Establishment Of PFGE Pattern Database Of Vibrio Cholerae Isolated In China And Optimization Of Experiment Protocol Of AFLP For Vibrio Cholerae Subtyping

Establishment of PFGE Pattern Database of Vibrio cholerae Isolated in China and Optimization of Experiment protocol of AFLP forVibrio cholerae SubtypingMaster candidate:Haijian ZhouSupervisor:Biao KanState Key Laboratory for Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,P.O.Box 5,Changping,Beijing 102206,People'sRepublic of ChinaPulseNet is a national molecular subtyping network for foodborne disease surveillance.PulseNet participants perform standardized molecular subtyping(or "fingerprinting") of foodborne disease-causing bacteria by pulsed-field gel electrophoresis(PFGE).In this study,we established the PFGE pattern database of Vibrio cholerae for PulseNet China and optimized the experiment condition of AFLP for V.cholerae subtyping in order to develop AFLP as a molecular subtyping method of V.cholerae in PulseNet.One thousand and seven hundred seventy-three isolates of V.cholerae Ol and O139 collected from 1961 to 2007 in China were analyzed by PFGE.Chromosomal DNAs from all V.cholerae isolates in agarose plugs were digested with the restriction enzyme NotI or(and) Sfil,resulting in 17 to 27 bands.One thousand and two hundred sixteen isolates of V.cholerae O1 and five hundred seventeen isolates of O139 were digested with Noil,722 and 169 PFGE types were identified,respective.Seven hundred and four isolates of V.cholerae O1 and one hundred and fifteen of O139 were digested with Sfil,532 and 53 PFGE types were identified,respective.Most of isolates(＞70%) have unique pattern,and also same types exist in different years or (and) different provinces. Through compared the discriminatory power between unrelated strains,possibly related strains and strains can't differentiated by ribotyping,we found that NotI and Sfil both were very suitable for V.cholerae O1 subtyping.But for V.cholerae O139, digested with Notl could gain higher level of discriminatory power than Sfil,the difference was obvious.Higher level of discriminatory power could be obtained by using the combination of the two restriction endonucleases.Based on the patterns of NotI-digested,we did cluster analysis of seven hundred and forty toxigenic serogroup O1 V.cholerae.There were twenty clusters after grouping under 87.5%similarity value.The result revealed a temporal pattern of change in the clone.Subcluster 4a consisted mainly of strains from 1980s,subcluster 4b consisted mainly of strains from 1960s and 1980s,and subcluster 4d consisted mainly of strains from 1960s.Cluster 5 consisted mainly of strains from 2005.Cluster 6 consisted mainly of strains from 1960s and 1970s.Cluster 8 consisted mainly of strains from 1990s.Other clusters and subclusters just consisted 1-5 strains.The strains isolated in 1961 showed the highest level of diversity,forty-one strains dispersed in nine clusters.And the strains isolated in 2005 was divided with other strains,suggest that it was a new clone.On the other hand,four hundred and seventeen toxigenic serogroup O139 V. cholerae were separated into 117 PFGE patterns.These patterns were so similar that couldn't be divided into two or more clusters.The results suggested that toxigenic V. cholerae serogroup O139 isolated in China between 1993 and 2007 was conservative.In this study,AFLP was optimized to analyze strains of V.cholerae,and also compared with PFGE to study the practical value of AFLP for V.cholerae subtyping. AFLP data were obtained by using fluorescently labeled PCR primers and analysis on an ABI PRISM automated DNA analysis platform.Three groups of restriction endonucleases were used to differentiate the strains of V.cholera.As the result, ApaⅠ-T/TaqⅠ-C(D=0.8964) and HindⅢ-A/TaqI-A(D=0.9796) were the highest discriminatory primer pairs of the two groups of restriction endonucleases ApaⅠ/TaqⅠand HindⅢ/Taql,respective.And the result also showed that the restriction endonucleases and primer combination EcoRⅠ-G,combined with /MseⅠ-T had the highest discriminatory power in the AFLP subtyping of V,cholerae(D=0.9408).Through comparing the categorization results of the two methods(AFLP and PFGE),it is found that PFGE showed the higher discriminatory power than AFLP. However,both methods had the D value greater than 0.9,which suggested that AFLP was also powerful in discrimination ability of V.cholerae subtyping.