HRM confirmation of Neisseria gonorrhoeae in clinical specimens by G → A (position 857) mutation detection in the 16S rRNA gene before sequencing and after porA confirmation

A total of 2273 specimens submitted to the Austin Hospital Pathology Service for Neisseria gonorrhoeae screening between September 1, 2009 and May 11, 2011 were used in this study. Specimens were simultaneously screened and confirmed with a previously published real time PCR assay for the opa gene (extra primers were included to increase sensitivity) and the porA gene respectively. The opa gene screen and initial porA gene confirmation yielded an N. gonorrhoeae positivity rate of 0.88% (20/2273) and 0.49% (11/2191) for specimens and patients respectively. A 16S rDNA High Resolution Melt confirmatory PCR was developed subsequently; this reduced the N. gonorrhoeae positivity rate to 0.35% (8/2273) and 0.27% (6/2191) for specimens and patients respectively (not altered by 16S sequencing). The higher rate of secondary confirmation (16S HRM) in patients compared with samples was due to the detection of species other than N. gonorrhoeae detected by the initial screening and confirmation test. This underlines the importance of performing the secondary confirmatory test that has been developed in this study.

► A method was developed to confirm N. gonorrhoeae by HRM RT-PCR of G857A. ► Screening by opaA RT-PCR gave an initial positivity rate of 0.88%. ► Initial confirmation by porA RT-PCR reduced the positivity rate to 0.49%. ► HRM of G857A reduced the positivity rate to 0.35% and 0.27%. ► Underlining the importance of HRM G857A N. gonorrhoeae secondary confirmation.