Establishment Of Standardized Method For Pulsed-field Gel Electrophoresis On Borrelia Burgdorferi And Preliminary Application

Background:Pulsed-field gel electrophoresis is invented by Schwartz and Canter in1980s andeventually become a key molecular biological technique after a series of development.Recently, PFGE is considered to be one of the most effevtive methods used todiscriminate bacterical typing and trace infectious source. PFGE has been widely used in thestudy of bacteria and has shown its strong value in controlling diseases outbreak. It not onlycan be used to track the source of infection alone, but also can support the relationship ofbacteria different species. Now it become an essential tool for tracking the source of disease.In addition, it is also used for dynamic detection of disease, and provide important referenceinformation for disease warning. At the present, PulseNet has been established as theinternational pathogen molecular typing detection network in the world. Based on strictlystandardized PFGE operation, a network platform for data exchange is established by thenetwork laboratory detection analysis and mutual comparison with type.Lyme disease is widely distributed in the worldwide, there are cases reported in the morethan70countries among the five continents. Lyme disease had prevailed severely in ourcountry, but it is not ranked among statutory reporting infectious diseases until today in China.Natural foci of Lyme disease almost throughout all of our mountains region, and there aretypical Lyme disease cases in the crowd. The crowd of nearly100million people are threatedby Lyme disease in China, estimated annual new cases of Lyme disease will not be less than20,000cases. Currently, the vast majority of Lyme patients can not get a clear diagnosis andeffective treatment,40%to50%to the middle and late, the serious consequences followed bylong-term repeated episodes.Recently, we found some species in B.burgdorferi can caused different disease and itshave strong correlation, in other words, different kinds of B.burgdorferi can lead to different disease. In such requirement, we need one molecular biology technique which is a simple,reliable and has strong ability to distinguish intra-and-inter species of B.burgdorferi, for theprevention and control of Lyme disease.In this study, we tried to use PFGE to B.burgdorferi, to establish the standardizedoperating procedures for B.burgdorferi PFGE, to analysis the classification feature atchromosomal DNA levels, to lay the foundation for genetic classification.On the base of the characteristics of strains and refer to the other SOPs of PFGE on otherpathogens provided by American CDC and PulseNet Asia Pacific, the genomic chromosomeDNA purification, restriction endonuclease digestion and the parameters for running PFGEwere optimized. Finally, the SOP of PFGE on B.burgdorferi was established, and thephylogenetic trees were achieved, including56strains from international standard strainsand Chinese strains, such as strains from Xinjiang, Inner Mongolia, Heilongjiang, Jilin,Liaoning, Hebei, Beijing, Sichuan and Guizhou, et al in China. The PFGE figures were highclear with high resolution, the fragments of Chinese strains were distributed equably to thatof international standard strains, each species had predominant molecular pattern. Thereexisted many distinction patterns among different species. There are a high degree ofconsistency between PFGE, RFLP and MLSA.Objective:In this study, we establish and optimize the B.burgdorferi PFGE, further enrich andimprove the research about B.burgdorferi molecular typing, to lay the foundation for controland prevention and epidemiological investigation on Lyme disease.Methods:On the base of the characteristics of strains and refer to the other SOPs of PFGE on otherpathogens provided by American CDC and PulseNet Asia Pacific, the genomic chromosomeDNA purification, restriction endonuclease digestion and the parameters for running PFGEwere optimized. The SOP of PFGE on B.burgdorferi was established, and the phylogenetic trees were achieved, including56strains from international standard strains and Chinesestrains.1、Preparating the BSKII medium, weighing the peptone, HEPES, yeast powder, sodiumpyruvate, and CMRL-1066, adjust the pH value after dissolution, adding the gelatin, BSAfractionⅤ and inactivated rabbit serum. Filtrating and sterilizing by nitrocellulose filter,packing;2、Culturing the international standard strains bought from ATCC with the BSKIImedium, and culturing the domestic viable strains coming from northwest China, northeastChina and so on, from human blood samples, ticks and host animal specimens, in33℃incubator. To observe the bacteria by dark field micrscopy, waiting the concentration ofbacteria up to+++to++++, we can prepare the gel block, extracting the chromosomal DNA;3、We used the gel block embedding method, embedding the bacteria into the gel block,lysising the bacteria, washing the gel block. Enzyme digestion, sample, electrophoresis,dyeing, image acquisition and data analysis. We choose the34B.burgdorferi total, choose thedifferent concentration, different restriction enzyme, and different electrophoresisparameters(EPs), and repeating the experiments above;4、We analysis the images and data obtained from experiment, and get the best operationscheme, best strain concentration, best endonuclease and best EPs;5、Another22strains B.burgdorferi was selected, we used the best operation schemeabove, got the phylogenetic tree. And we analyze the clustering results of this experiment,evaluate the methods mentioned above.Results:We established the PFGE standard operation of B.burgdorferi.Conclusions:1、the molecular genetic features of B.burgdorferi can be well reflected by PFGE;2、There is a good correlation of three typing methods, such as PFGE、RFLP and MLSA; 3、As a typing method, PFGE is less affected by external factors, and it can be used forroutine monitoring.Innovation:We tried to use PFGE to B.burgdorferi, to establish the standardized operatingprocedures for B.burgdorferi PFGE. To analysis the classification feature at chromosomalDNA levels, to lay the foundation for genetic classification. Providing a standard database inLyme disease prevention and control.