RESUMEN
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.
Asunto(s)
Haemophilus influenzae , Meningitis Bacterianas , Técnicas de Diagnóstico Molecular , Neisseria meningitidis , Técnicas de Amplificación de Ácido Nucleico , Streptococcus pneumoniae , Neisseria meningitidis/genética , Neisseria meningitidis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Humanos , Haemophilus influenzae/genética , Haemophilus influenzae/aislamiento & purificación , Meningitis Bacterianas/diagnóstico , Meningitis Bacterianas/microbiología , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y EspecificidadRESUMEN
Structural RNA is a challenging target for recognition by hybridization probes. This chapter addresses the recognition problem of RNA amplicons in samples obtained by multiplex nucleic acid sequence-based amplification (NASBA). The method describes the design of G-quadruplex binary (split) DNA peroxidase sensors that produces colorimetric signal upon recognition of NASBA amplicons.
Asunto(s)
Colorimetría , Replicación de Secuencia Autosostenida , Colorimetría/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/genética , ARN ViralRESUMEN
Visual detection of ssRNA and dsDNA amplicons was achieved at room temperature without the need for a probe-analyte annealing stage. This approach uses a DNA nanostructure equipped with two analyte-binding arms. Highly selective binding of the third arm leads to the formation of a G-quadruplex structure capable of changing the solution color.