焦磷酸测序检测铜绿假单胞菌的SHV基因点突变  

Detection of SHV gene point mutation of Pseudomonas aeruginosa by pyrosequencing

作　　者：黄源芳[1] 宋晓红[1] 尹亚非[2] 扎拉嘎白乙拉 姬小丽[1] 刘小康[1] 

机构地区：[1]四川大学华西基础医学与法医学院药理教研室,四川成都610041 [2]成都市第二人民医院检验科,四川成都610017

出　　处：《四川生理科学杂志》2012年第1期3-6,共4页Sichuan Journal of Physiological Sciences

摘　　要：目的:通过焦磷酸测序技术检测产超广谱β-内酰胺酶(ESBLs)铜绿假单胞菌的SHV基因点突变,探讨一种快速、准确的ESBLs基因分型方法。方法:双纸片法确定产ESBLs的铜绿假单胞菌,纸片扩散法(K-B法)进行药敏试验,聚合酶链反应(PCR)法扩增ESBLs的SHV基因片段,用焦磷酸测序法检测16株产ESBLs铜绿假单胞菌的SHV基因35位和43位密码子点突变。结果:焦磷酸测序发现,本地区分离出的16株产ESBLs铜绿假单胞菌有10株扩增出SHV基因片段,且在43位密码子基因没有突变,35位密码子有基因突变,核苷酸由T突变为A,亮氨酸变为谷氨酰胺,突变发生率达到70%(7/10)。16株产ESBLs的菌株对亚胺培南全部敏感。结论:焦磷酸测序技术可快速检测产ESBLs铜绿假单胞菌的SHV基因点突变,具有准确、快速、实时和高通量等优点,可应用于产ESBLs菌株的ESBLs基因分型。Objective. To investigate the method that can rapidly and accurately detect SHV gene point mutation and extendedspectrum β-lactamases （ESBLs） genotyping of ESBLs-producing Pseudomonas aeruginosa （PA） by pyrosequencing. Methods： Clinical isolated ESBLs producing strains were detected by double-disk method; antimicrobial susceptibility was tested by disk diffusion method （K-B method） ; SHV gene fragments of ESBLs were amplified by polymerase chain reaction（PCR） ; pyrosequencing was used to detect amino acids 35 and 43 of SHV gene of the 16 strains ESBLs producing PA. Results： As shown by pyrosequencing that 10 of the 16 locally isolated ESBLs-producing strains had SHV gene fragments, amino acids 43 had no gene mutation, while 35 had gene mutation, nucleotide mutated from T to A, amino acids from Leu to Gln, and mutation rate reached 70% （7/10）. All of the 16 stains producing ESI3Ls were sensitive to imipenem. Conclusions. Pyrosequencing can rapidly detect SHV gene point mutation of ESI3Ls producing PA with advantages of accuracy, speediness, real-time implementation and high-throughput, thus can be used in ESBLs geno- typing of clinical isolated strains producing ESBLs.

关 键 词：焦磷酸测序 铜绿假单胞菌 ESBLS SHV基因 点突变 

分 类 号：R440[医药卫生—诊断学]