Electropositive filter membrane as an alternative for the elimination of PCR inhibitors from sewage and water samples

PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorgani c compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in ad dition to fluorpentane treatment are quite efficient for virus concentratio n and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reve rse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted c reek water. Samples were subjected separately to each of the following proc edures: filtration through electropositive filter membranes (ZP60S), organi c extraction with Vertrel XF, and filtration through ZP60S followed by orga nic extraction. Afterwards, aliquots were experimentally inoculated with ro tavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of t he VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natura sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eigh t samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additi onal sample purification methods.