Quick detection of herpes viruses from skin vesicles and exudates without nucleic acid extraction using multiplex PCR.
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.
Sakai
Keisuke
K
Department of Dermatology & Plastic and Reconstructive Surgery, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan. ke0i4su0ke3@yahoo.co.jp
Wakasugi
Shoji
S
Muchemwa
Faith C
FC
Ihn
Hironobu
H
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*