Detection of Neisseria Meningitidis in clinical samples by a duplex real-time PCR targeting the porA and ctrA genes.
BACKGROUND
In recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused by Neisseria meningitidis.


METHODS
We developed and evaluated a N. meningitidis LightCycler real-time duplex PCR (NM-LCdPCR) capable of simultaneously detecting and distinguishing between two separate genes on the N. meningitidis genome.


METHODS
The 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 the porA and ctrA genes 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 the porA gene only (porA-LCPCR).


METHODS
One-hundred and forty-eight clinical samples from patients with suspected meningococcal infection were evaluated.


RESULTS
The results of the NM-LCdPCR and porA-LCPCR gave 100% agreement; N. meningitidis DNA 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.


CONCLUSIONS
By targeting two separate N. meningitidis genes, 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 different N. meningitidis genes within the same reaction mix offers a rapid means for confirming the presence of N. meningitidis DNA in clinical samples, thereby reducing the need for subsequent confirmatory assays to be performed.


CONCLUSIONS
The sensitivity and specificity of the NM-LCdPCR assay, combined with its ability to detect and discriminate both the N. meningitidis porA and ctrA genes, make it suitable for the diagnosis of N. meningitidis infections in the routine clinical laboratory.
Whiley
David M
DM
Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Herston, and Clinical Medical Virology Centre, University of Queensland, Brisbane, Queensland, Australia.
Crisante
Michelle E
ME
Syrmis
Melanie W
MW
Mackay
Ian M
IM
Sloots
Theo P
TP
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D008589:Meningococcal Infections / Q000175:diagnosis; D009345:Neisseria meningitidis / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D018272:Porins / Q000235:genetics*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; C097706:CtrA protein, Caulobacter; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018272:Porins; D014157:Transcription Factors; C057699:porin protein, Neisseria