实时荧光定量RT-PCR快速检测四种呼吸道病毒的研究

目的利用实时荧光定量RT-PCR建立快速检测人腺病毒(HADV)、人博卡病毒(HBoV)、人偏肺病毒(HMPV)、人呼吸道合胞病毒(HRSV)等四种呼吸道病毒的方法。方法寻找四种呼吸道病毒的相对保守序列,针对该序列分别设计4对引物对及其相应的Taqman探针,将此保守序列作为目的基因片段,使用一步法RT-PCR试剂盒建立、优化反应体系;采用10倍稀释体外转录RNA,检测建立的体系的灵敏度和重复性并建立反应体系标准曲线;通过临床样本检验体系的特异性。结果 HADV、HBoV、HMPV、HRSV四种病毒的检测灵敏度均为10 copies/μl,标准曲线的决定系数分别为:HADV R2=0.9998,HBoV R2=0.9981,HMPV R2=0.9981,HRSV R2=0.9947和0.9965,扩增效率分别为:HADV 105.08%,HBoV 107.81%,HMPV 102.08%,HRSV FAM荧光106.38%及CY5荧光107.95%。荧光RT-PCR反应体系检测体外转录的RNA的各个拷贝数浓度的变异系数均较低,对临床样本检验的特异性为100%。结论本研究建立的实时荧光定量RT-PCR体系可快速、准确地检测人HADV、HBoV、HMPV、HRSV等四种呼吸道病毒,且重复性较好,特异性高。

Rapid detection of four kinds of respiroviruses by real-time fluorescence quantitative RT-PCR

Objective To devolop a rapid method to detact human adenovirus （HADV）, human bocavirus （HBoV）, human metapneumovirus （HMPV） and human respiratory syncytial virus （HRSV） by real-time fluorescence quantitative RT-PCR. Methods According to conservative sequences of HADV, HBoV, HMPV and HRSV, four sets of primers and Taqman probes were designed. One Step PrimerScript RT-PCR Kit was used to set up and optimize the real-time fluorescence quantitative RT-PCR system for the rapid detection of HADV,HBoV,HMPV and HRSV.10-fold serial dilution of in vitro transcribed RNA was used for the sensitivity detection and the reproducibility detection of this system. Clinical samples were used for the specificity detection of this system. Results The sensitivity of detecting HADV, HBoV, HMPV and HRSV were all 10 copies/μl. The coefficients of determination were： HADV 0.9998, HBoV 0.9981, HMPV 0.9981,and HRSV 0.9947 and 0.9965.The amplification efficiencies were： HADV 105.08%, HBoV 107.81%, HMPV 102.08% ,and HRSV 106.38% and 107.95% respectively. The values of variation coefficients of real-time fluorescent quantitative RT-PCR all remain in a low level in the detection of 6 different concentrations of the vitro transcribed RNA, and the specificity of this assay reached 100% in clinical samples testing. Conclusion This system based on real-time fluorescence quantitative RT-PCR assay can be used as a rapid and sensitive method in the detection of HADV. HBoV. HMPV and HRSV.