assay reagent and above-described primer sets;
Reaction Mix, Enzyme Mix and Distilled Water all can be according to the existing method preparations of routine; At the Reaction Mix described in the specific embodiment of the present invention, Enzyme Mix from RNAAmplification Kit (RT-LAMP) (available from Japanese Rong Yan company).
Wherein, described RT-LAMP primer is by a pair of outer primer (H9-F3 and H9-B3), and inner primers (H9-FIP and H9-BIP) and a pair of ring primer (H9-LF and H9-LB) are formed; Said outer primer is shown in SEQ ID No.1 and the SEQ ID No.2 to sequence, and said inner primer is shown in SEQ ID No.3 and the SEQ IDNo.4 to sequence, and said ring primer is shown in SEQ ID No.5 and the SEQ ID No.6 to sequence.Said primer can be processed lyophilized powder according to existing method, and PAGE is pure or HPLC is pure.
In a specific embodiment of the present invention, described luciferase assay reagent is Fluorescent Detection Reagent, available from Japanese Rong Yan company.
The present invention also provides the purposes of described H9 subtype avian influenza virus RT-LAMP detection kit in the biological reagent of preparation detection or diagnosis H9 subtype avian influenza virus.
Another object of the present invention provides a kind of use of above-mentioned detection H9 subtype avian influenza virus RT-LAMP detection method, comprising:
1, following component is joined in the reaction tubes:
2, reaction tubes is placed PCR appearance or constant temperature water bath, loop parameter is 62.5 ℃ of reaction 1h, and 80 ℃ of effect 10min finish reaction;
3, detect:
(1) visual inspection: add 1.0 μ L Fluorescent Detection Reagent dyestuffs in the reactant, get final product direct display result: green fluorescence is sent in positive reaction, and negative reaction keeps former yellow constant;
(2) agarose gel electrophoresis: the agarose gel electrophoresis with 2.0% is analyzed: positive reaction presents distinctive scalariform band, and negative reaction does not then have band and occurs.
The present invention finds out HA gene conserved regions through the subtype influenza virus HA gene order of analyzing H9AIV in the influenza DB, has designed the RT-LAMP Auele Specific Primer on this basis.These gene orders have contained the HA gene of multiple poultry and wild bird, have ubiquity and representativeness.The RT-LAMP detection kit of utilizing this primer to set up detects other 7 stud bird disease pathogens such as H1～H16 subtype avian influenza virus and newcastle disease viruses; The result shows that this detection kit only can specific amplification H9 subtype avian influenza virus nucleic acid; And other viral nucleic acids that can not increase have excellent specificity.
The latent period of bird flu virus from several hours to a couple of days, length can reach 21 days.At the bird flu outbreak of epidemic initial stage, what do at first should be to forbid the circulation of poultry on market, carries out regional blockade, and seeks the source of virus.Therefore to the monitoring of live-bird and wild fowl particularly important (the Sakar et al that seems; 2010), AIV has cyst membrane, and is relatively more responsive to sterilizing agent; But in production reality; AIV often discharges from the nasal secretion that infects fowl and in the fecal matter, and virus is under the organic protection, can pass through air, contact and ight soil and propagate.It is reported that test chicken can be separated to virus after attacking malicious 18h from cloaca and larynx swab, last till and attack about the 7d of poison back; Attack poison back 36h and just can from the biased sample of brain, liver,spleen,kidney, pancreas, be separated to virus; Can continue until and attack about the 10d of poison back (LIAO, et al, 2004); This shows through cotton swab can not detect AIV when symptom occurring, can really accomplish early to find prevention and control early.The present invention infects through the intranasal inoculation natural imitation, and the cotton swab that different time after infecting is taked carries out parallel detection with viral separation, RT-PCR and RT-LAMP detection kit of the present invention.Wherein, virus is separated and the H9-RT-LAMP method can detect virus on the 1st day after infection, and three kinds of methods all can detect virus in the 3rd day.But it is different to detect required time, and using RT-LAMP detection kit detection cause of disease of the present invention only needs 60-30min; The virus separation method needs 2-3d at least; The RT-PCR method needs 6-7h, though short susceptibility of time spent is lower than RT-LAMP detection kit of the present invention.
The present invention is directed in the poultry popular comparatively extensively, the hemagglutinin gene of H9 subtype avian influenza virus that harm is bigger set up the RT-LAMP detection method; This method is limited to 0.1PFU to the minimum detection of H9AIV RNA; Its susceptibility is high 10 times than conventional RT-PCR, can be used for the detection of highly pathogenicity and low pathogenicity H9 hypotype AIV simultaneously.Through detection to 16 AIV subtype virus and other avian viruses disease pathogens RNA, show that this method can only specific amplification H9 hypotype AIV, have good specificity.Detection kit of the present invention have with appearance less, high specificity, susceptibility be high, quick and precisely wait characteristics, for the prevention and control early warning mechanism of H9 subtype avian influenza provides simple and direct, effectively early stage quick diagnosis approach.
Description of drawings
Fig. 1 carries out the susceptibility amplification of RT-LAMP and RT-PCR for the different concns substrate;
Figure 1A is the H9-RT-LAMP amplification; Figure 1B is the H9-RT-PCR amplification; The pairing viral RNA concentration of 1-8 is for being respectively 1000PFU, 100PFU, 10PFU, 1PFU, 0.1PFU, 0.01PFU, 0.001PFU, 0.0001PFU
Fig. 2 carries out the real-time monitoring figure display result of RT-LAMP amplification for the concentration of substrate of different concns;
The pairing viral RNA concentration of 1-8 is for being respectively 1000PFU, 100PFU, 10PFU, 1PFU, 0.1PFU, 0.01PFU, 0.001PFU, 0.0001PFU
Fig. 3 is for detecting the real-time monitoring figure display result of H1～H16 subtype avian influenza virus with RT-LAMP;
1.MD/HLJ/390/06(H1N1)；2.MD/HLJ/135/06(H2N2)；3.MD/HLJ/90/06(H3N2)；4.MD/HLJ/499/06(H4N6)；5.DK/AH/1/06(H5N1)；6.MD/HLJ/87/06(H6N1)；7.CK/HeB/1/02(H7N2)；8.A/wigeon/Denmark/77-66157-G8/04(H8N1)；9.CK/HuN/33/08(H9N2)；10.MD/HLJ/108/06(H10N2)；11.MD/HLJ/80/06(H11N2)；12.A/Duck/Alberta/60/76(H12N5)；13.A/gull/Maryland/704/77(H13N6)；14.A/mallard/Gurjer/263/82(H14N5)；15.A/duck/AUST/34/83(H15N8)；16.A/cormonant/Denmark/74-68899-G2/02(H16N3)；
Fig. 4 is for detecting the specificity test-results of H1～H16 subtype avian influenza virus with RT-LAMP;
1.MD/HLJ/390/06(H1N1)；2.MD/HLJ/135/06(H2N2)；3.MD/HLJ/90/06(H3N2)；4.MD/HLJ/499/06(H4N6)；5.DK/AH/1/06(H5N1)；6.MD/HLJ/87/06(H6N1)；7.CK/HeB/1/02(H7N2)；8.A/wigeon/Denmark/77-66157-G8/04(H8N1)；9.CK/HuN/33/08(H9N2)；10.MD/HLJ/108/06(H10N2)；11.MD/HLJ/80/06(H11N2)；12.A/Duck/Alberta/60/76(H12N5)；13.A/gull/Maryland/704/77(H13N6)；14.A/mallard/Gurjer/263/82(H14N5)；15.A/duck/AUST/34/83(H15N8)；16.A/cormonant/Denmark/74-68899-G2/02(H16N3)；
Fig. 5 is the specificity test-results that detects other poultry diease viral nucleic acid with RT-LAMP;
M:DL2000DNAMarker; 1:H9N2; 2: newcastle disease virus (NDV); 3: avian infectious bronchitis virus (IBV); 4: infections chicken cloacal bursa virus (IBDV); 5: chicken Marek's disease virus (MDV); 6: avian infectious laryngotracheitis virus (ILTV); 7: CAV (CIAV); 8: fowl leukosis virus (ALv);
Fig. 6 is the real-time monitoring figure display result that detects other poultry diease viral nucleic acid with RT-LAMP.
1:H9N2; 2: newcastle disease virus (NDV); 3: avian infectious bronchitis virus (IBV); 4: infections chicken cloacal bursa virus (IBDV); 5: chicken Marek's disease virus (MDV); 6: avian infectious laryngotracheitis virus (ILTV); 7: CAV (CIAV); 8: fowl leukosis virus (ALV).
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
1 test materials and method
1.1 strain and reagent
This tests used bird flu virus H1～H16 hypotype canonical reference strain (available from China Veterinery Drug Inspection Office);
H9 subtype avian influenza virus and newcastle disease virus thereof (NDV), avian infectious bronchitis virus (IBV), infections chicken cloacal bursa virus (IBDV), chicken Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), CAV (CIAV), fowl leukosis virus (ALV) (available from China Veterinery Drug Inspection Office).
RNA Amplification Kit (RT-LAMP) and Fluorescent Detection Reagent are available from Japanese Rong Yan company; RNA
Total RNA Isolation system and Access RT-PCR system are all available from Promega company.10 age in days SPF chicken embryos, 6 the week age SPF chicken provide by Harbin Veterinary Medicine Inst., China Academy of Agriculture's SPF Experimental Animal Center, all SPF chickens are all raised in the negative pressure shield retaining.
1.2LAMP primer design is with synthetic
Analyzed the homology of H9 subtype influenza virus HA gene order in the influenza DB; Find out HA gene conserved regions; 6 primers have been designed; Be respectively outer primer (H9-F3 (shown in the SEQ ID No.1) and H9-B3 (shown in the SEQ ID No.2)), inner primer H9-FIP (shown in the SEQ ID No.3) and H9-BIP (shown in the SEQ ID No.4)) and ring primer (H9-LF (shown in the SEQ ID No.5) and H9-LB (shown in the SEQ ID No.6)) (table 1).
Table 1 RT-LAMP primer sequence
1.3RNA extract
Trizol LS test kit (inVitrogen company) is adopted in the extraction of RNA.Virus allantoic fluid or oropharynx cloaca swab are by after the conventional processing, and sample thief 250ul adds 750ul Trizol LS lysate, mix concussion 5 minutes (as far as possible guarantee cracking after liquid-transparent); Add the 200ul chloroform, 12000 change, 4 ℃ centrifugal 15 minutes, get supernatant; Add the 500ul Virahol, precipitation at room temperature 10-15 minute, 12000 changeed, and 4 ℃ centrifugal 15 minutes; Abandon supernatant, slowly add 75% ethanol and wash deposition once, be inverted filter paper and blot the surplus liquid of tube wall; Vacuum is drained or 37 ℃ of oven dry, adds the water 20ul of no RNA enzyme, and-70 ℃ of preservations are subsequent use.
1.4RT-LAMP reaction system with RT-PCR
H9-RT-LAMP reaction TV is 25 μ L, and moity is following: Reaction Mix 12.5 μ L; 100p mol H9 FIP 0.4 μ L, 100p mol H9 BIP 0.4 μ L, 10p mol H9 F3 0.5 μ L, 10p mol H9 B30.5 μ L, 100p mol H9 LF 0.2 μ L, 100p mol H9 LB 0.2 μ L; Enzyme Mix 1 μ L; The RNA that adds 5.0 μ L extractings acquisition adds Distilled Water to 25 μ L in system, place turbidimeter, PCR appearance or constant temperature water bath; Loop parameter is 62.5 ℃ of reaction 1h, and 80 ℃ of effect 10min finish reaction.Add the Fluorescent Detection Reagent of 1.0 μ L in the reactant, get final product direct display result:
Green fluorescence is sent in positive reaction, and negative reaction keeps former yellow constant; Or analyze with 2.0% agarose gel electrophoresis: positive reaction presents distinctive scalariform band, and negative reaction does not then have band and occurs.
The primer sequence of H9-RT-PCR is:
H9Up：5’TCAACAAACTCCACCGAAACTGT3’；
H9Low：5’TCCCGTAAGAACATGTCCATACCA3’；
The amplified fragments size is 732bp.The reaction TV is 25 μ L, and moity is following: the sterilization distilled water that 5.0 μ L 5X reaction buffers, 0.5 μ L 10M dNTP, 1.0 μ L 15M sal epsom, 0.25 μ L 20pmol H9 Up, 0.25 μ L20p mol H9 Low, 0.5 μ L A MV ThermoScript II, 0.5 μ L Taq archaeal dna polymerase, 14 μ L DEPC handle.Add 2.5 μ L RNA to system, place the reaction of PCR appearance, loop parameter is 45 ℃ of rt 45min, 94 ℃ of preparatory sex change 2min, and 94 ℃ of 30s, 52 ℃ of 45s, 68 ℃ of 45s, 35 circulations, last 68 ℃ are extended 8min.The PCR product is analyzed (Hongmei, et al, 2009) with 1.0% agarose gel electrophoresis.
1.5 sensitivity test
To measuring H9 hypotype canonical reference strain (available from China Veterinery Drug Inspection Office) (the Garten et al of viral level; 1985) allantoic fluid extracts nucleic acid, and the total RNA of the H9AIV of extracting gained is carried out 10 times of doubling dilutions (being respectively 1000PFU, 100PFU, 10PFU, 1PFU, 0.1PFU, 0.01PFU, 0.001PFU, 0.0001PFU).Above-mentioned each concentration RNA is detected with H9-RT-LAMP and H9-RT-PCR method simultaneously.
1.6 specificity test
Detect H1～H16 subtype avian influenza canonical reference strain respectively with this H9-RT-LAMP method, detect between the different subtype virus whether have non-specific amplification.Detection newcastle disease virus (NDV), avian infectious bronchitis virus (IBV), infections chicken cloacal bursa virus (IBDV), chicken Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), CAV (CIAV), fowl leukosis virus (ALV) wait other 7 stud bird disease pathogens, detect the specificity of H9-RT-LAMP method.The H9AIV strain is as positive control.
2 experimental results
2.1RT-LAMP susceptibility result
By viral level is 10
3The H9RNA of PFU; Carry out carrying out parallel detection with RT-LAMP detection kit of the present invention with the RT-PCR method behind 10 times of doubling dilutions; Find that the RT-PCR method can detect the virus (shown in Figure 1B) of 1PFU titre, the stepped band brightness of characteristic of RT-LAMP reaction reduces with concentration has gradually dark change (shown in Figure 1A), and final virus still can amplify tangible stepped purpose band (Figure 1A and B) when 0.1PFU; Can detect the viral nucleic acid of 0.1PFU sensitively; Than responsive 10 times of conventional RT-PCR method (Figure 1B), the real-time monitoring figure of RT-LAMP amplification shows (as shown in Figure 2), along with dilution increase; The starting point of response curve is passed backward gradually, shows that this method is very sensitive to the variation of concentration.
2.2RT-LAMP specificity result
Specificity for the primer that detects H9-RT-LAMP detects H1～H16 subtype avian influenza virus canonical reference strain with RT-LAMP, and the result only shows the H9 subtype avian influenza virus is amplified the stepped band of characteristic and presents green fluorescence (as shown in Figure 4).The real-time monitoring figure of H9-RT-LAMP amplification also shows only have the H9 subtype avian influenza virus that amplification curve is arranged, other subtype virus result (as shown in Figure 3) that then is negative.Simultaneously other 7 kinds of poultry dieases such as newcastle disease are also detected, other poultry diease nucleic acid do not amplify the stepped band of characteristic yet and demonstrate green fluorescence (as shown in Figure 5)., the real-time monitoring figure of H9-RT-LAMP amplification also shows only have the H9 subtype avian influenza virus that amplification curve is arranged, other poultry diease viral nucleic acid result (as shown in Figure 6) that then is negative.Show that RT-LAMP detection kit of the present invention can specific amplification H9 subtype avian influenza virus, and not with the AIV and other poultry diease viral nucleic acid generation cross reaction of other hypotype.In addition, through the front and back that add the ring primer are compared discovery, the adding of ring primer can be shortened the reaction times greatly, can shorten to 30min by original 60min.
Claims (7)
1. one kind is used for the RT-LAMP primer sets that the H9 subtype avian influenza virus detects, and it is characterized in that described RT-LAMP primer sets by a pair of outer primer, and inner primers and a pair of ring primer are formed; Said outer primer is respectively shown in SEQ ID No.1 and the SEQ ID No.2 sequence, and said inner primer is respectively shown in SEQ ID No.3 and the SEQ ID No.4 sequence, and said ring primer is respectively shown in SEQ ID No.5 and the SEQ ID No.6 sequence.
2. a method that detects the H9 subtype avian influenza virus is characterized in that: use the described primer sets of claim 1 that testing sample is carried out the RT-LAMP amplification.
3. according to the described method of claim 2, it is characterized in that described amplified reaction is that reaction tubes is placed PCR appearance or constant temperature water bath, loop parameter is 62.5 ℃ of reaction 1h, and 80 ℃ of effect 10min finish reaction.
The described RT-LAMP primer sets of claim 1 preparation detect or the biological reagent of diagnosis H9 subtype avian influenza virus in purposes.
5. one kind is used for the test kit that H9 subtype avian influenza virus RT-LAMP detects, and comprises Reaction Mix, Enzyme Mix and Distilled Water, it is characterized in that also comprising the described primer sets of luciferase assay reagent and claim 1.
6. according to the described H9 subtype avian influenza virus of claim 5 RT-LAMP detection kit, it is characterized in that: described luciferase assay reagent is Fluorescent Detection Reagent, available from Japanese Rong Yan company.
Claim 5 or 6 described H9 subtype avian influenza virus RT-LAMP detection kit preparation detect or the biological reagent of diagnosis H9 subtype avian influenza virus in purposes.
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| CN102634607A (en) * | 2012-04-18 | 2012-08-15 | 中国检验检疫科学研究院 | H9 subtype avian influenza virus loop-mediated isothermal amplification micro total analysis method |
| CN103614490A (en) * | 2013-10-22 | 2014-03-05 | 山东省农业科学院家禽研究所 | Application of RT-LAMP primer group composition used for detecting H4 subtype avian influenza virus, and kit used for detecting the H4 subtype avian influenza virus |
| CN105986042A (en) * | 2016-01-29 | 2016-10-05 | 中国动物卫生与流行病学中心 | Method for quickly detecting nucleic acid of H9 subtype avian influenza virus |
| CN106702028A (en) * | 2017-02-27 | 2017-05-24 | 解码（上海）生物医药科技有限公司 | Real-time fluorescent PCR detection kit for H10N7 avian influenza virus and detection method of real-time fluorescent PCR detection kit |
| CN106702028B (en) * | 2017-02-27 | 2020-05-19 | 解码（上海）生物医药科技有限公司 | Real-time fluorescence PCR detection kit and detection method for H10N7 avian influenza virus |
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