title: Quick detection of herpes viruses from skin vesicles and exudates without nucleic acid extraction using multiplex PCR.
abstract: 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 degrees 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.
authors.affiliation: Department of Dermatology & Plastic and Reconstructive Surgery, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan. ke0i4su0ke3@yahoo.co.jp
authors.forename: Keisuke
authors.initials: K
authors.lastname: Sakai
authors.forename: Shoji
authors.initials: S
authors.lastname: Wakasugi
authors.forename: Faith C
authors.initials: FC
authors.lastname: Muchemwa
authors.forename: Hironobu
authors.initials: H
authors.lastname: Ihn
mesh_terms: D004587:Electrophoresis, Agar Gel; D006564:Herpesviridae / Q000235:genetics* / Q000302:isolation & purification*; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012867:Skin / Q000821:virology*