猫疱疹病毒I型环介导等温扩增检测方法的建立-The Establishment of a ...

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英文关键词 : loop-mediated isothermal amplification feline herpes virus I SYBR Green I thymidine kinase (TK) gene detection method 基金项目 : 国家科技支撑计划项目 (2015BAI07B02) ；吉林省科技发展计划项目 (20170623093-09TC)

作者	单位
冀中豪	吉林大学动物科学学院，吉林长春 130062
任文陟	吉林大学动物科学学院，吉林长春 130062
郝杨	吉林大学动物科学学院，吉林长春 130062
张铭锦	吉林大学动物科学学院，吉林长春 130062
高巍	吉林大学动物科学学院，吉林长春 130062

摘要点击次数 : 733 全文下载次数 : 48 中文摘要 : 建立一种环介导等温扩增（LAMP）检测方法，实现对猫疱疹病毒Ⅰ型（FHV-1）的快速、准确、简便检测。根据GenBank中FHV-1的TK基因设计引物，优化反应条件，通过琼脂糖凝胶电泳和SYBR GreenⅠ染色观察分析扩增效果。该方法特异性好，敏感性检测实验表明该检测方法最低能够检测到的模版是10 ¹ copies/μL，是PCR检测方法灵敏度的10倍。金属浴、烘箱、恒温水浴锅与PCR仪四种不同的扩增反应仪器均可达到LAMP的要求，扩增出梯形条带。建立了针对FHV-1的LAMP检测方法，该种检测方法具有高特异性的扩增引物，对检测的仪器、反应条件及检测人员技术要求比较宽松，从扩增开始到通过SYBR GreenⅠ实现的结果可视化解读所用时间不到1 h，实现了快速、准确、简便检测FHV 1的目的。 英文摘要 : A detection method using loop mediated isothermal amplification (LAMP) for fast, accurate, and convenient detection of feline herpes virus I (FHV-1) was established. According to TK gene of FHV-I in Genebank primers were designed, optimized the reaction conditions the amplification effects were observed and analyzed through agarose gel electrophoresis and SYBR Green staining. This method had fine specificity, the sensitivity detection experiment suggested that the detection method could detect the template at the minimum 10 ¹ copies/μL, being 10 times sensitivity of PCR detection method. Four different amplification reaction instruments metal bath, oven, constant temperature water bath, and PCR instrument all could meet the requirement of LAMP, and showed amplification trapezoid bands. The establishment of LAMP detection for FHV-I, with its amplification primers of high specificity, the technical requirements for detection instruments, reaction conditions, as well as personnel was fairly ample, the time used from the starting of the amplification through the results of the realization of SYBR Green I to visibly unscramble was less than one hour, it really realized the goal of the detection of FHV-I with rapid, accurate, and simple. 查看/发表评论下载PDF阅读器