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Primer and probe for detecting rift valley fever virus

A Rift Valley fever virus and probe technology, applied in the field of primer sets and probes for the detection of Rift Valley fever virus, can solve problems such as inability to meet field detection, expensive instruments and equipment, etc.

Inactive Publication Date: 2015-10-28
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several real-time fluorescent RT-PCR methods have been established to detect the disease, but these methods generally require expensive equipment and cannot meet the needs of field detection

Method used

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  • Primer and probe for detecting rift valley fever virus
  • Primer and probe for detecting rift valley fever virus
  • Primer and probe for detecting rift valley fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: The establishment of RPA detection RVFV method

[0015] Primers and probes were designed according to the genome sequence of Rift Valley fever 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).

[0016] Table 1: Primers and probes used for RPA detection of RVFV

[0017]

[0018] Note: K in RVFV-RPAf stands for G or T, and Y in RVFV-RPAr stands for T or C. The 27th base in RVFV-RPAp was modified with a BHQ1 quenching group, the 28th base was replaced with tetrahydrofuran, the 29th base was marked with a FAM luminescent group, and the 3' end was phosphorylated.

[0019] The sensitivity and specificity of the primer...

Embodiment 2

[0030] Example 2 Detection application of RPA detection technology in clinical samples

[0031] Using the method established in Example 1, from July to September 2014, 30 sheep mouth and nose swabs and clinical samples of tissue disease materials submitted for inspection in Yunnan, Guangxi and Guangdong provinces were sampled and tested. Use a commercial kit (High Pure PCR Template Preparation kit from Roche Company) to extract the total nucleic acid template in the sample, and use the established RPA method to detect whether these samples contain RVFV; simultaneously refer to the Real- time RT-PCR method for detection. Results The detection results of the two detection methods were consistent, only the positive control had a positive amplification curve, but no positive samples were detected in the clinical samples.

[0032]

[0033]

[0034]

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PUM

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Abstract

The invention aims at providing a primer and probe for detecting rift valley fever virus by applying an RPA technology. The sequence of a forward primer body is showed in SEQ ID NO:1, the sequence of a reverse primer body is showed in SEQ ID NO:2, and the sequence of a probe is showed in SEQ ID NO:3. According to the primer and probe for detecting the rift valley fever virus by applying the RPA technology, a great number of RPA primers and probes are designed according to a rift valley fever virus genomic sequence, and a primer and probe combination which can rapidly and effectively detect rift valley fever virus nucleic acid can be screened out from the great number of the RPA primers and probes. Rapid detection is conducted by utilizing the set of the primer and probe, an obvious amplification curve can be obtained by taking rift valley fever virus nucleic acid RNA in vitro transcription as a positive control, and other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue virus which serve as a template to conduct the reaction do not have the amplification curve.

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 Rift Valley fever virus. That is, the primers and detection methods used in the detection of Rift Valley fever virus by using Recombinase Polymerase Amplification (RPA) technology. Background technique [0002] Rift valley fever (RVF) is a zoonotic disease caused by Rift valley fever virus (RVFV) in ruminants and humans. The Rift Valley fever virus can be spread by mosquitoes or by contact with blood, body fluids, tissues, or sick animals. The disease affects sheep, goats, and cattle more seriously, and can cause abortion of pregnant animals and death of new animals. Older non-pregnant animals are also more susceptible, but have stronger clinical disease resistance. The susceptibility of animals is related to their own genetic differences. Animals from outside Africa and non-endemic areas o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 李林吴晓东刘春菊邹艳丽王华王清华王志亮
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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