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Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes. 总被引:10,自引:10,他引:0
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The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments. 相似文献
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Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes 总被引:2,自引:0,他引:2
X Jiang M K Estes T G Metcalf J L Melnick 《Applied and environmental microbiology》1986,52(4):711-717
The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments. 相似文献
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Chantal Jehl-Pietri B. Hugues M. Andre J.M. Diez † A. Bosch † 《Letters in applied microbiology》1993,17(4):162-166
Immune electron microscopy (IEM), radioimmunoassay (RIA) and molecular hybridization with a digoxigenin-labelled cDNA probe were compared for the detection of wild-type human hepatitis A virus (HAV) in raw and treated sewage. In the same experiments, classic tests for culturable enteroviruses were carried out. With the hybridization probes, HAV was detected in three of the 13 amuent samples (23%) and in eight out of 13 effluent samples (61%). For four of the emuent samples, positivity revealed by IEM was confirmed by the cDNA probe. In contrast, two of the samples shown as positive by IEM were negative with the probes. Detection of HAV by RIA was negative in all cases. Demonstration of HAV was higher in emuent than in affluent. No particular relationship was established between demonstration of HAV, on the one hand, and the various concentrations of enteroviruses observed in the same samples on the other. Overall, if all the results, irrespective of the type of water (affluent or effluent), are taken together, 50% of the sewage samples tested were found to contain HAV by one or another method of detection. 相似文献
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Borchardt MA Bertz PD Spencer SK Battigelli DA 《Applied and environmental microbiology》2003,69(2):1172-1180
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Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination. 总被引:4,自引:3,他引:1
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Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts). 相似文献
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A.M. Silva H. Vieira N. Martins A.T.S. Granja M.J. Vale F.F. Vale 《Journal of applied microbiology》2010,108(3):1023-1031
Aims: To assess the presence of viral pathogens in bathing water samples and to evaluate the interdependency of bacterial indicator counts and viral detection. Methods and Results: Bathing water samples of 16 beaches collected along a Portuguese Coastal area were screened for the hepatitis A virus (HAV) and norovirus genogroup I (NVGI) using RT‐PCR technique. Bacteriological water quality was also assessed, according to European regulations. HAV and NVGI were detected in 95% and 27% of the water samples, respectively, whereas bacteriological quality was good in all but one sample, according to current water quality regulations. Conclusions: All water samples would be considered of excellent quality according to the most recent European regulations. No relationship between viral detection and regulatory‐based bacterial indicators was found. Significance and Impact of the Study: The current results reinforce the importance of increased surveillance for pathogenic viruses in bathing waters. 相似文献
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Virus-Contaminated Oysters: a Three-Month Monitoring of Oysters Imported to Switzerland 总被引:5,自引:2,他引:3
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Molluscan shellfish are known to be carriers of viral and bacterial pathogens. The consumption of raw oysters has been repeatedly linked to outbreaks of viral gastroenteritis and hepatitis A. Switzerland imports over 300 tons of oysters per year, 95% of which originate in France. To assess the level of viral contamination, a 3-month monitoring study was conducted. Therefore, the sensitivities of several previously described methods for virus concentration were compared, and one protocol was finally chosen by using dissected digestive tissues. Eighty-seven samples consisting of five oysters each were analyzed for Norwalk-like viruses (NLVs), enteroviruses, and hepatitis A viruses from November 2001 to February 2002. The oysters were exported by 31 French, three Dutch, and two Irish suppliers. Eight oyster samples from six French suppliers were positive for NLVs, and four samples from four French suppliers were positive for enteroviruses; two of the latter samples were positive for both viral agents. No hepatitis A viruses were detected. The sequences of NLV and enterovirus amplicons showed a great variety of strains, especially for the NLVs (strains similar to Bristol, Hawaii, Mexico, and Melksham agent). The data obtained indicated that imported oysters might be a source of NLV infection in Switzerland. However, further studies are needed to determine the quantitative significance of the risk factor within the overall epidemiology of NLVs. 相似文献
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Virus-contaminated oysters: a three-month monitoring of oysters imported to Switzerland 总被引:6,自引:0,他引:6
Molluscan shellfish are known to be carriers of viral and bacterial pathogens. The consumption of raw oysters has been repeatedly linked to outbreaks of viral gastroenteritis and hepatitis A. Switzerland imports over 300 tons of oysters per year, 95% of which originate in France. To assess the level of viral contamination, a 3-month monitoring study was conducted. Therefore, the sensitivities of several previously described methods for virus concentration were compared, and one protocol was finally chosen by using dissected digestive tissues. Eighty-seven samples consisting of five oysters each were analyzed for Norwalk-like viruses (NLVs), enteroviruses, and hepatitis A viruses from November 2001 to February 2002. The oysters were exported by 31 French, three Dutch, and two Irish suppliers. Eight oyster samples from six French suppliers were positive for NLVs, and four samples from four French suppliers were positive for enteroviruses; two of the latter samples were positive for both viral agents. No hepatitis A viruses were detected. The sequences of NLV and enterovirus amplicons showed a great variety of strains, especially for the NLVs (strains similar to Bristol, Hawaii, Mexico, and Melksham agent). The data obtained indicated that imported oysters might be a source of NLV infection in Switzerland. However, further studies are needed to determine the quantitative significance of the risk factor within the overall epidemiology of NLVs. 相似文献
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Suffredini E Corrain C Arcangeli G Fasolato L Manfrin A Rossetti E Biazzi E Mioni R Pavoni E Losio MN Sanavio G Croci L 《Letters in applied microbiology》2008,47(5):467-474
Aims: To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. Methods and Results: Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8·3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. Conclusions: The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. Significance and Impact of the Study: The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events. 相似文献
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Detection of hepatitis A virus in sewage sludge by antigen capture polymerase chain reaction. 总被引:7,自引:4,他引:3
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Antigen capture polymerase chain reaction (PCR) was tested as a sensitive and rapid method for detecting hepatitis A virus (HAV) in raw sewage sludge. The antigen capture PCR was performed both with and without solid-phase virus-catching monoclonal antibodies. Similar results proved that both methods were equally sensitive. Sewage sludge samples from different regions in Germany were examined for evidence of HAV contamination by antigen capture PCR. This method of detection was compared with that used in a previous study of these sewage sludge samples, in which the HAV was detected through indirect immunofluorescence after cell culture inoculation. The results obtained by antigen capture PCR matched those obtained in the earlier cell culture investigations, when HAV was detected in raw as well as digested sewage sludge samples. The advantage of the PCR method, however, lies in the fact that it needs only two days while the cell culture propagation of HAV takes about 8 to 10 weeks. 相似文献
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