实时荧光定量PCR鉴定小麦矮腥黑穗菌技术研究

 【目的】建立荧光定量PCR体系以准确灵敏的鉴定小麦黑穗菌(Tilletia controversa Kühn,TCK)【方法】根据筛选的TCK独有差异基因片段（1 322 bp）设计特异性引物对CQUTCK4/CQUTCK5和TaqMan 探针CQUP1,建立SYBR GreenⅠ荧光染料法和TaqMan水解探针法定量PCR检测体系,并对体系进行优化。【结果】建立的两套定量PCR检测体系的检测下限相当,可达到0.1fg,对应的拷贝数为2.31×104个,检测灵敏度比常规PCR高2～3个数量级,均可成功鉴别出TCK与小麦网腥黑穗病菌(Tilletia caries (DC)Tul,TCT),并可快速准确检测小麦矮腥黑穗菌冬孢子和检测罹病小麦植株体内的侵染菌丝体。【结论】建立的2种定量PCR检测技术可运用于小麦矮腥黑穗病的早期诊断。

Detection of Tilletia controversa Kuhn by Real Time Quantitative PCR

Objective] To detection of Tilletia controversa Kühn (TCK) sensitively and accurately,real-time PCR systems were developed.Method]The species-specific primer pair CQUTCK_4/CQUTCK_5 and probe CQUP_1 were designed based on a selected specific fragment (Ⅰ322 bp) specific for TCK,and the SYBR Green Ⅰ and TaqMan quantitative PCR detection systems were established with optimized reaction conditions.Result]The detection limit of the two systems were 0.1fg,equal to 2.31×10~4 copies,which was 10~2-10~3 fold higher than conventional PCR.By the constructed detection systems,the TCK and Tilletia caries (DC)Tul (TCT) could be distinguished.The teliospore and mycelium of TCK in the infected wheat plant tissue also could be identified accurately and rapidly.Conclusion]The earlier diagnosis approaches of wheat durwf bunt pathogen were set up using the twe real-time PCR systems.