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Development and Clinical Validation of a Multiplex Real-time PCR Assay for Herpes Simplex and Varicella Zoster Virus

Tan, Thean Yen MBBch*; Zou, Hao PhD*; Ong, Danny Chee Tiong MSc*; Ker, Khor Jia MBBS; Chio, Martin Tze Wei MBChB, MRCP, MSc; Teo, Rachael Yu Lin MBBS, M Med, MRCP; Koh, Mark Jean Aan MBBS, MRCPCH

doi: 10.1097/PDM.0b013e3182914291
Original Articles

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are related members of the Herpesviridae family and are well-documented human pathogens causing a spectrum of diseases, from mucocutaneous disease to infections of the central nervous system. This study was carried out to evaluate and validate the performance of a multiplex real-time polymerase chain reaction (PCR) assay in detecting and differentiating HSV1, HSV2, and VZV from clinical samples. Consensus PCR primers for HSV were designed from the UL30 component of the DNA polymerase gene of HSV, with 2 separate hydrolysis probes designed to differentiate HSV1 and HSV2. Separate primers and a probe were also designed against the DNA polymerase gene of VZV. A total of 104 clinical samples were available for testing by real-time PCR, conventional PCR, and viral culture. The sensitivity and specificity of the real-time assay was calculated by comparing the multiplex PCR result with that of a combined standard of virus culture and conventional PCR. The sensitivity of the real-time assay was 100%, with specificity ranging from 98% to 100% depending on the target gene. Both PCR methods detected more positive samples for HSV or VZV, compared with conventional virus culture. This multiplex PCR assay provides accurate and rapid diagnostic capabilities for the diagnosis and differentiation of HSV1, HSV2, and VZV infections, with the presence of an internal control to monitor for inhibition of the PCR reaction.

Departments of *Laboratory Medicine

Dermatology, Changi General Hospital

Department of STI Control, National Skin Centre, Singapore, Singapore

Funded by internal funding from the Department of Laboratory Medicine, Changi General Hospital.

The authors declare no conflict of interest.

Reprints: Thean Yen Tan, MBBch, Department of Laboratory Medicine, Changi General Hospital, 2 Simei Street 3, Singapore 529889, Singapore (e-mail:

© 2013 by Lippincott Williams & Wilkins.