Standardization of two multiplex real-time PCRs for the diagnosis of bacterial meningitis and detection of Neisseria meningitidis serogroups

Abstract

Introduction: Bacterial meningitis is a serious neurological infection that mainly affects children, caused primarily by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. In Peru, there is no surveillance system for Neisseria meningitidis and its serogroups. Methods: A multiplex real-time PCR (qPCR) assay using Taqman probes was standardized for the detection of N. meningitidis, S. pneumoniae, and H. influenzae. Additionally, three duplex SYBR Green-based qPCR assays were performed to detect the six most significant Neisseria meningitidis serogroups, using DNA from ATCC reference strains. Results: The multiplex qPCR assay showed amplification efficiencies between 100.2% and 109.1%, with a detection limit of one genome copy/reaction for each bacterium. The duplex qPCR assays made it possible to differentiate serogroups using a temperature melting analysis. Conclusions: Two qPCR assays were standardized for the etiological study of bacterial meningitis, enabling prompt diagnosis and supporting epidemiological surveillance in public health studies and interventions.