Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

Abstract

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R² values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.

Keywords: Equine; multiplex real-time PCR; respiratory viruses.

Figure 1.

Amplification curves obtained from monoplex (red) and multiplex forms (blue) of A. EHV-1, B. EHV-4, and C. EIV. Amplification curves of monoplex and multiplex forms show similarity in Ct values.

Figure 2.

Amplification curves obtained from the multiplex RT-rtPCR for the detection of EHV-1 and EHV-4 in mixed reaction. Amplification curves for EHV-1 (red) and EHV-4 (blue) indicated that both can be detected in samples from coinfection cases.

Figure 3.

Comparison between standard curves of the monoplex and multiplex forms of A. EHV-1, B. EHV-4, and C. EIV. Standard curves of monoplex (blue line) and multiplex (red line) forms are linear. Mean Ct values of monoplex (blue dot) and multiplex (red square) forms are similar.