检测非洲猪瘟病毒快速EIS-qPCR方法的建立

Establishment of rapid EIS-qPCR assay for detection of African swine fever virus

为满足对非洲猪瘟病毒(ASFV)的早期、超快速、精准基因检测的市场需求,本研究根据ASFV的p72基因保守区和猪内源性内标(endogenous internal standard,EIS)cytb基因序列设计双重qPCR的引物和探针,并在反应体系中加入UNG酶防污染系统,建立了用于检测ASFV的快速、高灵敏EIS-qPCR方法。结果显示,该方法时间短,可在30 min内完成ASFV的qPCR检测;灵敏度高,最低检测限为4.12拷贝/μL;特异性强,对ASFV p72基因呈现特异性扩增,对猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)、猪圆环病毒2型(PCV2)均无扩增;重复性好,变异系数(Cv)小于2%;防污染能力强,可有效消除低剂量气溶胶污染产生的假阳性扩增。146例临床样本的检测结果与商业化ASFV检测试剂盒检测结果比较,符合率为100%。本研究建立的EIS-qPCR技术与现有同类技术相比,具有检测更快速、灵敏度更高等特点,适合ASFV感染早期的快速诊断,可为非洲猪瘟的监测和疫情的精准防控提供可靠的技术支撑。

In order to meet the market demand for the fast and accurate genetic detection of African swine fever virus(ASFV),a new method for EIS-qPCR detection was established with a rapid,high sensitivity,and pollution prevention.Primers and probes for a duplex qPCR were designed based on the conserved region of ASFV virus p72 gene and the endogenous internal standard(EIS)cytb gene sequence in pigs.An anti-contamination system was established with uracil DNA N-glycosylase enzyme in the reaction system.The results showed that the method can finish the rapid qPCR detection of ASFV within 30 min with a minimal detection limit of 4.12 copies/μL.Moreover,the method only detected the ASFV p72 gene,and no amplifications of classical swine fever virus(CSFV),pseudorabies virus(PRV),porcine parvo virus(PPV),porcine reproductive and respiratory syndrome virus(PRRSV)and porcine circovirus type 2(PCV2)were observed.Repetitive results showed a coefficient of variation below 2%.With strong anti-pollution capacity,the method can effectively eliminate false-positive amplification caused by low-dose aerosol pollution.Detection results of 146 clinical samples showed a 100%consistence with the results of the commercial ASFV detection kit.Compared with similar technologies,the EIS-qPCR established in this study was faster,sensitive,and suitable for the rapid diagnosis of ASFV infection in the early stage,which provided the tool for the monitoring and precise prevention and control of African swine fever.