耐氧氟沙星鸡大肠杆菌gyrA基因QRDR的扩增与测序  

Detection of the Quinolone Resistance-Determining Region of the gyrA Gene of Ofloxacin Resistant Chicken Escherichia coli

作　　者：雷连成[1] 韩文瑜[1] 王兴龙[1] 王世若[1] 冯现伟[1] 江文正[1] 陈伟[2] 

机构地区：[1]解放军军需大学动物科技系,吉林长春130062 [2]新疆塔里木农垦大学动物科技学院,新疆阿拉尔843300

出　　处：《中国兽医学报》2001年第3期266-269,共4页Chinese Journal of Veterinary Science

基　　金：吉林省自然科学基金!资助项目 ( 980 2 13 )

摘　　要：取临床分离的 1 3株氧氟沙星 ( OFL)耐药菌 ,提取其质粒 DNA,经纯化后作为模板 ,用 PCR扩增 gyr A基因喹诺酮耐药决定区 ( QRDR) ;PCR阳性质粒的菌株 ,再扩增其染色体 gyr A基因 QRDR,直接测定质粒和染色体 DNA扩增产物序列并进行分析。只有 CE0 1菌株的质粒 DNA和染色体 DNA可扩增出长度为 668bp的 PCR产物片段 ,两者基因序列相同率为 98.1 7% ,相同位点突变相同的碱基有 5个 ,相同位点突变不同的碱基有 2个。与基因数据库中的大肠杆菌 gyr A基因相应序列比较 ,该菌质粒 DNA PCR产物同源率为97.80 % ,有 1 3个位点发生突变 ,3个氨基酸被替换 ;染色体 DNA PCR产物同源率 98.0 0 % ,有 1 2个位点发生突变 ,2个氨基酸被替换 ;都包括了国外资料报道的突变率较高的第 83位氨基酸的突变。结果表明 ,该菌株质粒和染色体上都存在喹诺酮耐药基因 ,对Thirteen ofloxacin resistant strains of chicken pathogenic E.coli were isolated from clinical samples. After plasmid extraction and purification, the quinolone resistance determining region (QRDR) of the gyrA gene was amplified by PCR with the plasmid templates. The plasmid PCR products were obtained from one strain, QRDR of the gyrA gene was also amplified by PCR from the templates of chromosomal DNA of this strain, then the PCR products were sequenced and analyzed. A expected 668 bp gyrA fragments was amplified from both plasmid DNA and chromosomal DNA of strain CEO1. The nucleotide sequences of the PCR products of plasmid DNA and chromosomal DNA showed 98.17% homology. When compared to the corresponding sequences of gyrA of E.coli from the nucleotide sequence data reported by Swanberg S L and Wang J C, 13 mutant sites were found in the nucleotide sequence of PCR product from plasmid DNA, and 3 amino acids changed; while 12 mutant sites were found in that from chromosomal DNA, and 2 amino acids changed. The results showed that the quinolone resistant gene occured both in the plasmid and chromosome of strain CE01 would be associated with quinolone resistance of strain CE01.

关 键 词：大肠杆菌 耐药基因 氧氟沙星 PCR扩增 测序 喹诺酮耐药决定区 点突变 

分 类 号：S859.796[农业科学—临床兽医学] S188[农业科学—兽医学]