BackgroundIn recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused byNeisseria meningitidis.Study designWe developed and evaluated aN. meningitidisLightCycler real-time duplex PCR (NM-LCdPCR) capable of simultaneously detecting and distinguishing between two separate genes on theN. meningitidisgenome.MethodsThe NM-LCdPCR was developed on the LightCycler platform (Roche Diagnostics, Castle Hill, NSW, Australia) and comprised two primer pairs and two hybridization probe sets, enabling the detection of both theporAandctrAgenes within the same reaction mix. To distinguish between the fluorescence emitted by each hybridization probe set, each downstream probe was labeled with a different fluorophore (either LC-Red640 or LC-Red705). The results obtained by the NM-LCdPCR were then compared with the results obtained by a mono-specific LightCycler assay targeting theporAgene only (porA-LCPCR).PatientsOne-hundred and forty-eight clinical samples from patients with suspected meningococcal infection were evaluated.ResultsThe results of the NM-LCdPCR and porA-LCPCR gave 100% agreement;N. meningitidisDNA was detected in 25 samples whereas 123 samples were negative by both assays. The breakdown of the NM-LCdPCR results show that both genes were detected in 26 of the 28 positive samples.DiscussionBy targeting two separateN. meningitidisgenes, the NM-LCdPCR has the potential to prevent the false-positive results which may arise from sequence variation. In addition, the ability to detect and discriminate between the two differentN. meningitidisgenes within the same reaction mix offers a rapid means for confirming the presence ofN. meningitidisDNA in clinical samples, thereby reducing the need for subsequent confirmatory assays to be performed.ConclusionsThe sensitivity and specificity of the NM-LCdPCR assay, combined with its ability to detect and discriminate both theN. meningitidis porAandctrAgenes, make it suitable for the diagnosis ofN. meningitidisinfections in the routine clinical laboratory.