不同荧光定量聚合酶链反应法基因检测肺炎链球菌的比较研究  

Comparison of different fluorescent quantitation polymerase chain reaction for genes detection of Streptococcus pneumonia

作　　者：董佳波 傅应裕 

机构地区：[1]浙江省上虞市人民医院检验科,312300 [2]浙江省第二人民医院检验科

出　　处：《中国医师进修杂志》2013年第34期29-32,共4页Chinese Journal of Postgraduates of Medicine

摘　　要：目的 比较不同荧光定量聚合酶链反应法（PCR）基因检测肺炎链球菌的敏感性和特异性.方法 通过设计5种针对肺炎链球菌不同基因的引物[自溶素基因（lytA）、溶菌素基因（ply）、黏附基因（spn9802）、荚膜多糖生物形成蛋白基因（psaA）、编码DNA片段的基因（cspA）],采用荧光定量PCR检测37株肺炎链球菌、28株非肺炎链球菌以及80例临床痰液标本,评价这5种基因检测肺炎链球菌的敏感性和特异性.结果 5种引物均具有较好的检测灵敏度,扩增效率均高于90％.5种引物的Ct值变化均在0.5以内,在PCR扩增时具有可靠的稳定性.利用这5种引物对37株肺炎链球菌和28株非肺炎链球菌进行荧光定量PCR检测,lytA 、ply和spn9802特异性较强（检出阳性率和检出阴性率均为100％）.但是lytA 、ply的检测下限较spn9802高一个数量级（101CFU/ml比102 CFU/ml）.saA和cspA特异性较弱[psaA检出阴性率为89％（25/28）,cspA检出阳性率为97％（36/37）].对80例临床痰液标本进行检测表明,荧光定量PCR在检测特异性上明显优于传统培养法.5种引物在检测临床痰液标本中存在差异,lytA和ply显示出较好的检出率[阳性率分别为92.50％（74/80）、90.00％（72/80）],spn9802次之[86.25％（69/80）],psaA和cspA特异性最弱[76.25％（61/80）和78.75％（63/80）].结论 临床常用的5种荧光定量PCR基因在检测肺炎链球菌感染中的特异性和敏感性各不相同,lytA和ply特异性最强,spn9802次之,psaA和csPA特异性最弱.Objective To compare the sensitivity and specificity of different fluorescent quantitation polymerase chain reaction（PCR） for genes detection of Streptococcus pneumonia.Methods By designing five sets of fluorescent quantitation PCR primers targeting different genes of Streptococcus pneumonia （lytA,ply,spn9802,psaA,cspA）,the sensitivity and specificity of fluorescent quantitation PCR was determined through detecting 37 strains of Streptococcus pneumonia and 28 strains of non-Streptococcus pneumonia,as well as 80 sputum specimens from suspected pneumonia cases.Results These five primers had excellent sensitivity with 90% amplification efficiency through analysis of the standard curve.The change of Ct value was less than 0.5,indicating that these five primers have good stability.By detecting 37 strains of Streptococcus pneumonia and 28 strains of non-Streptococcus pneumonia,it showed that the specificity of lytA,ply and spn9802 was better than the other two primers （positive rate and negative rate was all 100%）.But the detection limit of lytA,ply was one order of magnitude than spn9802 （101 CFU/ml vs.102 CFU/ml）.The specificity of psaA and cspA was worse [negative rate of psaA was 89% （25/28）,positive rate of cspA was 97% （36/37）].By detecting 80 sputum specimens from suspected pneumonia cases,it showed that the specificity of fluorescent quantitation PCR were better than the bacterial culture.The specificity of lytA and ply was the best two [positive rate：92.50%（74/80）,90.00%（72/80）],and next was spn9802 [86.25%（69/80）],the specificity ofpsaA and cspA was the worst two for detection [76.25%（61/80） and 78.75%（63/80）].Conclusions The sensitivity and specificity of fluorescent quantitation PCR genes used in clinic for detecting Streptococcus pneumonia is different.LytA and ply are the best primers in specificity,next is spn9802,and psaA and cspA are the worst primers for in specificity detection.

关 键 词：聚合酶链反应 基因 DNA引物 链球菌 肺炎 

分 类 号：R440[医药卫生—诊断学]