BioScience Trends. 2008;2(4):164-168. (DOI: )
Quick detection of herpes viruses from skin vesicles and exudates without nucleic acid extraction using multiplex PCR.
Sakai K, Wakasugi S, Muchemwa FC, Ihn H
Distinguishing herpes virus infection from other skin diseases is sometimes difficult. This study aims to detect herpes virus DNA by multiplex real-time PCR without nucleic acid extraction in a short period of time. Specimens of cutaneous vesicles and swabs were obtained from 23 patients suspected of having herpes virus infection. These specimens were stored at -80°C after dissolving them in sterilized water. DNA extraction was not performed. Specific real-time PCR primers for herpes simplex virus (HSV) 1 and 2 and varicella-zoster virus (VZV) were designed. These primers were used to perform realtime PCR with the frozen solution as template. Results clearly revealed a type-specific dissociation curve. Agarose gel electrophoresis was also performed and produced a single band of the expected size. In addition to using multiplex PCR, other steps were used to reduce the time even further. Each experiment took only 2 h to complete; the type of Herpes virus was successfully detected by multiplex real-time PCR without nucleic acid extraction in a short period of time. In conclusion, omission of the nucleic acid extraction step prior to real-time PCR does not negatively affect downstream reactions. Using multiplex PCR may allow more rapid qualitative analysis of HSV1, 2 and VZV.