Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR.

Human noroviruses (NoV) are major agents of foodborne outbreaks. Because of the lack of a standardized cell culture method, real-time reverse transcriptase PCR is now commonly used for the detection of NoV in foodstuffs and environmental samples. However, this approach detects the viral nucleic acids of both infectious and non-infectious viruses and needs to be optimized to predict infectivity for public health risk assessment. The aim of this study was to develop a viability PCR method to discriminate between native and heat-treated virus, for both NoV and its surrogate, murine norovirus (MNV). To this end, screening of viability markers (monoazide dyes, platinum and palladium compounds) was performed on viral RNA, native virus or heat-treated virus, and incubation conditions were optimized with PtCl4, the most efficient viability marker. Multiple MNV molecular models were designed: no impact of amplicon length was observed on inactivated MNV genomic titer; but the 5'NTR, ORF1 and 3'UTR regions resulted in higher reductions than central genomic regions. The optimal viability PCR conditions developed (incubation with 2.5 mM PtCl4 in PBS for 10 min at 5 °C) were finally applied to MNV by performing heat inactivation studies and to native and heat-treated NoV clinical strains. The viability PCR discriminated efficiently between native and heat-inactivated MNV at 72 °C and 80 °C, and efficiently reduced the genomic titer of heat-treated NoV strains. This viability PCR method could be useful to study heat inactivation kinetics of NoV and MNV. It could also be evaluated for the identification of infectious enteric viruses in foodstuffs and environmental samples.

Digital RT-PCR method for hepatitis A virus and norovirus quantification in soft berries.

Raw fruits may harbour many pathogens of public health concern including enteric viruses, which are the leading cause of foodborne outbreaks. Recently, consumption of soft berries has been associated with increasing reports of norovirus and hepatitis A virus outbreaks in Europe. Due to their low infectious doses and low concentrations in food samples, an efficient and sensitive analytical method is required for virus detection. In this study we explored two different ways to improve the reference method for the detection of enteric viruses in soft fruits (ISO/TS 15216-1; 15216-2): an additional purification step after RNA extraction; and the detection of enteric viral genome by an absolute quantification method (microfluidic digital RT-PCR). Both of these approaches led to an improvement of enteric virus detection in soft berries by greatly lowering PCR inhibition, raising viral extraction efficiencies and enabling validation of controls using pure RNA extracts. The PCR inhibitor removal step can be easily included in the routine method. Absolute quantification by digital RT-PCR may be a relevant alternative method to standardize quantification of enteric viruses in foodstuffs.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System.

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.

A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples.

Hepatitis A virus subgenotyping based on RT-qPCR assays.

BACKGROUND: The hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is recognized as one of the most widespread foodborne pathogens. HAV genotypes and subtypes differ in their geographic distribution and the incidence of HAV infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. Phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of HAV strains. In food microbiology, this approach is complicated by the low contamination levels of food samples. To date, real-time reverse-transcription PCR has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide HAV subtyping information. RESULTS: Six subtype-specific RT-qPCR assays were developed for human HAV. The limit of detection of HAV was 50 genome copies/assay for subtype IIB, 500 genome copies assay for IA, IB, IIA and IIIB and 5000 genome copies/assay for IIIA. The specificity of the assays was evaluated by testing reference isolates and in vitro HAV RNA transcripts. No significant cross reactivity was observed. Subtyping results concordant with sequencing analysis were obtained from 34/35 clinical samples. Co-infection with a minor strain of a different subtype was suggested in 5 cases and a recombinant event in one case. CONCLUSIONS: These RT-qPCR assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples.

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

BACKGROUND: Human 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. RESULTS: Once 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. CONCLUSIONS: We 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.