Neisseria gonorrhoeae

While many of the Gram-negative and non-motile Neisseria species are part of the normal flora in humans and animals, N. gonorrhoeae is the causative agent of the sexually-transmitted disease gonorrhea. Infection primarily affects the skin and mucous membranes - including the mouth and genitals - involving a rash and inflammation.Today the treatment of gonorrhoea is more difficult since antimicrobial resistance of N. gonorrhoeae occurs to penicillins, tetracyclines, spectinomycin, and recently to fluoroquinolones.

Targets for the Real-Time-PCR detection are the genes for 16S RNA1,2, C-Methylase, porA2 or gyrA1,2, the opacity genes opaC or opaD3, and the plasmid based cppB2,4 gene. The latter has been found to detect also other Neisseria species. The Australian guidelines present a good overview

Description

A 166 bp fragment of the gyrA gene of the Neisseria gonorrhoeae genome is amplified with specific primers and detected with probes labeled with LightCycler® Red 640 (detected in channel 640). The PCR product is identified by running a melting curve with a specific melting point (Tm) of 63°C and a shoulder at 57°C in channel 640.

An additional PCR product of 278 bp is formed from the internal positive control DNA. This control does not interfere with the Neisseria gonorrhoeae specific reactions. The amplification will usually fail in the presence of higher concentrated Neisseria gonorrhoeae DNA samples (1,000 copies or higher) while displaying an amplification signal in negative and low-concentrated samples. The hybridization probes are

labeledwith LightCycler® Red 690 (recorded in channel 705). The IC is supplied separately to allow running the assay in the presence or absence of the IC.

The use of a color compensation file generated with TIB MOLBIOL Color Compensation Kit 530/640/690 or the Roche Diagnostics ‘LightCycler®-Color Compensation Set’ or with the Roche Diagnostics ‘LightCycler® Multicolor Demo Set’ is a prerequisite to run the duplex reaction.

The supplied standard row allows to determine the linear range of the reaction and to estimate the quantity of the target sequence in unknown samples.

1 Verification of clinical samples, positive in AMPLICOR Neisseria gonorrhoeae polymerase chain reaction, by 16S rRNA and gyrA compared with culture. Airell et al. Int J STD AIDS. 2005 Jun;16(6):415-9

2 A comparison of three real-time PCR assays for the confirmation of Neisseria gonorrhoeae following detection of N. gonorrhoeae using Roche COBAS AMPLICOR. Chui et al., Clin Microbiol Infect 2008; 14: 473–479

3 Evaluation of opa-based real-time PCR for detection of N. gonorrhoeae. Tabrizi et al. Sex Transm Dis. 32 (2005) 199-202

4 A real-time PCR assay for the detection of N. gonorrhoeae by LightCycler. Whiley et al.. D.Microb.Infect 42 (2002) 85-9

5 Guidelines for the use and interpretation of nucleic acid detection tests for N. gonorrhoeae in Australia: Smith et al. (2006)

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