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H9 and H10 subtype avian influenza virus triple RT-PCR detection primer group, kit and method

A technology of RT-PCR and avian influenza virus, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of mixed infection, difficult identification and diagnosis, etc., and achieve fast and simple operation, easy operation, highly specific effect

Pending Publication Date: 2020-01-10
GUANGXI VETERINARY RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because AIV-infected animals have similar clinical symptoms, and the phenomenon of mixed infection is also common, it is difficult to make a timely and accurate differential diagnosis in daily diagnosis
In recent years, with the development of molecular biology technology, the application of multiplex PCR technology is also increasing, especially in the field of avian influenza diagnosis, but the method of triple RT-PCR detection to distinguish H9 and H10 subtype AIV mixed infection see also report

Method used

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  • H9 and H10 subtype avian influenza virus triple RT-PCR detection primer group, kit and method
  • H9 and H10 subtype avian influenza virus triple RT-PCR detection primer group, kit and method
  • H9 and H10 subtype avian influenza virus triple RT-PCR detection primer group, kit and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Primer Development and Synthesis

[0023] According to the specific conserved regions of the M gene sequences of different subtypes of avian influenza viruses, and the HA gene sequences of H9 and H10 subtype AIVs, 3 pairs of amplified genes for the M gene, H9 and H10 subtype AIV HA genes were designed and screened through validation. Added specific primers, including primer M-F, primer M-R, primer H9-F, primer H9-R, primer H10-F and primer H10-R. The sequence of primer M-F is shown in SEQ ID NO.1, the sequence of primer M-R is shown in SEQ ID NO.2, the sequence of primer H9-F is shown in SEQ ID NO.3, and the sequence of primer H9-R is shown in SEQ ID NO. .4, the sequence of primer H10-F is shown in SEQ ID NO.5, and the sequence of primer H10-R is shown in SEQ ID NO.6. Primers were synthesized by Treasure Bio (Dalian) Co., Ltd.

Embodiment 2 3

[0024] The establishment and optimization and specificity test of embodiment 2 triple RT-PCR reaction system

[0025] 1. Strains: AIV strains (H1N2, H3N2, H6N2, H9N2, H9N6, NDV, IBV, ARV and ILTV are all preserved by Guangxi Key Laboratory of Veterinary Biotechnology; cDNA templates of AIV (H7N2, H14N5, H15N9 and H16N3) Donated by Pennsylvania State University; AIV strain (H5N1) cDNA templates were donated by Connecticut State University; other AIV strains (H2N3, H4N5, H8N4, H10N3, H11N3, H12N5 and H13N5) strains or cDNA templates were provided by Hong Kong university gift;

[0026] 2. Reagents: SPF chicken embryos used in the experiment were purchased from Beijing Boehringer Ingelheim Viton Biotechnology Co., Ltd., DL1000bp DNA Marker, competent cells, PCR product quick ligation vector, gel recovery kit and small amount of plasmid extraction kit were all Purchased from Beijing Quanshijin Biotechnology Co., Ltd. MLV reverse transcriptase, random primers, 10mmoldNTP, RNA extr...

Embodiment 3

[0031] Embodiment 3 sensitivity test

[0032] 1. Preparation of standard products: refer to HA in [Hoffmann E, Stech J, Guan Y, et al. Universal primerset for the full-length amplification of all influenza A viruses [J]. ArchVirol, 2001, 146:2275-2289] The primers of the full-length gene, using the cDNA of the H9N2 and H10N3 strains as templates to amplify the M gene and the HA gene by RT-PCR, obtain the full-length target fragments, and connect the target fragments to the vector and send them to the company for sequencing . The recombinant plasmids inserted with full-length fragments of HA gene and M gene of H9 and H10 subtype AIV and sequenced correctly were named M-T, H9-T and H10-T, respectively. Use a commercial plasmid extraction kit to extract plasmids containing M-T, H9-T and H10-T respectively, and measure their concentration with a micro-nucleic acid detector, and calculate the corresponding copy number of the sample according to the relevant formula. H9-T and H10-...

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Abstract

The invention provides an H9 and H10 subtype avian influenza virus triple RT-PCR detection primer group, a kit and a method. The design is directed at specific primers of H9 and H10 subtype AIV HA genes and M genes, different primer combinations are tested and screened to obtain a primer combination with a relatively good amplification effect; the concentration of the primers, the proportion amongthe primers, the annealing temperature and the amplification time in a reaction system are continued to be optimized; and finally, a method for simultaneously detecting H9 and H10 subtype AIV is established. The primers and the detection method can be used for simultaneously determining the situation of mixed infection and single infection of the H9 and H10 subtype AIV in samples through one-timePCR reaction; and the method has the advantages of strong specificity, high detection sensitivity and the like, is quick, convenient and easy to operate, is suitable for application and popularization in grass-roots departments, and provides technical support to supervision, prevention and control of the H9 and H10 subtype AIV.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a triple RT-PCR detection primer set, kit and method for H9 and H10 subtype avian influenza viruses. Background technique [0002] Avian influenza virus (AIV) is a member of Orthomyxoviridae. AIV can be divided into 18 different HA subtypes (H1-H18) and 11 different NA subtypes according to the differences in their surface antigens, hemagglutinin (HA) and neuraminidase (NA). Type (N1 ~ N11). In addition, according to its pathogenicity to chickens, AIV can also be divided into highly pathogenic AIV (HPAIV) and low pathogenic AIV (LPAIV). H9 subtype AIV was first discovered in Wisconsin, USA in 1966, and has caused serious economic losses to the poultry industry worldwide. In 1998, the first case of direct human infection by H9N2 subtype AIV was discovered in Guangdong, China, and there have been reports of human cases of H9N2 subtype AIV infection since then. H10 subty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 谢芝勋李丹李孟谢丽基罗思思张艳芳张民秀黄娇玲范晴王盛谢志勤邓显文曾婷婷
Owner GUANGXI VETERINARY RES INST
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