Establishment of rapid EIS-qPCR assay for detection of African swine fever virus
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-gly-cosylase 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.Moreo-ver,the method only detected the ASFV p72 gene,and no amplifications of classical swine fever vi-rus(CSFV),pseudorabies virus(PRV),porcine parvo virus(PPV),porcine reproductive and re-spiratory syndrome virus(PRRSV)and porcine circovirus type 2(PCV2)were observed.Repeti-tive 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 com-mercial 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.
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