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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。
Streptococcus pneumoniae is a pathogenic and mortality of high pathogenic bacteria, without extensive clinical testing experience from the clinical criteria for the isolation and identification of this bacteria more difficult, the experiment oligonucleotide primers YH1 YH2, YH7 YH8 were amplified Streptococcus pneumoniae autolysin and hemolysin gene 35 4bp and 30 7bp of DNA fragments, by changing the reaction conditions, the establishment of the PCR method of detection of these two etiological factor genes. 20 strains of Streptococcus pneumoniae and 7 strains of control were identified by this method. The amplified products were digested with restriction endonucleases TthHB81 and AccI to confirm whether the amplification products were correct. The detection sensitivity of the PCR method was determined by extracting the purified whole cell DNA, and 28 clinical isolates were identified by this method. Results The DNA fragments of 35 4bp and 30 7bp were amplified by primers YH1 YH2 and YH7 YH8 in all 20 Streptococcus pneumoniae strains, while the control strains were negative. The amplified products of autolysin and hemolysin were The fragments generated after digestion with restriction endonuclease TthHB81 and AccI were exactly as expected and both pairs of products were capable of amplifying the target DNA fragment from 10 fg of whole cell DNA. Two sets of PCR systems were established to identify 28 clinical isolates, of which 15 PCR-positive isolates were identified as S. pneumoniae by biochemical characterization. The test set up two sets of PCR detection system has the advantages of strong specificity, high sensitivity and easy operation, can be used for the identification of Streptococcus pneumoniae.