Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strain

A technology of African swine fever virus and fluorescent probe, applied in the field of quadruple quantitative fluorescent probe primer combination, can solve the problems of complex immune evasion mechanism, lack of neutralizing antibody, and no treatment measures

Pending Publication Date: 2021-12-07
FUJIAN AONONG BIOLOGICAL TECH GRP CO LTD +3
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no effective treatment after the onset of African swine fever, and the virus has a complex immune evasion mechanism, lacks typical neutralizing antibodies, and there is no effective vaccine for epidemic prevention

Method used

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  • Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strain
  • Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strain
  • Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strain

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Experimental program
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Embodiment 1

[0097] 1. Preparation of positive standard

[0098]The positive standards of B646L, EP402R and MGF360-14L were used to extract DNA from the positive samples of known ASFV, and the total DNA was extracted according to the DNA virus extraction kit of Tiangen, using a 25 μL system, and the reaction system included: 2×PCRMix 10 μL , 1 μL upstream primer, 1 μL downstream primer, 2 μL DNA, 0.8 μL TRUEscript Enzyme Mix and RNasefree H 2 O (nuclease-free water) 10.2 μL. PCR amplification program: 94°C for 5min; cycle 94°C for 30s, 55°C for 30s, 72°C for 30s, a total of 30 cycles; then extend at 72°C for 10min. After amplification, all products were identified by 1% agarose gel electrophoresis. PCR products identified as positive were purified and recovered with Tiangen’s glue recovery kit, connected to the pEASY-T1 vector and transformed into DH5ɑ competent cells, and positive clones were picked and amplified by shaking the bacteria with LB culture medium. Sent to Shanghai Sangon B...

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Abstract

The invention discloses a quadruple quantitative fluorescent probe primer combination, a kit and an identification method for identifying an African swine fever wild virus and a vaccine strain, and belongs to the technical field of molecular biology. The quadruple quantitative fluorescent probe primer combination for identifying the African swine fever wild virus and the vaccine strain comprises: (a) a first primer probe combination as shown in SEQ ID NO:1-SEQ ID NO:3; (b) a second primer probe combination as shown in SEQ ID NO:4 to SEQ ID NO:6; (c) a third primer probe combination as shown in SEQ ID NO:7-SEQ ID NO:9; and (d) a fourth primer probe combination as shown in SEQ ID NO:10 to SEQ ID NO:12. The kit and the identification method can simultaneously identify four genes of the African swine fever wild strain and the vaccine strain, primers of different genes do not interfere with each other, and the kit and the identification method have the characteristics of high sensitivity, strong specificity, good repeatability and good stability.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strains. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus (ASFV). Domestic pigs and wild boars are generally susceptible, and soft ticks are the storage host and medium of ASFV. The incubation period of ASFV is 5-15 days. Once an outbreak occurs, the course of disease is short and the case fatality rate is high, which can reach 100%. The on-site symptoms are complex and difficult to distinguish from other diseases, mostly manifested as high fever, skin congestion, miscarriage, edema, and organ hemorrhage. However, there is no effective treatment after the onset of African swine fever, and the virus has a compl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11C12N15/70C12R1/93
CPCC12Q1/686C12Q1/701C12N15/70C12Q2600/16C12Q2563/107C12Q2545/114C12Q2561/113
Inventor 张蓉毛旭明凌勇丁能水龙毅龙小敏张志刚吴有林
Owner FUJIAN AONONG BIOLOGICAL TECH GRP CO LTD
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