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Duck flavivirus and duck plague virus duplex RT-PCR kit

A RT-PCR, detection kit technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve complex problems

Active Publication Date: 2013-09-25
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 duplex PCR method is much more complicated than that of a single-plex PCR method, which has higher requirements on reagents and primers, and at the same time, it is necessary to ensure that there is no mutual interference between different primers
At present, there is no report on the simultaneous detection and diagnosis of duck yellow virus and duck plague virus using double PCR technology

Method used

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  • Duck flavivirus and duck plague virus duplex RT-PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, design and synthesis of primers

[0033]According to the conserved sequences of the E gene of BYD and the UL6 gene of DPV in GenBank, DNAStar software was used for multiple sequence alignment, and primer5.0 was used to design primers in the conserved regions. Primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (Table 1).

[0034] Table 1 Primer information

[0035]

Embodiment 2

[0036] Embodiment 2, establishment of double RT-PCR detection method

[0037] 1. Extraction of nucleic acid and reverse transcription of RNA

[0038] According to the instructions of the DNA / RNA extraction kit, extract the DNA of duck plague virus, Muscovy duck parvovirus, duck circovirus and egg drop syndrome virus; simultaneously extract duck yellow virus, duck hepatitis virus, H9 subtype avian influenza virus, The RNA of duck-derived Newcastle disease virus was reverse-transcribed into cDNA according to the reverse transcription instructions, as follows: the following reverse transcription system was established, with a total reaction volume of 20 μL, and the above viral RNA was reverse-transcribed according to the reverse transcription instructions (Beijing Quanshi Jin , AT301-02), 8 μL of RNA, 1 μL of random primers, 10 μL of 2×TS Reaction Mix, 1 μL of TransScript RT / RI Enzyme Mix, 10 min at 25 °C, 30 min at 42 °C, 5 min at 85 °C, and end at 4 °C to obtain duck yellow vir...

Embodiment 3

[0049] Embodiment 3, the assembly of detection kit

[0050] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

[0051] Solution A: PCR reaction solution, containing 12.5 μL of 2×Taq PCR Mix (Beijing Quanshijin, AS111-12), 0.5 μL of BYD upstream and downstream primers (both primer concentrations are 50 μmol / mL, and the final concentrations in the reaction system are 1 μmol / mL), 0.5 μL of DPV upstream and downstream primers (both primer concentrations are 50 μmol / mL, and the final concentration in the reaction system is 1 μmol / mL), add ddH 2 O8.5 μL.

[0052] Solution B: Contain 1 μL each of BYD+DPV template as a positive control.

[0053] Solution C: Contains ddH 2 O1μL, as a negative control.

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PUM

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Abstract

The invention discloses a duck flavivirus and duck plague virus duplex RT-PCR kit. The kit contains two pairs of specific primers. The kit has the advantages of simple operation, high sensitivity, strong specificity, good repeatability and the like, can establish a duck flavivirus and duck plague virus duplex RT-PCR detection method, can simultaneously detect and identify the two pathogens of the duck flavivirus and the duck plague virus, can realize the time cost saving and pollution reduction when the kit is used in the duck plague virus mixed infection caused by the immunity reduction caused by the duck flavivirus infection.

Description

technical field [0001] The invention belongs to the technical field of RT-PCR detection kits, in particular to a dual RT-PCR detection kit for duck yellow virus and duck plague virus. Background technique [0002] Bai yang dian virus (BYD), also known as duck Tembusu virus, mainly causes a sharp loss of appetite, a sharp drop in egg production, follicle 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 an outbreak of a disease that caused a sharp decline in duck egg production, causing great economic losses to the laying duck farming industry. Duck plague virus (DPV) is a herpes virus that causes acute septicemia infectious diseases of ducks, geese and other waterfowl. All can be infected, but Muscovy ducks, Shelduck ducks, and Sheep ducks are the most susceptible. In natural epidemics, adult ducks and laying ducks are mor...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
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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