Impact of Various Humic Acids on EMA-RT-qPCR to Selectively Detect Intact Viruses in Drinking Water

Abstract

To assess the potential risk of viral infection through drinking water, a rapid and effective method to quantify pathogenic viruses is necessary. Ethidium monoazide (EMA) combined with reverse transcription qPCR (EMA-RT-qPCR) is a currently widely accepted method to assess the integrity of viruses. However, this technique can be hampered by humic acids which are co-concentrated during virus concentration processes (VCPs). Co-concentration of four commercially available humic acids (Ald, Wa, Na, and IH) during VCPs and their impacts on the subsequent EMA-RT-qPCR over spiked intact Aichi virus 1 and its naked RNA were studied. The recoveries of Ald, Wa, Na, and IH during VCPs were less than 8.9%, 5.4%, 7.2%, and 0.7%, respectively, indicating that IH was much less concentrated than other humic acids. In the concentrates, Ald, Wa, and Na caused severe inhibition of EMA-RT-qPCR, whereas only a slight inhibitory effect on IH was observed. Tests of the influence of actual drinking water concentrates on EMA-RT-qPCR indicated the absence of severe inhibition. Therefore, EMA-RT-qPCR has a high potential for monitoring enteric viruses in drinking water.