Multiplex SYBR Green real-time RT-PCR assays for the improved detection of viral pathogens in horses: equine infectious anemia, West Nile and Japanese encephalitis viruses

Abstract

Equine infectious anemia virus (EIAV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are mosquito-borne viruses that are of great concern for the equine industry worldwide. These viruses produce similar clinical signs and veterinarians need to be able to differentiate between them quickly. In this study, multiplex known as duplex and triplex SYBR Green real-time RT-PCR assays were developed to detect these three viruses and to discriminate between them based on differences in the melting temperatures (Tm) of their amplification products. Three melting peaks, generated simultaneously at temperatures of 78.5±0.5°C, 83.5±0.5°C and 85.0±1.0°C, indicated the presence of EIAV, JEV and WNV, respectively. The optimal melting rate for duplex was 0.5°C/sec and a slower rate of 0.1°C/sec for triplex to improve resolution of the melting temperature. The optimal combinations of primers for multiplex were 600 or 900 nM for EIAV and WNV and 300 or 600 nM for JEV detection. The assays all demonstrated high sensitivities, ranging from 3.29X10-2 to 3.29X106copies/μl which was similar to those obtained based on agarose gel analysis of the RT-PCR products. No cross-reaction was observed with the other closely related viruses, which indicated a high specificity of these assays. Less than 0.5% of the coefficient of variation (CV) of intra-specific assay and inter-specific assay were considered acceptable to generate reproducible results. These results indicated that the multiplex, duplex and triplex, SYBR Green real-time RT-PCR assays described in this paper could be used for the detection of EIAV, WNV and JEV and would provide a valuable addition to the methods currently available for the routine diagnosis and surveillance of these zoonoses which are listed by the World Animal Health Organization (OIE) as important for international trade.