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

• Published in: Journal of veterinary diagnostic investigation 2018-11, Vol.30 (6), p.924-928
• Main Authors: Ghoniem, Shimaa M., El Deeb, Ayman H., Aggour, Mohammed G., Hussein, Hussein A.
• Format: Article
• Language: English
• Subjects: Animals; Brief Communications; DNA Primers; Herpesviridae Infections - diagnosis; Herpesviridae Infections - veterinary; Herpesviridae Infections - virology; Herpesvirus 1, Equid - genetics; Herpesvirus 1, Equid - isolation & purification; Herpesvirus 4, Equid - genetics; Herpesvirus 4, Equid - isolation & purification; Horse Diseases - diagnosis; Horse Diseases - virology; Horses; Influenza A virus - genetics; Influenza A virus - isolation & purification; Multiplex Polymerase Chain Reaction - methods; Multiplex Polymerase Chain Reaction - veterinary; Orthomyxoviridae Infections - diagnosis; Orthomyxoviridae Infections - veterinary; Orthomyxoviridae Infections - virology; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - veterinary; Respiratory Tract Diseases - diagnosis; Respiratory Tract Diseases - veterinary; Respiratory Tract Diseases - virology; Sensitivity and Specificity
• ISSN: 1040-6387; 1943-4936
• DOI: 10.1177/1040638718799388

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.