非洲猪瘟病毒RPA等温检测方法的建立

摘要

为建立一种简单、快速的非洲猪瘟病毒（ASFV）分子检测方法，基于ASFV P72基因保守序列，设计并合成引物，建立了一种ASFV重组酶聚合酶扩增（RPA）等温检测方法。结果表明，所建立的RPA方法在38℃水浴锅中恒温反应30 min，即可实现对目的片段的有效扩增；以包含ASFV P72基因的ORF质粒为模板， RPA的检测限达到102 copies，同世界动物卫生组织（OIE）推荐的实时荧光PCR方法检测限一致，但比OIE推荐方法的检测限高10倍；RPA仅特异性扩增ASFV P72基因，对FMDV、CSFV、PRRSV、PRV和PCV-2基因组cDNA或DNA没有扩增。本研究所建立的RPA方法操作简单、反应快速、检测成本低、结果确实可靠，为非洲猪瘟的一线防控提供了一种新的、可靠的技术支持。%To develop an convenient and rapid molecular diagnostic method for the African swine fever virus(ASFV), the recombinase polymerase amplification(RPA)was developed using primers specific for the conserved region of the ASFV P72 gene. The RPA reaction was performed successfully at 38℃for 30 min in a water bath. By using recombinant plasmid DNA carrying the P72 gene as template.The results showed that the detection limit of RPA was 102 copies of plasmid DNA,which was the same as the real-time PCR recommended by OIE,and it was 10 times more sensitive than it. RPA could not amplify templates of FMDV,CSFV,PRRSV,PRV or PCV-2,demonstrating high specificity.The ASFV RPA developed in the study is simple,rapid,cost-effective and reliable,which can be an novel and reliable tool for the control of ASF.