Abstract
The rapid and accurate diagnosis of meningitis is critical for preventing severe complications and fatalities. This study addresses the need for accessible diagnostics in the absence of specialized equipment by developing a novel diagnostic assay. The assay utilizes dual-priming isothermal amplification (DAMP) with unique internal primers to significantly reduce non-specificity. For fluorescence detection, the dye was selected among Brilliant Green, Thioflavin T, and dsGreen. Brilliant Green is preferred for this assay due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay was developed for the detection of the primary causative agents of meningitis (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae), and tested on clinical samples. The developed method demonstrated high specificity, no false positives, sensitivity comparable to that of loop-mediated isothermal amplification (LAMP), and a high S/B ratio. This versatile assay can be utilized as a standalone test or an integrated assay into point-of-care systems for rapid and reliable pathogen detection.