Real-time polymerase chain reaction species specific for Neisseria Gonorrhoeae

Gonorrhea is a sexually transmitted disease caused by bacteria called gonococci. The disease was described in ancient times, but until the late nineteenth century it was mixed up with syphilis. Eventually microscopy could differ between the organisms and give a reliable diagnosis. Human is the only known reservoir for gonorrhea. It is transferred during all acts of sexual activity, and an infection cause characteristic symptoms if established. Reliable diagnostic tools are important not only because gonorrhea is a severe disease but also because the means of spreading will always have a negative social impact on people given the diagnosis. Gonococci are fastidious bacteria, and survive poorly outside its human host. Thus diagnostic methods have been challenged by the problem of retrieving living bacteria for phenotypic tests. There have been several attempts to identify gonococci by specific characteristic in its genome, but these have been less successful. Due to its fluctuating genome it has been difficult to find a specific target for the gonococci. Here I present an attempt to establish a reliable diagnostic tool for reproducible detection and specific identification of gonococci in samples from different body sites

Appropriate Time for Test-of-Cure when Diagnosing Gonorrhoea with a Nucleic Acid Amplification Test

This article is part of Stig Ove Hjelmevolls doctoral thesis. Available in Munin at http://hdl.handle.net/10037/3816</a

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

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