实时定量PCR检测肺炎支原体方法的建立及临床应用  

Development and dinical application of real-time quantitative PCR for the detection of mycoplasma pneumonia

作　　者：胡文娟[1] 郭东星[1] 王红[2] 黄慧[2] 姜越[1] 王良玉[1] 辛德莉[1] 

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,热带病防治研究北京市重点实验室,100050 [2]北京大学民航临床医学院,100123

出　　处：《国际儿科学杂志》2015年第5期570-574,共5页International Journal of Pediatrics

基　　金：北京市科技计划课题（Z131100004013029）;首都卫生发展科研专项重大项目（首发2011-2009-05）

摘　　要：目的利用实时定量PCR技术（SYBR Green染料法）建立一种快速、特异、敏感、经济、准确检测肺炎支原体的方法，并评价其在临床上的应用价值。方法选择肺炎支原体的23S rRNA耐药相关基因作为扩增区，设计特异性引物，然后提取相应质粒作为标准品，建立绝对定量的标准曲线。比较荧光定量PCR方法与传统巢式PCR方法和市售试剂盒的特异度和灵敏度；分别利用上述三种方法和培养法检测临床咽拭子样本，进行相关系数、符合率及kappa系数的分析。结果实时定量PCR和巢式PCR检测肺炎支原体的灵敏度为10拷贝；市售试剂盒为10^2拷贝。特异性检验中，肺炎支原体样本检测为阳性，人型支原体和其他4种细菌均检测为阴性。临床样本检测中，实时定量PCR、巢式PCR、市售试剂盒和培养法的阳性率分别为55．49％、52．75％．47．25％和39．0l％。统计学分析结果显示，新建实时定量PCR与传统巢式PCR的结果总符合率为89．6％，Kappa系数为0．790。实时定量PCR方法与市售试剂盒检测结果的相关系数R=0．923，P〈0．001；两种方法的总符合率为89．6％，Kappa系数为0．792。实时定量PCR与培养法的结果总符合率为83．5％，Kappa系数为0．678。结论采用实时定量PCR方法检测肺炎支原体与其它几种方法相比，快速经济，且污染少，特异性强，灵敏度高，具有很重要的科研和临床实用价值。Objective To develope a new Real-time quantitative PCR assay using SYBR green as fluo- rescence reporter, which is rapid, specific, sensitive, cheap and accurate for the detection of mycoplasma pneu-moniae（MP） ,and evaluated its clinical application value. Methods The sequence of the 23S rRNA gene in MP type strain FH was selected as amplified regions,and specific primers were designed. Then the related plasmids were extracted as standards, and the absolute quantitative standard curve was established. The sensitivity, specificity of the fluorescence quantitative PCR assay was compared with the nest-PCR and kit; To calculate correlation coefficient, coincidence rate and kappa coefficient, clinical samples were detected using above-mentioned methods and cultivation,respectively. Results The detection sensitivity of the new real-time PCR and nest-PCR was 10 copies of FH DNA,while the kit 100 copies. In the specificity tests,the MP sample was positive,while mycoplasma hominis and other four bacteria were all negative. We applied this real-time PCR assay, nest-PCR, kit and cultivation to 182 clinical specimens, and the detection rates were 55.49% ,52.75% ,47. 25% and 39.01% ,respectively. The total consistency rate and Kappa coefficient of the new real-time PCR method and nest-PCR were 89. 6% ,0. 790,respectively; while those of the new method and cultivation were 83.5% ,0. 678, respectively. The total consistency rate and Kappa coefficient of the new real-time PCR method and the kit were 89. 6% ,0. 792 ,respectively; and the correlation coefficient of these two methods was 0. 923 ,P 〈0. 001. Conelusion Compared with other methods, the new real-time PCR assay could be used to detect mycoplasma pneu- moniae quickly and economically, with high sensitivity and specificity ,revealing great utility value on varied in-strumentation platforms.

关 键 词：肺炎支原体 儿童 荧光定量PCR 诊断 

分 类 号：R725.6[医药卫生—儿科]