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RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) primer group for detecting Seneca valley virus as well as kit and application

A technology of RT-LAMP and primer set, which is applied in the direction of DNA/RNA fragments, microorganisms, recombinant DNA technology, etc., can solve the problems of expensive instruments, difficulty in detecting Seneca Valley virus, and long time consumption, and achieve short amplification time, Low cost, good repeatability and stability

Active Publication Date: 2018-08-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the shortcomings and deficiencies such as difficulty in detecting Seneca Valley virus in the prior art, time-consuming and expensive instruments, the primary purpose of the present invention is to provide a kind of RT-LAMP primer set for detecting Seneca Valley virus, which primer set can Rapid, convenient and efficient detection and identification of Seneca Valley virus with good specificity and high sensitivity

Method used

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  • RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) primer group for detecting Seneca valley virus as well as kit and application
  • RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) primer group for detecting Seneca valley virus as well as kit and application
  • RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) primer group for detecting Seneca valley virus as well as kit and application

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Experimental program
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Effect test

Embodiment 1

[0061] 1. Primer design

[0062] According to the reference and comparison of Seneca Valley virus gene sequences in GenBank (Acession No.MF893200, Accession: NC-011349.1, Accession: KC667560.1, Accession: DQ641257.1, Accession: KT321458.1, Accession: KY419132.1, Accession: KY038016.1, Accession: KX377924.1, Accession: MG428685.1, Accession: MG428684.1, Accession: MG428683.1, Accession: KX857728.1, Accession: KX173339.1, Accession: KY747512.1, Accession: KY747510.1, Accession: KU954090.1, Accession: KU954089.1, Accession: KT757282.1, Accession: KT757281.1, Accession: KR063109.1, Accession: KR063108.1, etc.), using MegAlign software for sequence alignment, Determine the conserved nucleotide sequence as the amplification region, and use the RT-LAMP method primer-assisted design software PrimerExplorer V5 (http: / / primerexplorer.jp / e / ) software to design three sets of detection Seneca according to the determined conserved nucleotide sequence RT-LAMP primer set for Valley virus gen...

Embodiment 2

[0071] (1) Extraction of viral genome RNA

[0072] Use viral genome DNA / RNA extraction kit (Corning Life Sciences (Wujiang) Co., Ltd., catalog number 08317KC5) to extract Seneca Valley virus (Seneca Valley virus SVV HN16 strain) viral RNA, and control virus (Seneca Valley virus (SVV) HN16 strain, foot-and-mouth disease virus (FMDV) vaccine strain, porcine D-coronavirus (PDCoV) Ch-A strain, porcine epidemic diarrhea virus (PEDV) CH / GDGZ / 2012 strain, porcine transmissible gastroenteritis virus ( Genomic RNA of TGEV) CN12 strain, porcine blue ear disease virus (PRRSV) YA strain and standard positive serum of classical swine fever virus).

[0073] (2) RT-LAMP reaction system establishment and system optimization

[0074] Construct 25 μ L RT-LAMP reaction system (table 2), successively to reaction temperature, reaction time, Mg 2+ , dNTP, BstDNA Polymerase concentration, AMV concentration, RT-LAMP inner primer and outer primer ratio are optimized, and the results obtained are det...

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Abstract

The invention relates to the technical field of biotechnologies and in particular relates to an RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) primer group for detecting a Seneca valley virus as well as a kit and application. The RT-LAMP primer group for detecting the Seneca valley virus has nucleotide sequences shown as SEQ ID NO. 1-6. The invention further provides a kitcomprising the primer group. The kit adopts a reverse transcription-loop-mediated isothermal amplification technology, depends on primers capable of identifying six specific regions on a target sequence and BstDNA polymerase which has an unwinding function and enables the target sequence to be in loop-mediated isothermal amplification, and is capable of efficiently, rapidly and specifically amplifying the target sequence under isothermal conditions. The primer group and the kit disclosed by the invention are applicable to export quarantine, food hygiene and field detection of livestock farms and are convenient for wide-range popularization and application.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to an RT-LA MP primer set, kit and application for detecting Seneca Valley virus. Background technique [0002] Porcine Seneca Valley virus (Seneca Valley vires, SVV) is a newly emerged virus that can infect piglets and sows and cause death of piglets. The virion is an icosahedral non-enveloped single strand, positive strand, non-segmented RNA virus with a diameter of about 27 nm, and is the only member of the genus Seneeavirus of the family Picornaviridae. The viral genome contains 7280 nucleotides. [0003] The clinical symptoms of porcine Seneca Valley virus disease are specifically manifested as obvious blisters in the snout and crown of the hooves of sick animals, accompanied by symptoms such as walking, anorexia, lethargy and fever. The disease is highly contagious and has a high incidence rate, and the mortality rate of newborn piglets at the age of 1 to 3 days can re...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 贺东生李锦辉
Owner SOUTH CHINA AGRI UNIV
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