建立设有内对照的Real-time PCR方法检测人血清中细小病毒B19  

Establishment of internally controlled Real-time PCR for the detection of human parvovirus B19 DNA in serum

作　　者：李萌[1] 邹小辉 王敏 于修平[1] 鲁茁壮 洪涛 

机构地区：[1]山东大学医学院,济南250012 [2]中国疾控中心病毒病预防控制所

出　　处：《中华实验和临床病毒学杂志》2010年第6期479-481,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家科技支撑计划（No.2008BAI56B01）

摘　　要：目的 建立设有内对照的TaqMan探针Real-time PCR方法 ,用于检测人血清中细小病毒B19.方法 人工合成2段DNA序列,克隆到T载体,线性化后进行DNA定量,分别作为模板标准品和内对照;合成1对引物和不同荧光素标记的2条探针,2条探针分别能与阳性模板或内对照结合,建立设有内对照的B19 Real-time PCR检测方法 .对方法 的稳定性进行了评价,并应用于人血清B19病毒检测.结果 获得了B19检测标准DNA和内对照DNA,确定了内对照的添加量,建立了使用内对照的Real-time PCR检测方法 .对160份血清标本进行了检测,检出2份阳性标本,病毒DNA浓度分别为2.1 × 105Geq/ml和3.6×103Geq/ml.结论 成功建立了人细小病毒B19 Real-time PCR检测方法 ,可用于人血清B19检测;由于使用了内对照,该方法 有利于排除假阴性检测结果 .Objective To establish a TaqMan-based Real-time PGR assay with internal control for the detection of parvovirus B19 DNA in human serum. Methods Two DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control,respectively. One pair of primers and two probes were included in the Real-time PGR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19DNA. Results The standard curve was constructed using the quantified standard B19 DNA. The Real-time PGR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus （HBV）. Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2. 1 × 105 Geq/ml and 3.6 × 103 Geq/ml,respectively. Conclusion The Real-time PGR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.

关 键 词：聚合酶链反应 细小病毒B19 人 血清学试验 内对照 

分 类 号：R4[医药卫生—临床医学] R73