Assessment of PCR Inhibitor Removal Methods to Monitor Viruses in Environmental Water Samples: DAX-8 Outperforms Competitors

Abstract

Complex water matrices or viral elution buffers can interfere with the qPCR leading to an underestimation of the potential public health hazards of waterborne viral pathogens. Here, we assessed different approaches to mitigate inhibitory impact of complex water samples during RT-qPCR of murine norovirus (MNV), as an inhibition control. The dilution of extracted samples, the use of qPCR additives, a commercial PCR inhibitor removal kit, and polymeric adsorbents such as Supelite DAX-8 and polyvinylpyrrolidone (PVP) were all investigated in this context. Data indicated that the maximum amplification of MNV by RT-qPCR could be obtained by pre-dilution of samples. However, the dilution factor may depend on inhibitors concentration, primer length, probe sequence, and binding capacity. Interestingly, PCR inhibitor removal kits do not seem to be adequate for removing all PCR inhibitors. In comparison to other approaches studied here, the application of 5% DAX-8 led to an increase in MNV qPCR concentrations. DAX-8 can permanently eliminate humic acids from the extracted nucleic acids from the environmental water samples, and it has the potential to considerably improve the accuracy of the obtained non-detects and measured concentrations by qPCR. Further research is required to understand the performance of polymeric adsorbents with enveloped viruses.