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

M. J. Espy, P. N. Rys, A. D. Wold, J. R. Uhl, L. M. Sloan, G. D. Jenkins, D. M. Ilstrup, F. R. Cockerill, Robin Patel, J. E. Rosenblatt, T. F. Smith

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

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 (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

Original languageEnglish (US)
Pages (from-to)2233-2236
Number of pages4
JournalJournal of Clinical Microbiology
Volume39
Issue number6
DOIs
StatePublished - 2001

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Simplexvirus
Polymerase Chain Reaction
Skin
DNA
Nucleic Acids
Cell Culture Techniques
Viruses
Costs and Cost Analysis
Research

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

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. / Espy, M. J.; Rys, P. N.; Wold, A. D.; Uhl, J. R.; Sloan, L. M.; Jenkins, G. D.; Ilstrup, D. M.; Cockerill, F. R.; Patel, Robin; Rosenblatt, J. E.; Smith, T. F.

In: Journal of Clinical Microbiology, Vol. 39, No. 6, 2001, p. 2233-2236.

Research output: Contribution to journalArticle

Espy, M. J. ; Rys, P. N. ; Wold, A. D. ; Uhl, J. R. ; Sloan, L. M. ; Jenkins, G. D. ; Ilstrup, D. M. ; Cockerill, F. R. ; Patel, Robin ; Rosenblatt, J. E. ; Smith, T. F. / 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. In: Journal of Clinical Microbiology. 2001 ; Vol. 39, No. 6. pp. 2233-2236.
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abstract = "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 (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2{\%}) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76{\%}) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.",
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AU - Espy, M. J.

AU - Rys, P. N.

AU - Wold, A. D.

AU - Uhl, J. R.

AU - Sloan, L. M.

AU - Jenkins, G. D.

AU - Ilstrup, D. M.

AU - Cockerill, F. R.

AU - Patel, Robin

AU - Rosenblatt, J. E.

AU - Smith, T. F.

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