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Duplex fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu virus and egg drop syndrome virus

A technology for duck Tembusu virus and egg reduction syndrome, which is applied to the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc. high effect

Active Publication Date: 2015-01-14
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 fluorophores labeled by different probes. The fluorescent quantitative PCR instrument used with corresponding multiple detection channels
At present, there is no report on the detection and diagnosis of duck Tembusu virus and egg drop syndrome virus using multiplex fluorescent quantitative PCR technology

Method used

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  • Duplex fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu virus and egg drop syndrome virus
  • Duplex fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu virus and egg drop syndrome virus
  • Duplex fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu virus and egg drop syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, design and synthesis of primers and Taqman probes

[0037] According to the conserved sequences of duck Tembusu virus and egg drop syndrome virus in GenBank, multiple sets of probes and primers were designed using Primer Express 3.0 software, and two pairs of specificity were selected by analyzing the dimer between the primers Primers and two Taqmam probes (Table 1).

[0038] Table 1 is the primer and TaqMan probe sequence (5'-3')

[0039]

Embodiment 2

[0040] Embodiment 2, establishment of fluorescence quantitative RT-PCR detection

[0041] 1. Determination of fluorescent quantitative RT-PCR detection method

[0042] 1. Sample preparation

[0043] 1), nucleic acid extraction

[0044] According to the instructions of the TIANamp virus genome DNA / RNA extraction kit, extract the RNA of duck Tembusu virus, Newcastle disease virus and H9 subtype avian influenza, and extract egg drop syndrome virus, duck circovirus, goose plague virus and duck plague The DNA of the virus.

[0045] 2), reverse transcription of RNA

[0046] Duck Tembusu virus reverse transcription synthesized cDNA, specifically as follows: establish the following reverse transcription system, the total reaction volume is 20 μL) of duck Tembusu virus AV2111 RNA is 2 μL (about 20 μg), 4 μL 8mM MgCl 2 (Dalian Bioengineering Co., Ltd., catalog number: DRR0019A), 2 μL 10×PCR buffer (Dalianbao Bioengineering Co., Ltd., catalog number: DRR0019A), 2 μL 10 mM dNTP (four ...

Embodiment 3

[0076] Embodiment 3, the assembly of detection kit

[0077] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

[0078] Solution A: PCR amplification buffer, primer 1, primer 2, probe A, primer 3, primer 4, probe B; among them, PrimeScript TM Reverse Transcriptase (Dalian Bao Biological Engineering Co., Ltd., catalog number: 2680A); Premix Ex Taq TM (Probe qPCR) (Dalian Bao Biological Engineering Co., Ltd., catalog number: RR390A); the final concentrations of primer 1, primer 2, and probe A were all 0.5 μmol / L, and the final concentrations of primer 3, primer 4, and probe B were all 0.2μmol / L;

[0079] Solution B: DTMUV+EDSV template, as a positive control;

[0080] Solution C: ddH 2 O, as a negative control.

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Abstract

The invention discloses a duplex fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu virus and egg drop syndrome virus. The detection kit comprises a primer group and a probe group, wherein the primer group comprises primers 1-4, which have base sequences as shown in SEQ. ID. No. 1 and 2, as well as SEQ. ID. No. 4 and 5 in a sequence table respectively; the probe group comprises a probe A and a probe B, which have the base sequences as shown in SEQ. ID. No. 3 and 6 in the sequence table respectively. Experiments prove that the detection kit disclosed by the invention has the advantages of short detection time, high detection sensitivity and strong specificity, and can realize simultaneous detection and identification of two pathogens of duck tembusu virus and egg drop syndrome virus and quantify the corresponding pathogen content, so that the detection kit can be used for evaluating curative effects of duck tembusu virus and egg drop syndrome virus vaccines and medicaments and researching pathogenic mechanisms and other aspects, and further has important significance in prevention and treatment of duck tembusu virus and egg drop syndrome virus.

Description

technical field [0001] The invention belongs to the technical field of RT-PCR detection kits, in particular to a double fluorescent quantitative RT-PCR detection kit for duck Tembusu virus and egg drop syndrome virus. Background technique [0002] Duck Tembusu Virus (DTMUV) is a harmful pathogen. The disease mainly affects laying ducks and ducklings, causing a sharp decline in egg production and a small number of deaths in laying ducks, resulting in a high rate of elimination of laying ducks. Since the outbreak in 2010, it has seriously endangered the duck industry. Egg Dropping Syndrome Virus (EDSV) also causes a decline in egg production in laying ducks. Duck Tembusu virus and egg drop syndrome virus infect laying ducks, causing similar symptoms, that is, a decline in egg production. Therefore, it is urgent to establish a rapid and sensitive detection method for duck Tembusu virus and egg drop syndrome virus, so that These viruses can be detected in the early stage of in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/70C12Q2563/107C12Q2537/143
Inventor 谢芝勋曾婷婷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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