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Journal of Clinical Microbiology, June 1999, p. 1941-1947, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

Alexander J. Ryncarz,1,dagger James Goddard,1 Anna Wald,2,3 Meei-Li Huang,1,4 Bernard Roizman,5 and Lawrence Corey1,2,4,*

Departments of Laboratory Medicine,1 Medicine,2 and Epidemiology,3 University of Washington, and Program in Infectious Diseases, Fred Hutchinson Cancer Research Center,4 Seattle, Washington, and Departments of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois5

Received 5 October 1998/Returned for modification 14 December 1998/Accepted 4 March 1999

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 µl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

* Corresponding author. Mailing address: 1100 Fairview Ave. North (D3-100), P.O. Box 19024, Seattle, WA 98109-1024. Phone: (206) 667-6702. Fax: (206) 667-4411. E-mail: lcorey{at}u.washington.edu.

dagger Present address: Phase-1 Molecular Toxicology, Inc. Santa Fe, NM 87505.

Journal of Clinical Microbiology, June 1999, p. 1941-1947, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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