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J Clin Virol. 2004 May;30(1):32-8.

Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction.

Author information

  • 1Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital & Health Service District, Herston Road, Herston, Queensland 4029, Australia.

Abstract

BACKGROUND:

In recent years polymerase chain reaction (PCR) has proven to be a highly sensitive and specific method for the diagnosis of herpes simplex virus (HSV) infections. The advent of real-time HSV PCR protocols now enables rapid result turnaround times with minimal hands-on time.

OBJECTIVES:

In this study, we developed a real-time duplex PCR assay (HSVgD-dPCR) comprising of HSV and internal control PCR reactions.

STUDY DESIGN:

Using the LightCycler, the HSVgD-dPCR targeted the HSV glycoprotein D gene and HSV typing was performed by melting curve analysis. The internal control PCR reaction targeted sequences of the DNA of the human endogenous retrovirus (ERV-3). In total, 300 swab specimens, from patients with suspected HSV infection, were tested by the HSVgD-dPCR assay. The results were then compared to the results obtained by another HSV LightCycler assay, which utilized published primer and probe sequences targeting the HSV DNA polymerase gene (Dpol-HSV-LCPCR).

RESULTS:

Overall, 91 (30.3%) specimens were positive and 204 (68.0%) specimens were negative for HSV by both LightCycler assays. In addition, four (1.3%) specimens were positive by Dpol-HSV-LCPCR and negative by HSVgD-dPCR, whereas one (0.3%) specimen was positive by HSVgD-dPCR and negative by Dpol-HSV-LCPCR. The presence of HSV in these five specimens was confirmed by conventional PCR. Melting curve analysis by the HSVgD-dPCR assay enabled all HSV positive specimens to be typed, whereas sequence variation prevented three HSV positive specimens from being typed by the Dpol-HSV-LCPCR. Using the ERV-3 PCR, 5% specimens were found to contain inhibitory substances.

CONCLUSIONS:

By developing the HSVgD-dPCR we have enhanced the diagnostic utility of real-time detection of HSV by incorporating an internal control reaction and by accurately typing a greater proportion of HSV positive specimens.

PMID:
15072751
[PubMed - indexed for MEDLINE]
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