人博卡病毒TaqMan探针实时定量PCR检测方法的建立及初步应用

目的:建立人博卡病毒(HBoV)核酸特异、快速、敏感的TaqMan探针实时定量PCR检测方法,并对临床样本进行检测。方法:比对编码HBoV非结构蛋白NP-1的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量PCR检测方法,并与传统PCR方法进行比较,然后分别对两者的灵敏性、特异性、稳定性及临床样本检验的适用性等进行评价。结果:所建立的实时定量PCR检测方法可用于HBoV的特异性检测;相对于传统PCR所达到的250拷贝/反应的检测灵敏度,实时定量PCR的检测灵敏度可高达10拷贝/反应,检测范围为109～101拷贝/反应,且具有良好的特异性和重复性;初步用于76份临床呼吸道标本检测,检出阳性5例,高于普通PCR方法(3/76)。结论:建立了HBoV TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,为开展HBoV流行病学监测及早期临床诊断提供了技术手段。

Development and Application of Real-Time PCR Assay for Detection of Human Bocavirus

Objective： To develop a specific,rapid,sensitive TaqMan based real-time quantitation PCR（Q-PCR） assay for detection and quantitation of human bocavirus（HBoV）.Methods： The specific primers and fluorescence-labeled probe to develop a real-time Q-PCR assay for detection of HBoV were designed according to the conser-vative gene sequence.Absolute viral copy was achieved through the standard curve.Subsequently,experiments were undertaken to assess diagnostic criteria such as specificity,sensitivity and reproducibility then compared with con-ventional PCR.Results： The analytical detection limit of this real-time Q-PCR assay was 10 DNA copies per re-action mixture and allowed quantitation of HBoV ranging from 109～101 copies per reaction mixture.Compared with conventional PCR analytical detection limit of 250 copies per reaction mixture,this method was more sensitive.A total of 76 nasopharyngeal swab specimens derived from Shenyang were screened for the presence of HBoV by us-ing real-time Q-PCR and identified 5 specimens positive for HBoV.The positive detection rage of HBoV by Q-PCR assays was higher than that by conventional PCR（3 / 76）,which were used to assess in parallel by the Q-PCR assay.Conclusion： A real-time QPCR assay for detection and quantitation of HBoV has been developed.The assay described was proved to be more sensitive than conventional PCR and showed a good reproducibility.This assay maybe applied for surveillance and clinical diagnosis of HBoV.