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Extramural Research

PCR Based Detection of Cytopathogenic and Non-Cytopathogenic Viruses in Water

EPA Grant Number: R824756
Title: PCR Based Detection of Cytopathogenic and Non-Cytopathogenic Viruses in Water
Investigators: Pepper, Ian L. , Gerba, Charles P. , Reynolds, Kelly A.
Institution: University of Arizona
EPA Project Officer: Manty, Dale
Project Period: October 1, 1995 through September 1, 1997
Project Amount: $227,258
RFA: Human Health Risk Assessment (1995)
Research Category: Health Effects , Human Health

Description:

A novel research strategy has been developed combining cell culture and PCR technology to rapidly detect infectious enteroviruses in environmental samples. Conventional cell culture methods have proven to be costly and time consuming, while direct PCR is complicated by the detection of nonviable viruses, small sample volumes assayable, and the presence of inhibitory compounds. The use of a combined cell culture/PCR technique utilizes the major advantages of each separate methodology while overcoming many of their disadvantages. Samples are first assayed on cells for 24-48 hours followed by PCR on cell harvests prior to CPE. The combined technologies increase the equivalent volumes examined while reducing the effects of toxicity in cell culture, reducing inhibitory effects on the PCR, and finally, maximizing the detection of infectious viruses using PCR. Studies in our laboratory of poliovirus type 1 (strain LSc-2ab) inoculated distilled water have shown positive PCR amplification after only 24 hours of incubation on BGM cells, compared to 3 days to more than two weeks using cell culture alone. For low theoretical levels of viruses (less than 1 PFU), CPE was visible only after a second passage on BGM cells but could be confirmed positive as soon as 24 hours using the integrated approach. This technology has also been applied to environmental sewage and marine water concentrates with similar results. In addition, environmental samples which were highly inhibitory to direct PCR were successfully evaluated using the integrated approach. Therefore, the integrated approach allows for more rapid and sensitive detection of low levels of enteric viruses in large volumes of water concentrates, compared to cell culture alone, while eliminating the effects of inhibitory substances to the PCR. In the future, the reduced time and lower cost associated with the integrated cell culture/PCR technology may allow for routine assay of water samples for infectious viruses.

Publications and Presentations:

Publications have been submitted on this project: View all 19 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 2 journal articles for this project

Supplemental Keywords:

RFA, Health, Scientific Discipline, Water, Health Risk Assessment, Risk Assessments, Ecology and Ecosystems, Drinking Water, ecological risk assessment, waterborne disease, enteric virus, rapid detection, infectious organisms, PCR, microbial risk management, cytopathogenic viruses, cell culture

Progress and Final Reports:
Final Report

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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