Date of Award

Spring 1994

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Aaron Margolin

Abstract

Potable water is a primary need of humankind. In the interest of public health, the water should be free of infectious agents, such as bacteria, intestinal parasites, and viruses. The current method for assaying water for viruses is cell culture, which is expensive, labor intensive, and time consuming. Nucleic acid probes offer an alternative method which is less costly, more rapid, and requires less training and experience. Consequently, this study provides alternatives to viral monitoring of water sources.

Three nonradioactive nucleic acid probe assays were developed and evaluated by colormetric and chemiluminescent signals. A biotin-tailed probe was able to detect 100 pg of homologous cDNA and 9 $\times$ 10$\sp5$ poliovirus plaque forming units (pfu), while the horseradish peroxidase (HRP) probe was able to detect 10 pg of cDNA and 9 $\times$ 10$\sp4$ pfu, and the digoxigenin probe detected 0.1 pg of cDNA and 9 $\times$ 10$\sp2$ pfu. No differences in sensitivity were seen between colormetric and chemiluminescent detections. Since the digoxigenin probe was the most sensitive of the nonisotopic assays, it was compared to $\sp{32}$P-cDNA, $\sp{32}$P-ssRNA, and cell culture for the detection of viruses from environmental samples. The digoxigenin probe and $\sp{32}$P-ssRNA probes detected a higher number of positive samples than the other two assays.

An in situ assay was developed to detect only infectious viruses. Cells were incubated with viral dilutions, fixed with a 4% solution of formalin, and then probed. The detection limit of in situ hybridizations was 0.9 pfu. The increased sensitivity over cell culture was most likely due to a subset of polioviruses which can begin the replication cycle but do not form cytopathic effects.

Poliovirus was then disinfected with chlorine and chlorine dioxide at pH 6, pH 7, and pH 9 as well as ozone, and ultraviolet light to determine the correlation between cell culture and digoxigenin probe on disinfected samples. No correlation existed using any disinfectant as the number of plaque forming units was reduced to zero and the digoxigenin probe assay did not lose any signal intensity. The only loss of digoxigenin probe sensitivity was seen at pH 9.

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