小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。

Selection of differential DNA fragment of Tilletia controversa Kühn and establishment of molecular detection approach

A reliable and simple polymerase chain reaction method for TCK pathogen was established firstly. A 1322bp unique fragment of TCK was amplified and identified by the technique of semi-specific random amplified polymorphism (RM-PCR). Two pairs of species-specific primers CQUK_2/ CQUK_3 and CQUK_4/ CQUK_5 were designed according to the unique fragment of TCK. The first pair primers were capable to stably amplify target DNA band of 747bp from chromosomal DNA of 18 strains of TCK isolates without any DNA bands obtained from 29 strains of TCT. The second pair primers could produce a 200bp target DNA band stably, while no band was amplified from teliospore or mycelium DNA of TCT of strains. Tilletia genus primers were used as internal control of molecular detection system, which can detect whether the PCR inhibitors exist in testing sample or avoid pseudo-negative and pseudo-positive of PCR reaction. The molecular detection approach could rapidly, accurately detect and identify the DNA of teliospore or mycelium of TCK from wheat tissues.