Detection of herpes simplex and varicella-zoster virus in clinical specimens by multiplex real-time PCR and melting curve analysis

Abstract

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), and varicella-zoster virus (VZV) are common agents resulting in various forms of clinical manifestation from skin vesicle to disseminated viral infection. The aim of the present study was to develop a real-time PCR and melting curve analysis which detect and differentiate HSV-1, HSV-2, and VZV, to compare with PCR-RFLP using clinical specimens, and to introduce the 4-year experience in the clinical laboratory. Three pairs of primers for HSV-1, HSV-2, and VZV were designed. Primers for human endogenous retrovirus-3 (HERV-3), an internal control, were adopted. A hundred selected specimens and many clinical specimens were tested for methods comparison and assay validation. Increased sensitivity and specificity were obtained from real-time PCR. In review of results of clinical specimens submitted to clinical laboratory, a total of 46 of 3,513 specimens were positive in cerebrospinal fluids, blood, skin vesicles, genital swabs, aqueous humor, and ear discharge. Thus, this method could be a rapid and accurate alternative to virus culture and other molecular tests for detection and typing of HSV-1, HSV-2, and VZV.

Figure 1

Differentiation of HSV-1, HSV-2, and VZV by multiplex real-time PCR-melting curve analysis. Melting peaks of HSV-1, HSV-2, and VZV positive samples by melting curve analysis. The HSV-1 amplicon has a melting temperature around 86.89°C, HSV-2 around 89.08°C, and VZV around 78.58°C, and internal control has a melting temperature around 82.41°C. HSV: herpes simplex virus; VZV: varicella-zoster virus; HERV-3: human endogenous retrovirus; I.C.: internal control.