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

Abstract

PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the 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 natural 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). Eight 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 additional sample purification methods.

FIG. 1

Results of snPCR of in natura sewage water collected on 17 June 1998. Nonautoclaved (A) and autoclaved (B) samples were subjected to different purification methods. Lanes: 1, in natura water; 2, in natura water plus rotavirus; 3, water treated with Vertrel XF; 4, water treated with Vertrel XF plus rotavirus; 5, water filtrated through ZP60S membranes; 6, water filtrated through ZP60S membranes and treated with Vertrel XF; 7, water filtrated through ZP60S membranes plus rotavirus; 8, water filtrated through ZP60S membranes and treated with Vertrel XF plus rotavirus. Cp and Cn, positive and negative controls; M, molecular size marker (100-bp DNA ladder).

FIG. 2

Results of snPCR of in natura creek water collected on 28 January 1998. Nonautoclaved (A) and autoclaved (B) samples were subjected to different purification methods. Lanes: 1, in natura water; 2, in natura water plus rotavirus; 3, water treated with Vertrel XF; 4, water treated with Vertrel XF plus rotavirus; 5, water filtrated through ZP60S membranes; 6, water filtrated through ZP60S membranes and treated with Vertrel XF; 7, water filtrated through ZP60S membranes plus rotavirus; 8, water filtrated through ZP60S membranes and treated with Vertrel XF plus rotavirus. Cp and Cn, positive and negative controls; M, molecular size marker (100-bp DNA ladder).

FIG. 3

Detection of naturally occurring rotavirus in sewage and creek water by the two-step concentration method and duplex snPCR. Lanes: 5, 6, 7, 9, and 11, sewage water samples; 2, 8, and 10, creek water samples; 1, positive control; 3 and 12, mixture of positive controls for rotavirus genogroups G1 to G4. M, molecular size marker (100-bp DNA ladder).