K196R gene-based African swine fever virus detection kit and detection method

A technology of African swine fever virus and detection kit, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of high specificity, low sensitivity, and low cost, and achieve The effect of strong specificity and high sensitivity

Active Publication Date: 2019-01-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescent antibody test and antigen capture test are used to detect African swine fever virus antigen, which is fast and accurate, but has low sensitivity and is prone to false negative results; conventional PCR method has high specificity, low cost, but low sensitivity; Fluorescent quantitative PCR technology has the advantages of high efficiency, high sensitivity, and strong specificity, and has been applied in the detection of African swine fever virus nucleic acid

Method used

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  • K196R gene-based African swine fever virus detection kit and detection method
  • K196R gene-based African swine fever virus detection kit and detection method
  • K196R gene-based African swine fever virus detection kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0066] The design of embodiment 1 primer and probe

[0067] A pair of specific primers and a probe were designed for the nucleotide sequence of the K196R gene with reference to the Georgian pig ASFV genome series published in GenBank. The specific primers are shown in SEQ ID No.3 and SEQ ID No.4; the probe sequence is shown in SEQ ID No.2, and the 5' end of the probe is marked with FAM, and the 3' end is marked with BHQ. Primer sequences and probes were synthesized by BGI. The K196R gene sequence is shown in SEQ ID No.1.

Embodiment 2

[0068] Preparation of positive control in embodiment 2 kit

[0069] The positive control recombinant plasmid pUC57-ASFV-K196R was synthesized by BGI and sequenced. After the sequencing results were compared with the GenBank sequence, the concentration and purity of the plasmid were determined by an automatic microplate reader.

Embodiment 3

[0070] Example 3 Virus Genomic DNA Extraction

[0071] According to the operating instructions of the virus genome DNA extraction kit, extract the whole vaccine of pseudorabies live vaccine (Kartha-K61 strain), porcine parvovirus disease inactivated vaccine (WH-1) and porcine circovirus type 2 inactivated vaccine (LG strain) Genome.

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Abstract

The invention discloses a K196R gene-based African swine fever virus detection kit and a detection method, and relates to the technical field of virus detection. The invention provides a group of newQPCR (Quantitative Polymerase Chain Reaction) composition used for detecting an African swine fever virus. The QPCR composition comprises the following primers and a probe used with the primers: a K196R upstream primer having a nucleotide sequence as shown by SEQ ID No.3, a K196R downstream primer having a nucleotide sequence as shown by SEQ ID No.4, and a probe having a nucleotide sequence as shown by SEQ ID No.2. The composition can be used for efficiently and quickly detecting whether a sample contains the African swine fever virus or not, and is high in detection sensitivity and high in specificity. The invention further provides an African swine fever virus detection kit and a method for detecting the African swine fever virus by using the QPCR composition provided by the invention.

Description

technical field [0001] The invention relates to the technical field of virus detection. More specifically, it relates to a K196R gene-based African swine fever virus detection kit and detection method. Background technique [0002] African swine fever (ASF) is an acute, highly contagious disease of pigs caused by African swine fever virus (ASFV). African swine fever virus belongs to the African swine fever virus family (Asfarviridae). It is a single-molecule linear double-stranded DNA virus with an envelope. It is also the only DNA virus among arboviruses. 200 kinds of proteins, the genome length difference of different virus strains is located in the variable region of 38-47kb at the left end of the genome and 13-22kb at the right end of the genome, and the conserved region in the center. Wild boars and soft ticks are wild storage hosts and vectors of African swine fever virus. African swine fever has similar clinical symptoms and pathological changes to classical swine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2527/101
Inventor 李霆崔贝贝王娜仇松寅冯春燕韩雪清吴绍强林祥梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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