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NOVEL METHODS OF DETECTION OF F+RNA PHAGES AS INDICATORS TO ASSESS THE MICROBIAL QUALITY OF IRRIGATION WATER

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Title: NOVEL METHODS OF DETECTION OF F+RNA PHAGES AS INDICATORS TO ASSESS THE MICROBIAL QUALITY OF IRRIGATION WATER
Author: Yongheng Yang
Department: Department of Food Science
Program: Food Science
Advisor: Griffiths, Mansel
Abstract: The microbial quality of irrigation water is critical to the prevention of contamination of fresh fruits and vegetables with enteric pathogens since polluted irrigation water is an important source of these pathogens contaminating fruits and vegetables in the field. This work examined the use of F+RNA phages as indicators of fecal contamination to assess the microbial quality of irrigation water. The different propagation profiles among subgroups of F+RNA phages may contribute to their distribution in the environment and the difference in their survival their persistence would not hinder their use for source tracking. It is rapid and sensitive to detect F+RNA phages based on monitoring phage-mediated release of β-galactosidase by a fluorescent or luminescent method. The later was more sensitive and detected 1.6 PFU/ml of F+RNA phages within three hours in the presence of Mg2+. The luminescent method was able to differentiate MS2 and Qβ when combined with anti-MS2 coat protein antibody. Disruptor filtration was an efficient and easy-to-perform approach to concentrate F+RNA phages, and more than 99% of phage particles in distilled water, wastewater treatment plant effluent and river water were removed after Disruptor filtration. As low as 0.16 PFU of F+RNA phages/100 ml of water were detected within three hours when Disruptor filtration was combined with the luminescent method. This allows rapid detection of potentially low levels of fecalcontamination, as well as reducing rates of false-negative results. Enzyme treatment with proteinase K and RNase A more readily degraded naked RNA or RNA from inactivated phage particles, and the enzyme treatment RT-PCR was effective at reducing false-positive signals encountered by RT-PCR alone. This provides more reliable information on the real risk of fecal contamination to human health in terms of the presence of infectious enteric pathogens. The ability of F+RNA phages to attach to isolated F pili provides the potential to selectively concentrate F+RNA phages by immobilized F pili (or their structural protein subunits), or to determine the infectivity of phage particles in combination with a detection method, such as RT-PCR. This is an area that requires further study.
URI: http://hdl.handle.net/10214/7783
Date: 2013-12


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