Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses

A duck hepatitis A virus, dual fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of inconvenient control, unreported content and dynamic distribution, and single weight.

Inactive Publication Date: 2014-09-17
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

Single and mixed infection of type 1 duck hepatitis A virus DHAV-1 (Duck hepatitis A virus type 1, DHAV-1) and type 3 duck hepatitis A virus DHAV-3 (Duck hepatitis A virus type 3, DHAV-3) in duck populations in China The p...

Method used

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  • Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses
  • Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses
  • Dual fluorescence quantitative method for quickly identifying type 1 and type 3 duck hepatitis A viruses

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Embodiment Construction

[0026] 1 Primer design

[0027] The present invention compares the sequences of DHAV-1 and DHAV-3 published on the reference GenBank, selects the conserved regions of the gene sequences of these two viruses, and designs a Taqman probe Probe1 and a pair of DHAV-1 probes respectively. Specific detection primers SEQ1 / SEQ2 for DHAV-1; one Taqman probe Probe2 for DHAV-3, and a pair of specific detection primers SEQ3 / SEQ4 for DHAV-3. In order to carry out sensitivity measurement to the 3 double fluorescence quantitative RT-PCR methods of establishment, designed respectively a pair of DHAV-1 standard substance primers SEQ5, SEQ6 (to prepare DHAV-1 nucleic acid template standard substance to detect the sensitivity of establishment method) and A pair of DHAV-3 standard primers SEQ7, SEQ8 (used to prepare DHAV-3 nucleic acid template standard to detect the sensitivity of the established method).

[0028] SEQ1: 5'-AGACACATGTTGCTGAAAAACT-3',

[0029] SEQ2: 5'-AGAACCAGTTGTCGTTTGGTC-3', ...

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Abstract

The invention provides a dual fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) specific primer for detecting type 1 and type 3 duck hepatitis A viruses. A dual fluorescence quantitative method comprises the following steps: comparing sequences of DHAV-1 and DHAV-3 published on GenBank, and designing a pair of specific detection primers SEQ1 and SEQ2 aiming at DHAV-1 and a Taqman probe (probe1) as well as a pair of specific detection primers SEQ3 and SEQ4 aiming at DHAV-3 and a Taqman probe (probe2) respectively in a region with conservation and relatively large difference between gene sequences of the two viruses; confirming the concentration of the dual fluorescence quantitative RT-PCR specific primer and the Taqman probe aiming at DHAV-1 and DHAV-3. The dual fluorescence quantitative method built by virtue of the group of primers is good in specificity and high in sensitivity, and can be used for quick serum type identification and real-time quantitative analysis of the type 1 and type 3 duck hepatitis A viruses.

Description

(1) Technical field [0001] The invention relates to a double fluorescence quantitative RT-PCR method for quickly identifying mixed infection of type 1 duck hepatitis A virus and type 3 duck hepatitis A virus, belonging to the field of virus molecular biology. (2) Background technology [0002] Duck hepatitis virus includes three different serotypes of duck hepatitis A virus (DHAV), duck astrovirus (DAstV) type 1 (DAstV-1) and type 2 (DAstV-2). There is no antigenic cross-reactivity between them. DHAV, as the only member of the genus Avian Hepavirus in the Picornaviridae family, is currently divided into three genotypes and serotypes: DHAV-1, DHAV-2 and DHAV-3, and there is almost no cross protection between the three serotypes. The main prevalent ones are DHAV-1 and DHAV-3. In recent years, the mixed infection of DHAV-1 and DHAV-3 has become increasingly serious, leading to the fact that many areas cannot effectively control the occurrence and prevalence of duck viral hepa...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2561/101C12Q2537/143Y02A50/30
Inventor 姜世金林少莉张瑞华
Owner SHANDONG AGRICULTURAL UNIVERSITY
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