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Fluorescent PCR detection kit for identifying infection and immunization of African swine fever

A technology of African swine fever and African swine fever virus, applied in the field of fluorescent quantitative PCR detection reagents, can solve the problems of ASFV prevention and control

Active Publication Date: 2020-05-19
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the current ASF epidemic situation in China and the history of the purification and eradication of African swine fever epidemics abroad, vaccine prevention and control is the most effective and urgent method for the prevention and control of African swine fever epidemics in my country. According to the current research progress, there will be in the near future Gene deletion vaccines are launched on the market, but when these gene deletion vaccines are launched on the market, it will bring new ASFV prevention and control problems. How to accurately identify whether the antibodies in pigs are caused by wild strain infection or vaccine strain immunity? , will become the top priority of ASFV prevention and control

Method used

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  • Fluorescent PCR detection kit for identifying infection and immunization of African swine fever
  • Fluorescent PCR detection kit for identifying infection and immunization of African swine fever
  • Fluorescent PCR detection kit for identifying infection and immunization of African swine fever

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Establishment of CD2v Gene Fluorescent Quantitative PCR Detection Method

[0075] 1. Materials and reagents: Inactivated ASFV cell culture medium was provided by the Polish National Veterinary Research Institute; DNA samples of PCV3, PCV2, porcine pseudorabies virus, porcine parvovirus, iridovirus, pox virus, and foot-and-mouth disease virus were provided by the Chinese Academy of Inspection and Quarantine Preserved by the Institute of Animal Quarantine. Wizard Genomic DNA Purification Kit was purchased from Promega, and 2×ExTaq Mix for qPCR (TaKaRa) was purchased from Beijing Liuhetong Economic and Trade Co., Ltd.

[0076] 2. Extraction of virus DNA from inactivated cell culture medium

[0077]Take 100 μl of inactivated ASFV cell culture medium to extract DNA according to the instructions of the Wizard Genomic DNA Purification Kit kit, and finally dissolve the DNA with 50 μl of DNA lysis solution, and store it at -20°C for future use.

[0078] 3. Primer Des...

Embodiment 2

[0093] Example 2 Establishment of eGFP gene fluorescence quantitative PCR detection method

[0094] 1. Materials and reagents: ASFV recombinant vaccine virulent cell culture fluid DNA was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army; PCV3, PCV2, porcine pseudorabies virus, porcine parvovirus, iridescent virus, pox virus, and foot-and-mouth disease virus DNA samples were inspected and quarantined by China Preserved by the Institute of Animal Quarantine, Academy of Sciences. Wizard Genomic DNA Purification Kit was purchased from Promega, and 2×ExTaq Mix for qPCR (TaKaRa) was purchased from Beijing Liuhetong Economic and Trade Co., Ltd.

[0095] 2. Primer Design

[0096] According to the sequence of the eGFP gene, a set of fluorescent PCR primers and probes were designed using Beacon Design software. The specific primer sequences are as follows:

[0097] eGFPF: 5'-CAGGAGCGCACCATCTTC-3'

[0098] eGFPR: 5'-AAGTCGATGCCCTTCAGC-3'

...

Embodiment 3

[0110] Example 3 Establishment of ASFV P72 / CD2v dual fluorescent quantitative PCR detection method

[0111] 1. Materials and reagents: The inactivated ASFV cell culture medium was provided by the Polish National Veterinary Research Institute, and the nucleic acid DNA of samples 1, 2, 3, and 4 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army. Wizard Genomic DNA Purification Kit was purchased from Promega, and 2×ExTaq Mix for qPCR (TaKaRa) was purchased from Beijing Liuhetong Economic and Trade Co., Ltd.

[0112] 2. Extraction of DNA from inactivated ASFV cell culture fluid

[0113] Take 100 μl of inactivated ASFV cell culture medium to extract DNA according to the instructions of the Wizard Genomic DNA Purification Kit kit, and finally dissolve the DNA with 50 μl of DNA lysis solution, and store it at -20°C for future use.

[0114] 3. Establishment of dual fluorescent quantitative PCR detection method for ASFV P72 / CD2v

[0115] ...

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Abstract

The invention provides a fluorescent quantitative PCR detection reagent, detection kit and detection method for identifying and diagnosing infection and immunization of African swine fever, and belongs to the technical field of biology. Specific primers and probes are designed aiming at CD2v of the African swine fever and eGFP of a fluorescent protein. An ASFVP72 / CD2v dual fluorescent PCR detection method or ASFVP72 / CD2v / eGFP triple fluorescent PCR detection method formed through combination with a P72 gene can be used for detecting the infection of the African swine fever and identifying a wild strain of a African swine fever virus and a CD2v gene deletion type recombinant vaccine strain, and thus, infected animals and immunized animals can be differentiated. The fluorescent PCR detectionkit has the advantages of simplicity and convenience in operation, high sensitivity, strong specificity, good repeatability and the like, and an important monitoring measure is provided for epidemicsituation detection and disease prevention and control.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection reagent, a detection kit and a detection method for differentially diagnosing African swine fever virus infection and immunity, and belongs to the field of biotechnology. Background technique [0002] African swine fever (African Swine fever) is an acute, hemorrhagic and severe infectious disease caused by African swine fever virus (African Swine fevervirus) infecting domestic pigs and various wild boars (such as African wild boar, European wild boar, etc.). The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 100%, clinical manifestations are fever (up to 40-42 ℃), rapid heartbeat, dyspnea, partial cough, serous or mucous purulence in eyes a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 冯春燕王彩霞林祥梅吴绍强
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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