An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp.

BACKGROUND: Vibrio spp. are a diverse group of ecologically important marine bacteria responsible for several foodborne outbreaks of gastroenteritis around the world. Their detection and characterization are moving away from conventional culture-based methods towards next generation sequencing (NGS)-based approaches. However, genomic methods are relative in nature and suffer from technical biases arising from library preparation and sequencing. Here, we introduce a quantitative NGS-based method that enables the quantitation of Vibrio spp. at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantification via digital PCR (dPCR). RESULTS: We developed six DNA standards, called Vibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA libraries via dPCR. To enable Vibrio-Sequin quantification, we validated three duplex dPCR methods to quantify the six targets. LOQs were ranging from 20 to 120 cp/µl for the six standards, whereas the limit of detection (LOD) was ~ 10 cp/µl for all six assays. Subsequently, a quantitative genomics approach was applied to quantify Vibrio-DNA in a pooled DNA mixture derived from several Vibrio species in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR. CONCLUSIONS: We significantly advance existing quantitative (meta)genomic methods by ensuring metrological traceability of NGS-based DNA quantification. Our method represents a useful tool for future metagenomic studies aiming at quantifying microbial DNA in an absolute manner. The inclusion of dPCR into sequencing-based methods supports the development of statistical approaches for the estimation of measurement uncertainties (MU) for NGS, which is still in its infancy.

Genetic Diversity of Hepatitis E Virus Type 3 in Switzerland-From Stable to Table.

Hepatitis E caused by hepatitis E viruses of the genotype 3 (HEV-3) is a major health concern in industrialized countries and due to its zoonotic character requires a "One Health" approach to unravel routes and sources of transmission. Knowing the viral diversity present in reservoir hosts, i.e., pigs but also wild boars, is an important prerequisite for molecular epidemiology. The aim of this study was to gain primary information on the diversity of HEV-3 subtypes present along the food chain in Switzerland, as well as the diversity within these subtypes. To this end, samples of domestic pigs from slaughterhouses and carcass collection points, as well as from hunted wild boars, were tested for HEV RNA and antibodies. HEV positive meat products were provided by food testing labs. The HEV subtypes were determined using Sanger and next generation sequencing. The genetic analyses confirmed the predominance of a Swiss-specific cluster within subtype HEV-3h in pigs, meat products, and wild boars. This cluster, which may result from local virus evolution due to the isolated Swiss pig industry, supports fast differentiation of domestic and imported infections with HEV.

Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver.

BACKGROUND: In the last years, the number of notified hepatitis E cases in humans has continuously increased in Europe. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Undercooked liver and raw sausages containing the liver of pigs and wild boar are at high risk of containing HEV. However, so far, no standardized method for the detection of HEV-RNA in pig liver is available. METHODS: An international collaborative study on method reproducibility involving 11 laboratories was performed for an HEV-RNA detection method, which consists of steps of sample homogenization, RNA extraction and real-time RT-PCR detection, including a process control. Naturally contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. RESULTS: Valid results were retrieved from 10 laboratories. A specificity of 100% and a sensitivity of 79% were calculated for the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. CONCLUSIONS: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, therefore, be applied to routine food control as well as in monitoring studies.

Interlaboratory Validation of a Method for Hepatitis E Virus RNA Detection in Meat and Meat Products.

Increasing numbers of hepatitis E cases are currently recognized in many European countries. The zoonotic hepatitis E virus (HEV) genotype 3 mainly circulates in domestic pigs and wild boars, and can be transmitted to humans via consumption of insufficiently heated meat or meat products produced from those animals. Here, a detailed protocol for detection of HEV RNA in meat products is provided, which is based on the method originally described by Szabo et al. (Intl J Food Microbiol 215:149-156, 2015). It consists of a TRI Reagent®/chloroform-based food matrix homogenization, a silica bead-based RNA extraction and a real-time RT-PCR-based RNA detection. The method was further validated in a ring trial with nine independent laboratories using pork liver sausage samples artificially contaminated with different amounts of HEV. The results indicate sufficient sensitivity, specificity, and accuracy of the method for its broad future use in survey studies, routine food control or outbreak investigations.

Screening of Ready-to-Eat Meat Products for Hepatitis E Virus in Switzerland.

Seroprevalence data for pig herds suggested that there must be a relevant reservoir for hepatitis E virus (HEV) in Switzerland. To know more about the viral presence in ready-to-eat meat products, we screened pork liver sausages and raw meat sausages from the Swiss retail market for the presence of HEV. Testing was performed with a detection method where the virus extraction step was optimized. As for the performance of the improved method, the mean recovery rate for the mengovirus process control was 24.4%, whereas for HEV-inoculated sample matrices between 10.4 and 100% were achieved. The limit of detection was about 1.56 × 103 and 1.56 × 102 genome copies per gram for liver sausages and raw meat sausages, respectively. In the screening programme, HEV-RNA was detected in 10 of total 90 (11.1%) meat products, 7 of 37 (18.9%) liver sausages, and 3 of 53 (5.7%) raw meat sausages. Virus loads of up to 5.54 log10 HEV genome copies per gram were measured. All sequences retrieved from positive samples belonged to HEV genotype 3. The significance of the presented work was a current overview of the HEV prevalence in ready-to-eat meat products on the Swiss retail marked and an improvement of the extraction efficiency of the HEV detection method.

Sensitive and semi-quantitative TaqMan™ real-time polymerase chain reaction systems for the detection of beef (Bos taurus) and the detection of the family Mammalia in food and feed.

Consumers distrust beef or products that could contain beef because of the BSE (bovine spongiform encephalopathy) and vCJD (variant Creutzfeld Jacob Disease) cases during recent years. Cows could be fed with meat and bone meal-containing food. To regain consumer confidence methods are needed that allow the detection of smallest amounts of beef in the most different kind of products. Polymerase chain reaction (PCR) analysis can be used to detect the smallest amounts of even highly degraded DNA. In this work two methods are presented that allow the detection of mammal DNA and beef DNA, respectively, even in highly degraded DNA. The amplification of fragments as short as 66 and 76 bp, respectively, allow a determination of mammal or beef DNA even in meat and bone meal products.