Dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, kit and application of dual fluorescent probe primer combination

Abstract

The invention discloses a dual fluorescent probe primer combination for identifying African swine fever type I strains and type II strains, a kit and application of the dual fluorescent probe primer combination and the kit, and belongs to the technical field of molecular biology. The dual fluorescent probe primer combination comprises a primer probe group for detecting an African swine fever virus type I strain and a primer probe group for detecting a universal P72 gene of the African swine fever virus. According to the invention, genomes are analyzed and compared in a conserved region of the African swine fever virus, the gene B117L is finally screened out, fragments with great difference between a type I virus strain and a type II virus strain are selected, and probe primer design and reaction condition optimization are carried out, so that the amplification efficiencies of the two virus genes are similar, and a kit convenient for diagnosis is developed. The kit can detect the African swine fever virus I type infection, the African swine fever virus II type infection or co-infection of the two viruses at one time, has the advantages of high sensitivity, strong specificity and good repeatability, and provides technical support for prevention and control of the African swine fever in a pig farm.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a dual fluorescent probe primer combination, a kit and an application thereof for distinguishing African swine fever type I strains and type II 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. African swine fever virus has only one serotype, and the genome of ASFV can be divided into 24 different genotypes according to the B646L gene (encoding P72 protein) in the conserved region. In 1957, the African swine fever virus genotype I was first spread from the African continent, and it has been popular in Europe and Latin America, and it is still endemic in Africa and Sardinia, Italy. In 2007, African swine fever virus genotype II spread ...

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Problems solved by technology

In addition, OURT88 / 3 has a frameshift in EP402R, EP153R and other genes, resulting in the inability to express the virulence protein CD2v, so it cannot cause clinical symptoms and viremia after infecting domestic pigs

[0004] At present, the vaccines developed in China are mainly based on the African swine fever type II strain as the parent, and the MGF360-505R gene deletion and CD2V (EP402R) and MGF360-505R double-gene combined deletion live attenuated vaccines are constructed. The main missing virulence gene Multigene family MGF360-505 (MGF360-9L, MGF360-10L, MGF360-11L, MGF360-12L, MGF360-13L, MGF360-14L, 505-1R, 505-2R, 505-3R) and EP402R, therefore vaccinated None of the pigs infected with the attenuated type I strain had obvious clinical symptoms and viremia, which brought great difficulties to clinical diagnosis and epidemic prevention and control. In addition, most of the current PCR detection kits use vaccine-deficient Gene fragments were used to design probe primers to identify wild virus and vaccine strains. However, the deletion fragments of African swine fever type I natural attenuated strains overlapped with the deletion fragments of vaccine strains, and this method could not identify type I African swine fever Natural attenuated strain infection, so it is particularly important to establish a fast, sensitive and accurate method for distinguishing African swine fever type I strains and type II strains

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