实时定量PCR检测流感嗜血杆菌感染实验室诊断方法的建立  

Laboratory diagnostic method establishment of real-time quantitative PCR for detecting Haemophilus influenza infection

作　　者：蔚然 王良玉 高琦[2] 胡文娟 窦海伟 向莉[2] 辛德莉 郭东星 WEI Ran;WANG Liangyu;GAO Qi;HU Wenjua;DOU Haiwei;XIANG Li;XIN Deli;GUO Dongxing(Affiliated Beijing Friendship Hospital of Capital Medical University/Beijing Tropical Medicine Research Institute,Beijing 100050,China;Affiliated Beijing Children Hospital of Capital Medical University,Beijing 100050,China)

机构地区：[1]首都医科大学附属北京友谊医院/北京热带医学研究所,北京100050 [2]首都医科大学附属北京儿童医院,北京100050

出　　处：《检验医学与临床》2018年第6期737-739,743,共4页Laboratory Medicine and Clinic

基　　金：首都市民健康培育项目(Z161100000116088;Z131100006813044)

摘　　要：目的建立SYBR-Green染料法实时定量聚合酶链反应(PCR)检测流感嗜血杆菌(Hi)感染的实验室诊断方法。方法设计合成针对Hi fucK基因的引物;扩增Hi fucK基因,构建质粒,10倍梯度稀释后,以10~108 copy/反应作为标准品绘制标准曲线,并检验新建实验室诊断方法的敏感度;采用实验室保存的肺炎支原体标准株、人型支原体标准株、阴沟肠杆菌标准株、金黄色葡萄球菌标准株、肺炎克雷伯菌标准株和大肠埃希菌标准株的DNA标本,检验新建实验室诊断方法的特异度;将新建立的实验室诊断方法,应用于临床标本的检验。结果新建实验室诊断方法敏感度较高,其可检测到拷贝数为10copy的Hi DNA;具有较高特异度,可成功区别于几种病原体DNA;将该方法应用于226份临床咽拭子标本的检测,检出Hi感染92份,阳性率为40.70%。结论该研究建立的实时定量PCR检测Hi感染的方法,具有较高的敏感度和特异度,操作简单,用时较短,可用于临床Hi感染的实验室诊断。Objective To establish a laboratory diagnostic method of SYBR Green dye method of real-time quantitative PCR for detecting Haemophilus influenza(Hi)infection.Methods The primers aiming at L-fuculokinase(fucK)gene were designed and synthesized.The Hi fucK gene was amplified,the plasmid was constructed,after 10 fold gradient dilution,the standard curve was drawn with 10-10 8 copy of reaction as the standard substance,the sensitivity of newly established laboratory diagnostic method was tested;the DNA samples of Mycoplasma pneumoniae standard strain,Mycoplasma hominis standard strain,Enterobacter cloacae standard strain,Staphylococcus aureus standard strain,Klebsiella pneumoniae standard strain and Escherichia coli standard strain preserved in the laboratory were adopted for detecting the specificity of newly established laboratory diagnostic method;then the newly established laboratory diagnostic method was used in the detection of clinical samples.Results This newly established laboratory diagnostic method had higher sensitivity,its detectable copy number was 10 copy Hi DNA;this method had higher specificity and could successfully distinguish several pathogenic DNA;in the application of this method for 226 clinical throat swab samples,92 cases of Hi infection were detected out with the positive rate of 40.70%.Conclusion The real-time quantitative PCR for detecting Hi infection established by this study has higher sensitivity and higher specificity,is easy to operate,uses short time and can be used in the laboratory diagnosis of clinical Hi infection.

关 键 词：流感嗜血杆菌 儿童 呼吸道感染 实时定量聚合酶链反应 

分 类 号：R446.5[医药卫生—诊断学]