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Title: Development of polymerase chain reaction techniques for the detection of waterborne pathogens in environmental waters 
Author: Roll, Bruce M.
Date: 1995
Abstract: Currently water quality indicator bacteria (Escherichia coli, enterococci) are
utilized to determine the sanitary quality of recreational waters. These indicators
are normal inhabitants of the intestinal tract of warm-blooded animals and their
presence in recreational water is considered indicative of fecal contamination.
However, in tropical climates E. coli and enterococci appear to be naturally present
in tropical fresh waters and soil. This has questioned the use of E. coli and
enterococci as water quality indicators for tropical climates and demonstrates the
need for alternative means to determine the sanitary quality of recreational waters.
The development of the polymerase chain reaction (PCR) method has offered an
attractive alternative to the problematic cultural isolation of indicator bacteria and
pathogens. The study utilize PCR to detect waterborne pathogens and indicator
bacteria in environmental waters. In addition survival experiments were conducted
to determine the persistence of PCR-target gene sequences in ocean water. A pair
of PCR primers were chosen targeting a Salmonella spp. gene sequence. These
primers were specific for 25 Salmonella spp. and did not cross react with 27
different non Salmonella bacteria. In seeding experiments the sensitivity of these
primers was 12 CFU (Salmonella typhimurium) in primary treated sewage and 1
CFU in ocean water. In addition, field studies were conducted using PCR and
standard culture method. A total of six field samples were tested with these primers
and 3 demonstrated the presence of Salmonella spp. Two of the PCR positive
samples were positive for culturally viable Salmonella spp. A set of PCR primers
were developed to detect Bacteroides spp., the most common inhabitant of the
human intestinal tract. These primers were specific for 4 Bacteroides spp. and did
not cross react with 27 non Bacteroides isolates. These primers were able to detect
Bacteroides spp. in a variety of environmental waters. Survival experiments using
E. coli and Salmonella typhimurium were conducted in ocean water and
demonstrated a loss of E. coli and S. typhimurium gene sequences after exposure
for 14 and 16 days, respectively. In summary this research supports the use of PCR
as a water quality monitoring tool.
Description: Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. Includes bibliographical references (leaves 120-131). Microfiche. xiii, 131 leaves, bound ill. 29 cm
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Keywords: Polymerase chain reaction, Marine microbiology, Salmonella typhimurium, Waterborne infection

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