PCR primer group, kit and detection method for detecting African swine fever virus MGF 360-505R gene

An African swine fever virus, 505R-P1 technology, applied in the biological field, can solve the problems of inability to identify and detect virus strains and vaccine strains, and achieve high-throughput, accurate and rapid detection, prevention of aerosol pollution, and simple operation Effect

Pending Publication Date: 2020-01-24
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

Once the attenuated vaccine is put into clinical use, the existing fluorescence quantitative detection method of African swine fever virus will not be used for the differential detection of clinical epidemic strains and vaccine strains

Method used

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  • PCR primer group, kit and detection method for detecting African swine fever virus MGF 360-505R gene
  • PCR primer group, kit and detection method for detecting African swine fever virus MGF 360-505R gene
  • PCR primer group, kit and detection method for detecting African swine fever virus MGF 360-505R gene

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Effect test

Embodiment 1

[0035] Design and synthesis of embodiment 1PCR primer

[0036] According to the principle of primer design, aiming at the conserved region sequence of African swine fever virus MGF 360-505R gene, Primer5 software was used to design PCR primers and probes, ensuring that the GC content of the primers was between 40% and 60%. After screening multiple sets of primers and probe combinations, the sequence of the primer set of the present invention was finally obtained as shown in Table 1. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the synthesized primers were diluted with sterilized triple distilled water At a concentration of 10 μM, store at -20°C.

[0037] Table 1 Primer sets for dual fluorescent quantitative PCR

[0038]

Embodiment 2

[0039] Application of embodiment 2 fluorescent quantitative PCR primers in detecting the virus to be tested

[0040] 1. Preparation of Nucleic Acid Standards

[0041] After measuring the nucleic acid concentration of the African swine fever virus MGF 360-505R gene synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., it was diluted with ultrapure water to a concentration of 0.4ng / μL (1.2×10 5 copy) as a nucleic acid standard.

[0042] 2. Establishment of fluorescent quantitative PCR detection method

[0043] Each concentration was 0.4ng / μL (1.2×10 5 copy) of the African swine fever virus MGF 360-505R gene DNA as a template, using the primers and probes obtained by screening (as shown in Table 1), configure the reaction system according to Table 2, and the reaction conditions are 37 ° C for 2 min; Denaturation for 5 minutes; 95°C, 10s; 60°C, 30s, 45 cycles. The result obtained is as figure 1Shown: 1 is a blank control, 2 is African swine fever virus M...

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Abstract

The present invention discloses a PCR primer group, a kit and a detection method for detecting an African swine fever virus MGF 360-505R gene. The primer group comprises primers and a probe with nucleotide sequence shown as SEQ ID NO.1-3; the kit comprises the primers, the probe and a reaction reagent. When the kit is used, an S-type amplification curve appearing on an ROX channel is positive to the African swine fever virus, a non-S-type amplification curve is negative to the African swine fever virus infection, so that the kit can be expected to be used for differential diagnosis of a deletion strain. The provided kit can effectively prevent aerosol pollution, and is also simple to operate, low in cost, high in sensitivity and good in specificity, and suitable for export quarantine, foodsanitation and African swine fever virus identification and detection in livestock farms.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fluorescent quantitative PCR primer set, a kit and a detection method for detecting African swine fever virus MGF360-505R gene. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious infectious disease caused by African swine fever virus (ASFV) infection. The World Organization for Animal Health (OIE) lists it as a legally notifiable animal disease, and China lists it as a first-class animal disease. African swine fever is not pathogenic to humans and is not a zoonotic disease, but the morbidity and fatality rate of pigs after infection can be as high as 100%. [0003] At present, the laboratory detection methods of ASF mainly include virus isolation, indirect immunofluorescence test, ELISA, PCR and so on. Although virus isolation and indirect immunofluorescence test are standard detection methods, they are cumbersome and diffi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q1/6848C12Q2600/16C12Q2531/113C12Q2563/107C12Q2537/143C12Q2521/531
Inventor 勾红潮李春玲卞志标孙铭飞蔡汝健宋帅蒋智勇李艳杨冬霞徐民生
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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