Development and evaluation of multiplex real‐time RT‐PCR assays for the detection and differentiation of foot‐and‐mouth disease virus and Seneca Valley virus 1

Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically important vesicular disease in cloven-hoofed animals that is clinically indistinguishable from symptoms caused by Seneca Valley virus 1 (SVV-1). To differentiate SVV-1 from FMDV infections, we developed a SVV-1 real-time RT-PCR (RT-qPCR) assay and multiplexed with published FMDV assays. Two published FMDV assays (Journal of the American Veterinary Medical Association, 220, 2002, 1636; Journal of Virological Methods, 236, 2016, 258) targeting the 3D polymerase (3D) region were selected and multiplexed with the SVV-1 assay that has two targets, one in the 5' untranslated region (5' UTR, this study) and the other in the 3D region (Journal of Virological Methods, 239, 2017, 34). In silico analysis showed that the primers and probes of SVV-1 assay matched 98.3% of the strain sequences (113/115). The primer and probe sequences of the Shi FMDV assay matched 85.4% (806/944), and that of the Callahan FMDV assay matched 62.7% (592/944) of the sequences. The limit of detection (LOD) for the two multiplex RT-qPCR assays for SVV-1 was both 9 copies per reaction by cloned positive plasmids and 0.16 TCID50 per reaction by cell culture. The LOD for FMDV by both multiplex assays was 11 copies per reaction using cloned positive plasmids. With cell cultures of the seven serotypes of FMDV, the Shi assay (Journal of Virological Methods, 236, 2016, 258) had LODs between 0.04 and 0.18 TCID50 per reaction that were either the same or lower than the Callahan assay. Interestingly, multiplexing with SVV-1 increased the amplification efficiencies of the Callahan assay (Journal of the American Veterinary Medical Association, 220, 2002, 1636) from 51.5%-66.7% to 89.5%-96.6%. Both assays specifically detected the target viruses without cross-reacting to SVV-1 or to other common porcine viruses. An 18S rRNA housekeeping gene that was amplified from multiple cloven-hoofed animal species was used as an internal control. The prevalence study did not detect any FMDV, but SVV-1 was detected from multiple types of swine samples with an overall positive rate of 10.5% for non-serum samples.