BACKGROUND: Herpes simplex virus (HSV) is the most common cause of acute sporadic encephalitis in the United States. PCR of DNA extracted from cerebrospinal fluid (CSF) allows for reliable diagnosis of herpes simplex encephalitis (HSE). A faster turnaround time for HSE testing would improve patient management and lead to better outcomes. The aims of this study, therefore, were to develop a microchip-based electrophoretic method for rapid detection of HSV PCR products, and to compare the performance characteristics of liquid hybridization/gel retardation as an established clinical PCR product detection method with the new microchip-based method. METHODS: The study examined archival DNA from 33 selected CSF specimens submitted for HSV PCR testing to the clinical laboratory. One aliquot of the HSV PCR product was analyzed by liquid hybridization/gel retardation analysis, and a second PCR aliquot was analyzed directly with a microchip capillary electrophoresis system using an instrument built in-house. PCR samples were introduced directly into the microchip without a desalting step by use of a novel fluidic interface. Channel surfaces on the glass microchip were silanized, followed by derivatization with polyvinylpyrrolidone. RESULTS: Of the 33 CSF specimens tested by liquid hybridization analysis of HSV PCR products, 10 tested positive for HSV DNA, 2 gave a weakly positive result, and 21 tested negative. Total analysis time for detection of HSV DNA by gel retardation assay was 18 h. Microchip electrophoresis provided identical results in <110 s/sample, achieving 100% sensitivity and specificity compared with the established method. CONCLUSIONS: Microchip-based electrophoresis can rapidly and accurately separate HSV PCR products, giving results identical to those obtained by liquid hybridization but with substantially decreased turnaround time. Clinical implementation of the new method will help to improve patient management and outcomes.
BACKGROUND: Herpes simplex virus (HSV) is the most common cause of acute sporadic encephalitis in the United States. PCR of DNA extracted from cerebrospinal fluid (CSF) allows for reliable diagnosis of herpes simplex encephalitis (HSE). A faster turnaround time for HSE testing would improve patient management and lead to better outcomes. The aims of this study, therefore, were to develop a microchip-based electrophoretic method for rapid detection of HSV PCR products, and to compare the performance characteristics of liquid hybridization/gel retardation as an established clinical PCR product detection method with the new microchip-based method. METHODS: The study examined archival DNA from 33 selected CSF specimens submitted for HSV PCR testing to the clinical laboratory. One aliquot of the HSV PCR product was analyzed by liquid hybridization/gel retardation analysis, and a second PCR aliquot was analyzed directly with a microchip capillary electrophoresis system using an instrument built in-house. PCR samples were introduced directly into the microchip without a desalting step by use of a novel fluidic interface. Channel surfaces on the glass microchip were silanized, followed by derivatization with polyvinylpyrrolidone. RESULTS: Of the 33 CSF specimens tested by liquid hybridization analysis of HSV PCR products, 10 tested positive for HSV DNA, 2 gave a weakly positive result, and 21 tested negative. Total analysis time for detection of HSV DNA by gel retardation assay was 18 h. Microchip electrophoresis provided identical results in <110 s/sample, achieving 100% sensitivity and specificity compared with the established method. CONCLUSIONS: Microchip-based electrophoresis can rapidly and accurately separate HSV PCR products, giving results identical to those obtained by liquid hybridization but with substantially decreased turnaround time. Clinical implementation of the new method will help to improve patient management and outcomes.
Authors: Viliam Kolivoska; Victor U Weiss; Leopold Kremser; Bohuslav Gas; Dieter Blaas; Ernst Kenndler Journal: Electrophoresis Date: 2007-12 Impact factor: 3.535