Genotyping Study On RAPD And AFLP Patterns Of Escherichia Coli O157:H7 Strains

These pathogens are an important threatening to the human health. Enterohemorrhagic Escherichia coli (EHEC) is the one of them. EHEC, an important and emergent pathogen, has been associated with blood and nonblood diarrhea and hemolytic-uremic syndrome (HUS). O157:H7 and O157:H- are the most important serotypes of EHEC. Molecular biological techniques were used to identify the pathogen of different source, different place and different period. It is very important to conform the infectant source and outbreak. The mechanism of pathogenic diversity and the mode of transmission and the difference of place are not clear at present. The pancity of knowledge in these areas is in part due to the lack of a reliable universal classification method. We must establish the classification method in order to the further study the prevalent ruleand distributing character. In the period from March to July 2000, an outbreak of O157:H7 occurred in one county of Henan Province in China. Animal reservoirs were suspected to be the cause. So RAPD and AFLP tests were applied to analysis the DNA polymorphic and identify the subtypes of E. coli O157:H7 strains isolated from animals such as goats, pigs, cows etc. Cluster analysis was applied to understand the data from RAPD and AFLP fingerprinting. At last, I compared RAPD with AFLP.METHODS1 The identification of EHEC O157:H7: O157 specific serum and H7specific serum were used to identify the O157:H7. At the same time the biochemical examinations were used to identify the O157:H7, too. If it passed the both examinations, it proved to be theO157:H7.2 Genotyping study on RAPD of O157:H7: The optimal primer was selected from six primers. The cycle parameter and the concentration of Mg2+ and Taq enzyme were optimized. The DNA was obtained by conventional method. After determining the optimal program and conditions, the molds were amplified. Amplified products were visualized by ethidium bromide staining after electrophoresis. Cluster analysis was applied to understand the data from RAPD.3 Genotyping study on AFLP of O157:H7: The DNA was obtained by conventional method. The genome DNA was digested with restriction enzyme PstI for 12 hours in 37℃. The digested DNA and synthetic adapter were connected by T4 DNA ligase for 12 hours in 16℃. The optimal primer was selected from two primers. Then templet and primer were amplified. The results were visualized by ethidium bromide staining after electrophoreSis. Cluster analysis was applied to understand the data from RAPD.4 Genotyping study on RAPD and AFLP of O157:H7: The result of RAPD incorporated with the result of AFLP, then to performing cluster analysis with the data from RAPD and AFLP fingerprinting.RESULTS1 The identification of EHEC O157:H7 After the biochemical examinations and specific serum tests, all of these germs accord with the standard of EHEC O157:H7. They were the EHEC O157:H7.2 The results of RAPD tests: The optimal program and conditions were obtained. 14 patterns were identified from the 32 O157:H7 isolates. According to the results of RAPD and cluster analysis, the transmit of O157:H7 existed among different animal reservoirs. Goats may be the reservoir which had transmitted the pathogen topersons.3 The results of AFLP tests: The optimal program and conditions were obtained. 16 patterns were identified from the 32 O157:H7 isolates. The results of AFLP were similar with the results of RAPD. According to the results of AFLP and cluster analysis, the transmit of O157:H7 existed among different animal reservoirs. Goats may be the reservoir which had transmitted the pathogen to persons.4 The results of RAPD and AFLP: Combined the results of RAPD with the results of AFLP, 18 patterns were identified from the 32 O157:H7 isolates. It still supports the above results.CONCLUSIONS1 Transmittion of O157:H7 existed among different animal reservoirs.2 Transmittion of O157:H7 existed among different animal reservoirsand persons. Goats may be the reservoir which had transmitted the pathogen to persons.3 A standard method to typing O157:H7 strains with RAPD fingerprinting was set up. The results indicate that RAPD is useful in the molecular typing of E.coli O157:H7.4 A standard method to typing O157:H7 strains with AFLP fingerprinting was set up. The results indicate that AFLP is usefulin the molecular typing of E.coli O157:H7. 5 The standard method to typing O157:H7 strains with AFLP isbetter than RAPD.