Dual PCR primer, detection method and kit for detecting porcine mouth-foot disease virus and Seneca valley virus

A porcine foot-and-mouth disease virus and detection kit technology, which is applied in the field of animal disease detection, can solve the problems that it is difficult to accurately distinguish between Seneca Valley virus infection and foot-and-mouth disease virus infection, complicated operations, etc.

Inactive Publication Date: 2018-04-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are relatively complicated to operate, and it is difficult to accuratel

Method used

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  • Dual PCR primer, detection method and kit for detecting porcine mouth-foot disease virus and Seneca valley virus
  • Dual PCR primer, detection method and kit for detecting porcine mouth-foot disease virus and Seneca valley virus
  • Dual PCR primer, detection method and kit for detecting porcine mouth-foot disease virus and Seneca valley virus

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0034] Example 1

[0035] 1. Preparation of porcine foot-and-mouth disease virus and Seneca Valley virus nucleic acid templates

[0036] 1) Take clinical samples at -80°C for 3 times of freezing and thawing for viral nucleic acid extraction. The total viral RNA was extracted according to the Trizol method.

[0037] 2) The total viral RNA extracted in the above step 1) was used to prepare cDNA with a reverse transcription kit, and stored at -80°C for later use. The reaction system is as follows:

[0038] first step

[0039] Random primer 1μL

[0040] RNA 5.75μL

[0041] 70°C water bath for 10 min and ice bath for 2 min to obtain RNA primer mix

[0042] second step

[0043]

[0044] 30°C water bath for 10min, 42°C water bath for 1h, ice bath cooling, after reverse transcription, the synthesized cDNA was placed on ice for subsequent experiments or stored at -20°C.

[0045] 2. Screening of specific primers for swine foot-and-mouth disease virus and Seneca Valley virus dou...

Example Embodiment

[0064] Example 2

[0065] Performance verification of the kit of the present invention

[0066] 1) Specificity test

[0067] The optimized multiplex RT-PCR method was used to detect swine foot-and-mouth disease virus (FMDV), porcine Senegal valley virus (SVV), porcine epidemic diarrhea virus (PEDV), porcine vesicular disease virus (SVDV), vesicular stomatitis virus ( VSV), porcine blue ear virus (PRRSV), porcine circovirus (PCV), and swine fever virus (CSFV) were amplified, and sterile water was selected as a negative control to test the specificity of the established method.

[0068] The result is as Figure 4 As shown, the size of the target fragment of swine foot-and-mouth disease virus (FMDV) is 312 bp; the size of the target fragment of swine Seneca Valley virus (SVV) is 599 bp; , 312bp and 599bp, respectively; no specific band amplification was found for PEDV, SVDV, VSV, PRRSV, PCV, and CSFV viruses.

[0069] 2) Sensitivity test

[0070] a) Singleplex RT-PCR sensiti...

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Abstract

The invention provides a dual PCR primer, detection method and kit for detecting porcine mouth-foot disease virus and Seneca valley virus. The dual PCR primer is designed according to a highly-conserved specific sequence for detecting porcine mouth-foot disease virus and Seneca valley virus, and the detection method for monotube synchronous detecting of porcine mouth-foot disease virus and Senecavalley virus is established. The kit has the advantages of quickness, simplicity, convenience and high specificity, sensitivity and reliability, sample analysis on a large scale can be performed at the same time, whether a sample is infected with the porcine mouth-foot disease virus or the Seneca valley virus or the two can be identified and diagnosed through single reaction, powerful technical support is provided for monitoring, preventing and controlling porcine mouth-foot disease virus and Seneca valley virus epidemic, and the kit has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of animal disease detection, and in particular relates to a double PCR primer, a detection method and a kit for detecting porcine foot-and-mouth disease virus and Senega Valley virus. Background technique [0002] Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is a member of the genus Aphthovirus. It is a single-stranded positive-sense RNA without an envelope. So far, 7 serotypes, more than 80 subtypes and many distinct virus strains have been discovered. O-type, A-type and Asian I-type are mainly popular in our country. [0003] Seneca Valley virus (SVV) is a typical representative of the genus Senecavirus in the family Picornaviridae. After analyzing the genetic sequence of SVV-001, Hales et al. listed Senecavirus as a new virus genus of Picornaviridae (Hales et al., 2008), which is a single-stranded positive-sense RNA without an envelope. SVV was initially thought to be a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 贺东生徐帅飞苏丹萍罗天霞俞丽
Owner SOUTH CHINA AGRI UNIV
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