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 $1000 CCD-based fluorescence imaging system. Application of a highly fluorogenic Syto-82 dye and CCD exposure control to microRTf-LAMP assays, increased their signal-to-noise ratios by 10-fold and reduced their threshold times to 10-50-fold, providing the single DNA copy level sensitivity. A novel digital LAMP method to track the in situ bacterial growth by gene amplification directly from gram-negative (Escherichia coli) and gram positive (Enterococcus faecalis) bacterial cells was developed. Propidium monoazide-based LAMP assay was performed to confirm the gene amplification was only from viable cells. Digital LAMP assays were rapid (20 min) and sensitive to single cell. Cost reduction of 50-fold was achieved by using polyester material for digital LAMP assays. To reduce the time of antibiotic susceptibility testing (AST), a microchip-based dynamic method for E. coli growth monitoring in the presence of a membrane intercalating fluorogenic dye (FM5-95) was developed. A variety of dye concentrations and CCD exposure times were tested. A combination of 20 μg/mL FM5-95 dye and 10 s of CCD exposure provided the detection of 1 ×104 E. coli cells in only 60 min. MicroAST of ampicillin and tetracycline on E. coli strain provided the minimum inhibitory concentration (MIC) of 16 μg/mL and 1.6 μg/mL respectively, which were consistent with the MIC values reported in literature. 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.
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