人博卡病毒实时荧光定量PCR检测方法的建立

Real-time fluorescent quantitative PCR assay for detection of human Bocavirus

目的建立人博卡病毒(human Bocavirus,HBoV)的实时荧光定量PCR(real-time fluorescent quanti-tative PCR,FQ-PCR)检测方法,检测呼吸道感染患儿标本的HBoV。方法设计HBoV NP1基因的引物和Taqman探针,扩增NP1基因片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品,建立FQ-PCR检测方法,进行敏感性、特异性试验,检测195份临床标本。结果所建立的FQ-PCR方法对临床其他呼吸道病毒不出现特异性扩增曲线,特异性好,线性范围为10～108copies/μl。195份临床标本中HBoV阳性12份,阳性率为6.2%。结论成功建立了HBoV实时荧光定量PCR检测方法并检测临床标本,为人博卡病毒感染的临床治疗提供快速、可靠的诊断依据。

Objective To establish the method for detecting human Bocavirus （ HBoV） by a real-time fluorescent quantitative PCR （ FQ-PCR） and to assay HBoV from nasopharyngeal samples of children with acute respiratory tract infections. Methods The specific primers and Taqman probes of targeted HBoV NP1 gene were designed. The aimed fragment of NP1 gene was amplified with PCR and ligated into a pGEM-T Easy vector to set up the standards. The method for detection of NP1 gene using FQ-PCR was established. The sensitivity and specificity of the method had been tested. Then 195 clinical specimens were subsequently tested. Results The specificity of this method was excellent. The linear amplification of the assay ranges from 10 copies / μl to 108 copies / μl. Twelve specimens were tested positive for HBoV in 195 clinical specimens （6.2%）. Conclusions The method for detection of HBoV NP1 gene by real-time fluorescent quantitative PCR was established successfully. It can offer fast and reliable diagnosis for HBoV infection.