Full Description

Combining the use of conventional cell culture techniques with molecular methods for the detection of human enteric viruses in surface and groundwater allows detection of infectious enteroviruses producing cytopathic effects (CPE), as well infectious non-CPE enteroviruses. The objective of this study was to assay surface and groundwater samples for the presence of enteroviruses by combining cell culture and molecular techniques. Critical to the success of this study was the implementation and evaluation of a Quality Assurance/Quality Control (QA/QC) plan and determining if additional steps were necessary to increase the rigorousness of the plan and to ensure the validity of the results. Over 240 surface and groundwater samples were collected and processed using a modification of the Information Collection Rule for virus analysis. Samples assayed using Buffalo green monkey kidney (BGMK) and human colonic carcinoma (CACO-2) cell lines were observed for CPE over a period of 10 to 14 days. Positive CPE sample flasks were confirmed by secondary cell culture passage prior to molecular analysis, while negative CPE flasks were assayed by molecular techniques. Following total RNA extraction, RT-PCR was carried out using primers targeting the human enteroviruses. Confirmation of PCR results was accomplished by Southern blotting, hybridization of an internal digoxigenin-labeled probe, and chemi-luminescent detection. Controls incorporated during analyses included blind coding of samples, periodic positive and negative spike controls, cell culture positive and negative controls, PCR positive and negative controls, and incorporation of dUTP and UDG to prevent carry-over contamination during PCR. Includes 14 references, table, figures.

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Edition: Vol. - No. Published: 11/01/2002 Number of Pages: 11File Size: 1 file , 280 KB