Multiplex real-time PCR for the detection and differentiation of equid gammaherpesvirus 2 and 5.

The research article presents a study on the development of a multiplex real-time PCR (rtPCR) for detecting and differentiating Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) in horses. The assay proves to be an efficient and swift tool for identifying these viruses, vital for conducting further studies on their clinical significance.

Introduction to the Equid gammaherpesvirus

• Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are prevalent in horses worldwide.
• Their complete pathogenic potential is not yet clear, though they are suggested to be a factor involved in equine diseases such as equine multinodular pulmonary fibrosis.
• Identifying and understanding these viruses is essential for exploring their role in the horse diseases in more depth.

Development of the Real-time PCR (rtPCR)

• The researchers designed a multiplex real-time PCR (rtPCR) to detect the DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5), indicative of the presence of the viruses.
• The rtPCR’s analytical specificity was determined by testing DNA of other herpesviruses.
• This testing was done using SYBR Green rtPCR and melting curve analysis, and through Sanger sequencing of positive field samples.

Evaluation of the rtPCR

• The rtPCR’s analytical sensitivity was evaluated using standard curves generated from serial plasmid dilutions containing the target gene for each virus.
• Specific detection of the viruses was confirmed through analysis of the melting curves and BLAST analysis of the sequences.
• The lower limit of detection, which refers to the smallest quantity of a substance that can be reliably measured by the assay, was found to be 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively.

Comparison to Other Assays

• When compared with singleplex assays (tests that detect a single target in one reaction), the new multiplex rtPCR showed minimal differences for a selection of positive field samples.
• The minimal differences suggest a comparable performance level between the new multiplex rtPCR and existing singleplex assays.

Conclusion

• The developed multiplex rtPCR offers sensitive and specific detection of EHV-2 and EHV-5.
• This new assay provides a convenient and rapid method for further investigations on the clinical importance of these viruses in equine diseases.