应用TaqMan MGB探针检测小麦印度腥黑穗病菌和黑麦草腥黑穗病菌

 以小麦印度腥黑穗病菌9个菌株和黑麦草腥黑穗病菌5个菌株及其近似种或相关种:稻粒黑粉菌、狼尾草腥黑粉菌、狗尾草腥黑粉菌、苏玛特腥黑粉菌、狐尾草腥黑粉菌、小麦网腥黑穗病菌和小麦矮化腥黑穗病菌共9种22个菌株为研究对象,通过序列比对分析,设计了检测小麦印度腥黑穗病菌及黑麦草腥黑穗病菌的TaqMan MGB实时荧光PCR引物和探针,优化了反应条件,筛选出特异性探针,分别建立了小麦印度腥黑穗病菌和黑麦草腥黑穗病菌实时荧光单重PCR和实时荧光双重PCR检测方法,其中实时荧光双重PCR检测方法实现了在同一PCR管中仅用5μL的反应体系,进行1次PCR反应就能特异性检测出小麦印度腥黑穗病菌或黑麦草腥黑穗病菌。本研究所建立的检测方法特异性强、结果可靠、检测速度快、成本明显降低,在文际应用中具有推广价值。

Detection of Tilletia indica and Tilletia walkeri by 5'-nuclease assays using TaqMan MGB probe

The 9 species including 9 strains of T. indica,5 strains of T. walkeri and their similar or related species, Tilletia horrida, Tilletia barclayana, Tilletia setariae, TiUetia sumatii, Tilletia fusca, Tilletia tritici and Tilletia controversa were studied. The primers and TaqMan MGB probes were designed for detection of T. indica and T. walked based on nucleotide differences within the target fragment sequences. The fluorescent monoplex PCR and doubleplex PCR were respectively developed. By the fluorescent doubleplex PCR, T. indica or T. walkeri was specifically detected simultaneously through one PCR reaction with 5 μL reaction volume per tube. These two methods, with the cost being obviously lower, are rather specific, reliable, rapid and suitable for application in routine quarantine.