title: Evaluation of conventional and real-time PCR assays using two targets for confirmation of results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae test for detection of Neisseria gonorrhoeae in clinical samples.
abstract: Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non-N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae-positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.
authors.affiliation: Laboratory for Medical Microbiology, PAMM Laboratories, P.O. Box 2, NL-5500 AA Veldhoven, The Netherlands. e.boel@pamm.nl
authors.forename: C H E
authors.initials: CH
authors.lastname: Boel
authors.forename: C M C
authors.initials: CM
authors.lastname: van Herk
authors.forename: P J M
authors.initials: PJ
authors.lastname: Berretty
authors.forename: G H W
authors.initials: GH
authors.lastname: Onland
authors.forename: A J C
authors.initials: AJ
authors.lastname: van den Brule
mesh_terms: D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000145:classification / Q000302:isolation & purification*; D017931:DNA Primers; D006801:Humans; D007124:Immunoenzyme Techniques; D009344:Neisseria gonorrhoeae / Q000145:classification / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers