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-time-PCR,MRTPCR）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10copies/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61.1%（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。
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 MRT-PCR and single fluorescence quantitative PCR. Results The MRT-PCR detection system were successfully established which showed fine specificity. The detection limit of the assays was10 copies / μL. Coefficient of variation was from 0. 99% to 2. 50%,which conducted to assess the reproducibility of the assays. Among 530 clinical specimens,the detectable rates of MRT-PCR and single fluorescence quantitative PCR were 59. 2%（ 314 /530） and 61. 1%（ 324 /530）, respectively.Conclusion We developed a fast,specific,sensitive and stable MRT-PCR detection assay,which possessed good clinical application prospect.
出处
《河北医科大学学报》
CAS
2016年第11期1302-1306,共5页
Journal of Hebei Medical University
基金
河北省医学科学研究重点课题(20150147)
关键词
呼吸道感染
聚合酶链反应
敏感性与特异性
espiratory tract infections
polymerase chain reaction
sensitivity and specificity
