Melting curve analysis-based multiplex real-time PCR assay mediated by universal primers for detection of multiple...

Multiplex quantitative real-time PCR (M-qPCR) is increasingly becoming a commonly used technique in molecular detection of pathogens due to its advantages in quantification and throughput. However, imbalanced amplification is still a potential problem affecting performance of M-qPCR assays. In this study, a novel universal primer multiplex quantitative real-time PCR (UP-M-qPCR) assay using EvaGreen and melting curve analysis was developed, using five swine diarrhea-related viruses as a case study. UPs and the chimeric primers (CPs) were introduced in this assay for sequential virus-specific and universal amplification to produce large amounts of amplicons for five target viruses with similar efficiency. Subsequent melting analysis of the amplicons displayed five distinguishable target-specific melting peaks with characteristic melting temperatures (T_ms), while no non-specific amplification or melting peak was observed for five non-target viruses. The assay achieved a limit of detection (LOD) of 5 copies /μL of viral plasmids in various settings including high-background co-infections. Meanwhile, the coefficient of variation of T_m value and Ct values fell within 0-0.57% and 0.12-4.37% respectively, suggesting good repeatability of the assay. The UP-M-qPCR and corresponding singleplex qPCR were used in parallel to detect field samples for five viruses. The diagnostic sensitivity and specificity of the UP-M-qPCR were 90.9-100% and 99.4-100%, respectively, with a kappa value of 0.95-1, indicating good consistency. Overall, this study presents a melting curve analysis-based UP-M-qPCR assay, enabling the concurrent detection of multiple viruses with high sensitivity and specificity, which provides a technical platform easily adaptable for multiple pathogens detection.