Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

快速、特异且灵敏地检测细菌病原体对于传染病的临床管理至关重要。然而，传统的病原体检测方法往往需要耗费大量时间的细菌培养和扩增程序。在此，我们展示了一种微粒增强型双链DNA探针，用于快速、物种特异性地检测细菌16S rRNA。在这种分子检测方法中，靶序列与荧光团共轭探针的结合会在热力学作用下置换猝灭探针，从而使荧光团发出荧光。通过引入包被链霉亲和素的微粒来固定生物素化探针，该检测方法的灵敏度可提高3个数量级。该检测方法无需靶标扩增，检测限低至仅8个细菌，并且对其他常见病原体具有高度特异性。通过直接鉴定尿路感染患者尿液样本中的细菌病原体，证明了该方法在临床诊断中的适用性。