Development of a simplex polymerase chain reaction-based assay for the detection of Neisseria meningitidis

Abstract

This study was undertaken to develop a sensitive and specific polymerase chain reaction (PCR)-based assay for Neisseria meningitidis and Neisseria gonorrhoeae that could ultimately be incorporated into a multiplex assay designed to screen cerebrospinal fluid (CSF) for a panel of pyogenic bacterial species associated with bacterial meningitis. N.meningitidis-specific primers were designed from porA gene sequences with the aid of a commercial software program and were used to develop and validate a clinical assay using a liquid hybridization-gel retardation detection system. The analytic sensitivity of the assay was determined using CSF spiked with N. meningitidis and comparing the PCR-based results with culture. Analytic sensitivity experiments showed the assay's limit of detection to be 100 fg purified input target DNA and 0.0125 colony-forming units of N. meningitidis spiked into CSF. Specificity experiments showed the assay could detect all strains of N. meningitidis and N. gonorrhoeae tested, but did not support amplification of the commensal neisserial species or a panel of other human bacterial pathogens. This PCR-based assay for pathogenic neisserial species is sensitive and specific and suitable for incorporation into a multiplex assay for the clinical differentiation of aseptic and septic meningitis.