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Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique

A technology of Peste des petits ruminants and reverse primers, applied in the direction of recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as difficult to meet detection needs, tedious test procedures of thermal cyclers, etc.

Inactive Publication Date: 2015-11-04
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main method of virus nucleic acid detection is PCR method. Conventional PCR detection requires a special thermal cycler and cumbersome test procedures, which is difficult to meet the detection needs under field or field conditions.

Method used

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  • Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique
  • Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique
  • Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1: the establishment of detection method

[0013] Primers and probes were designed according to the genome sequence of Peste des petits ruminants virus in GenBank. The length of the primer is about 30-35nt. Since there is no primer design software for RPA at present, a large number of primers were designed and synthesized in the previous work of the present invention, and a pair of primers with high sensitivity and good specificity were screened out. The primer and probe sequence SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3 (Table 1).

[0014] Table 1: Primers and probes for RPA detection of PPRV

[0015]

[0016] Note: R stands for G or A, Y stands for T or C, the 30th base in PPRV-RPAp marks the BHQ1 quenching group, the 31st base is replaced by tetrahydrofuran, and the 32nd base marks FAM The luminescent group is modified by phosphorylation at the 3' end.

[0017] 1. Materials and methods

[0018] (1) Materials Peste des petits ruminants Tibet strai...

Embodiment 2

[0027] Application of Example 2 in Clinical Samples

[0028] The RPA method established in Example 1 was used to detect the sheep mouth and nose swabs and tissue disease samples of 12 clinical suspected cases in China. Use the High Pure PCR Template Preparation kit from Roche to extract the total nucleic acid templates in the samples to be tested, and then use the RPA method to detect whether these samples contain PPRV. At the same time, 12 samples were tested in parallel according to the common RT-PCR method recommended by the World Organization for Animal Health (OIE). Results The results of the two detection methods were consistent, and all 12 samples were positive for PPRV nucleic acid. The above results show that the results obtained by the primer set, probe and detection method of the present invention are consistent with the results of RT-PCR, which proves the reliability of the present invention.

[0029]

[0030]

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Abstract

The invention aims at providing a primer and a probe for detecting peste des petits ruminants virus by virtue of a RPA (Recombinase Polymerase Amplification) technique. According to the primer and the probe, a forward primer sequence is represented by SEQ ID NO:1, a reverse primer sequence is represented by SEQ ID NO:2, and a probe sequence is represented by SEQ ID NO:3. According to a method, a large number of primers and probes are designed according to genomic sequences of the peste des petits ruminants virus, and a pair of primer and probe combinations capable of rapidly detecting nucleic acid of the peste des petits ruminants virus are screened out; obvious amplification curves can be obtained by carrying out rapid detection by virtue of the primer and the probe and taking nucleic acid RNA of the peste des petits ruminants virus of domestically separated Tibet strains and Xinjiang strains as a template, and no amplification curve is obtained through a reaction by taking other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue disease virus as the template.

Description

technical field [0001] The invention belongs to the technical field of veterinary pathogenic microorganism detection, and in particular relates to a primer set and a probe for detecting Peste des Petits Ruminants virus. That is, primers, probes and detection methods for the rapid detection of Peste des petits ruminants virus by using Recombinase Polymerase Amplification (RPA) technology. Background technique [0002] Peste des petits ruminants (PPR) is a severe infectious disease infecting goats and sheep caused by Peste des petits ruminants virus (PPRV), which mainly infects sheep, goats and wild small ruminants animal. Peste des petits ruminants is mainly endemic in Central Africa, West Africa, East Africa, the Middle East and South Asia. The disease was first discovered in my country in 2007, and then there were several small-scale outbreaks, but only in the Ngari area of ​​Tibet. Since it was re-introduced to my country in 2013, it has spread rapidly to most provinces...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/70
Inventor 李林吴晓东邹艳丽刘春菊李金明包静月王志亮
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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