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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。
Random amplified polymorphic DNA (RAPD) primer-mediated semi-specific PCR (RAPD primer mediated hemi-specific PCR, RM-PCR) was used in 18 wheat genitalia collected from different regions The total genomic DNA of Tilletia controversa Kühn (TCK) and 29 strains of Tilletia caries (DC) Tul, TCT were screened and identified as 1322 bp differential genomic fragments unique to TCK. Two pairs of specific primers CQUTCK2 / CQUTCK3 and CQUTCK4 / CQUTCK5 were designed and screened according to the sequence of the fragment. Stable single-stranded DNA of 747 bp and 200 bp was stably amplified from mycelia and teliospore DNA of 18 TCK strains, However, no amplification products were found in mycelia or teliospore DNA of 29 TCT strains. Taking the common primers of Corynebacterium spp. To CQUK6 / CQUK7 as a built-in control, it is possible to determine whether the sample to be tested contains PCR inhibitors and determine whether the detection system is correct or not, and to effectively eliminate the false positives and false negatives of the test results. The establishment of TCK specific PCR detection system, to achieve a simple and rapid identification of wheat dwarf typhim winter infected spores or diseased tissue mycelium purposes.