Development of a cell culture-based method for detecting infectious tick-borne encephalitis virus (TBEV) in milk products.

Tick-borne encephalitis outbreaks have been reported in Europe after consumption of raw milk products from infected animals. While molecular methods are commonly used in viral foodborne outbreak investigations due to their sensitivity, specificity and rapidity, there are very few methods to detect infectious tick-borne encephalitis virus (TBEV) in milk products for routine use/analyses. To address this gap, we developed a cell culture-based method to detect infectious TBEV in artificially contaminated raw goat milk and raw goat cheese, and evaluated the sensitivity of TBEV infectivity assays. Raw goat milk samples were spiked with TBEV to achieve inoculation levels ranging from 106 to 100 TCID50/mL, and Faisselle and Tomme cheese samples were spiked so their TBEV concentrations ranged from 9.28 × 105 to 9.28 × 101 TCID50 per 2.5g. To detect infectious TBEV, Vero cells were infected by raw goat milk. For cheese samples, after homogenisation and membrane filtration, Vero cells were infected with samples adsorbed on the filter (method A) or with samples eluted from the filter (method B). After 5 days, cytopathic effects (CPEs) were observed and TBEV replication in Vero cells was confirmed by an increase in the number of genome copies/mL that were detected in cell supernatant. Infected Vero cells exhibited CPEs for both milk and cheese samples. Infectious TBEV was detected to 103 TCID50/mL in raw milk samples and to 9.28 × 101 TCID50 from Faisselle samples using both methods A and B. For Tomme samples, method A was able to detect TBEV to 9.28 × 102 TCID50/2.5g and method B to 9.28 × 103 TCID50/2.5g. The number of positive samples detected was slightly higher with method A than with method B. To conclude, this qualitative cell culture-based method can detect infectious TBEV artificially inoculated into raw milk and cheese; it should be further evaluated during foodborne outbreak investigations to detect infectious TBEV from naturally contaminated milk and cheese.

Method for tick-borne encephalitis virus detection in raw milk products.

The transmission of tick-borne encephalitis virus (TBEV) through food is rare, but can occur through the consumption of raw milk products from animals infected by tick bites. In 2020, France faced a TBEV outbreak linked to the consumption of unpasteurized goat cheese. The aim of this study was to develop and characterize a molecular method for the detection of TBEV in raw milk products based on the recent international standard PR ISO/DIS 16140-4. The TBEV recovery rates varied with the inoculation level and settings. The LOD50 and LOD95 of TBEV were 6.40 × 103 genome copies per g or per mL and 2.84 × 104 genome copies per g or per mL, respectively. The percentages of RT-qPCR inhibitions were lower than 75% and the murine norovirus (MNV-1), used as process control, was detected in all samples with a recovery rate higher than 1%, as recommended in ISO 15216. We conclude that the described method is appropriate to detect TBEV in raw milk products for routine diagnosis, and to assess potential health risks.

Virological analyses in collective catering outbreaks in France between 2012 and 2017.

Enteric viruses cause the majority of foodborne illnesses and common symptoms of many foodborne illnesses include vomiting, diarrhea, abdominal pain, and fever. Among the enteric viruses, human Norovirus (NoV) and hepatitis virus (HAV and HEV) are the main viruses suspected to cause foodborne outbreaks and represent a serious public health. The study presents survey tools of viruses in a wide variety of foodstuffs and results obtained during 56 foodborne outbreaks investigation in France between 2012 and 2017. 246 suspected foods were examined for the presence of four human enteric viruses (NoV GI and NoV GII, HAV or HEV) either using methods described in the EN ISO 15216-1 or in house methods. All viral analysis of food samples were performed with the implementation of process control and an external amplification controls. Eighteen of 56 foodborne outbreaks investigated included at least one positive food sample (16/18 NoV, 1/18 HAV and 1/18 HEV). The genomic levels of four viruses detected ranged from < 102 to 107 genome copies per g or per L. This study showed the interest to develop methods for the extraction of viruses in different foodstuffs to increase the possibility to identify the association between viral illness and food consumption.

Evaluation of methods for elution of HEV particles in naturally contaminated sausage, figatellu and pig liver.

Foodborne transmission of HEV is a growing public health concern in industrialised countries, where the disease is mainly autochthonous, caused by zoonotic HEV of either genotype 3 or 4. Foodstuffs containing pig's liver were suspected on several occasions to be the cause of autochthonous cases of HEV infection, while the transmission was associated with animal contact and the ingestion of raw or uncooked meat, especially liver. In assessing the risk related to the presence of HEV in food, detection methods were previously developed but HEV detection rates seem to vary with the type of samples and methods. As foodstuff containing pig liver can be contaminated with HEV internally, an efficient virus extraction procedure is required. The aim of this study was to evaluate six methods for their efficiency in releasing HEV viral particles from figatelli, pig liver sausages and liver samples previously tested positive for the presence of HEV. The ratio weight to volume of elution buffer (1:5) and the FastPrep®-24 homogeniser showed to significantly improve the quantity of HEV genomes released per gram of figatelli and pig liver sausages. To our knowledge, this study is the first to evaluate several methods for elution of HEV particles from naturally contaminated pig liver products, and may be extended for quantifying other viral genomes from food of animal origin.

Comparison of three extraction methods to detect noroviruses in dairy products.

Noroviruses (NoV) are currently the most common cause of viral foodborne diseases and RT-qPCR is widely used for their detection in food because of its sensitivity, specificity and rapidity. The ISO/TS (15216-1, 15216-2) procedures for detecting NoV and HAV in high-risk food categories such as shellfish, bottled water and vegetables were published in 2013. Milk products are less implicated in foodborne viral outbreaks but they can be contaminated with fruit added to these products or by the food handler. Thus, the development of sensitive and reliable techniques for the detection of NoV in dairy products is needed to ensure the safety of these products. The aim of this study was to develop a RT-qPCR based method for the detection of NoV in milk products. Three methods were tested to recover NoV from artificially contaminated milk and cottage cheese. The selected method was based on the use of proteinase K and the recovery efficiencies ranged from 54.87% to 98.87% for NoV GI, 61.16%-96.50% for NoV GII. Murine norovirus and mengovirus were used as process controls and their recovery efficiencies were respectively 60.59% and 79.23%. The described method could be applied for detecting NoV in milk products for routine diagnosis needs.

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.

A Useful Method to Provide Infectious and Cultivable In Vitro Naked Viral Particles of Hepatitis A Virus.

Hepatitis A virus (HAV) is an enteric virus mainly transmitted by the faecal-oral route. Belonging to the Picornaviridae family, HAV was first described as small naked particles, like all viruses of this family. However, for about a decade, it was demonstrated that HAV particles can exist surrounded by a lipid bilayer. This type of particle, called enveloped HAV (eHAV), acquires its lipid bilayer by hijacking a part of cell membranes during the virion egress in the last steps of the viral cycle. In vitro culture systems produce mainly eHAV, and so, to date, most of the studies on HAV have been carried out using this type of viral particle. In this study, a method based on lipid bilayer removal by chemical delipidation is proposed for the production of naked HAV particles. The resulting naked HAV particles conserve their infectivity and are therefore fully cultivable in vitro. By using this method, naked HAV particles can easily be produced in vitro and can be useful to perform further studies such as inactivation processes for the food industry, as HAV is a main concern for food safety.

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.

Development of an Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus, and Noroviruses in Fish Products.

Viruses are a leading cause of foodborne disease worldwide. Hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) and human norovirus are recognized as the main viruses of public health concern in food hygiene. ISO 15216 approved procedures are not validated for detection of HAV and human norovirus in foodstuffs, such as fishes, leading to an inability to ensure the safety of these products. This study aimed to provide a rapid and sensitive method for detecting these targets in fish products. An existing method that includes proteinase K treatment was selected for further validation using artificially contaminated fish products, according to the recent international standard ISO 16140-4. Recovery efficiencies in pure RNA extracts of viruses ranged from 0.2% to 66.2% for HAV, 4.0% to 100.0% for HEV, 2.2% to 100.0% for norovirus GI, and 0.2% to 12.5% for norovirus GII. LOD50 values were between 144 and 8.4 × 104 genome copies/g for HAV and HEV, and 104 and 2.0 × 103 copies/g for norovirus GI and norovirus GII, respectively. LOD95 values were between 3.2 × 103 and 3.6 × 105 genome copies/g for HAV and HEV, and between 8.8 × 103 and 4.4 × 104 genome copies/g for norovirus GI and norovirus GII, respectively. The method developed here was successfully validated in various fish products and can be applied for routine diagnostic needs.

Evaluation of a Proteinase K-Based Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus and Norovirus in Artificially Contaminated Dairy Products.

Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are leading causes of foodborne disease worldwide. Among the various food products, different types of dairy products can be implicated in viral foodborne outbreaks and contamination can occur at different stages, such as preparation, contact with contaminated equipment or via other foods. The aim of this study was to characterise a proteinase K method adapted from the ISO 15216 method for the detection of HAV, HEV and norovirus in artificially contaminated dairy products, based on the recent international standard of ISO 16140-4. Results showed that the recovery yields obtained from pure RNA in dairy products ranged from 5.76% to 76.40% for HAV, from 35.09% to 100.00% for HEV, from 25.09% to 100.00% for norovirus GI and from 47.83% to 100.00% for norovirus GII. The process control MNV-1 was detected in all RNA extracts, with recovery yields between 36.83% and 100.00%. The limit of detection (LOD) of the method was between 184 and 642 genome copies/mL (or/g) for the LOD50 and 802 and 2800 genome copies/mL or/g for the LOD95 according to the virus analysed. This method proved to be suitable for detecting viruses in dairy products for routine diagnostic needs.

Clustered Cases of Waterborne Hepatitis E Virus Infection, France.

The identification of seven cases of hepatitis E virus infection in a French rural hamlet in April 2015 led to investigations confirming the clustering and identifying the source of the infection. Laboratories and general practitioners in the area actively searched for other cases based on RT-PCR and serological tests. The environment, including water sources, was also checked for HEV RNA. Phylogenetic analyses were performed to compare HEV sequences. No other cases were found. Six of the seven patients lived in the same hamlet, and the seventh used to visit his family who lived there. All HEV strains were very similar and belonged to the HEV3f subgenotype, confirming the clustering of these cases. All the patients drank water from the public network. A break in the water supply to the hamlet was identified at the time the infection probably occurred; HEV RNA was also detected in a private water source that was connected to the public water network. The water flowing from the taps was quite turbid during the break. The private water supply containing HEV RNA was the likely source of the contamination. Private water supplies not disconnected from the public network are still frequent in rural areas, where they may contribute to public water pollution.

Method for detecting norovirus, hepatitis A and hepatitis E viruses in tap and bottled drinking water.

Viruses are a leading cause of foodborne disease worldwide. Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are recognised to be the main viruses of importance to public health. The ISO 15216 procedure describes molecular methods for detecting HAV and norovirus in bottled water by using an electropositive filter to concentrate viruses. The aim of this study was to validate the Zeta Plus 1MDS membrane (1MDS) for detecting enteric viruses from tap and bottled water using the recent international standard ISO/DIS/16140-4:2018, which describes the protocol for validating methods for microbiology in the food chain. Method with direct lysis of viruses from the 1MDS filter, and RNA extraction was used for detecting noroviruses, HAV and HEV from different tap and bottled drinking water. By taking into account virus's inoculation levels above the LOD, the recovery rates of noroviruses and HAV obtained from pure RNA extracts ranged from 2.50% to 14.31% and for HEV from 27.87% to 53.54% according to the water samples analysed. The virus recovery rates did not differ according to the operator or drinking water analysed but did according to the virus inoculated. The LOD95 values were respectively 50 genome copies/mL for HAV and 2.8 genome copies/mL for HEV, 420 genome copies/mL for norovirus GI and 134 genome copies/mL of water sample for norovirus GII. LOQs were determined for HAV and HEV by the total error approach and were 15.8 genome copies/mL for HAV and 2.8 genome copies/mL of water sample for HEV. The described method could be used for detecting viruses from tap and bottled water for routine diagnosis needs.

Detection of norovirus, hepatitis A and hepatitis E viruses in multicomponent foodstuffs.

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD95 was 102 genome copies/g for HAV, HEV and norovirus GII and 103 genome copies/g for norovirus GI. The LOQ was 2.90 × 104, 1.40 × 103, 1.60 × 104 and 1.30 × 104 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs.

Evaluation of three different filters and two methods for recovering viruses from drinking water.

Among the enteric viruses implicated in waterborne outbreaks, human norovirus and hepatitis A virus (HAV) are a serious public health issue. Most foodborne viruses are difficult or currently unlikely to cultivate. Because of the lack of a cell culture method, real-time reverse transcriptase PCR is commonly used for the detection of norovirus in foodstuffs and environmental samples. Due to low infectious doses in humans and low virus concentration in water sample, filter adsorption methods were used for concentrating viruses from water. The ISO (Anonymous, ISO 15216-1, 2017) describes standardized molecular methods for detecting HAV and norovirus in bottled water. This method includes a two-step procedure: concentrating the virus using a microporous electropositive filter (47 mm diameter, 0.45 µm pore size) then molecular detection. The Zetapor filter, which had a charged membrane with a pore size of 0.45 µm, was commonly used in the past to concentrate viruses from water or from salad leaves following virus elution. But, unfortunately, the Zetapor filter is no longer marketed and it is therefore necessary to assess an alternative filter. The aim of this study was to compare the ability of two electropositive filters with a pore size of 0.45 µm or 0.22 µm and one uncharged filter (0.45 µm) to recover norovirus and HAV from two different types of drinking water (bottled water and tap water) with the adsorption-elution method proposed by ISO (Anonymous, ISO 15216-1, 2017) (method A) and with direct viral extraction using filters (method B). The mean extraction yields for norovirus and HAV calculated with RNA extracts ranged from 0.2 % - 4.81 % with method A and from 5.05 % - 53.58 % with method B, and did not differ significantly between the two types of drinking water tested. For method B, the mean extraction yields for HAV and norovirus were evaluated according to results from the three filters used. The recovery rate of HAV and norovirus ranged between 3.47 % and 62.41 % with the 0.45 µm electropositive filter and were higher than the other filters. The 0.45 µm electropositive filter could be used to concentrate viruses for routine viral monitoring of drinking water for researchers who want to adopt the method in their lab routine.

Development of an extraction method to detect enteric viruses in dressed vegetables.

Food-borne viral infections are caused mainly by noroviruses (NoV) and the hepatitis A virus (HAV), which respectively cause gastroenteritis and hepatitis. Various foods have been implicated in viral outbreaks, including vegetables that are consumed in a variety of forms, often with salad dressing. NF EN ISO procedures (15216-1:2017) propose standard methods for quantifying NoV and HAV in high-risk food categories, such as vegetables, based on viral elution and PEG concentration methods, but these methods are not suitable for composite meals like salads dressed with oily, fatty or emulsified food ingredients. The development of sensitive and reliable techniques for the detection of viruses in these products is therefore needed to ensure the safety of these products. The aim of this study was to develop an RT-qPCR based method for the detection and quantification of NoV and HAV in various vegetables with different dressings. Three methods for recovering NoV and HAV from artificially contaminated dressed vegetables were evaluated. The selected method was based on the use of Trizol reagent and, according to the type of dressing, the limit of detection ranged from 104 to 106 genome copies/g for NoV and from 102 to 103 PFU/g for HAV. The described method can be applied for detecting NoV and HAV in food containing salad dressing for routine diagnosis needs.

Development of a Real-Time Cell Analysis (RTCA) Method as a Fast and Accurate Method for Detecting Infectious Particles of the Adapted Strain of Hepatitis A Virus.

Hepatitis A virus (HAV) is one of the most common agents causing acute liver disease worldwide. HAV has been increasingly reported as the cause of foodborne disease outbreaks. The standard method currently available for detection of the genome of HAV in vulnerable foodstuffs is by RT-qPCR (ISO 15216). Despite its usefulness in the investigation of foodborne viruses, the use of RT-qPCR in food virology has been shown to overestimate the quantity of infectious virus or to highly underestimate the effect of the treatment on virus inactivation. The gold standard methods currently used for evaluating the efficacy of inactivation treatments on the adapted strain of HAV (HM175/18f) are either the plaque assay or the end-point dilution assay (TCID50). However, both assays are labor-intensive and time-consuming. The aim of this study was to evaluate the use of the xCELLigence real-time cell analysis (RTCA) system for detecting the infectivity of the adapted strain of HAV. Kinetics of cell impedance showed that HAV induced a decrease in cell index (CI) correlated with the onset of HAV-induced cell death. In addition, the time to which the HAV-induced CI drop occurred was dependent on the viral concentration. An inverse linear relation could be established over a range of 5 log10 between the concentration of HAV and the time to reach 50% of CI decrease (TCI50), showing that the RTCA assay could be used as a titration method for HAV. In addition, the RTCA-based assay could be performed in less than 6 days instead of 12 to 14 days with the gold standard methods. Therefore, the RTCA-based titration method is a powerful and suitable tool for high-throughput screening of anti-viral treatments. Its usefulness in HAV inactivation studies will improve the assessment of viral risk in food virology, as controlling transmission of viruses through their removal from foodstuffs is also an important challenge in reducing the burden of viral foodborne illnesses.

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.