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Journal of Clinical Microbiology, November 2005, p. 5653-5659, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5653-5659.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Multidisciplinary Laboratory of Molecular Diagnostics and Regional Laboratory of Medical Microbiology, Jeroen Bosch Hospital,¹ Medical Microbiology and Infectious Diseases, Gelre Hospitals, Location Lukas,Apeldoorn ,² GG&GD, Municipal Health Service, Amsterdam,³ Laboratory for Pathology and Microbiology, PAMM, Veldhoven, The Netherlands⁴

Received 4 March 2005/ Returned for modification 19 May 2005/ Accepted 11 August 2005

Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae in clinical samples. The target sequence is a 76-bp fragment of the 5' untranslated region of the opa genes that encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were negative 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 opa positive and 16S rRNA negative, which may be due to the higher sensitivity of the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific, semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for confirmation of less specific tests.

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