多种呼吸道病原体的多重实时荧光定量PCR检测方法的建立及应用.pdfVIP

第 37 卷第 11 期 河北医科大学学报 Vo l. 37 No.ll • 1302. 2016 年 11 月 JOURNAL OF HEBEI MEDICAL UNIVERSITY Nov. 2016 ·论著· 多种呼吸道病原体的多重实时荧光定量 PCR 检测方法的建立及应用 邸红芹，杨永辉，康丽菲，安晓颖，李晓霞，张 辉替 (河北省胸科医院检验科，河北石家庄 05004 1) [摘要] 目的建立多种呼吸道病原体的多重实时荧光定量聚合酶链反应(multiplex real-tim e-PCR , MRT- PCR)检测方法。方法根据生物信息学分析结果选定各种呼吸道病原体的靶序列，建立 MRT-PCR 检测体系。构 建质粒标准品，检测建立体系的敏感度、特异度和重复性，并运用 MRT-PCR 和单荧光定量 PCR 检测临床样本进 行对比分析。结果成功建立了多种呼吸道病原体的 MRT-PCR 检测体系;该方法特异度较好，最低检测限为 10 copies/μL ，重复性良好，变异系数为0.99 %~ 2.50 %, 530 例临床样本中，MRT-PCR 检测的阳性率为59.2 %(314/ 530) ，单荧光定量 PCR 检测的阳性率为6 1.1 %(324/530) ，阳性检出率差异不大。结论建立快速、敏感、特异、稳定 的 MRT-PCR 检测体系，具有良好的临床应用前景。 [关键词] 呼吸道感染;聚合酶链反应;敏感性与特异性 doi: 10.3969/i.issn.1007-3205 .20 16.1 1.016 [中图分类号] R517.6 [文献标志码] A [文章编号] 1007-3205(2016)11-1302-05 Establishment and application of multiplex real唰time fluorescence quantitative PCR assay for detecting various respiratory pathogens DI Hong-qin , YANG Yong-hui , KANG Li-fei , AN Xiao-ying , LI Xiao-xia , ZHANG Hui 锋 (Deþartment of Clinical Laboratory , the Chest Hosρital of Heibei Province ,Shijiazhuang 050041 , China) [Abstract] Objective To develop multiplex real-time fluorescence quantitative PCR (MRT- PCR) assay for detecting various respiratory pathogens. Methods MRT-PCR assay was established with kinds of respiratory pathogenic genes , selected based on their relevant reference strains by bioinformatics analysis , as target genes. Standard products and quality control samples were built to testing and analysising the sensitivity , specificity and reproducibility of this system. Clinical samples were used to evaluating 肌ifRT-PC