人偏肺病毒TaqMan-MGB探针实时定量RT-PCR检测方法的建立及初步应用

Development and Application of Real-Time RT-PCR Assay for Detection of Human Metapneumovirus in Beijing

目的:建立人偏肺病毒(hMPV)核酸特异的快速、敏感的TaqMan-MGB探针实时定量RT-PCR检测方法。方法:分别设计hMPV特异的引物与荧光标记探针,合成hMPV绝对定量RNA模板,建立实时荧光定量PCR方法,并与常规RT-PCR平行比较,对其灵敏性、特异性和可重复性,以及用于临床样本的适用性等进行评价。结果:本方法可对hMPV进行特异性诊断,检测灵敏度可达10拷贝/25μL,检测线性范围至少可达101～106拷贝/反应,且实验重复性好,初步应用于北京地区采集的158份临床鼻咽拭子标本,定量RT-PCR检出31份标本阳性,明显高于常规RT-PCR方法(22/158)。结论:建立了人偏肺病毒TaqMan-MGB探针定量RT-PCR检测方法,并初步证实可用于临床鼻咽拭子标本的检测,为开展hMPV的流行监测及临床早期诊断提供了技术手段。

Objective： To develop a rapid and sensitive TaqMan-MGB based, real-time quantitation reverse transcriptase PCR（qRT-PCR） assay for detection and quantitation of human metapneumovirus（hMPV）. Methods： The specific primers and fluorescence-labeled probe to develop a real-time qRT-PCR assay for detection of hMPV were designed. Absolute viral copy was achieved through the construction of in-hous hMPV standard for the generation of a standard curve. Subse- quently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. Resuits： The analytical detection limit of this real-time qRT-PCR assay was 10 RNA copies per reaction mixture. This assay allowed quantitation of hMPV over 6-log-unit span ranging from 10^1～10^6 copy. The variations of intra-assay were less than 5%. A total of 158 nasopharyngeal swab specimens derived from Beijing were screened for the presence of hMPV by using real-time qRT-PCR and identified 31 specimens positive for hMPV. The positive detection rage of hMPV by qRT-PCR assays was higher than that by conventional RT-PCR （22/158）, which were used to assess in parallel by the qRT-PCR assay. Conclusion： A real-time qRT-PCR assay for detection and quantitation of hMPV has been developed. The assay described was proved to be more sensitive than conventional RT-PCR and showed a good reproducibility. This assay maybe applied for surveillance and clinical diagnosis of hMPV.