肺炎链球菌自溶素和溶血素基因的PCR法鉴定  

Identification of Streptoccocus pneumoniae by polymerase chain reaction for amplification of autolysin and pneumolysin gene

作　　者：杨永红[1] 朱保权[1] 宁淑敏[1] 安真光[1] 王棣[2] 王兴民[2] 

机构地区：[1]兰州医学院第二附属医院,兰州730030 [2]兰州生物制品研究所,兰州730046

出　　处：《微生物学免疫学进展》2000年第2期49-53,共5页Progress In Microbiology and Immunology

基　　金：甘肃省自然科学基金!号为 :ZS991 A2 3 0 73Y

摘　　要：肺炎链球菌是一种致病率和致死率很高的病原菌 ,若无丰富临床检测经验从临床标准中分离鉴定此菌较困难 ,本实验以寡核苷酸引物YH1 YH2、YH7 YH8分别扩增肺炎链球菌自溶素和溶血素基因的 35 4bp和 30 7bp的DNA片段 ,通过改变各种反应条件 ,建立了这两种病原因子基因的PCR检测方法。用此方法对 2 0株肺炎链球菌标准菌株及 7株对照菌株进行了鉴定 ;其扩增产物分别经限制性内切酶TthHB81和和AccI进行酶切以确认扩增产物是否正确 ;用酚 氯仿抽提纯化的全细胞DNA对PCR方法的检测灵敏度进行了测定 ;并利用此方法对 2 8份临床标本分离物进行了鉴定。结果　所有 2 0株肺炎链球菌均可分别用引物YH1 YH2、YH7 YH8扩增出 35 4bp和 30 7bp的DNA片段 ,而对照菌株均呈阴性 ;自溶素及溶血素基因的扩增产物分别经限制性内切酶TthHB81和AccI消化后产生的片段和预期的完全一致 ;两对产物均可从 10fg的全细胞DNA中扩增出目的DNA片段。所建立的两套PCR系统对 2 8份临床标本分离物进行鉴定 ,其中PCR阳性的 15份分离物经生化学特性检查被鉴定为肺炎链球菌。本试验所建立的两套PCR检测系统具有特异性强 ,灵敏度高及操作简单等优点 ,均可用于肺炎链球菌的鉴定。Streptoccocus pneumoniae is a major cause of morbidity and mortality worldwide.It is difficult to isolate the organisms from clinical specimens without enough technical skill.It is the aim that a set of polymerase chain reaction(PCR) methods will be developed to identify the S.pneumoniae strains for this study.By altering various conditions,the PCRs were established by using primer sets YH1 YH2 and YH7 YH8 which respectively designed to amplify the DNA fragment in size of 354 by for autolysin gene and 307 bp for pneumolysion gene.Twenty strains of S.pneumoniae and 7 control strains were identified with the PCRs.The amplified products were digested respectively with restriction endonuclease Tth HB81 and AccI to verify whether the fragments would be amplified correctly.The sensitivities were determined with the whole cell DNA purified with Phenol Chloroform.At the last,28 clinical isolates were identified by these PCR systems. The target fragments in size of 354 bp and 307 bp respectively related to gene of autolysin and pneumolysin were amplified in all of the 20 strains of S.pneumoniae,whereas all the other bacterial strains tested were negative in the two PCR systems.The digest patterns of restriction endonucleases were in conformity with the predicted sizes completely.Clear DNA fragments could be amplified from 10 fg of whole cell DNA in all of these two PCR systems.Fifteen of 28 strains isolated from clinical specimens were positive in the two PCR systems.The strains that were positive in PCRs were also defined to S.pneumoniae by the biochemical properties.The PCRs established in this study were useful to identify the S.pneumoniae strains since they were specific,sensitive and simple.

关 键 词：肺炎链球菌 自溶素 溶血素 PC的鉴定 病原菌 

分 类 号：R378[医药卫生—病原生物学]