基于E184L基因的非洲猪瘟病毒实时荧光定量PCR检测方法的建立

非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)感染引起的一种高度接触性传染病。由于ASFV的感染机制极为复杂,基因型多,至今尚无有效疫苗用于防控,防止该病暴发主要依赖于早期快速诊断和控制。为建立一种高效快速、特异的ASFV检测方法,根据ASFV的E184L基因序列,设计了TaqMan荧光定量PCR引物及探针,建立了检测ASFV的TaqMan荧光定量PCR方法。结果表明,该方法设计的引物具有高度特异性,以构建的重组质粒为标准品建立的TaqMan荧光定量PCR方法的标准曲线具有良好的线性关系,线性相关系数为0.992,对ASFV核酸最低检测限为1.51拷贝,且与伪狂犬病病毒、猪细小病毒、猪圆环病毒2型等不存在交叉反应。建立的基于ASFV E184L基因实时荧光定量PCR检测方法能够快速、准确、特异地对ASFV核酸进行定量分析,丰富了ASFV的检测方法。

Establishment of a Real-time PCR for Detection of African Swine Fever Virus E184L Gene

African swine fever(ASF)is a highly contagious disease caused by African swine fever virus(ASFV).The infection mechanism of ASFV is extremely complicated and there are many genotypes,there is no effective vaccine for prevention and control.The prevention of the outbreak of the disease mainly depends on early rapid diagnosis and prevention.In order to establish an efficient,rapid and specific method for early quantitative detection of ASFV infection,the TaqMan real-time PCR primers and probes were designed based on the ASFV E184 Lgene sequence,and the method of TaqMan real-time PCR for rapid detection of ASFV was established.The results showed that the primers designed by this method were highly specific.The standard curve of the TaqMan real-time PCR method established by using the constructed recombinant plasmid as a standard has a good linear relationship,with a linear correlation coefficient of0.992.The detection limit of the ASFV nucleic acid is 1.51 copies,and there is no cross-reactivity with pseudorabies virus,porcine parvovirus,porcine circovirus type 2 and so on.The established ASFV E184 L gene-based real-time PCR detection method can quickly,accurately,specifically quantify ASFV nucleic acids,enriching the detection method of ASFV.