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

Abstract

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.

Development of a real‐time PCR assay for detection of African swine fever virus with an endogenous internal control

“…The real‐time PCR tests for diagnosis were performed as described (Wang, Das, et al, 2019; Wang, Feng, et al, 2019). Briefly, a master mix of a 20 µl reaction is composed of 5 µl of template DNA, 0.25 µM of each primer, 0.25 µM of each probe and 10 µl of 2X iQ™ Multiplex Powermix (Bio‐Rad).…”

Development of a differential multiplex real-time PCR assay for porcine circovirus type 2 (PCV2) genotypes PCV2a, PCV2b and PCV2d

“…In our design, the three sets of primers and probes can match 75.3–86.3 % strains of each genotype. In general, an assay with single nucleotide mismatches occurring in the middle, especially in the 5′ end of a primer, or in the two ends of a probe may still amplify and generate a signal ( Bru et al, 2008 ; Forney et al, 2004 ; Wang et al, 2020a ). Therefore, when single nucleotide mismatch in such situations is considered in the design of the primers and probes, strain coverage can potentially increase to 93.4 %–95.1 %.…”

Development and Evaluation of Duplex TaqMan Real-Time PCR Assay for Detection and Differentiation of Wide-Type and MGF505 Gene-Deleted African Swine Fever Viruses

“…However, it is not well comparable when the Ct value is greater than 35. It makes sense that in most PCRs the higher the Ct value, the lower the repeatability when Ct value is greater than 36 [18].…”