Specific and Sensitive Detection of Neisseria gonorrhoeae in Clinical Specimens by Real-Time PCR

Earlydiagnosis of Neisseria gonorrhoeae infections is importantwith regard to patients' health and infectivity. We report thedevelopment of a specific and sensitive TaqMan assay for the detectionof N. gonorrhoeae in clinical samples. The target sequence isa 76-bp fragment of the 5' untranslated region of theopa genes that encode opacity proteins. A panel of 448well-defined N. gonorrhoeae isolates was used to evaluate andoptimize the assay. The method employs two minor-groove binding probes,one of them recognizing a newly identified sequence in the opagenes. Testing a large panel of related and unrelated microorganismsrevealed that other Neisseria strains and other microorganismstested negative in the opa test. With a lower detection limitof one genome per reaction, the opa test appeared moresensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV,Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of apanel of 122 COBAS AMPLICOR-positive samples revealed that 68% werenegative in both the 16S rRNA test and the opa assay(confirming that the COBAS AMPLICOR test produces false positives),while 30% were positive in both assays. Three samples were opapositive and 16S rRNA negative, which may be due to the highersensitivity of the opa assay. We conclude that theopa gene-based real-time amplification assay offers asensitive, specific, semiquantitative, and reliable assay suitable forthe detection of N. gonorrhoeae in clinical specimens and/orfor confirmation of less specifictests.