超分支滚环扩增法检测小麦矮腥黑穗菌

 【目的】建立小麦矮腥黑穗病菌（Tilletia controversa Kühn,TCK）的超分支滚环扩增（hyper- branched rolling cycle amplification,HRCA）检测体系,为小麦矮腥黑穗病的鉴定以及早期诊断提供了一种新的稳定、可靠的检测技术。【方法】锁式探针包含一个公共连接序列和在探针两端与靶DNA序列互补的2个序列。根据TCK的差异序列设计锁式探针两端序列,以此为基础建立了TCK超分支滚环扩增反应体系。以优化的HRCA反应条件为基础,确定检测体系的特异性和灵敏度。比较HRCA体系和常规PCR体系的性能,并利用这2种体系对来自中国出入检验检疫局截获的51个小麦样品进行了验证检测。【结果】HRCA体系能专一地检出小麦矮腥黑穗菌的DNA靶带,而TCT、TCL等近源种及健康小麦样品都不能扩增。HRCA检测TCK靶序列质粒DNA的下限为1 fg?μl-1,检测基因组DNA的下限为10 pg?μl-1,检测灵敏度比常规PCR检测体系高10倍。HRCA检测体系具有很好的特异性、灵敏度和准确性,更适合于TCK的检测及鉴定。【结论】稳定、灵敏、特异的小麦矮腥黑穗菌的HRCA检测体系的建立,为小麦矮腥黑穗病的早期诊断及其近源种的多靶标检测提供了同步检测技术。

Detection of Tilletia controversa with H RCA Approach

【Objective】 To establish a detection system of Tilletia controversa (TCK) with hyper-branched rolling cycle amplification (HRCA) method, which provides a stable , reliable and novel technique for early diagnosis of wheat dwarf bunt disease and identification of pathogen. 【Method】 The padlock probe consists of a universal linking sequence and the two target complementary regions at 5′and 3′ends, which was designed based on the unique fragment sequence of 1 322 bp of TCK. Detection system of HRCA was established and optimized. The specificity and limitation of HRCA was determined and compared with conventional PCR. A total of 51 samples intercepted at Customs or collected from different wheat cultivation areas in USA and China were examined with HRCA. 【Result】 HRCA is capable of amplifying mycelial and teliosporal DNA of TCK , while not detecting related smut species of Tilletia and other plant pathogen. The detection sensitivity of HRCA is as low as 1 fg?μl-1 for plasmid DNA and 10 pg?μl-1 for genomic DNA of TCK which is 10-fold higher than that of conventional PCR. The results show HRCA method to be sensitive, specific and accurate. 【Conclusion】 The detection system of HRCA for TCK was successfully established, which provided a new approach for the simultaneous diagnosis of wheat dwarf bunt disease and identification of TCK pathogen.