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

Moreno, Luis Pérez; Aznar, Rosa; Sánchez, Glòria

doi:10.1016/j.ijfoodmicro.2015.02.012

Cited by 69 publications

(60 citation statements)

References 42 publications

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“…Photoactivatable intercalating dyes have begun to show promise in being able to selectively detect infectious HAV (Sanchez et al, 2012; Coudray-Meunier et al, 2015; Moreno et al, 2015; Fuster et al, 2016; Randazzo et al, 2018b) and human NoV (Parshionikar et al, 2010; Randazzo et al, 2016, 2018a; Jeong et al, 2017). Recently, Fraisse et al (2018) proposed PtCl 4 as a successful viability marker for human NoV.…”

Section: Resultsmentioning

confidence: 99%

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

et al. 2018

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The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To this end, different concentrations of viability markers (PMAxx and platinum chloride, PtCl4) were screened firstly on purified viral RNA using different RT-qPCR assays. Reductions of HEV RNA signals of >17.5, >15.0, and >15.5 quantification cycles (Cq) were reported for PtCl4 and 1.6, 2.9, and 8.4 Cq for PMAxx, clearly indicating a better performance of PtCl4 than PMAxx irrespective of the RT-qPCR assay used. The most efficient viability pretreatment (500 μM PtCl4 incubated at 5°C for 30 min) was then assessed on native, heat-, and HPP-treated HEV suspension. The optimized viability RT-qPCR discriminated successfully between native, heat-, and HPP-treated HEV, to different extents depending on the experimental conditions. In particular, approximately 2-log10 reduction was reported by PtCl4-RT-qPCR at both 72 and 95°C compared to the control. Additionally, both viability pretreatments were tested for HPP-treated HAV without success, while PtCl4-RT-qPCR completely eliminated (>5.6-log10 reduction) the RT-qPCR signals of HPP-treated HEV. Although this viability procedure may still overestimate infectivity, the PtCl4 pretreatment represents progress to better interpreting the quantification of intact HEV, and it could be included in molecular procedures used to quantify enteric viruses in food and environmental samples.

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Section: Resultsmentioning

confidence: 99%

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

et al. 2018

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“…manure, sewage and shellfish, which may have to be diluted to improve the performance of intercalating dye treatments (Moreno et al . ; Leifels et al . ).…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study, our group reported that PMA pretreatment combined with Triton was quite effective to discriminate between infectious and thermally inactivated HAV in vegetable samples, but not in shellfish concentrates (Moreno et al . ). Therefore, the main goal of this study was to further explore the potential of new intercalating dyes to differentiate between infectious and thermally inactivated HAV suspensions using the RT‐qPCR assay proposed in the framework of the ISO 15216‐1, and to assess its applicability in vegetables and shellfish.…”

Section: Introductionmentioning

confidence: 97%

Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples

et al. 2017

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Aim To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food. Methods and Results Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT‐qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT‐qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx–Triton pretreatment resulted in complete removal of the RT‐qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT‐qPCR method reduced the signal of inactivated HAV between 1·7 and 2·2 logs at high inoculation level, and signal was completely removed at low inoculation level. Conclusions This study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT‐qPCR. It was shown that PMAxx–Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish. Significance and Impact of the Study The PMAxx–Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection.

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“…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”).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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The increase of foodborne viral outbreaks highlights the need for a rapid and sensitive method for the prediction of viral infectivity in food samples. This study assesses the use of propidium monoazide (PMA) coupled with real-time PCR methods (RT-qPCR or qPCR for RNA or DNA viruses, respectively) in the determination of viral infectivity in complex animal-related food matrices. Clam and Spanish fermented sausage (“chorizo”) samples were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) and mengovirus (vMC0). PMA-qPCR/RT-qPCR discriminated infective virus particles, with significant reductions (>2.7 log10 or 99.7%). Additionally, infectious HAdV-2 and vMC0 were quantified by plaque assay (in plaque forming units, PFU), and compared with those in virus genomes copies (GCs) quantified by PMA-qPCR/RT-qPCR. A consistent correlation (R2 > 0.92) was showed between PFU and GCs along serial 10-fold dilutions in both DNA and RNA virus and in both food matrices. This study shows the use of PMA coupled to qPCR/RT-qPCR as a promising alternative for prediction of viral infectivity in food samples in comparison to more expensive and time-consuming methods and for those viruses that are not able to grow under available cell culture techniques.

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

Cited by 69 publications

References 42 publications

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples

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

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