非洲猪瘟病毒PCR检测试剂盒的研制

Development of a PCR kit for the detection of African swine fever virus

选取非洲猪瘟病毒(ASFV)结构蛋白基因VP73中保守性强的区域,人工合成该基因片段;再根据GenBank中公布的ASFV的VP73基因序列,设计特异性引物,利用PCR扩增该VP73基因片段,克隆入pET-32a(+)载体,以构建的重组质粒为模板建立了检测ASFV的PCR检测方法。优化扩增条件,并组装成PCR试剂盒。结果显示,PCR扩增出429bp的ASFV VP73基因片段;该试剂盒与猪伪狂犬病病毒、猪瘟病毒(疫苗株)、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪乙型脑炎病毒、猪细小病毒、健康猪和蜱的基因组无交叉反应。试剂盒敏感性可达0.1fg。试剂盒批内和批间检测结果无明显差异,稳定性良好。置于4℃和-20℃条件下保存12个月,试剂盒稳定性无明显改变。结果表明,研制的ASFV PCR试剂盒为ASFV的快速检测及流行病学调查提供了技术手段。

The nucleotide sequences of VP73 gene of different African swine fever virus（ASFV） strains available in the GenBank were aligned with DNAStar software,and the highly conserved region was synthesized artificially and amplified with a pair of specific primers. A polymerase chain reaction（PCR） kit was then developed for the detection of ASFV with the amplification conditions optimized. Specificity test showed that there was no cross-reaction with genome of healthy pig or tick,and with different kinds of viruses isolated from swine,including pseudorabies virus, classical swine fever virus（vaccine strain）, porcine parvovirus,porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus and porcine circovirus type 2. Sensitivity test proved that the sensitivity of the PCR kit was up to 0.1 fg,and the intra- and inter-repeatability test showed no significant difference,and no significant change was found after being conserved at 4 ℃ or -20 ℃ for 12 months. In conclusion,the developed PCR kit provided technical tools for rapid detection and epidemiological investigation of ASFV.