Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded, positive-sense RNA virus. PHEV mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. However, our recent findings provide strong evidence that PHEV can also cause respiratory disease in older pigs. Genomic analysis of new PHEV strains identified in our former study further classifies PHEV into three genotypes. Detection and differentiation of these new mutants are critical in monitoring PHEV evolution in the field. In the present study, we report the development of a triplex real-time RT-PCR assay for detection and differentiation of three PHEV genotypes, 1, 2, and 3. Three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the 3' end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of NS2 with different patterns of deletions for detection of both genotypes 1 and 3, or genotype 3 only. Genotype 1 was positive when two probe dyes showed signals, genotype 2 was positive when only one probe dye showed a signal, and genotype 3 was positive when all three probes showed signals. The detection limit of the developed triplex real-time RT-PCR was as low as 8 or 9 DNA copies for three sets of primers and probes. The specificity test showed no cross reaction with other porcine viruses. Positive field-samples were correctly typed by this new assay, which was further confirmed by DNA sequencing. The triplex real-time RT-PCR provides a rapid and sensitive method to detect and differentiate all three US genotypes of PHEV from clinical samples.