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Duck Flavovirus and Duck Circovirus Duplex RT-PCR Detection Kit

A technology of RT-PCR and duck circovirus, applied in the field of dual RT-PCR detection kits for duck yellow virus and duck circovirus, can solve complex problems

Active Publication Date: 2016-03-09
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment of a double or multiplex PCR method is much more complicated than that of a single multiple PCR method, which has higher requirements on reagents and primers, and it is necessary to ensure that there is no mutual interference between different primers

Method used

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  • Duck Flavovirus and Duck Circovirus Duplex RT-PCR Detection Kit
  • Duck Flavovirus and Duck Circovirus Duplex RT-PCR Detection Kit
  • Duck Flavovirus and Duck Circovirus Duplex RT-PCR Detection Kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, design and synthesis of primers

[0036] According to the conserved sequences of BYD's E gene and DuCV's REP gene in GenBank, DNAStar software was used for multiple sequence alignment, and PrimerPremier5.0 was used to design primers in the conserved regions. Primers (see Table 1) were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0037] Table 1 Primer Information

[0038]

Embodiment 2

[0039] Embodiment 2, establishment of double RT-PCR detection method

[0040] 1. Extraction of nucleic acid and reverse transcription of RNA

[0041] Refer to the instructions of the DNA / RNA extraction kit to extract the DNA of duck circovirus, muscovy duck parvovirus, duck plague virus and egg drop syndrome virus; simultaneously extract duck yellow virus, duck hepatitis virus, muscovy duck reovirus, For the RNA of H9 subtype avian influenza virus and duck-derived Newcastle disease virus, the RNA was reverse-transcribed into cDNA according to the reverse transcription instructions, as follows: the following reverse transcription system was established, the total reaction volume was 20 μL, and the above-mentioned viral RNA was reverse-transcribed Instruction manual (Beijing Quanshijin, AT301-02), 8 μL of RNA, 1 μL of random primers, 10 μL of 2×TSReactionMix, 1 μL of TransScriptRT / RIEnzymeMix, 10 min at 25 °C, 30 min at 42 °C, 5 min at 85 °C, and end at 4 °C to obtain duck yello...

Embodiment 3

[0052] Embodiment 3, the assembly of detection kit

[0053] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

[0054] Solution A: PCR reaction solution, containing 12.5 μL of 2×TaqPCRMix (Beijing Quanshijin, AS111-12), 0.5 μL of BYD upstream and downstream primers (both primer concentrations are 50 μmol / mL, and the final concentration in the reaction system is 1 μmol) / mL), each 0.5 μL of DuCV upstream and downstream primers (primer concentration is 50 μmol / mL, and the final concentration in the reaction system is 1 μmol / mL), add ddH 2 O8.5 μL;

[0055] Solution B contains 1 μL each of BYD+DuCV template as a positive control;

[0056] Solution C is 2μLddH 2 O, as a negative control.

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Abstract

The invention discloses an amphimorphic RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit of a duck flavivirus and a duck circovirus. The kit contains two pairs of specific primers. The kit is simple and convenient to operate, high in sensibility, strong in specificity, good in repeatability and the like. A detection method of the amphimorphic PCR of the duck flavivirus and the duck circovirus can be established by applying the kit, so that two pathogens, namely, the duck flavivirus and the duck circovirus, can be detected and identified at the same time. When the kit is used for detecting mixed infection of the duck circovirus due to the hypoimmunity caused by the infection of the duck flavivirus, the time cost is saved and the pollution is reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double RT-PCR detection kit for duck yellow virus and duck circovirus. Background technique [0002] Baiyangdianvirus (BYD), also known as duck Tembusu virus, mainly causes a sharp loss of appetite, a sharp drop in egg production, follicular degeneration, and theca hemorrhage in breeding ducks and laying ducks. Since 2010, Fujian, Zhejiang, Jiangsu, Shandong, Anhui, Henan, Hebei, Guangdong and other places in China have successively reported the outbreak of a disease that caused a sharp decline in duck egg production, causing great economic losses to the laying duck farming industry. Duck circovirus (Duck Circovirus, DuCV) is a member of the Circovirus genus in the Circoviridae family and was first reported in Germany. In recent years, there have been reports of duck infection or disease caused by duck circovirus in Hungary, the United States, Taiwan, my country, Shandong, Liaonin...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2531/113
Inventor 谢芝勋张艳芳谢丽基刘加波庞耀珊范晴罗思思邓显文谢志勤
Owner GUANGXI VETERINARY RES INST
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