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Primer and method for detecting peste des petits ruminant virus and bluetongue virus

A technology for Peste des petits ruminants and bluetongue virus, applied in biochemical equipment and methods, microbe measurement/testing, DNA/RNA fragments, etc., can solve problems such as no effective treatment drugs, uncertain results, and difficulty in multiple distinctions , to achieve the effects of convenient reading, suppression of false positives, and good specificity

Active Publication Date: 2019-08-23
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different from the PCR-specific target band, the LAMP product electrophoresis shows a ladder-like band; no matter whether it is single-weight or multiple-weight, the sediment and color change of the LAMP detection, the positive results are presented in the same way, and it is not sure which one it is. It is difficult to make multiple distinctions as a result of pathogenic reactions
[0004] Peste des petits ruminants and bluetongue have similar symptoms and are difficult to distinguish. It is easy to be misdiagnosed clinically. At present, bluetongue and peste des petits ruminants are spreading rapidly in my country, with a large span, high risk and severe situation. The two diseases There is no effective treatment yet, only vaccines can be used for prevention

Method used

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  • Primer and method for detecting peste des petits ruminant virus and bluetongue virus
  • Primer and method for detecting peste des petits ruminant virus and bluetongue virus
  • Primer and method for detecting peste des petits ruminant virus and bluetongue virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1, the establishment of a multiple fluorescent RT-LAMP method for simultaneous detection of BTV and PPRV

[0067] (1) Primer design

[0068]Download the NS3 gene of BTV and the N gene sequence of PPRV registered on Genebank, and use MEGA5.0 for alignment analysis, and use primer premier5.0 and Primer explore V4 to design 2 sets of LAMP-specific primers. Each set of primers targets 6 sites, including 4 primers: outer primers F3 and B3, inner primers FIP (FIP=F1c+F2) and BIP (BIP=B1c+B2), the 5' ends of the two inner primers are labeled respectively Different fluorescent groups, PPRV-FIP labeled FAM fluorescence, yellow-green at 520nm wavelength, BTV-FIP labeled CY5 fluorescence, bright red at 670nm wavelength. Primers were synthesized by Dalian Bao Biological Company and purified at HPLC grade. The specific nucleotide sequences of the primers are shown in Table 1.

[0069] Table 1

[0070]

[0071] In Table 1, the nucleotide sequences of primers PPRV-F3, P...

Embodiment 2

[0082] Embodiment 2, verification experiment of method

[0083] (1) Specificity experiment

[0084] In order to assess the specificity of the multiple fluorescent RT-LAMP method established in Example 1, the strains of common sheep virus diseases in Table 2 were amplified using the multiple fluorescent RT-LAMP reaction system and reaction process described in Example 1, The experimental results are shown in Table 2 and figure 1 shown.

[0085] Table 2 and figure 1 The experimental results show that the multiple fluorescent RT-LAMP primers and methods provided in Example 1 only amplify BTV and PPRV nucleic acids, and the detection results of FMDV, VSV, BVDV, MV, IBRV, EHDV, RPV, GTPV, and SPPV are all negative. This result shows that the multiple fluorescent RT-LAMP primers and method provided in Example 1 have good specificity.

[0086] Table 2

[0087]

[0088]

[0089] GVRI: Guangxi Veterinary Research Institute; YNCIQ: Yunnan Entry-Exit Quarantine Inspection Bure...

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Abstract

The invention provides a primer and method for detecting a peste des petits ruminant virus and a bluetongue virus. According to the invention, a screened primer combination is designed, and finally itis achieved for the first time that in the same reaction tube, the peste des petits ruminant virus (PPRV) and the bluetongue virus (BTV) are simply, rapidly and accurately identified through a multiple-fluorescence RT-LAMP detection method. The multiple-fluorescence RT-LAMP primer and the method for detecting the peste des petits ruminant virus and the bluetongue virus are good in specificity, high in sensitivity and small in interference, can detect 100 mixed template copies / reactions at the minimum, and can effectively inhibit false positive results. By adopting the primer and the method, visual and accurate result judgment is achieved through differences of colors displayed by fluorescent groups, amplification can be completed only by one water bath kettle, the cost is low, the operation is convenient, the method is simple, convenient, rapid and low-cost diagnosis method, and the primer and the method are suitable for large-scale epidemiological investigation of the BTV and PPRV.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a primer and a method for detecting Peste des Petits Ruminants virus and bluetongue virus Background technique [0002] Peste des petits ruminants and bluetongue are two important acute infectious diseases of ruminants. Peste des petits ruminants is caused by Peste despetitsruminants (PPRV) of the paramyxoviridae family (paramyxoviridae) and the genus morbillivirus. It can be transmitted through contact and mainly infects small ruminants such as goats, sheep, and deer. It can be as high as 90% to 100%, and the fatality rate can reach 50% to 100% in severe cases. There is only one serotype of PPRV, and the virus is divided into four lines (I, II, III, and IV) according to the gene evolution tree. In 2007, the PPRV epidemic was first discovered in Tibet, China. In 2014, PPRV re-emerged. The epidemic has spread to nearly 20 provinces, causing huge economic loss...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2537/143C12Q2521/107
Inventor 谢芝勋范晴谢志勤熊文婕黄娇玲张艳芳曾婷婷谢丽基
Owner GUANGXI VETERINARY RES INST
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