DNA Microchip-Based Microbial Monitoring

Microbial monitoring of closed systems such as spacecraft and space stations is crucial to maintaining the health of crew members. Molecular methods of microbial detection and identification are superior to culture-based techniques, direct counts, and biochemical assays. In particular, rRNA-based methods are a powerful tool, providing a robust molecular-phylogenetic framework for the taxonomic classification of bacteria. Traditional forms of rRNA analysis include hybridizations in membrane or whole-cell format. DNA microchip technology for determinative and environmental microbiology provides advantages over these formats for rRNA analysis. This method utilizes a high-density polyacrylamide gel micro-array, consisting of a matrix of hundreds or thousands of individual gel elements bound to the surface of a glass slide. Each gel element contains DNA probes complementary to the 16S rRNA sequence of a selected bacterial species, genus, or taxonomic group. This format provides for massively parallel hybridization, allowing for simultaneous hybridization to hundreds or even thousands of probes. A microchip can be used up to 20 or 30 times without a noticeable deterioration of the hybridization signal.
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We propose to develop DNA microchip technology to detect, identify, and quantify microbial contaminants of potable and wastewater systems. We will accomplish this by (a) designing probes for targeted bacterial groups and species, (b) optimizing chip hybridization conditions for these probes, (c) optimizing RNA extraction and recovery from water samples, (d) determining working detection limits of DNA microchips, and (e) using the microchip to distinguish between live, viable but nonculturable (VNC), and dead cells. The DNA microchip should have broad application in the areas of human health and environmental monitoring. Since a microchip can be designed to target specific organisms, such as pathogens, it should have immediate application in medical diagnostics. And, since a microchip can also be designed to monitor large populations of microorganisms simultaneously within biologically complex settings, it should have many applications in monitoring the environmental "health" of natural and engineered systems that are dependent upon microorganisms for proper functioning.