白斑综合症病毒超分支滚环检测试纸的构建及应用

白斑综合症病毒超分支滚环检测试纸的构建及应用 Establishment and Application of the Hyper-Branched Rolling Circle Amplification Test Strip for White Spot Syndrome Virus 赵玉然 ZHAO Yuran 1 first-author 尹伟力 YIN Weili 2 岳志芹 YUE Zhiqin 3 李八方 LI Bafang 1 中国海洋大学食品科学与工程学院 中国海洋大学食品科学与工程学院 College of Food Science and Engineering,Ocean University of China College of Food Science and Engineering,Ocean University of China 烟台出入境检验检疫局 烟台出入境检验检疫局 Yantai Entry-Exit Inspection and Quarantine Bureau Yantai Entry-Exit Inspection and Quarantine Bureau 山东出入境检验检疫局技术中心 山东出入境检验检疫局技术中心 Technical Centre of Shandong Entry-Exit Inspection and Quarantine Bureau Technical Centre of Shandong Entry-Exit Inspection and Quarantine Bureau 依据白斑综合症病毒（white spot syndrome virus,WSSV）的囊膜蛋白VP28基因核苷酸序列设计特异的锁式探针,建立了WSSV超分支滚环扩增检测方法并构建试纸条.结果表明,WSSV超分支滚环扩增锁式探针在T4DNA连接酶作用下37℃连接30min,而后在Bst DNA聚合酶大片段作用下61℃反应50min,即能够实现病毒靶序列的有效检出.灵敏度实验表明,WSSV超分支滚环扩增试纸最低检出限为10拷贝/μL,是常规PCR（聚合酶链式反应）法灵敏度的100倍.特异性实验表明,该方法能够保证对WSSV的特异性检测.利用该试纸检测68份虾样本,与PCR方法检测结果基本一致,但更为灵敏和直观. Based on the capsid protein gene VP28of white spot syndrome virus（WSSV）,the padlock probe was designed,and the hyper-branched rolling circle amplification（HRCA）system and the test strip for WSSV was established.The result indicates that padlock probes are linked with the target sequence by the T4DNA ligase at 37℃for 30min,and then reacted by the Bst DNA polymerase large fragments at 61℃for 50min,then the target sequence can be effectively detected.Sensitivity test shows that the detection limit of the present HRCA test strip is close to 10 copies/μL,and 100times higher than the conventional polymerase chain reaction（PCR）.The specificity test shows that this method can ensure the specificity of WSSV.In practical applications,68patches of shrimps were collected to detect WSSV by the present test strip,and the results was basically identical with PCR but more sensitive and intuitive. 锁式探针 对虾白斑综合症病毒 超分支滚环扩增 检测试纸 padlock probe white spot syndrome virus hyper-branched rolling circle amplification test strip 10.14188/j.1671-8836.2014.01.009 S945.4 国家质量监督检验检疫总局科研项目（2012IK018）;国家质量监督检验检疫总局公益性项目（201210055） 2014-01-01 2021-04-01 1 60