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.
Hjelmevoll
Stig Ove
SO
Department of Microbiology and Infection Control, University Hospital of North Norway, 9038 Tromsø, Norway. stig.ove.hjelmevoll@unn.no
Olsen
Merethe Elise
ME
Sollid
Johanna U Ericson
JU
Haaheim
Håkon
H
Unemo
Magnus
M
Skogen
Vegard
V
D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D006069:Gonorrhea / Q000175:diagnosis*; D009344:Neisseria gonorrhoeae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D018272:Porins / Q000235:genetics*; D011544:Pseudogenes; D012680:Sensitivity and Specificity; D013048:Specimen Handling
D004269:DNA, Bacterial; D018272:Porins; C057699:porin protein, Neisseria