RESUMO
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.
RESUMO
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.
Assuntos
Vírus da Hepatite E/genética , Carne/virologia , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral , Virologia/normas , Animais , Limite de Detecção , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Sus scrofa/virologiaRESUMO
We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities.
