Concentration and detection of naturally occurring enteric viruses by hollow fiber ultrafiltration, cell culture and RT-PCR-ELISA.

摘要

The objective of this study was to evaluate the ability of ultrafiltration to concentrate viruses from large volumes of environmental water. Two ultrafiltration systems, hollow fiber and tangential flow, were evaluated in a small- (2 L) and large-scale (100 L) configuration for their ability to consistently and efficiently recover greater than 60% of multiple viruses from different water qualities. Both ultrafiltration systems proved capable of recovering greater than 50% of multiple viruses from different water qualities with the hollow fiber ultrafiltration capable of recovering greater than 60%. Tangential flow ultrafiltration in two configurations, screen channel and open channel, was capable of recovering greater than 70% of bacteriophage PP7 and T1 as a second-step concentration procedure. Clogging of the filters occurred when particulates exceeded 1.6 g/L in the screen channel and 5.5 g/L in the open channel. A two-step concentration procedure using two hollow fiber ultrafilters was developed to recover greater than 50% of viruses from 100 L of environmental water. Mean viral recoveries of 87% (T1), 53% (PP7) and 63% (poliovirus 2) and 119% (T1), 66% (PP7) and 56% (poliovirus 2) were observed in ground and surface water respectively. A cell culture and RT-PCR-ELISA-based detection method was incorporated into the two-step ultrafiltration method for the detection of naturally occurring enteric viruses. Water samples (90 L) were taken and tested for enteric viruses from six sites along the Rio Grande in southwest New Mexico and west Texas. Two cell lines, buffalo green monkey (BGM) and H-HeLa, were evaluated on their ability to detect naturally occurring enteric viruses. In cell culture, 18 of 29 (62.1%) samples were positive in BGM cells with concentrations of 23--722 pfu/L while 9 of 29 (31.0%) samples were positive in H-HeLa cells with concentrations of 8--450 pfu/L. The RT-PCR-ELISA had a detection sensitivity of 0.2 pfu/ml. By RT-PCR-ELISA, 15 of 29 (51.7%) samples were positive for enteroviruses while 6 of the 15 positive samples were positive by cell culture-PCR (CC-PCR).