A novel porA-based real-time PCR for detection of meningococcal carriage

Abstract

Real-time PCR based on the capsule transfer gene (ctrA) is a significant aid in the diagnosis of meningococcal infection but fails to detect a high proportion (60 %) of non-groupable strains associated with nasopharyngeal carriage. This study aimed to design a novel real-time (TaqMan) PCR that would detect more strains of meningococci and be suitable for large-scale carriage studies. Primer and probe sequences were based on the meningococcal porA gene and designed specifically to exclude the highly related porA pseudogene in Neisseria gonorrhoeae. The specificity of the assay was confirmed by testing strains of N. gonorrhoeae known to contain the porA pseudogene together with commensal strains of Neisseria lactamica and Neisseria sicca. None of these was detected in the assay. Neisseria meningitidis strains representing a wide range of serogroups together with non-groupable strains isolated from the nasopharynx were tested by ctrA assay and the novel porA-based TaqMan PCR. All carriage strains were detected by the porA-based assay including four that gave weak or no reaction with the ctrA assay. Comparison of ctrA and porA assays on 71 throat swabs obtained from university students showed that the porA assay detected meningococcal DNA in all samples that were ctrA positive plus three that were ctrA negative but culture positive. This novel porA-based TaqMan assay provides a highly specific method for detecting meningococcal DNA that is more sensitive than the ctrA assay for detecting meningococcal carriage and is particularly suitable for carriage studies where non-groupable strains and other Neisseria are present.