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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

AI Technical Summary

Problems solved by technology

However, these methods are relatively complicated to operate, and it is difficult to accurately distinguish between Seneca Valley virus infection and foot-and-mouth disease virus infection

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0036] 1) Take clinical samples and freeze and thaw them three times at -80°C for extraction of viral nucleic acids. Total virus RNA was extracted according to the Trizol method.

[0037] 2) Prepare cDNA from the total virus RNA extracted in the above step 1) with a reverse transcription kit, and store it at -80°C for future use. The reaction system is as follows:

[0038] first step

[0039] Random primer 1 μL

[0040] RNA 5.75 μL

[0041] Water bath at 70°C for 10 min, then ice bath for 2 min to obtain the RNA primer mixture

[0042] second step

[0043]

[0044] Water bath at 30°C for 10 minutes, water bath at 42°C for 1 hour, and cool in an ice bath. After reverse transcription, the synthesized cDNA was placed on ice for subsequent experiments or stored at -20°C.

[0045] 2. Screening of specific primers and system optimization of double PCR method for por...

Embodiment 2

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

[0066] 1) Specificity test

[0067] Porcine foot-and-mouth disease virus (FMDV), porcine senega valley virus (SVV), porcine epidemic diarrhea virus (PEDV), porcine vesicular disease virus (SVDV), vesicular stomatitis virus ( VSV), porcine blue ear disease virus (PRRSV), porcine circovirus (PCV), and swine fever virus (CSFV) were amplified, and sterilized 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 target fragment size of porcine foot-and-mouth disease virus (FMDV) is 312bp; the target fragment size of porcine senega valley virus (SVV) is 599bp; , respectively 312bp and 599bp; PEDV, SVDV, VSV, PRRSV, PCV, CSFV virus no specific band amplification.

[0069] 2) Sensitivity test

[0070] a) Single-plex RT-PCR sensitivity test

[0071] The plasmid (10 times) diluted with the mixed plasmid of SVV and FMDV respect...

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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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