Summary: | Methods for bacterial detection and identification has garnered renewed interest in
recent years due to the infections they may cause and the antimicrobial resistances
they can develop, the potential for bioterrorism threats and possible contamination of
food/water supplies. Therefore, the rapid, specific and accurate detection of pathogens
is crucial for the prevention of pathogen-related disease outbreaks and facilitating
disease management as well as the containment of suspected contaminated food
and/or water supplies. In this dissertation an integrated modular-based microfluidic
system composed of a fluidic cartridge and a control instrument has been developed for
bacterial pathogen detection. The integrated system can directly carry out the entire
molecular processing pipeline in a single disposable fluidic cartridge and can detect
sequence variations in selected genes to allow for the identification of the bacterial
species and even its strain. The unique aspect of this fluidic cartridge is its modular
format with a task-specific module interconnected to a fluidic motherboard to permit the
selection of a material appropriate for the given processing step(s). In addition, to
minimize the amount of finishing steps for assembling the fluidic cartridge, many of the
functional components were produced during the polymer molding step used to create
the fluidic network. The operation of the fluidic cartridge was provided by electronic,
mechanical, optical and hydraulic controls located off-chip and assembled into a small
footprint instrument. The fluidic cartridge was capable of performing cell lysis, solidphase
extraction of genomic DNA from the whole cell lysate, continuous flow PCR
amplification of specific gene fragments, continuous flow ligase detection reaction to
discriminate sequence variations and universal DNA array readout, which consisted of
DNA probes patterned onto a planar polymer waveguide for evanescent excitation. The
performance of the fluidic system was demonstrated through its successful application
to the genetic detection of bacterial pathogens, such as Escherichia coli O157:H7,
Salmonella, methicillin-resistant Staphylococcus aureus and multi-drug resistant
Mycobacterium tuberculosis, which are major threats for global heath. The modular
system, which could successfully identify several strains of bacteria in <40 min with
minimal human intervention and also perform strain identification, represents a
significant contribution to pathogen detection.
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