AWWA WQTC58786

There has been a long-standing need to develop autonomous pathogen detection systems for water samples that will eliminate the time, variability, and operator involvement needed to obtain accurate results. Construction and validation of an autonomous system for pathogen detection in water supplies is a significant challenge that must take into account: the representative volume of water that must be sampled and examined to have statistical confidence in the results; the methods used for concentrating the sample into reasonable volumes for analyses; the processing and purification steps needed for detection; the detection platform chosen (e.g. cell culture, PCR, fluorescent antibody); and, the engineering required to accomplish these tasks that will provide precise and accurate results with limited false positive and false negative error rates. The authors are currently testing a breadboard field device that was originally designed for aerosolized bioterrorism agents. However, the guiding biochemistry that drives the system can be readily adapted for water quality monitoring. The prototype field device is currently being developed for E. coli O157:H7. The device can autonomously sample up to 100 mL volumes of water. Processing to remove inhibitors is accomplished by flow-through immunomagnetic separation. The purified cells, immobilized on the magnetic beads, are then moved to a flow through PCR system, and PCR is performed directly off of the beads. The eluate is collected and the PCR sample is hybridized to an array with specific probes for the detection of these products. Both specificity and sensitivity of the automated protocol are excellent. Consistent recovery of 10 spiked cells into matrix water (Columbia River water) is routinely achieved. The system architecture and PCR thermal cycling protocols virtually eliminate PCR carryover allowing the system to be used many times before tubing and other easy user replaceable parts is required. The next generation system will be able to collect much larger volumes of water (up to 10 L), and link highly multiplexed PCR for multiple agents, with a flow through bead-array system for true autonomous detection of any waterborne pathogen. Includes 31 references, figures.