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A primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR

A real-time fluorescent quantitative and astrovirus technology, applied in the biological field, can solve the problems of primers, probes and their application methods that have a great impact on the development of the duck industry and have not yet seen fluorescent quantitative, and achieve high sensitivity, good repeatability, Easy to use effect

Active Publication Date: 2022-05-17
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

The antigenicity and genome sequence homology differences among the three DAstV are obvious
[0006] Duck astrovirus DAstV-3 infection in duck populations in my country has a wide distribution range and has a great impact on the development of the duck industry. At present, virus isolation and conventional PCR methods are mainly used to detect duck astrovirus DAstV-3 at home and abroad, and no real-time quantitative method has been found. Related research reports on fluorescent quantitative primers, probes and their application methods

Method used

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  • A primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR
  • A primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR
  • A primer probe set, kit and detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR

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preparation example Construction

[0032] In the present invention, the preparation method of the plasmid standard preferably comprises: using the duck astrovirus DAstV-3 nucleic acid extracted by the DNA / RNA extraction kit (EasyPure Viral DNA / RNA Kit) as a template, and using the upstream primer DAstV-3F2 Carry out RT-PCR amplification with the downstream primer DAstV-3R2, the size of the amplified fragment is 278bp, (the amplified fragment contains the target gene sequence detected by real-time fluorescence quantitative PCR). The above primers were synthesized in Fuzhou Boshang Biotechnology Co., Ltd.

[0033] In the present invention, the sequence of the upstream primer DAstV-3F2 is as the nucleotide sequence shown in SEQ ID No.5 in the sequence listing; the sequence of the downstream primer DAstV-3R2 is as shown in SEQ ID No.6 in the sequence listing The nucleotide sequence shown.

[0034] In the present invention, the nucleotide sequence shown in SEQ ID No. 5 is TGTATGGATAA TGAAATTGAT; the nucleotide sequ...

Embodiment 1

[0044] (1) Design and preparation of DAstV-3 fluorescent quantitative PCR primers and probes

[0045] Referring to the conserved region sequence of the duck astrovirus DAstV-3 non-structural protein nsp1a, a pair of primers with a 278bp amplification target fragment were designed using the primer design software Primer 5, and the amplified products were used to prepare standard products. A conservative sequence was selected, and the primer design software Beacon designer 7.5 was used to amplify 148bp fluorescent quantitative PCR primers (DAstV-3F1 and DAstV-3R1) and TaqMan fluorescent probes (DAstV-3P). The PCR amplification primers and probes The needle sequences are:

[0046] Upstream primer DAstV-3F1: 5'-CGCAATGAACAAATAGATCGGG-3'

[0047] Downstream primer DAstV-3R1: 5'-ATGTGTAAGAGGTGCCTGATCAC-3'

[0048] The probe DAstV-3P is: 5'-FAM-TCGCCCAGAAGAAAGCCCTTAAAA CT-TAMRA-3', wherein the 5' end of the probe is labeled with a fluorescent reporter group FAM, and the 3' end is l...

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Abstract

The invention provides a primer probe set for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR, including primers and probes, a kit and a detection method, and relates to the field of biotechnology. The primers and probes provided by the invention are suitable for fluorescent quantitative PCR amplification, and can be accurately identified from duck hepatitis A virus, avian influenza virus, duck Tembusu virus, duck plague virus, duck reovirus and duck parvovirus Duck Astrovirus type 3, with a specificity of 100%. The present invention also provides a real-time fluorescence quantitative PCR-based test kit for detecting duck astrovirus type 3 and a detection method thereof. The kit can conveniently and accurately identify the presence of duck astrovirus type 3, and is easy to operate and has high sensitivity , the detection limit was 21.3 copies, the coefficient of variation within the group was 0.58-2.21%, and the coefficient of variation between groups was 0.68-2.53%, with good repeatability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer probe set, a kit and a detection method for detecting duck astrovirus type 3 based on real-time fluorescent quantitative PCR. Background technique [0002] Duck astrovirus (DAstV) is one of the important pathogens causing duck viral hepatitis. It is a member of the genus Avastrovirus (Astroviridae) -1), duck astrovirus type 2 (DAstV-2) and duck astrovirus type 3 (DAstV-3). DAstV is a non-enveloped virus with a single-stranded positive-sense RNA genome of about 7.4kb in length, including three main open reading frames (ORFs) (ORF1a, ORF1b, ORF2), which encode two non-structural proteins of the virus, nsp1a, nsp1b, RNA-dependent RNA polymerase (RDRP) and viral capsid protein, etc., the 5' end and 3' end of the viral genome are non-coding regions, and the 5'-end non-coding region is 19 nt long, containing the CCGAA motif, The 3' non-coding region is 219nt, contain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/706C12Q1/6851C12Q2561/113C12Q2563/107C12Q2531/113Y02A50/30
Inventor 傅光华黄瑜傅秋玲程龙飞施少华万春和陈红梅刘荣昌林建生
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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