Detection of Herpes Simplex Virus DNA by Real-Time PCR

Abstract

Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 104 copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2 × 103 to 5 × 103 HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.

A Brief Review on Diagnosis of Foot-and-Mouth Disease of Livestock: Conventional to Molecular Tools

“…Recently, TaqMan technology has combined the 5 nuclease activity of the Taq DNA polymerase and forster resonance energy transfer to detect and quantify amplification product in a closed tube format. Using this technology real-time PCR has been developed to detect the nucleic acid [164,165]. This is most sensitive and rapid method to detect the nucleic acid.…”

<p>Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis</p>

“…The primers employed for the nested PCR with sequencing were designed in-house and selected according to the previously published literature. [26][27][28] A negative control was included for each PCR run to ensure the purity of the reagent. A specimen was considered positive for a given result if either of the two assays for that result were positive.…”

Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system

“…The latter are employed in the TaqMan (Fig. 3) (Holland et al, 1991;Higuchi et al, 1993;Heid et al, 1996) andLightCycler (Wittwer et al, 1997a,b) technology, that have both been described for quantitation of viral DNA in CSF (Kessler et al, 2000;Verstrepen et al, 2001;Gunther et al, 2001;Nagai et al, 2001;Read et al, 2001;Gautheret-Dejean et al, 2002;Aberle and Puchhammer-Stö ckl, 2002) (Table 4). Real-time PCR also allows simultaneous quantification, in the same tube, of different genomes (Read et al, 2001), as well as virus genotyping and mutational analysis.…”

“…Ando et al (1993),Revello et al (1997),Domingues et al (1998),Kessler et al (2000),Aberle and Puchhammer-Stö ckl (2002) HSV-2Real-time PCR Narrower range of level variation in patients with HSV-2 meningitis than in patients with HSV-1 encephalitis. Highest levels found in children with congenital infection (up to 10 6 copies/ml)Aberle and Puchhammer-Stö ckl (2002) VZV Semiquantitative PCR, real-time PCR Higher levels in patients with herpes zoster complications than in those with varicella Puchhammer-Stö ckl et al (1991), Aberle and Puchhammer-Stö ckl (2002) CMV Semiquantitative PCR, competitive PCR, branched DNA Association of high DNA levels with HIV associated VE or PRP, and with lesion extention in VE Decrease of DNA following antiviral therapy in HIV-infected patients Arribas et al (1995b), Cinque et al (1995levels with bad prognosis?…”