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Primer pair for authenticating North America-branch H7 subtype avian influenza virus and application thereof

A bird flu virus and primer pair technology, applied in the identification of North American branch H7 subtype bird flu virus primer pair and its application field, to achieve the effect of easy promotion, good specificity and simple operation

Inactive Publication Date: 2017-05-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been no reports of species infecting the H7 subtype avian influenza virus of the North American branch in China, but with the increase in international trade and the impact of migratory bird migration, the risk of the H7 subtype avian influenza virus of the North American branch being introduced into my country is increasing, so there is an urgent need to Establishment of a rapid detection method for North American clade H7 subtype avian influenza virus

Method used

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  • Primer pair for authenticating North America-branch H7 subtype avian influenza virus and application thereof
  • Primer pair for authenticating North America-branch H7 subtype avian influenza virus and application thereof
  • Primer pair for authenticating North America-branch H7 subtype avian influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the design of primer set

[0034] After a large number of sequence analysis, multiple primer pairs were obtained, and the specificity and sensitivity of each primer pair were tested through preliminary experiments, and finally a primer pair for identifying the H7 subtype avian influenza virus of the North American branch was screened out.

[0035] F1 (sequence 1 of the sequence listing): 5'-AATCACTAGGAGTCCAGAGTGATGC-3';

[0036] R1 (SEQ ID NO: 2 of the Sequence Listing): 5'-AGTTCTAGAGTTGATGTTTTGGAAT-3'.

[0037] The annealing temperature was 58°C.

Embodiment 2

[0038] Embodiment 2, establishment of method

[0039] 1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA.

[0040] The sample to be tested can be a tissue to be tested or a virus to be tested. The tissue to be tested can be liver tissue of poultry, spleen tissue of poultry, lung tissue of poultry, oropharyngeal swab or cloacal swab of poultry, etc.

[0041] 2. Using the cDNA obtained in step 1 as a template, the primer pair designed in Example 1 was used to perform fluorescent quantitative PCR, and the fluorescence intensity was monitored in real time.

[0042] Fluorescent quantitative PCR reaction system (20 μL): SYBR Premix Ex Taq (2X) 10 μL, primer F1 0.5 μL, primer R1 0.5 μL, cDNA 2 μL, add double distilled water to 20 μl.

[0043] The reaction program of fluorescent quantitative PCR: 95°C for 10min; 95°C for 15s, 58°C for 1min, 40 cycles.

[0044] 3. To judge the result

[0045] When the sample to be tested is a tissue to be tested, ...

Embodiment 3

[0047] Embodiment 3, establishment of standard curve

[0048] Gene template copy number = DNA mass concentration / DNA molecular weight.

[0049] 1. Preparation of standard plasmids

[0050] The DNA molecule shown in sequence 3 of the sequence listing is a gene fragment of the North American branch H7N2 subtype avian influenza virus. The DNA molecule shown in sequence 3 of the sequence listing was inserted into the multiple cloning site of the pUC57 vector to obtain a standard plasmid.

[0051] Second, the establishment of the standard curve

[0052] 1. Dilute the standard plasmid 10 times with double distilled water to get 1.3×10 8 -1.3×10 3 copies / μL of each dilution.

[0053] 2. Using the dilution solution obtained in step 1 as a template, the primer pair designed in Example 1 was used to perform fluorescent quantitative PCR, and the fluorescence intensity was monitored in real time.

[0054] Fluorescent quantitative PCR reaction system (20 μL): SYBR Premix Ex Taq (2X) ...

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Abstract

The invention discloses a primer pair for authenticating a North America-branch H7 subtype avian influenza virus and application thereof. The primer pair disclosed by the invention consists of a primer F1 and a primer R1, wherein the primer F1 is a single stranded DNA molecule as shown in sequence 1 in the sequence table; and the primer R1 is a single stranded DNA molecule as shown in sequence 2 in the sequence table. Use of the primer pair is as described in (b1) or (b2) as follows: (b1) to assist in authenticating the North America-branch H7 subtype avian influenza virus; (b2) to assist in authenticating whether the North America-branch H7 subtype avian influenza virus is contained in a sample to be detected. By adopting the primer pair and a kit disclosed by the invention for detection, good specificity and high sensibility are realized, a clinical sample can be detected by needing 3-5 hours only, the operation is simple and easy to popularize, and the primer pair disclosed by the invention is convenient for basic operation and application, and has a significant application value for the diagnosis of the North America-branch H7 subtype avian influenza virus and the survey of epidemiology.

Description

technical field [0001] The invention relates to a pair of primers for identifying North American branch H7 subtype avian influenza virus and its application. Background technique [0002] Since the occurrence of H7N3 subtype HPAI in British turkeys in 1963, H7N3, H7N7, H7N4, H7N1, and H7N2 subtypes of influenza viruses have successively occurred in chickens, turkeys, ducks, quails and other varieties of poultry. In addition to the diversification of subtype combinations and the variety of infected hosts, the geographical distribution of H7 subtype avian influenza virus is also very wide. Europe, Canada, the United States and other European and American countries are high incidence areas of H7 subtype avian influenza virus. In recent years, the reports of human infection by H7 subtype influenza virus, especially the novel H7N9 influenza virus, have aroused widespread concern. The H7 subtype influenza virus is a highly pathogenic influenza virus. According to the molecular ge...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/686C12Q2563/107
Inventor 蒲娟王晨曦孙洪磊张谞霄宋晶伟刘金华
Owner CHINA AGRI UNIV
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