16SrRNA基因快速测序法鉴定3种常见病原微生物  

Identification of three common pathogenic microorganisms by using rapid sequencing of 16S r RNA gene

作　　者：陈霖祥[1,2] 蔡颖[2] 周广彪[2] 周霓[3] 林昆[1] 

机构地区：[1]汕头大学医学院公共卫生与预防医学教研室,广东汕头515041 [2]汕头出入境检验检疫局,广东汕头515041 [3]汕头市中心医院,广东汕头515041

出　　处：《现代预防医学》2014年第17期3180-3183,3200,共5页Modern Preventive Medicine

基　　金：广东省汕头市科技项目(汕市财文[2009]387号-126);广东出入境检验检疫局科技项目(2012GDK45);广东省感染病与分子免疫病理重点实验室

摘　　要：目的改良传统Sanger测序技术,建立16S r RNA快速测序法,并应用于常见病原微生物的鉴定。方法以铜绿假单胞菌、金黄色葡萄球菌和大肠埃希菌标准菌株为实验对象,建立16S r RNA快速测序鉴定方法。再以自医院分离的,已经传统生化方法鉴定的铜绿假单胞菌、金黄色葡萄球菌和大肠埃希菌各30株进行方法验证。结果采用本方法进行细菌鉴定,可以正确、快速鉴定所有3种标准菌株。临床样品中,所有金黄色葡萄球菌和铜绿假单胞菌均可获得与传统生化方法完全一致的带定结果,而在30株大肠埃希菌中,只有8株(占26.7%)被鉴定为大肠埃希氏菌,有10株(占33.3%)被鉴定为宋氏志贺菌,12株(占40%)被鉴定为痢疾志贺菌。结论本方法适用于铜绿假单胞菌和金黄色葡萄球菌的快速准确鉴定,对大肠埃希菌则不完全适用。Objective By means of improving conventional Sanger sequencing, to propose a protocol for rapid sequencing of 16 S r RNA gene, and to apply it on identifying common pathogenic microorganisms. Methods Three reference strains, Pseudomonas aeruginosa, Staphyloccocus aureus and Escherichia coli, were targeted as subjects to establish this method. To validate this method,a total of 90 experimental strains from hospital, consisted of the three kinds of bacteria each of 30 strains, which had been previously recognized by using traditional biochemical tests, were also performed with this method. Results The three reference strains were correctly and rapidly recognized by using this method. In the 90 clinical samples, the identification results of all the Pseudomonas aeruginosa and Staphyloccocus aureus sample using this method were in agreement with that using traditional biochemical tests, but only 26.7%（8） of the Escherichia coli samples were recognized as Escherichia coli as expected, while 33.3% were identified as Shigella sonnei and 40% as Shigella dysenteriae. Conclusion The assay described here is suitable for identifying Pseudomonas aeruginosa and Staphyloccocus aureus fast and accurately, but it is not completely suitable for Escherichia coli.

关 键 词：16S r RNA GENE Sanger测序 病原微生物 SYBR GreenⅠPCR 

分 类 号：R117[医药卫生—公共卫生与预防医学]