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.

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications – A systematic review

“…The remainder of the articles described the samples being stored on ice or at 4–6 °C; none of them used higher temperatures as sometimes discussed for bacterial assays ( Codony et al., 2019 ). Studies that applied a long incubation time tended to use low temperatures ( Coudray-Mounier et al., 2013 ; Sangsanont et al., 2014 ; Prevost et al., 2016 ; Leifels et al., 2016 , 2019 ; Canh et al., 2018 , 2019 ; Oristo et al., 2018 ; Fraisse et al., 2018 ).…”

“…Early publications utilizing azo dyes for the removal of non-infectious virus particles exclusively applied 500- to 800-Watt halogen light sources used in stage lighting ( Bellehumeur et al., 2015 ; Canh et al., 2018 ; Escudero-Abarca et al., 2014 ; Graiver et al., 2010 ; Leifels et al., 2015 ; Parshionikar et al., 2010 ; Sangsanont et al., 2014 ). Besides their operational hazards such as light bulbs exploding due to long running times (a maximum of 5 min is recommended by the manufacturer), both the heat and light emission in the ultraviolet and infrared spectra could potentially harm the sample, thus subverting the purpose of the pretreatment altogether.…”

“…Various methods to inactivate viruses were used in the studies included in this review. The intention was either to evaluate disinfection efficiency as it is currently used in food safety and water treatment ( Jeong et al., 2017 ; Langlet et al., 2018 ; Leifels et al., 2016 ; Randazzo et al., 2018a ) or to act as controls to evaluate the efficacy of the capsid integrity protocol ( Canh et al., 2018 , 2019 ; Farkas et al., 2020 ; Leifels et al., 2015 ). Addition of chlorine, exposure to heat, and proteolysis are known to damage the viral capsid, thus rendering the genome accessible to azo dyes.…”

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications – a systematic review

“…The remainder of the articles described the samples being stored on ice or at 4 to 6°C; none of them used higher temperatures as sometimes discussed for bacterial assays (Codony et al, 2019). Studies that applied a long incubation time tended to use low temperatures (Coudray-Mounier et al, 2013; Sangsanont et al, 2014; Prevost et al, 2016; Leifels et al, 2016; Canh et al, 2018; Oristo et al, 2017; Fraisse et al, 2018; Canh et al, 2019; Leifels et al, 2019).…”

Removal of pepper mild mottle virus by full-scale microfiltration and slow sand filtration plants

“…14 In addition, there are methodological limitations to discriminate between infectious and noninfectious viruses in water samples. 15,16 Thus, the assessment of viral infection risk in drinking water supplies is essential for protecting public health.…”