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Primer and probe for real-time fluorescence quantification PCR detection of duck II hepatitis A virus

A real-time fluorescence quantitative, hepatitis A virus technology, applied in the field of animal epidemiology, to achieve the effect of rapid detection, good repeatability and high sensitivity

Inactive Publication Date: 2019-04-05
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant research reports on primers, probes and methods for detecting duck type 2 hepatitis A virus by real-time fluorescent quantitative PCR at home and abroad. The establishment of the present invention can fill in the gaps in related fields at home and abroad

Method used

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  • Primer and probe for real-time fluorescence quantification PCR detection of duck II hepatitis A virus
  • Primer and probe for real-time fluorescence quantification PCR detection of duck II hepatitis A virus
  • Primer and probe for real-time fluorescence quantification PCR detection of duck II hepatitis A virus

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Embodiment 1

[0032] 1. Design and synthesis of primers and probes

[0033] Referring to the gene sequence characteristics of duck type 2 hepatitis A virus (duck type 2 hepatitis A virus 90D strain, GenBank accession number is EF067924), specific primers (DHAV-2-F, DHAV -2-R) and probe (DHAV-2-P), the relevant primer sequences are:

[0034] Upstream primer DHAV-2-F: 5'-CTGTGATTTGTGCAGAAA-3',

[0035] Downstream primer DHAV-2-R: 5'- GGTCTGATTCAATTTCAAGA-3';

[0036] The probe sequence DHAV-2-P is: 5'-CTCGCAATCGCAGACCATTCAG-3', its 5'-end is labeled with a fluorescent reporter group FAM, and its 3'-end is labeled with a fluorescent quencher group Eclipse. Primers and probes were synthesized by Baoriyi Biotechnology (Beijing) Co., Ltd.

[0037] 1. Construction of standard products

[0038] In this study, the full-length sequence of DHAV-2 (90D strain) 2A2 gene was directly synthesized by the whole gene synthesis technology. The whole gene synthesis was carried out in Sangon Bioengineering ...

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Abstract

The invention relates to a primer and a probe for real-time fluorescence quantification PCR detection of duck II hepatitis A virus and belongs to the field of epizootiology. The invention includes design of specific primer and probe sequences, construction of standard plasmids, establishment and optimization for a real-time fluorescence quantification PCR amplification method, and detection and judgement for results. The method for real-time fluorescence quantification PCR detection of duck II hepatitis A virus established by the invention has high sensitivity, high stability, high specificityand high repeatability for detecting duck II hepatitis A virus, is capable of detecting at least 33.7 copies and can be used for detecting duck II hepatitis A virus infection in a clinical sample.

Description

technical field [0001] The invention relates to a real-time fluorescent quantitative PCR detection primer and probe of duck type 2 hepatitis A virus (DHAV-2), belonging to the field of animal infectious diseases. Background technique [0002] Currently, the pathogen causing duckling viral hepatitis is duck hepatitis A virus (DHAV), which is a member of the genus Avihepatovirus in the family Picornaviridae, and is divided into three genotypes: DHAV-1 , DHAV-2 and DHAV-3. The complete genome of DHAV virus is about 7.7kb long, and it is single-stranded positive-strand RNA. Genomic RNA is composed of 5' non-translated region (untranslated region, UTR), an open reading frame (ORF), 3' UTR and poly (A) tail. The complete viral polypeptide chain (polyprotein) (that is, the open reading frame ORF). The 5'UTR is 626nt long and contains abundant secondary structures. The 3′UTR of DHAV is 314~317nt long, which is the virus with the longest 3′UTR among known Picornaviridae members. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/706C12Q2561/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 万春和黄瑜陈翠腾施少华傅光华程龙飞陈红梅傅秋玲刘荣昌
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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