Double fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for duck hepatitis virus I and Muscovy duck parvovirus

A technology of Muscovy duck parvovirus and hepatitis virus, which is applied in the biological field and can solve the problems of high requirements and complexity of reagents and primers

Active Publication Date: 2013-10-02
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is much more complicated to establish a multiplex fluorescent PCR method than a single-plex one, and it has higher requirements on reagents and primers. At the same time, it is necessary to ensure that there is no mutual interference between the fluorescent groups labeled by different probes. The fluorescent quantitative PCR instrument used has Corresponding Multiple Detection Channels
[0005] At present, there are no reports on the detection and diagnosis of duck hepatitis I virus and Muscovy duck parvovirus using multiplex fluorescent quantitative PCR technology

Method used

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  • Double fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for duck hepatitis virus I and Muscovy duck parvovirus
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  • Double fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for duck hepatitis virus I and Muscovy duck parvovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, design and synthesis of primers and Taqman probes

[0045] According to the conserved sequences of duck hepatitis I virus and Muscovy duck parvovirus in GenBank, two pairs of specific primers and two Taqmam probes were designed using Primer Express3.0 software (Table 1).

[0046] Table 1 is the primer and TaqMan probe sequence (5'-3')

[0047]

Embodiment 2

[0048] Embodiment 2, fluorescence quantitative RT-PCR detection

[0049] 1. Determination of fluorescent quantitative RT-PCR detection method

[0050] 1. Sample preparation

[0051]1), nucleic acid extraction

[0052] Refer to the instructions of the TIANamp virus genome DNA / RNA extraction kit to extract the RNA of duck type I hepatitis virus AV2111 (DHV I), Newcastle disease virus and H9 subtype avian influenza, and extract Muscovy duck parvovirus, duck circovirus and goose plague Virus and DNA of duck plague virus AV1221 strain.

[0053] 2), reverse transcription of RNA

[0054] Duck Type I Hepatitis Virus AV2111 RNA was reverse-transcribed to synthesize cDNA, specifically as follows: the following reverse transcription system was established, the total reaction volume was 20 μL, the duck Type I Hepatitis Virus AV2111 RNA in the above 1) was 2 μL (about 20 μg), 4 μL 8mM MgCl 2 (Dalian Bioengineering Co., Ltd., catalog number: DRR0019A), 2 μL 10×PCR buffer (Dalianbao Bioe...

Embodiment 3

[0094] Embodiment 3, the detection of clinical disease material

[0095] The samples to be tested were 118 diseased materials (numbered 1-118) collected from duck populations in Guangxi, and the RNA and DNA of duck diseased materials (liver of sick duck, spleen of sick duck or lung of sick duck) were extracted respectively.

[0096] The DNA and RNA of each sample numbered 1-118 were mixed (volume ratio 1:1) as a template, and fluorescent quantitative RT-PCR was carried out according to 1-3 of Example 2.

[0097] If the reaction result under the 610nm excitation light (ROX) is an S-shaped curve, the sample to be tested contains duck hepatitis I virus; otherwise, the sample does not contain duck hepatitis I virus;

[0098] If the reaction result under the 530nm excitation light (FAM) is an S-shaped curve, the sample to be tested contains Muscovy duck parvovirus; otherwise, the sample does not contain Muscovy duck parvovirus;

[0099] If the reaction results under the 610nm exci...

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Abstract

The invention discloses a double fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for duck hepatitis virus I and Muscovy duck parvovirus. The invention provides a double fluorescence PCR primer group for detecting the duck hepatitis virus I and the Muscovy duck parvovirus. The primer group consists of a primer 1, a primer 2, a primer 3 and a primer 4, wherein the nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are the sequence 1, the sequence 2, the sequence 4 and the sequence 5 in the sequence table sequentially and respectively. The test proves that the primers and the method have the characteristics of short detection time, high sensitivity and high specificity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a dual fluorescence quantitative RT-PCR detection kit for duck type I hepatitis virus and Muscovy duck parvovirus. Background technique [0002] Duck Hepatitis Virus type I (DHV I) is the pathogen of a highly lethal viral infectious disease in ducklings. The disease mainly affects ducklings within 4 weeks of age, and the mortality rate is as high as more than 90%. It is one of the most serious infectious diseases that endanger the duck industry. Muscovy duckling parvovirus (MDPV) is an important pathogen that endangers muscovy duck breeding. It can cause disease in young muscovy ducks aged 1-3 weeks, and the fatality rate can reach 50%-80%. These two viruses are the ones ducklings are most susceptible to and have a very high fatality rate. Duck hepatitis I virus and Muscovy duck parvovirus are the most important pathogens that harm ducklings, and the mortality rate after infection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谢芝勋谢丽基邓显文谢志勤庞耀珊范晴刘加波
Owner GUANGXI VETERINARY RES INST
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