AWWA ACE59875

This paper presents a simple rapid screening assay for the detection of pathogens in water. The assay was developed, optimized and evaluated using a suite of representative waterborne pathogens that included: Cryptosporidium parvum; Giardia duodenalis; enteroviruses; and, Escherichia coli O157:H7. The assay consisted of nucleic acid amplification followed by hybridization with non-radioactively labeled probes in 96-well streptavidin-coated microplates and detection of the hybrids using colorimetric or fluorometric substrates. The streptavidin-coated 96-well microplate was coated with biotin-labeled amplicons, which were labeled during amplification by the incorporation of 5' biotin tagged primers, and hybridization was conducted using a 5'-end digoxigenin (DIG)-labeled probe Colorimetric detection was achieved using an anti-DIG antibody-alkaline phosphatase conjugate and a solution of nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP). C. parvum and Echovirus 1 were used as model organisms to determine the optimum assay conditions and to compare the rapid screening assay with conventional techniques. G. duodenalis and E. coli were used to evaluate the adaptability of the assay to detect other organisms. The microplate format offers high sample throughput, rapid reading and the potential for automation at low cost with the use of multi-well microplates and a microplate reader. The assay is broadly applicable to all types of pathogens so long as sequence information is available to allow the design of primers and probes. Since the vast majority of water samples are expected to be negative for pathogens, a rapid screening assay allows the option of eliminating negative samples from further analysis. More time-consuming and costly techniques for pathogen enumeration or infectivity determination can then be focused on positive samples only. Includes 5 references.