| Objective:To ascertain main popular serotype in Shenzhen area and understand the difference and popular trend of vibrio parahaemolyticus strains for providing a basis epidemiological survey. Meanwhile establishing molecular parting database of vibrio parahaemolyticus can provide the basis for tracking infection sources and finding early outbreak. This topic will be researched from serotyping, PFGE parting and MLVA parting. Gradually to improve PFGE database network, establish MLVA method system, evaluation the application and establish MLVA database for futher related cluster analysis.The results are analyzed by pulsed field gel electrophoresis imaging system and BioNumerics software.Metheds:(1) To collect and serotype the vibrio parahaemolyticus strains from strains save library in Microbial inspection of Shenzhen center for disease control and prevention from2006to2011. These strains are obtained by food pollutant monitoring network and Shenzhen infectious diarrhea pathogenic spectrum monitoring network.(2) To screen and culture the strains for the PFGE parting by cracking cell, embedding strains and enzyme method.(3) Through culturing the strains, extracting DNA with boiled method, polymerase chain reaction (PCR) amplification and sending out for sequence, the results can be analyzed by MLVA parting with BioNumerics software, which is contrast to PFGE parting method.Result:(1) This experiment is completed by serotyping197vibrio parahaemol yticus from2006to2011. There are31different serotype separated. The most mainstream serotype is O3:K6for the proportion of45.18%. Followed by the serotype is O4:K8for the proportion of10.15%. In the third place is01:K25for the proportion of7.11%.(2) There are90different PFGE belt type obtained by the pulsed field gel electrophoresis with99vibrio parahaemolyticus from food and external environment source of which parting efficiency is99.77%. There are26different PFGE belt type obtained by the pulsed field gel electrophoresis with98vibrio parahaemolyticus from foodborne disease outbreak source of which parting efficiency is87.71%.(3)99vibrio parahaemolyticus from food and external environment source are analysed by MLVA parting with6VNTR locus.99vibrio parahaemolyticus strains are classified into96MLVA type or90PFGE type of which the resolution factor is99.91%or99.77%.98vibrio parahaemolyticus from foodborne disease outbreak source are analysed by MLVA parting with6VNTR locus.98vibrio parahaemolyticus strains are classified into50MLVA type or26PFGE type of which the resolution factor is97.14%or87.71%.Combine with9outbreak cases determined and2outbreak cases suspected, MLVA has a better rate of clusters and aggregation than PFGE. It has a more sensitive on the judgment of the outbreak, stronger recognition ability, more persuasive of starting the epidemiological investigation.Conclusion:(1) The most mainstream serotype of vibrio parahaemolyticus strains is O3:K6which comes from clinical source.The second is O4:K8. The third is O3:K29. The fourth is O1:K25.Vibrio parahaemolyticus strains from food or external environment sources have diversified serotype, with which the trend is scattered.(2) All vibrio parahaemolyticus strains from clinical, food and external environment source can be parted accurately by MLVA clustering analysis method with6VNTR locus. MLVA method has a higher resolution, a more simple operation, a cheaper expense and a more accurate result.(3)6VNTR locus namely TR2,TR4,TR5,TR8,TR9and TRIO applied together are analysed by MLVA method,which is suitable for molecular parting and analysis of outbreak vibrio parahaemolyticus strains.Not only the parting is very accurate and it can save workload and work cost. MLVA method can better determine and judge the outbreak to trace the source comparing to PFGE method,which has a better prompt action in early detection outbreak source especially. |
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