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.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 290 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 6 |
| Published : | 06/17/2004 |
