Detection of Neisseria Meningitidis in clinical samples by a duplex read-time PCR targeting the porA and ctrA Genes

Whiley, David M ., Crisante, Michelle E., Syrmis, Melanie W., Mackay, Ian M. and Sloots, Theo P. (2003) Detection of Neisseria Meningitidis in clinical samples by a duplex read-time PCR targeting the porA and ctrA Genes. Molecular Diagnosis, 7 3: 141-145.

Author Whiley, David M .
Crisante, Michelle E.
Syrmis, Melanie W.
Mackay, Ian M.
Sloots, Theo P.
Title Detection of Neisseria Meningitidis in clinical samples by a duplex read-time PCR targeting the porA and ctrA Genes
Journal name Molecular Diagnosis   Check publisher's open access policy
ISSN 1084-8592
Publication date 2003
Sub-type Article (original research)
Volume 7
Issue 3
Start page 141
End page 145
Total pages 5
Place of publication Auckland, New Zealand
Publisher Adis International
Collection year 2003
Language eng
Subject CX
1103 Clinical Sciences
110316 Pathology (excl. Oral Pathology)
Formatted abstract
AB Background:
In recent years PCR has proven to be a highly sensitive and specific method for the diagnosis of infections caused by Neisseria meningitidis.

Study design:

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). Patients: 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. Discussion: 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. Copyright 2003 Adis Data Information BV
Q-Index Code CX

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Medicine Publications
 
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Created: Wed, 15 Aug 2007, 03:25:14 EST