Development of a quadruple PCR-based gene microarray for detection of vaccine and wild-type classical swine fever virus, African swine fever virus and atypical porcine pestivirus.

Abstract

Abstract Background: Classical swine fever (CSF), African swine fever (ASF) and atypical porcine pestivirus (APPV) are acute, virulent and contagious viral diseases currently hampering pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assay for the differentiation of infection and vaccination of CSFV in addition to the detection of ASFV and APPV are urgently required for better prevention, control and elimination of these viral diseases in China. Methods: A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. 42 laboratory confirmed samples including positive samples of other 10 swine viral diseases were tested using this assay to confirm its high specificity.Results: The limit of detections (LODs) of this assay for the wild type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 ×10 and 1.80×10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875-2018), ASFV (MARR issue No.172) or APPV（CN108611442A）using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity can specifically distinguish ASFV, APPV and CSFV including CSFV infection and immunization.Conclusion: This assay provides a practical, simple, economical and reliable test for the rapid detection and accurate diagnosis of the three viruses, and may have good prospects for application in epidemiological investigation, prevention and control and elimination of these three diseases.