Evaluation of an In-House Melting Curve Analysis Assay for HSV-1 and HSV-2 Detection via Multiplex Real-Time PCR

Background: Herpes simplex virus (HSV) has developed mechanisms that allow it to evade the immune system and persist in the body, thus causing diseases and conditions such as sexually transmitted diseases and serious neurological infections. Objective: To evaluate an in-house melting curve analysis (IHMCA) assay for multiplex real-time polymerase chain reaction (PCR) product to minimize false positive and false negative results in HSV detection. Methods: A total of 233 clinical specimens were subjected to analysis using the HSV1&2 VZV R-GENE® multiplex real-time PCR assay with melting curve analysis. This analysis was then compared to the Luminex ARIES HSV 1&2 assay. Results: HSV-1 and HSV-2 demonstrated sensitivity of 90.91% and 96.77%, and specificity of 99.50% and 96.55%. The positive predictive values were 96.77% and 81.08% , and the negative predictive values were 98.51% and 99.49%. The overall accuracy was 98.28% and 96.58%. The assay demonstrated strong agreement with the reference method with statistically significant results (HSV-1: P < .001, SE = 0.04, = 0.93 [95% CI, 0.86-1.00]; HSV-2: P < .001, SE = 0.05, = 0.86 [95% CI, 0.77-0.96]). Conclusions: Amplicon analysis of the multiplexed real-time PCR product was performed in the same reaction tube without interference from multiple TaqMan probe signals due to the use of a DNA binding dye. The integration of multiplex real-time PCR with melting curve analysis enhances the accuracy and efficiency of HSV detection while potentially reducing costs and turnaround time. Importantly, the IHMCA should be considered as a complementary post-PCR method rather than a replacement for standard assays.