Development of methods for concentration and detection of enteroviruses in water.
Committee ChairGerba, Charles P.
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PublisherThe University of Arizona.
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AbstractMethods to improve the speed and reduce the cost of enterovirus detection in water were developed. A new positively-charged filter was evaluated for its ability to concentrate enteroviruses from tapwater. Both viable and inactivated viruses were detected by RT-PCR. It was demonstrated that the concentrate from filters could be reduced in volume to increase the sensitivity of RT-PCR detection of viruses. Finally, combination of RT-PCR and cell culture methodology was applied to the detection of enteroviruses in groundwater. Microporous filters are used for the concentration of enteric VIruses from large volumes of water. A new type of electropositively charged filter, MK zeta-plus, was evaluated and compared to the commonly used IMDS filter. Recovery of poliovirus type 1 from tapwater using MK filter and IMDS filters was 73.2 ± 26% and 90.2 ± 5.9%, respectively. Recovery of coxsackievirus B3 from tapwater using MK and IMDS filters was 32.8 ± 34.5% and 95.8 ± 12.0%, respectively. Inactivation of poliovirus type 1 by 1 N HCI, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared using infectivity in cell culture and RTseminested PCR. A minimum contact time of 45 min with HCI, 3 min with NaOH, 3 and 6 with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render poliovirus undetectable by RT-seminested PCR. Viruses inactivated by UV light could still be detected by RT-PCR. Application of reverse transcriptase (RT)-polymerase chain reaction (PCR) to detect enteroviruses in concentrated tap water samples was studied. The final volume of concentrates of 378 liters of tap water was successfully reduced from 25 ml to 5 ml without loss of virus. Direct phenol-chloroform-isoamyl alcohol (PCl) extraction was found to be sufficient to remove inhibitory substances for RT-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 μl (0.2 PFU/liter of tap water). Groundwater samples were assayed by both cell culture and RT-PCR. Nine of 48 samples were enteroviruses positive by cell culture assay (19%). Cell culture harvests of groundwater samples were assayed by RT-PCR (for positive samples) or RT-seminested PCR (for negative samples) and the results were identical to those of cell culture assay. Fifteen out of 37 samples were positive by direct RT-PCR (40.5%), while only 8 of the 37 samples were positive by cell culture (21.6%). However, one method was not superior to the other in demonstrating the presence or absence of enteroviruses in groundwater based on statistical analysis (P>0.05). These data indicated that RT-PCR can be used as a confirmation procedure of cell culture assay, however, it can not yet be used independently of cell culture.
Degree ProgramMicrobiology and Immunology