CN103290144A - Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer - Google Patents
Primer for detecting H7N9 subtype avian influenza virus infected by humans though RT-LAMP (reverse transcription loop-mediated isothermal amplification) and application of primer Download PDFInfo
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Abstract
The invention relates to a primer for detecting virus and an application of the primer and particularly relates to a primer for detecting avian influenza virus and an application of the primer, belonging to the technical field of medicine. An RT-LAMP primer and a loop primer with subtype specificity against hemagglutinin (H7) and neuraminidase (N9) genes of H7N9 subtype avian influenza virus affected by humans are designed and synthesized through bioinformatics knowledge and related bioinformatics software by means of the RT-LAMP technology. The reaction time is shortened, and the target nucleic acid is quickly detected with high specificity. Furthermore, RT-LAMP visual fluorescent dyes are added to the reaction system so as to judge the reaction result by visual inspection simply and conveniently. The primer has the advantages of being high in detection sensitivity and specificity, short in reaction time, simple and convenient to operate and free of special demand on instrument and equipment, so that the primer is suitable for performing quick specific detection and early diagnosis on the H7N9 subtype avian influenza virus infected by humans.
Description
Technical field
The present invention relates to a kind of primer and application that detects virus, especially a kind of primer and application that detects avian influenza virus belongs to medicine technology field.
Background technology
Since in February, 2013, unknown cause severe pneumonia case successively takes place in Shanghai City, Anhui Province, Jiangsu Province, Zhejiang Province.The conditions of patients development is adult respiratory distress syndrome, Sepsis, septic shock but how the severe pneumonia rapid progress to occur at 5-7 days rapidly, even multiple organ dysfunction, and case fatality rate is high.Study on etiology shows its pathogenic agent behaviour infection H7N9 subtype avian influenza virus.Be different from H7N9 subtype virus popular in the bird, the people infects H7N9 subtype avian influenza virus genome inside reprovision has taken place, and makes it obtain the attribute of infected person.This also is the reported first of H7N9 subtype avian influenza virus infected person.
Because this disease state of an illness is dangerous, process is fast, has become the raising curative ratio to finding the morning of this disease, early diagnosing, and reduces the key of case fatality rate.Be that main foundation is early found and early diagnosed to this disease and detect at the etiology that the people infects the H7N9 subtype avian influenza virus.At present, the main detection method of avian influenza virus comprises viral separation, virus antigen and gene test and three aspects of Serological testing.Wherein, virus is separated length consuming time, and the technical requirements height is unfavorable for quick diagnosis; Serological testing can only be used for retrospective diagnosis; Consuming time, effort that virus antigen detects; In the virogene context of detection, the bird flu H7N9 real-time fluorescence quantitative PCR detection architecture that WHO recommends has become the main method that infects the detection of H7N9 subtype avian influenza etiology for the people at present.Adopt the method for real-time fluorescence quantitative PCR to detect bird flu and can accomplish high-throughput, quick and precisely, still, this system needs specific apparatus, and it is higher to detect cost, is not suitable for carrying out at the primary care health unit.
Summary of the invention
The technical problem to be solved in the present invention is the defective that exists at prior art, proposes a kind ofly to infect primer and the application of H7N9 subtype avian influenza virus by RT-LAMP detection people, but the rapid detection people infects the H7N9 subtype avian influenza virus.
The present invention is the technical solution problem by the following technical programs: a kind of primer that infects the H7N9 subtype avian influenza virus by RT-LAMP detection people, comprise degenerated primer and ring primer, described degenerated primer and ring primer are to carry out the homology comparison respectively according to H7 gene segment sequence and N9 gene segment sequence that people in the existing nucleic acid database infects the H7N9 subtype avian influenza virus, according to conserved sequence, get at each gene design, the sequence of described H7 gene degenerated primer is:
H7-F3TGTCTGTTATCCTGGGAAAT
H7-B3AGCATTATCTGTGTTTGACAG
H7-FIP?GTATGTGAATCCCATTGCTTCCTTGGTGAATGAAGAAGCTCTGAGG
H7-BIP?GGAATAAGAACTAATGGARCAACCAAGCCATTTCATTTCTGCATAG；
The sequence of described H7 gene ring primer is:
H7-LF?CCGCCTGATTCTCTGAGWATTT
H7-LB?GCATGTAGGAGATCAGGATCTTCA；
The sequence of described N9 gene degenerated primer is:
N9-F3CGAGGTCTGGATACGAGA
N9-B3TTATCYTCCTTGGGTCTTCC
N9-FIP?AGCGTTTARTACAATTGTCTGACCTTTAAAAGTGCCAAATGCATTGA
N9-BIP?CTGGAGTGGTTACAGTGGATCTYTCCACATAAAAACACGCTC；
The sequence of described N9 gene ring primer is:
N9-LF?TGAATGGGCTTTGATCTATCATCTG
N9-LB?TGGACTATTGGGCTGARGGGGAC。
The present invention detects the application that the people infects the primer of H7N9 subtype avian influenza virus by RT-LAMP, may further comprise the steps:
Step 1: the H7 gene segment and the N9 gene segment sequence that infect the H7N9 subtype avian influenza virus at the people, design and synthesize the primer that comprises the T7 promotor, target gene fragment to amplification is carried out in-vitro transcription, the transcription product purifying and quantitatively after, be 10 with its dilution
6-10
0Copy/μ l namely gets the standard rna template that the people infects H7 gene segment and the N9 gene segment of H7N9 subtype avian influenza virus, in contrast, is used for the relatively susceptibility of the inventive method;
Step 2: the HA and the NA gene that the people are infected the H7N9 subtype avian influenza virus carry out H7 hypotype RT-LAMP reaction and N9 hypotype RT-LAMP reaction respectively, in described RT-LAMP reaction system, add the range estimation fluorescence dye, finish the back in reaction and carry out interpretation as a result according to visual observation.
Further, in the described step 1, the sequence of H7 gene T7 promoter primer is:
HA:CCTGGTATTCGCTCTGATTGC
HA-T7:ACTCGTTAATACGACTCACTATAGGGAGGCACCGCATGTTTCCATTCT；
The sequence of N9 gene T7 promoter primer is:
NA:TCTATGCACTTCAGCCACTG
NA-T7:ACTCGTTAATACGACTCACTATAGGGAGCCATCAGGCCAGTTCCATTG。
The RT-LAMP reaction conditions of described H7 hypotype and N9 hypotype is 60～65 ℃, 30～60min.
In described step 2, the RT-LAMP reaction system of H7 hypotype is 25 μ l, contain H7-FIP primer 40～80pmol in the reaction system, H7-BIP primer 40～80pmol, H7-F3 primer 5～10pmol, H7-B3 primer 5～10pmol, H7-LF primer 2 0～40pmol, H7-LB primer 2 0～40pmol, sal epsom 200nmol, trimethyl-glycine 20 μ mol, every kind of 35nmol of dNTPs, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, 10 * Bst damping fluid, 2.5 μ l, luciferase assay reagent 1 μ l, RNA1～5 μ the l that add detected sample again continue to add the sterilization distilled water of process DEPC processing to cumulative volume 25 μ l; The RT-LAMP reaction system of N9 hypotype is 25 μ l, contain N9-FIP primer 40～80pmol in the reaction system, N9-BIP primer 40～80pmol, N9-F3 primer 5～10pmol, N9-B3 primer 5～10pmol, N9-LF primer 2 0～40pmol, N9-LB primer 2 0～40pmol, sal epsom 200nmol, trimethyl-glycine 20 μ mol, every kind of 35nmol of dNTPs, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, 10 * Bst damping fluid, 2.5 μ l, luciferase assay reagent 1 μ l adds the RNA1～5 μ l of detected sample again, continues to add the sterilization distilled water handled through DEPC to cumulative volume 25 μ l.
Wherein, described dNTPs comprises dATP, dCTP, dGTP and dTTP.Described luciferase assay reagent is LMP221.
During use, the people infects H7N9 subtype avian influenza virus RT-LAMP detection and carries out the detected result interpretation by range estimation, and green fluorescence is sent in positive reaction, and negative reaction keeps primary colors constant.
(loop mediated isothermal amplification LAMP) is a kind of new-type constant temperature nucleic acid amplification technology of Japanese scholar Notomi invention to loop-mediated isothermal amplification technology.
The present invention utilizes the RT-LAMP technology, use bioinformation to gain knowledge and relevant information biology software, design and synthesize the RT-LAMP primer and the ring primer that infect the hypospecificity of H7N9 subtype avian influenza virus hemagglutinin (H7) and neuraminidase (N9) gene at the people.By can high special 4 primers and a kind of archaeal dna polymerase (Bst DNA polymerase) that the strand displacement activity is arranged in site on the identification target sequence, can be under constant temperature (about 60-65 ℃) efficient (30-60min) amplification purpose nucleic acid fragment.Add simultaneously the ring primer of one group of subtype sepcific at H7 gene and N9 gene respectively, further shorten the reaction times, realize high special, testing goal nucleic acid fast.In addition, by in reaction system, adding RT-LAMP range estimation fluorescence dye, make its reaction result carry out interpretation by range estimation, simple and convenient.Its advantage is: detection sensitivity and specificity are high, and the reaction times is short, easy and simple to handle, plant and instrument is not had particular requirement.Be fit to that the people is infected the H7N9 subtype avian influenza virus and carry out special detection and early diagnosis fast; And provide foundation for the detection method rapidly and efficiently of developing other various subtype influenza viruses.
Description of drawings
Fig. 1 is RT-LAMP sensitivity Detection result.Among the figure, A is to 10
6-10
0The visual observation that the H7 gene standard form of copy/μ l detects the H7 hypotype with described method; B is to 10
6-10
0The visual observation that the H7 gene standard form of copy/μ l detects the N9 hypotype with described method.
The H7N9 that Fig. 2 announces for WHO detects Real-Time PCR system to 10
6-10
0The H7 gene standard form of copy/μ l detects the amplification collection of illustrative plates.
The H7N9 that Fig. 3 announces for WHO detects Real-Time PCR system to 10
6-10
0The H7 gene standard form of copy/μ l detects the amplification typical curve.
The H7N9 that Fig. 4 announces for WHO detects Real-Time PCR system to 10
6-10
0The N9 gene standard form of copy/μ l detects the amplification collection of illustrative plates.
The H7N9 that Fig. 5 announces for WHO detects Real-Time PCR system to 10
6-10
0The N9 gene standard form of copy/μ l detects the amplification typical curve.
Fig. 6 is RT-LAMP specific detection result.Among the figure, A is for carrying out the visual observation that the H7 hypotype detects with described method to H7N9, H5N1, H1N1, H3N2 subtype influenza sample and second type influenza virus sample; B is for carrying out the visual observation that the N9 hypotype detects with described method to H7N9, H5N1, H1N1, H3N2 subtype influenza sample and second type influenza virus sample.
Embodiment
Embodiment
Agents useful for same is this area common agents in the present embodiment, can be bought by the market.
Present embodiment prepares primer by the following method and uses primer:
Infect H7 gene segment sequence and the N9 gene segment sequence of H7N9 subtype avian influenza virus according to people in the existing nucleic acid database and carry out the homology comparison respectively, according to conserved sequence, obtain the primer that the people infects the H7N9 subtype avian influenza virus at each gene design, comprise degenerated primer and ring primer, wherein, the sequence of H7 gene degenerated primer is:
H7-F3TGTCTGTTATCCTGGGAAAT
H7-B3AGCATTATCTGTGTTTGACAG
H7-FIP?GTATGTGAATCCCATTGCTTCCTTGGTGAATGAAGAAGCTCTGAGG
H7-BIP GGAATAAGAACTAATGGARCAACCAAGCCATTTCATTTCTGCATAG; The sequence of H7 gene ring primer is:
H7-LF?CCGCCTGATTCTCTGAGWATTT
H7-LB?GCATGTAGGAGATCAGGATCTTCA；
The sequence of N9 gene degenerated primer is:
N9-F3CGAGGTCTGGATACGAGA
N9-B3TTATCYTCCTTGGGTCTTCC
N9-FIP?AGCGTTTARTACAATTGTCTGACCTTTAAAAGTGCCAAATGCATTGA
N9-BIP?CTGGAGTGGTTACAGTGGATCTYTCCACATAAAAACACGCTC；
The sequence of N9 gene ring primer is:
N9-LF?TGAATGGGCTTTGATCTATCATCTG
N9-LB?TGGACTATTGGGCTGARGGGGAC。
The application of above-mentioned primer, carry out according to the following steps:
Step 1: the H7 gene segment and the N9 gene segment sequence that infect the H7N9 subtype avian influenza virus at the people, design and synthesize the primer that comprises the T7 promotor, this primer sequence is entrusted the preparation of TaKaRa company, and sequence is as follows: the sequence of H7 gene T7 promoter primer is:
HA:CCTGGTATTCGCTCTGATTGC
HA-T7:ACTCGTTAATACGACTCACTATAGGGAGGCACCGCATGTTTCCATTCT；
The sequence of N9 gene T7 promoter primer is:
NA:TCTATGCACTTCAGCCACTG
NA-T7:ACTCGTTAATACGACTCACTATAGGGAGCCATCAGGCCAGTTCCATTG。With the primer of the above-mentioned T7 of comprising promotor the target gene fragment of amplification is carried out in-vitro transcription, the transcription product purifying and quantitatively after, be 10 with its dilution
6-10
0Copy/μ l namely gets the standard rna template that the people infects H7 gene segment and the N9 gene segment of H7N9 subtype avian influenza virus, in contrast, is used for the relatively susceptibility of the inventive method;
Step 2: the HA and the NA gene that the people are infected the H7N9 subtype avian influenza virus carry out H7 hypotype RT-LAMP reaction and N9 hypotype RT-LAMP reaction respectively, the RT-LAMP reaction system of H7 hypotype is 25 μ l, contain H7-FIP primer 40～80pmol in the reaction system, H7-BIP primer 40～80pmol, H7-F3 primer 5～10pmol, H7-B3 primer 5～10pmol, H7-LF primer 2 0～40pmol, H7-LB primer 2 0～40pmol, sal epsom 200nmol, trimethyl-glycine 20 μ mol, dNTPs(comprises dATP, dCTP, dGTP and dTTP) every kind of 35nmol, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, 10 * Bst damping fluid, 2.5 μ l, luciferase assay reagent (LMP221, EIKEN) 1 μ l adds the RNA1～5 μ l of detected sample again, continues to add the sterilization distilled water handled through DEPC to cumulative volume 25 μ l; The RT-LAMP reaction system of N9 hypotype is 25 μ l, contain N9-FIP primer 40～80pmol in the reaction system, N9-BIP primer 40～80pmol, N9-F3 primer 5～10pmol, N9-B3 primer 5～10pmol, N9-LF primer 2 0～40pmol, N9-LB primer 2 0～40pmol, sal epsom 200nmol, trimethyl-glycine 20 μ mol, every kind of 35nmol of dNTPs, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, 10 * Bst damping fluid, 2.5 μ l, luciferase assay reagent 1 μ l, RNA1～5 μ the l that add detected sample again, the sterilization distilled water that continues to add process DEPC processing is to cumulative volume 25 μ l, and reaction conditions is 63 ℃, 30min.Reaction finishes the back and carries out interpretation as a result according to visual observation, and green fluorescence is sent in positive reaction, and negative reaction keeps primary colors constant.
1. susceptibility and specificity analyses
Utilize the step of present embodiment that clinical doubtful sample is detected, and compare with the detected result of Real-Time PCR, concrete detected result is as follows:
(1) present embodiment detects the sensitivity testing of H7 hypotype and N9 hypotype
RNA template to the known copy number is compared with the method detection of present embodiment and with the H7N9 detection Real-Time PCR system that WHO announces, result such as Fig. 1, and A is to 10 among Fig. 1
6-10
0The H7 gene standard form of copy/μ l carries out the visual observation that the H7 hypotype detects with the method for present embodiment, and B is to 10
6-10
1The H7 gene standard form of copy/μ l carries out the visual observation that the N9 hypotype detects with the method for present embodiment.By range estimation interpretation reaction result, present embodiment is 10 to the detection limit that the people infects H7N9 subtype avian influenza virus H7 hypotype and N9 hypotype
1Copy/μ l.Wherein the method for present embodiment is identical with Real-Time PCR system to the detection sensitivity of H7 hypotype, and the method for present embodiment is better than order of magnitude of Real-Time PCR system to the detection sensitivity of N9 hypotype, also reaches 10
1Copy/μ l(sees Fig. 2～Fig. 5).
(2) present embodiment detects the specific assay of H7 hypotype and N9 hypotype
Adopt present embodiment to detect the people and infect H7N9, H5N1, H1N1, H3N2 subtype influenza sample and second type influenza virus sample, the results are shown in Figure A among 6, Fig. 6 is that present embodiment carries out the visual observation that the H7 hypotype detects to H7N9, H5N1, H1N1, H3N2 subtype influenza sample and second type influenza virus sample; B is that present embodiment carries out the visual observation that the N9 hypotype detects to H7N9, H5N1, H1N1, H3N2 subtype influenza sample and second type influenza virus sample.By range estimation interpretation reaction result, only the people infects H7N9 subtype avian influenza sample show as positive green fluorescence in the detection of H7 hypotype and N9 hypotype, all the other subtype influenza samples all show as negative primary colors in the detection of H7 hypotype and N9 hypotype constant, the specificity height.
(3) present embodiment detects the people and infects H7N9 virus in the clinical doubtful sample of H7N9 subtype avian influenza, and compares with detected result that H7N9 that WHO announces detects Real-Time PCR system.Collect clinical doubtful people and infect 127 parts in H7N9 subtype avian influenza patient lower respiratory tract secretory product sample, the detected result of present embodiment detected result with Real-Time PCR compared, see Table 1.H7 hypotype part wherein, the result of present embodiment and Real-Time PCR system is identical; And N9 hypotype part, Real-Time PCR system does not detect the sample of 2 parts of H7 positives, and present embodiment all detects, and in full accord with the result of H7.Therefore, described present embodiment is identical with Real-Time PCR system to the detection sensitivity of H7 hypotype, and the detection sensitivity of N9 hypotype is better than the Real-TimePCR system that WHO announces.
Table 1 people infects H7N9 subtype avian influenza virus RT-LAMP method with the detected result comparison of Real-Time PCR.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (7)
1. one kind is detected the primer that the people infects the H7N9 subtype avian influenza virus by RT-LAMP, comprise degenerated primer and ring primer, described degenerated primer and ring primer are to carry out the homology comparison respectively according to H7 gene segment sequence and N9 gene segment sequence that people in the existing nucleic acid database infects the H7N9 subtype avian influenza virus, according to conserved sequence, get at each gene design, the sequence of described H7 gene degenerated primer is:
H7-F3TGTCTGTTATCCTGGGAAAT
H7-B3AGCATTATCTGTGTTTGACAG
H7-FIP?GTATGTGAATCCCATTGCTTCCTTGGTGAATGAAGAAGCTCTGAGG
H7-BIP GGAATAAGAACTAATGGARCAACCAAGCCATTTCATTTCTGCATAG; The sequence of described H7 gene ring primer is:
H7-LF?CCGCCTGATTCTCTGAGWATTT
H7-LB?GCATGTAGGAGATCAGGATCTTCA；
The sequence of described N9 gene degenerated primer is:
N9-F3CGAGGTCTGGATACGAGA
N9-B3TTATCYTCCTTGGGTCTTCC
N9-FIP?AGCGTTTARTACAATTGTCTGACCTTTAAAAGTGCCAAATGCATTGA
N9-BIP?CTGGAGTGGTTACAGTGGATCTYTCCACATAAAAACACGCTC；
The sequence of described N9 gene ring primer is:
N9-LF?TGAATGGGCTTTGATCTATCATCTG
N9-LB?TGGACTATTGGGCTGARGGGGAC。
2. detect the application that the people infects the primer of H7N9 subtype avian influenza virus according to claim 1 is described by RT-LAMP, may further comprise the steps:
Step 1: the H7 gene segment and the N9 gene segment sequence that infect the H7N9 subtype avian influenza virus at the people, design and synthesize the primer that comprises the T7 promotor, target gene fragment to amplification is carried out in-vitro transcription, the transcription product purifying and quantitatively after, be 10 with its dilution
6-10
0Copy/μ l namely gets the standard rna template that the people infects H7 gene segment and the N9 gene segment of H7N9 subtype avian influenza virus, in contrast;
Step 2: the HA and the NA gene that the people are infected the H7N9 subtype avian influenza virus carry out H7 hypotype RT-LAMP reaction and N9 hypotype RT-LAMP reaction respectively, in described RT-LAMP reaction system, add the range estimation fluorescence dye, finish the back in reaction and carry out interpretation as a result according to visual observation.
3. detect the application that the people infects the primer of H7N9 subtype avian influenza virus according to claim 2 is described by RT-LAMP, it is characterized in that: in the described step 1,
The sequence of H7 gene T7 promoter primer is:
HA:CCTGGTATTCGCTCTGATTGC
HA-T7:ACTCGTTAATACGACTCACTATAGGGAGGCACCGCATGTTTCCATTCT; The sequence of N9 gene T7 promoter primer is:
NA:TCTATGCACTTCAGCCACTG
NA-T7:ACTCGTTAATACGACTCACTATAGGGAGCCATCAGGCCAGTTCCATTG。
4. detect the application that the people infects the primer of H7N9 subtype avian influenza virus according to claim 2 is described by RT-LAMP, it is characterized in that: the RT-LAMP reaction conditions of described H7 hypotype and N9 hypotype is 60～65 ℃, 30～60min.
5. detect the application that the people infects the primer of H7N9 subtype avian influenza virus according to claim 2 is described by RT-LAMP, it is characterized in that: in the described step 2, the RT-LAMP reaction system of H7 hypotype is 25 μ l, contain H7-FIP primer 40～80pmol in the reaction system, H7-BIP primer 40～80pmol, H7-F3 primer 5～10pmol, H7-B3 primer 5～10pmol, H7-LF primer 2 0～40pmol, H7-LB primer 2 0～40pmol, sal epsom 200nmol, trimethyl-glycine 20 μ mol, every kind of 35nmol of dNTPs, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, 10 * Bst damping fluid, 2.5 μ l, luciferase assay reagent 1 μ l adds the RNA1～5 μ l of detected sample again, continues to add the sterilization distilled water handled through DEPC to cumulative volume 25 μ l; The RT-LAMP reaction system of N9 hypotype is 25 μ l, contain N9-FIP primer 40～80pmol in the reaction system, N9-BIP primer 40～80pmol, N9-F3 primer 5～10pmol, N9-B3 primer 5～10pmol, N9-LF primer 2 0～40pmol, N9-LB primer 2 0～40pmol, sal epsom 200nmol, trimethyl-glycine 20 μ mol, every kind of 35nmol of dNTPs, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, 10 * Bst damping fluid, 2.5 μ l, luciferase assay reagent 1 μ l adds the RNA1～5 μ l of detected sample again, continues to add the sterilization distilled water handled through DEPC to cumulative volume 25 μ l.
6. detect the application that the people infects the primer of H7N9 subtype avian influenza virus according to claim 5 is described by RT-LAMP, it is characterized in that: described dNTPs comprises dATP, dCTP, dGTP and dTTP.
7. detect the application that the people infects the primer of H7N9 subtype avian influenza virus according to claim 5 is described by RT-LAMP, it is characterized in that: described luciferase assay reagent is LMP221.
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| CN103898237B (en) * | 2014-01-09 | 2019-08-06 | 吉林大学 | Degenerate primer RT-PCR detection method for Eimeria coccidia virus |
| CN103898237A (en) * | 2014-01-09 | 2014-07-02 | 吉林大学 | Degenerate primer RT-PCR detection method for eimeria viruses |
| CN103740863A (en) * | 2014-01-13 | 2014-04-23 | 华南农业大学 | RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9 |
| CN103740863B (en) * | 2014-01-13 | 2015-06-17 | 华南农业大学 | RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9 |
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| CN110551850A (en) * | 2019-09-02 | 2019-12-10 | 拱北海关技术中心 | RT-LAMP primer and method for detecting highly pathogenic H7N9 avian influenza virus |
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