Sequential treatment using a hydrophobic resin and gel filtration to improve viral gene quantification from highly complex environmental concentrates

Abstract

Assays based on the polymerase chain reaction (PCR) are widely applied to quantify enteric viruses in aquatic environments to study their fates and potential infection risks. However, inhibitory substances enriched by virus concentration processes can result in inaccurate quantification. This study aimed to find a method for improving virus quantification by mitigating the effects of inhibitory environmental concentrates, using previous knowledge of the properties of the inhibitory substances. Performances of anion exchange resins, gel filtration, and a hydrophobic resin (DAX-8) were comparatively evaluated using poliovirus and its extracted RNA spiked into humic acid solutions. These solutions served as good representatives of the inhibitory environmental concentrates. A sequential treatment using DAX-8 resin and gel filtration produced the most favorable results, i.e., low virus losses that were stable and a reduced inhibitory effect. Furthermore, the sequential treatment was applied to another set of 15 environmental concentrates. Without the sequential treatment, serious underestimation (>4.0 log10 to 1.1 log10) of a molecular process control (murine norovirus) was measured for eight samples. With the treatment, the control was detected with <1.0 log10 underestimation for all samples. The treatment improved the quantification of seven types of indigenous viruses. In summary, the sequential treatment is effective in improving the viral quantification in various of environmental concentrates.