耐多药大肠埃希菌质粒介导的耐药基因探讨.pdfVIP

Study to Plasmid-Mediated Genotypes in Multiple Drug- Resistance Escherichia coli Abstract Objective To investigate ESBLs-producing genotypes and drug-resistance of CTX-M-22,CTX-M-24 ，CTX-M,CTX-M-3,SHV,TEM in plasmid-mediated multiple –drug resistance Escherichia coli(E.coli),which were isolated from the patients in Affiliated Hospital of LuZhou Medical College,and to provide theoretical basis. Methods 1. The Kerby-Bauer disk diffusion method from the National Committee for clinical Laboratory Standards，NCCLS ）of America identified what kinds of drug-resistance in 7 1 E.coil. Antibacterials included cephalosporins, aminoglycosides and fluoquinolones, control group from ATCC 25922 of standard E.coil strains. 2. PCR technique: Firstly, the plasmid DNA were refined according to specification. CTX-M-22, CTX-24, CTX–M , CTX-M-3, SHV, TEM genes were amplified by PCR technique. Genes size was detected by agarose gel electrophoresis and sequencing . The volume of PCR reaction is 35μl (15μl of 2×PCR MasterMix, 7μl of refined DNA template , 5ul of each primer , 8ul of ddH O). Amplification was carried out as follows: initial denaturation at 94 ℃ 2 for 3min and denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30s, amount to 30 cycles, and a final step of 72 ℃ extension again for 5min. PCR products were visualized in the ultraviolet light after 2% agarose gels electrophoresis made in 0.5×TBE ethidium bromide, and photographed with gelatin camera. 3. Sequencing: The PCR products were sequenced, and then compared with known sequences. Results 1. A total of 71 strains of E.coil were