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This paper presents a simple rapid screening assay for the detection of pathogens inwater. The assay was developed, optimized and evaluated using a suite of representativewaterborne pathogens that included: Cryptosporidium parvum; Giardia duodenalis; enteroviruses; and,Escherichia coli O157:H7. The assay consisted of nucleic acid amplification followed byhybridization with non-radioactively labeled probes in 96-well streptavidin-coated microplatesand detection of the hybrids using colorimetric or fluorometric substrates. The streptavidin-coated96-well microplate was coated with biotin-labeled amplicons, which were labeled duringamplification by the incorporation of 5' biotin tagged primers, and hybridization was conductedusing a 5'-end digoxigenin (DIG)-labeled probe Colorimetric detection was achieved using ananti-DIG antibody-alkaline phosphatase conjugate and a solution of nitroblue tetrazolium saltand 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP). C. parvum and Echovirus 1 were usedas model organisms to determine the optimum assay conditions and to compare the rapidscreening assay with conventional techniques. G. duodenalis and E. coli were used to evaluatethe adaptability of the assay to detect other organisms. The microplate format offers high samplethroughput, rapid reading and the potential for automation at low cost with the use of multi-wellmicroplates and a microplate reader. The assay is broadly applicable to all types of pathogens solong as sequence information is available to allow the design of primers and probes. Since thevast majority of water samples are expected to be negative for pathogens, a rapid screening assayallows the option of eliminating negative samples from further analysis. More time-consuming andcostly techniques for pathogen enumeration or infectivity determination can then be focused onpositive samples only. Includes 5 references.

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Edition: Vol. - No. Published: 06/17/2004 Number of Pages: 6File Size: 1 file , 290 KB