Detection of Herpes Simplex Virus DNA in Genital and Dermal Specimens by LightCycler PCR after Extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 Methods

Abstract

The LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) is an automated instrument which can monitor the development of amplified target nucleic acid by fluorescence resonance energy transfer after each amplification cycle. The instrument provides rapid (30 to 40 min) PCR results by precise air-controlled temperature cycling; most importantly, the amplification and detection of an amplified product occur in a closed system, which virtually eliminates the likelihood of carryover contamination. In three evaluations that involved 877 dermal and genital and a few ocular specimens and yielded 285 herpes simplex virus (HSV)-and 44 varicella-zoster virus (VZV)-positive results, LightCycler PCR produced a greater sensitivity for the detection of HSV (22%) and VZV (91%) than did the shell vial cell culture assay for the laboratory diagnosis of these viral infections (5, 6, 7). Based on these performance characteristics, together with comparable cost analysis for LightCycler PCR and shell vial cell culture, we implemented the molecular amplification procedure in May 2000 for the routine diagnosis of HSV dermal and genital and VZV dermal infections.Preliminary to LightCycler PCR, we extracted nucleic acids from dermal and genital specimens by the manual IsoQuick (Orca Research, Inc., Bothell, Wash.) procedure. In the present study, we evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method with the goal of implementing a cost-efficient, standardized system for the processing of clinical specimens. MATERIALS AND METHODSSpecimens and shell vial assay. Genital (n ϭ 152) and dermal (n ϭ 46) swab specimens from patients suspected of having HSV infections were extracted into 2-ml volumes of serum-free medium; the specimen extracts were then divided into four equal aliquots. Each of two shell vial MRC-5 cell cultures received 200 l of inoculum from one aliquot. The vials were centrifuged, incubated overnight at 36°C, and stained by the indirect immunofluorescence test as previously described (10). Nucleic acids were extracted from the remaining aliquots by three different extraction techniques and processed for amplification of HSV DNA by LightCycler PCR. Nucleic acid extracts obtained by each method were stored at 4°C for a maximum of 2 weeks before PCR amplification.IsoQuick nucleic acid extraction. Nucleic acids were extracted manually from a 200-l volume of serum-free extract of genital or dermal swab specimens by the IsoQuick procedure (Orca Research, Inc.), which utilizes guanidine thiocyanate and a noncorrosive extraction reagent, in accordance with the manufacturer's instructions (4, 7).MagNA Pure nucleic acid extraction. A second aliquot (200 l) was extracted by the MagNA Pure LC automated extractor (Roche Molecular Biochemicals) by using the DNA isolation extraction kit produced by the same manufacturer.Qiagen BioRobot 9604 nucleic acid extraction. Another 200-l ...

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