title: Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.
abstract: Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Nipaporn
authors.initials: N
authors.lastname: Sankuntaw
authors.forename: Saovaluk
authors.initials: S
authors.lastname: Sukprasert
authors.forename: Chulapan
authors.initials: C
authors.lastname: Engchanil
authors.forename: Wanlop
authors.initials: W
authors.lastname: Kaewkes
authors.forename: Wasun
authors.initials: W
authors.lastname: Chantratita
authors.forename: Vantanit
authors.initials: V
authors.lastname: Pairoj
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
mesh_terms: D020805:Central Nervous System Viral Diseases / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000821:virology*; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D006564:Herpesviridae / Q000145:classification / Q000235:genetics*; D006566:Herpesviridae Infections / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000235:genetics; D018258:Herpesvirus 2, Human / Q000235:genetics; D014645:Herpesvirus 3, Human / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004279:DNA, Viral