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JMD 2006, Vol. 8, No. 5
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

Stig Ove Hjelmevoll^*, Merethe Elise Olsen^*, Johanna U. Ericson Sollid, Håkon Haaheim^*, Magnus Unemo and Vegard Skogen

From the Department of Microbiology and Infection Control * and Department of Medicine, University Hospital of North Norway; Department of Microbiology and Virology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway; National Reference Laboratory for Pathogenic Neisseria, Örebro University Hospital, Örebro, Sweden; and Department of Internal Medicine B, University Hospital of North Norway, and Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway

Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.