DNA Probe Design for Preflight and Inflight Microbial Monitoring

Crew health is a dominant issue in manned space flight. Microbiological concerns, in particular, have repeatedly emerged as determinants of flight readiness. For example, in at least one case, suspected contamination of the potable water supply nearly forced a launch delay. In another instance, a crew member's urinary tract infection nearly led to early termination of the mission, in part due to the difficulty of accurately diagnosing the nature of the infection in-flight. Microbial problems are an increasing concern with the trend towards longer-duration missions. It is essential to the success of such missions that systems that deliver acceptable quality of air and water during the anticipated lifetime of the spacecraft be available. As mission duration and re-supply intervals increase, it will be necessary to rely on advanced life support systems which incorporate both biological and physical-chemical recycling methods for air and water as well as provide food for the crew. It is therefore necessary to develop real-time, robust, in-flight monitoring procedures that are sensitive enough to detect less than 100 CFU (colony forming units) of bacteria per 100 milliliters of water. It would be desirable if the monitoring system could be readily "reprogrammed" to identify specific pathogens if an in-flight incident were to occur.
The long-term objective of the proposed work is to develop technology that can be used to monitor microbial populations and, when necessary, to identify pathogens in air and water samples during long-duration space missions. The initial focus is on developing a prototype water analysis system. The prototype system is being developed as a sandwich assay, which utilizes both a capture probe and detector probe(s). The surface capture probe selectively acquires the target rRNA from a total RNA preparation. The detector probe provides a labeling group and assists in the denaturation of the target molecule by disrupting the secondary structure of the target rRNA in the region where it binds. The availability of both kinds of probes allows implementation of the assay on hybridization arrays ("DNA chips") or, using just the detector probes, in a solution format. Our specific aims in the current year were (1) to design a set of probes that can in principle be used to simultaneously monitor the levels of total bacteria, fecal coliforms, Escherichia coli, Pseudomonas aeruginosa, Ralstonia, Acinetobacter, Enterococcus and Burkholderia; (2) to synthesize and test the probes needed to implement the prototype system using a microtiter-format assay; (3) to develop sample processing procedures that could be used in the space environment; and (4) to examine the utility of solution hybridization methods as an alternative format.
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Additional probes were designed and tested. These included successful probes for Gram negative bacteria and enteric bacteria. We now have useful probes for essentially all the organisms needed to devise an assay system for monitoring water quality. This now includes probes for total bacteria, Gram negative bacteria, enteric bacteria, Escherichia coli, Vibrio proteolyticus, Burkholderia cepacia, and Acinetobacter. We also extended our non-toxic method of purifying DNA from RNA using compaction agents to also purify RNA from DNA. Finally, we showed that molecular beacon technology could be successfully used to detect specific sequences in ribosomal RNA. A molecular beacon assay would be implemented by astronauts who would mix predetermined reagents with a sample and then analyze the sample's color change using a simple spectrophotometer. The technique can readily detect up to five key organism types and is considered promising for use in routine daily monitoring.
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Similar approaches to those being developed here might be used on Earth to simultaneously screen samples for multiple bacterial species. Such instrumentation might be useful in either environmental or clinical laboratories.