Ultra-efficient multiple cross displacement amplification-lateral flow biosensor (MCDA-LFB) for serogroup identification of prevalent Neisseria meningitidis

Abstract

Meningococcal disease caused by Neisseria meningitidis remains a major global public health concern. Serogroup A, B, C and W135 were the major disease-causing serogroups. It is vital to timely and efficiently detect and differentiate these four serogroups. Herein, we developed multiple cross displacement amplification-lateral flow biosensor (MCDA-LFB) assays targeting ctrA, sacB, siaD, siaD and synG gene respectively for detection and subtyping of four N. meningitidis serogroups. This assay utilizes LFB to detect FITC and biotin-labeled target amplicons produced by MCDA through double antibody sandwich principle, to allow sensitive and specific detection under a constant temperature. The detection limit was as low as 10 fg or 100 fg genomic DNA in pure cultures and 5.5 CFUs or 36 CFUs in spiked cerebrospinal fluid (CSF) specimens, which were overall 100 to 1000-fold more sensitive than conventional PCR. High specificity of these assays was also validated through type strains and clinical isolates, with no cross-reactions. MCDA-LFB testing procedure can be finished within 1 h. In conclusion, the N. meningitidis- and serogroup-MCDA-LFB assays established in this study are simple, rapid and efficient, providing valuable molecular methods for diagnosis and surveillance of meningococcal disease, especially in resource-limited regions and when specimen culture fails.