Detection Of Escherichia Coli O157 By Duplex Real-Time PCR And Molecular Polymorphic Analysis Of O157 Isolates

Escherichia coli O157 is an important serotype of the Enterohemorrhagic Escherichia coli(EHEC) associated with hemolyticuremic syndrome (HUS), thrombotic thrombocytopenicpurpura and hemorrhagic colitis in people . Study of epidemic shows that it can cause infectioneven in case of extremely little number. So that, developing of a sensitive detection method isparticularly important.A duplex real-time PCR, based on Taqman hybridization probes technology, were developed and applied to detect enterohemorrhagic Escherichia coli O157.Two pairs of primer and two probes were designed for detection rfbE and stx2 genes .the rfbE probe was 5'end labeled with FAM and 3'end labeled with Taqman-MGB. The stxl probe was 5'end labeled with VIC and 3'end labeled with Taqman-MGB. The detection limits of the sensitivity assays were 10⁰ copies/μL of DNA; the qualitative consensus PCR assay indicated all Escherichia coli O157 were found rfbE positive and did not detect rfbE from non- O157 isolates and other Gram positive and negative bacteria. In the duplicated experiment, coefficients of variation intra-assay and inter-assay over the dynamic range of the MGB probe assays were lower than 70% and 80%, respectively. These results show that this duplex real-time PCR can detect the Escherichia coli O157 and stxl gene which was one of key virulence factors for Escherichia coli O157 pathogenicity from samples rapidly.The developed duplex real-time PCR target rfbE and stxl was used to identify the 64 isolates recovered from meat or fecal during 2000-2004. Meanwhile, the real-time PCR was applied to detect the simulated samples. Before the real-time PCR, the meat samples were mixed into EC broth after treated with asepsis and the concentration of pure culture was measured and diluted by 10 times. The diluted pure culture was inoculated into EC broth to obtain different concentration of bacterium. The genome were extracted from the EC broths compound for real-time PCR.The real-time PCR results show that 10 isolates were amplified while 54 isolates did not produce considerable amplification. So that , the 10 isolates were identified as E coli O157 and the 54 isolates were non- E coli O157, which comply with the results of serotyping.Furthermore, the results indicate that sensitivity of the real-time PCR was 3.05×10⁰CFU/μL. In a word, this method can detect as little as 30.5 CFU/g, which suggested that the real-time PCR is a new potential method for practice.To understand the characteristic of the strains isolated from tissue or fecal, RAPD (randomly amplified polymorphic DNA) and plasmid profile were developed. Meanwhile, detection of virulence factors and antimicrobial susceptibility test were conducted to verify the relationship of the strains. All the 10 strains were typed into three types by RAPD at the genetic distance of 0.89 and 0.973: 11^# and 13^# were typed into one category according to their RAPD profiles. while the others were typed into another two types.The ten strains were typed into five types by plasmid profile at different genetic distance: 11^# and 13^# are of high homology. Comparing the profiles of the two methods, we find that they show similar results: 11^# and 13^#, O157-æ ‡andé—µ-66,é—µ-27 and ;表-4,were typed into three categories ,respectively. Plasmid profile results were more sensitive which reflect the genetic relationship of the strains. The genes hly,eae,uid and flich₇ were detected from all the 10 strains, and antimicrobial susceptibility test results implied that drug resistant types of 1 l^#and 13^# were different from the other 8 strains, which comply with the RAPD and plasmid profile.Consequently, plasmid profile is a suitable method for typing which is rapid,convenient and stable.