Real-time PCR assays are highly sensitive, specific and rapid techniques for the identification of ASF virus (ASFV) (Section 3.8, OIE Terrestrial Manual, 2019). Although an ASFV p72 gene based real-time PCR assay (a.k.a. the Zsak assay) (Zsak et al. J. Clinical Microbiology, 43, 2005, 112) has been widely used for ASFV detection, several more ASFV sequences have become available in the 15 years since the design of the Zsak assay. In this study, we developed a new ASFV p72 gene based real-time PCR after analysis of all currently available sequences of the p72 gene and multiplexed the new assay with a modified Zsak assay aiming to have a broader coverage of ASFV strain/isolates. To reduce false negative detections, porcine house-keeping gene, beta actin (ACTB), was applied as an internal control. Eight ACTB sequences from the GenBank and 61 partial ACTB sequences generated in this study, and 1,012 p72 sequences from the GenBank and 23 p72 sequences generated at FADDL, were used for ACTB and ASFV primer and probe designs, respectively, to ensure broader host and ASFV coverage. Multiplexing ACTB in the reaction did not inhibit ASFV amplification. The multiplex assay was evaluated for strain/isolate coverage, sensitivity and specificity. The in silico analysis showed high ASFV strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available. The limit of detection (LOD) was 6 plasmid copies or 0.1-1 TCID50 /ml of ASFV isolates per reaction. Only targeted ASFV isolates and the viruses in the positive clinical samples were detected, indicating that the assay is highly specific (100% specificity). The test results of 26 ASFV isolates with different country origins showed that this newly developed multiplex assay performed better than the Zsak assay that has been widely accepted and used worldwide, indicating that it may be used as an alternative assay for ASFV detection.
Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control.