Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR

Abstract

BackgroundHuman enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR.ResultsOnce the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 μM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 μM) for RV (WA) and PMA (50 μM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37°C, 68 C, 72°C, 80°C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37°C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68°C to 80°C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV.ConclusionsWe concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples.

Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples

“…Recently, the utility of viability RT-qPCR has been expanded by modifications to this basic strategy. Surfactant cotreatment was reported to facilitate PMA and EMA penetration in thermally inactivated HAV and rotavirus, thereby improving the ability to discern their viability (Coudray-Meunier et al, 2013). However, reports on the application of this methodology in environmental samples are somewhat limited.…”

“…Moreover, Coudray-Meunier et al (2013) found that treatments combining viability dyes and surfactants were effective for differentiating infectious and thermally inactivated HAV suspensions. The purpose of this work was then to assess the applicability of viability dyes for the detection and quantification of infectious HAV by RT-qPCR in lettuce wash water, as an example of the fresh-cut industry water, as well as in artificially inoculated foods including vegetables and shellfish.…”

Propidium Monoazide Integrated with qPCR Enables the Detection and Enumeration of Infectious Enteric RNA and DNA Viruses in Clam and Fermented Sausages

“…Although coupling PMA with qPCR or RT-qPCR has been successfully applied to discriminate infectious and inactivated virus particles from viral suspension, river water, vegetables, swine raw manure, swine effluent from anaerobic biodigesters and biofertilized soil (Parshionikar et al, 2010; Sanchez et al, 2012; Coudray-Meunier et al, 2013; Moreno et al, 2015; Fongaro et al, 2016) little is known about the applicability of this technique in complex animal origin foods. In this study we chose two different food matrices: clam and Spanish fermented sausage (“chorizo”).…”

Advances and Challenges in Viability Detection of Foodborne Pathogens

“…It was concluded that EMA was less effective than PMA in selective amplification of DNA from viable cells and PMA was a useful alternative (van Frankenhuyzen et al, 2011). Besides viable cells of bacterial pathogens (Nocker and Camper, 2006, 2009; Nocker et al, 2007a; Pan and Breidt, 2007; Luo et al, 2010; Li and Chen, 2012, 2013), the PMA-qPCR has been applied to detect viable cells of fungi (Vesper et al, 2008), parasites (Brescia et al, 2009; Cancino-Faure et al, 2016), and viruses (Fittipaldi et al, 2010; Parshionikar et al, 2010; Kim and Ko, 2012; Sanchez et al, 2012; Coudray-Meunier et al, 2013; Fuster et al, 2016). …”