title: 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: 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.
authors.affiliation: Division of Clinical Microbiology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Espy
authors.forename: P N
authors.initials: PN
authors.lastname: Rys
authors.forename: A D
authors.initials: AD
authors.lastname: Wold
authors.forename: J R
authors.initials: JR
authors.lastname: Uhl
authors.forename: L M
authors.initials: LM
authors.lastname: Sloan
authors.forename: G D
authors.initials: GD
authors.lastname: Jenkins
authors.forename: D M
authors.initials: DM
authors.lastname: Ilstrup
authors.forename: F R
authors.initials: FR
authors.lastname: Cockerill
authors.forename: R
authors.initials: R
authors.lastname: Patel
authors.forename: J E
authors.initials: JE
authors.lastname: Rosenblatt
authors.forename: T F
authors.initials: TF
authors.lastname: Smith
mesh_terms: D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification*; D005835:Genitalia / Q000821:virology*; D006561:Herpes Simplex / Q000821:virology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012371:Robotics; D018139:Simplexvirus / Q000235:genetics / Q000302:isolation & purification*; D012867:Skin / Q000821:virology*
chemical_list: D004279:DNA, Viral