| The potential for microbial diagnostics to impact health care in developing regions of the world is well recognized. However, there is a large gap in the use of such technologies due to the unavailability of low cost point of care genetic screening tools. The device developed within this project fills this gap, with the potential for providing genetic screening at a cost that is well within reach of resource-limited regions (< ;Validation of primers and probes was accomplished using an existing high throughput real time thermocycler for RT-PCR, and an in situ synthesized microarray for hybridization-based studies. The limits of the hybridization-based approach for detection of closely related fragments will be useful in developing assays for mutation detection. In addition to validating 220 primers sets targeting 30 microbial pathogens, a predictive equation for quantification of starting copies was experimentally established based on the sequence characteristics of primers and target organisms (as an efficient alternative to generating standard curves). Validated primers can be used in the gene analyzer after it is fully developed.;Constraints in the development of truly portable and low cost gene analyzers are typically associated with the means of detection. Less costly components such as light emitting diodes (LEDs) and photodiodes are promising alternatives to charge coupled devices and lasers; however, simultaneous detection of multiple channels with spot detectors typically requires movement or assigning independent detectors to each channel on the device. To overcome this issue, an array of sixteen individually controlled LEDs (one LED for each reaction well) was used with optical fibers sending fluorescent signal to a single photodiode detector. Only one LED/well/optical fiber is turned "on" at a time and identity is determined by the associated sequence and timing of the photodiode reading. Optical cross talk between reaction wells was avoided using a thin-wall (shelled) microfluidic chip. The disposable chips were pressed in 100-mum thin Zeonor film by hot embossing with a male mold fabricated using stereolithography and a thermal plastic counter tool.;Components and control was embedded into a hand-held footprint through design of a custom printed circuit board, control with a microcontroller, and design of a cartridge to package the device. Comparable results were observed between the gene analyzer and a state-of-the-art real-time PCR instrument, demonstrating that this simple, small, low component cost system has the potential to play a key role in propagating the use of gene based screening devices in resource-limited settings. |
