超分支滚环扩增法检测传染性脾肾坏死病毒  

Detection of Infectious Spleen and Kidney Necrosis Virus(ISKNV) by HRCA Approach

作　　者：孔铖将[1] 史雨红[1] 黎昊雁 陈炯[1] 

机构地区：[1]宁波大学生命科学与生物工程学院,浙江宁波315211 [2]浙江出入境检验检疫局微生物实验室,浙江杭州310012

出　　处：《水产科学》2011年第9期575-579,共5页Fisheries Science

基　　金：长江学者和创新团队发展计划(IRT0734);浙江省科技厅重点项目(2008C22049)

摘　　要：用超分支滚环扩增法检出传染性脾肾坏死病毒。超分支滚环扩增法包括锁式探针的连接及超分支滚环扩增法扩增两部分,通过研究锁式探针的最佳连接体系和连接条件,并进行超分支滚环扩增法反应条件的优化,得出的本研究最适反应条件为:锁式探针在T4 DNA连接酶作用下37℃连接20 min,接下来的超分支滚环扩增法在Bst DNA聚合酶大片段作用下61℃反应40 min,即能够实现靶序列的有效检出。灵敏度试验表明,超分支滚环扩增法所能检测到的最低模板量接近1 copy。在4种病毒中进行的特异性试验表明,本方法能够保证对传染性脾肾坏死病的特异性检测。In this paper,a 60-min hyper-branched rolling circle amplification（HRCA） method was established for rapidly and simply visual detection of infectious spleen and kidney necrosis virus（ISKNV） as an important viral pathogen in aquaculture.The padlock probe consists of a universal linking sequence and two target complementary regions at 5＇ and 3＇ ends,which was designed based on the DPOL sequence of ISKNV.Detection system of HRCA was established and optimized with the ISKNV detection limit of 1 copy.The results showed that the HRCA method had advantages of high sensitivity,and speed and equipment-independent outcompeting the standard PCR-AGE for specifically detecting the low levels of ISKNV DNA,which can be useful in field as a routine diagnostic tool.

关 键 词：传染性脾肾坏死病毒 DNA聚合酶 超分支滚环扩增 锁式探针 

分 类 号：S917.1[农业科学—水产科学]