ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples.
This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.
Reynolds
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KA
Department of Soil, Water and Environmental Science, The University of Arizona, Tucson 85745, USA. reynolds@u.arizona.edu
Gerba
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CP
Abbaszadegan
M
M
Pepper
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LL
D018929:Cell Culture Techniques / Q000379:methods; D004770:Enterovirus / Q000302:isolation & purification*; D004783:Environmental Microbiology; D005618:Fresh Water / Q000382:microbiology; D006507:Hepatovirus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012722:Sewage / Q000382:microbiology
D012722:Sewage