RT-RPA detection reagent kit for quickly detecting pest des petits ruminant viruses and application thereof

A technology of RT-RPA and Peste des petits ruminants, which is applied in the field of detection kits for Peste des petits ruminants virus and RT-RPA detection kit for rapid detection of Peste des petits ruminants virus, which can solve the problems of diagnosing Peste des petits ruminants virus and achieve saving The effect of test time, portability, and simple method

Active Publication Date: 2016-05-11
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the diagnosis of Peste des petits ruminants virus based on RPA at home and abroad

Method used

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  • RT-RPA detection reagent kit for quickly detecting pest des petits ruminant viruses and application thereof
  • RT-RPA detection reagent kit for quickly detecting pest des petits ruminant viruses and application thereof
  • RT-RPA detection reagent kit for quickly detecting pest des petits ruminant viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Establishment of real-timeRT-RPA detection kit and detection method for rapid detection of Peste des petits ruminants virus

[0037] 1. Preparation of Sequences

[0038] Primers and probes were designed for the N region of Peste des petits ruminants virus. At the same time, we compared the N gene homologous sequences of X74443, L39878, EU267274, EU267273, AJ849636, FJ905304, AY560591 and AJ563705 from GenBank to further determine the conserved region of Peste des petits ruminant virus, in order to detect as many small Peste ruminant virus. All primers and probes were synthesized by Sangon Biotech (Shanghai, China).

[0039] 2. Virus strains, cells and clinical samples

[0040] All strains used in this study are preserved by our laboratory: PPRV / Nigeria75 / 1, ORFV / Vaccine / CHA, ORFV / Gansu / CHA, ORFV / HB / CHA, GPVAV40, SPVJingtai2011 and foot-and-mouth disease virus (FMDVtypeA / CHA / 2009; FMDV / O / CHA; FMDV / Asia1 / CHA). The PPRV-positive samples (n=14) and PPRV-nega...

Embodiment 2

[0063] Example 2 Application of PPRVreal-timeRT-RPA detection kit in field detection

[0064] 1. Sample

[0065] 18 samples were collected from sheep with suspected PPRV infection, and 5 tissue samples were from healthy sheep. Virus genome extraction was the same as in Example 1.

[0066] 2. Detection method

[0067] (1) PPRVreal-timeRT-PRA method

[0068] The extracted and purified viral RNA was used as a template for amplification, and the reaction system was as follows: 13.75 μL hydrolysis buffer (rehydration buffer, TwistDxexoKit, Cambridge, United Kingdom), 1.05 μL upstream primer (10 μM), 1.05 μL downstream primer (10 μM), 0.3 μL RPAexo probe (10 μM), 2 μL of viral RNA template, 4.6 μL of ddH 2 O and 1.25 μL of magnesium acetate (magnesium acetate, 280 mM). The sequences of the primers and probes are as follows:

[0069] Upstream primer: 5'-CCAAGGCGGTTACGGCACCGGATACGGCAGCTGAC-3' (shown in SEQ ID NO.1);

[0070] Downstream primer: 5'-ACTGCGTCCAGCCACCCTTTGTCAAGGCGAAA...

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Abstract

The invention discloses an RT-RPA detection reagent kit for quickly detecting pest des petits ruminant viruses and application thereof. The reagent kit comprises a pair of primers and a probe. The sequences of the primers are as shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the probe is as shown in SEQ ID NO.3. Experiments prove that the reagent kit can specifically detect the pest des petits ruminant viruses and does not amplify sheep pox viruses, goat pox viruses, contagious pustular dermatitis sheep viruses, foot and mouth disease viruses and other important sheep infection viruses under the same condition, and it is illustrated that the method has good specificity. Sensitivity experiments show that the RT-RPA primers and the probe for quickly detecting the pest des petits ruminant viruses can at least detect 150 copied templates at the temperature of 40 DEG C and under the condition that amplification is conducted for 20 min and have high conformity with RT-qPCR. Accordingly, the reagent kit can quickly, efficiently and sensitively detect the pest des petits ruminant viruses, and an effective technical means is provided for identification and diagnosis of the pest des petits ruminant viruses.

Description

technical field [0001] The invention relates to a kit for detecting Peste des petits ruminants virus and its use, in particular to an RT-RPA detection kit for rapidly detecting Peste des petits ruminants virus and its use. The invention belongs to the field of preventive veterinary inspection. Background technique [0002] Thirty years have passed since the birth of PCR technology. From classic PCR, real-time quantitative PCR to the current digital PCR, this technology is constantly changing but has never faded out of our field of vision. However, this technology requires special and expensive thermal cycle instruments and skilled operators, which is time-consuming and labor-intensive, and it is difficult to be used in on-site diagnosis and places with poor experimental conditions. Recombinase Polymerase Amplification (RPA) has been known as a nucleic acid detection technology that can replace PCR since it was developed by the British company TwistDx Inc in 2006. based on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2561/113C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 杨洋张志东秦晓东宋一鸣胡高维窦永喜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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