Establishment and application of triplex RT-qPCR for detection of bluetongue virus, epizootic hemorrhagic disease virus and Palyam serogroup virus

Objective  To establish triplex RT-qPCR detection technique for rapid screening and diagnosis of bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV) and Palyam serogroup virus (PALV) infection.

Method  NS3 gene of BTV, NS1 gene of EHDV and VP7 gene of PALV were used as target genes to design three sets of primers and probes for the establishment of a triplex RT-qPCR assay. The sensitivity, specificity and repeatability of this assay were evaluated, meanwhile different BTV, EHDV, PALV serotype strains isolated in China and their corresponding positive blood samples, as well as reference strains of 24 BTV serotypes and six EHDV serotypes were used to verify detection effects of the assay.

Result  The amplification efficiencies of the triplex RT-qPCR assay were all more than 90%, and the copy number detection limits of in vitro transcribed ssRNAs were all low to 10 μL⁻¹, which was equivalent to those of single plex RT-qPCR assay for each virus. There was no cross reaction with akabane virus, peste des petits ruminants virus, foot-and-mouth disease virus and bovine ephemeral fever virus. The intra-assay and inter-assay variable coefficients of Ct value were all less than 2%. The established triplex RT-qPCR successfully detected viral strains belonging to 24 BTV serotypes, six EHDV serotypes and three PALV serotypes, indicating that this assay possessed remarkable group specificity. The established triplex RT-qPCR assay could effectively detect the positive blood samples collected from animals infected by BTV, EHDV and PALV, and the results were consistent with those of monoplex RT-qPCR assay.

Conclusion  The triplex RT-qPCR detection assay for BTV, EHDV and PALV has good sensitivity, specificity and repeatability, and can be used for the simultaneous diagnosis and screening of these three viruses in clinical samples.