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Molecular Alternatives to Indicator and Pathogen Detection (Paperback, Illustrated Ed)
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Molecular Alternatives to Indicator and Pathogen Detection (Paperback, Illustrated Ed)
Series: WERF Research Report Series
Expected to ship within 12 - 17 working days
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Microbial quality of water is a prime public health concern in
today's world. To protect public health, the World Health
Organization and the U.S. Environmental Protection Agency have
established microbial pollution indicator standards and recommended
routine monitoring of water for both total and fecal coliforms
(Dufour, 1984, U.S. EPA 1986,1994, WHO 1993). However, the adequacy
of current water quality standards to indicate the presence or
absence of human pathogens is still questionable. For example,
human viruses are more resistant to sewage treatment processes and
environmental conditions than bacterial indicators and therefore
may pose a substantial threat. It is now recognized that the
absence, or a low concentration, of indicator organisms in water
may not adequately reflect the absence of human viruses. In our
previous study of southern California coastal waters, we found over
30% of coastal waters tested contained human viruses, and the
presence of these viruses did not correlate with an elevated level
of bacterial indicators (Jiang et al. 2000). The goal of this
research is to develop and validate a molecular method for rapid
and specific detection of microbial contaminants including human
viruses and bacterial indicators in treated sewage effluents and
receiving waters. Research Outcomes An extensive search and review
of current state of technology for molecular alternatives to
indicator and viral pathogen detection was conducted. The results
indicate that although real-time PCR methods have been widely
applied in the clinical research for detection of human viruses,
environmental application of this method is very limited. In
addition, there is an urgent need for a method for efficient
concentration and purification of human viruses in complex
environmental matrixes. During this study, we have designed, tested
and optimized real-time quantitative PCR method using specific and
degenerate primers and probes targeting at adenoviruses and
enterococci, respectively. Experimental testing of real time PCR
primers and probes for adenoviruses demonstrated reproducible
results at efficiency greater than 90% over a 5-log dynamic range
of target concentration. The enterococci real time PCR was
efficient at 99% of the time and over a 7-log dynamic range of
target concentration. Application of these methods to sewage
effluents and coastal waters demonstrated that real-time PCR
methods are more sensitive than culturing methods at detection of
targets, suggesting a great potential for real-time quantification
of microbial contaminants in environments. However, real-time PCR
based method, like other genome based detection technology,
overestimates the concentration of infectious viral concentration
in the environment. In addition to the method development for
real-time PCR detection, we have also sampled and isolated human
adenoviruses from Newport Bay, California using human embryonic
kidney cells, 293A. Cloning and sequencing of selective
environmental adenovirus hexon gene suggested that most of the
viruses recovered from the environment belong to adenovirus
serotype 40 (66%). This result is in agreement with clinical data
on the load of viral shedding in feces. Since adenovirus serotypes
40 and 41 are the major cause of childhood diarrhea, the result of
this investigation indicates the importance of monitoring water for
viral quality. Comparison of four different tissue culture cell
lines for their sensitivities to adenoviruses infection has
demonstrated that the genetically engineered 293A cells are the
most efficient at recovery of adenoviruses 40. This result is a
significant contribution to our ability to assay for infectious
adenoviruses in environmental samples.
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