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.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 280 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 11 |
| Published : | 11/01/2002 |
