Rapid detection of pathogens and their antibiotic susceptibility using simple microfluidics and CCD imaging.

摘要

Emergence of virulent and antibiotic resistant bacterial strains is posing a serious threat to human health. In developing countries, deaths associated with infectious diseases (e.g., diarrheal pathogens) are often due to the delayed diagnostics rather than effective and economical treatment options. Gold standard methods for pathogen/resistance identification rely on either polymerase chain reaction (PCR) or culture-based approaches, which are time consuming and require either manual intervention(s) or expensive instrumentation. Microfluidic PCR-based diagnostic systems require dedicated sample processing steps, thermal cycling, and sophisticated microfabrication processes, thus are slow and expensive. Microfluidic loop-mediated isothermal amplification (LAMP) is a promising alternative as it requires simpler instrumentation and inexpensive detector due to its isothermal character and high amplicon yield. Therefore, for this dissertation, novel fluorogenic dyes were applied to isothermal genetic and cellular assays for rapid and real-time detection of pathogens and their antibiotic susceptibility by using simple microfluidics and charge-coupled device (CCD) imaging.;A critical and quantitative review on miniaturized nucleic acid amplification systems was performed for the selection of an appropriate molecular assay, low cost material for microchip, detection system, and parameters that influence the gene amplification time. Real-time fluorescence LAMP (microRTf-LAMP) assays of 12 virulence genes of 6 diarrheal pathogens were performed in cyclic olefin copolymer microchips and monitored by a ;The methods developed for this dissertation would eliminate the requirement of complex sample preparation steps, facilitating the development of sensitive, rapid, low-cost, and integrated diagnostic systems. The methods developed here for digital LAMP and microAST when integrated on a single microfluidic chip will enable faster detection of AST in clinical settings. The overall approach of faster detection of pathogens and their antibiotic resistance based on pathogen growth in the presence of antibiotic(s) using digital LAMP directly on cells can be extended to many different fields.