【摘要】 为了建立禽呼肠孤病毒(Avian reovirus, ARV)微滴数字PCR(droplet digital PCR,ddPCR)的检测方法，试验根据ARV S1133毒株S1基因序列(GenBank登录号为L39002.1)的保守区设计特异性标准品引物BQ-F/BQ-R,检测引物ARV-F/ARV-R及特异性探针(ARV-Probe);以ARV S1133毒株反转录的cDNA为模板，使用引物BQ-F/BQ-R进行PCR扩增，构建标准品质粒pMD18-T-ARV;以标准品质粒pMD18-T-ARV为模板，使用引物ARV-F/ARV-R及特异性探针进行ddPCR试验，优化引物、探针反应浓度及退火温度，并考察ddPCR检测方法的敏感性、特异性及重复性。结果表明：筛选得到引物和探针的最佳反应浓度分别为1 000 nmol/L和250 nmol/L,最佳退火温度为57.1℃。建立的ARV ddPCR最低能检测到的模板浓度为8 copies/μL;同时检测禽流感病毒(AIV)、新城疫病毒(NDV)、鸡细小病毒(ChPV)、鸡传染性贫血病毒(CIAV)、禽腺病毒(FAdV)及ARV,仅ARV为阳性，其他均为阴性；重复检测试验样品及临床样品3次，变异系数均小于15%。说明ddPCR可用于ARV的临床检测中。更多还原

【Abstract】 In order to establish droplet digital PCR(ddPCR) detection method for Avian reovirus(ARV), in this experiment, specific standard primers BQ-F/BQ-R, detection primers ARV-F/ARV-R and specific probe(ARV-Probe) were designed according to the conservative region of the S1 gene sequence(GenBank registration number L39002.1) of the ARV S1133 strain. With the use of the reverse transcription cDNA of ARV S1133 strain as a template, PCR amplification was performed by using the primers BQ-F/BQ-R to construct the standard plasmid pMD18-T-ARV. With the use of the standard plasmid pMD18-T-ARV as the template, the ddPCR test was performed by using the primers ARV-F/ARV-R, and specific probe to optimize the primer, probe reaction concentrations and annealing temperature, and the sensitivity, specificity and repeatability of the ddPCR detection method were investigated. The results showed that the optimal reaction concentrations of primers and probes were 1 000 nmol/L and 250 nmol/L, respectively, and the optimal annealing temperature was 57.1 ℃. The minimum detectable template concentration of established ARV ddPCR was 8 copies/μL; avian Influenza virus(AIV), Newcastle disease virus(NDV), Chicken parvovirus(ChPV), Chicken anemia virus(CAV), Fowl adenovirus(FAdV) and ARV were simultaneously detected; only ARV was positive, and all others were negative. The test samples and clinical samples were repeated 3 times, and the coefficient of variation was less than 15%. The results suggested that ddPCR could be used in the clinical detection of ARV.更多还原