G亚属猴腺病毒荧光定量PCR检测方法的建立与初步应用

Establishment of real-time fluorescence quantitative(FQ)-PCR for the detection of simian adenovirus and application

目的建立G亚属猴腺病毒(simian adenovirus,SAdV)特异的荧光定量PCR(FQ-PCR)检测方法,并研究实验猴群和猴源性生物制品中SAdV感染或污染情况。方法根据G亚属SAdV基因保守区,设计特异性引物和TaqMan探针。制备重组质粒pGEM-TEasy-SAdVpol标准品,建立标准曲线。经灵敏度、特异性、准确性和重复性评价后,检测33份猴粪便样本和4批次脊髓灰质炎疫苗。结果建立的FQ-PCR检测方法特异检测G亚属,与A、F亚属无交叉反应,准确性及重复性较好。标准曲线相关系数0.999,线性范围6lg(60～6×106拷贝),最低检出限可达6拷贝/μl。33份猴粪便样本中15份为阳性(45.5%),4批脊髓灰质炎疫苗均为阴性。结论建立的FQ-PCR检测方法可定量检测G亚属SAdV拷贝数,初步应用表明我国实验猴群中G亚属SAdV流行率较高,应加强监测避免人类感染的潜在风险。

Objective To establish real-time fluorescence quantitative（FQ）-PCR methods for the detection of simian adenovirus（SAdV）and apply this method to study the infectious status of SAdV in monkeys.Methods Primers and probe-primers were designed according to the SAdV sequence.Recombinant plasmid pGEM-TEasy-SadVpol was prepared as the reference to establish the standard curve.The sensitivity,specificity,accuracy and repeatability of the FQ-PCR were evaluated.Thirty-three monkey stool samples and 4 batches of polio vaccines were used to test this method.Results The established FQ-PCR method showed high specificity for the subgroup G of SAdV with sensitivity up to 6 copy/μl.R2 is 0.999.The linear detection scope ranges from 60 to 6×106 copies.Conclusions The established FQ-PCR can be used to quantitatively detect the copy number of subgroup G SAdV.Preliminary applications showed this subgroup of virus has a higher prevalence in Chinese experimental primates.