实时荧光 PCR 检测瓜炭疽病菌

摘要

采用实时荧光 PCR 技术建立了瓜炭疽病菌（Colletotrichum orbiculare ）的检测方法。根据瓜炭疽病菌甘油醛-3-磷酸脱氢酶（GAPDH）基因和谷氨酰胺合成酶（GS）基因序列，设计了该病菌特异性引物和TaqMan 探针，并对所设计的引物和探针的反应条件进行了优化。采用本试验建立的实时荧光 PCR 方法对瓜上的其他菌株及近似菌株进行检测，可将瓜炭疽病菌与其他病原菌区分开。灵敏度试验表明，25μL 体系中只要有39．6 pg 的核酸量就可以被检测到，检测灵敏度达到1．584 pg／μL，比普通 PCR 检测灵敏度高100倍。同时对田间采集的病株和未知样品进行的检测证明了引物和TaqMan 探针的特异性。%The detection of Colletotrichum orbiculare by real-time PCR was established.The specific primers and Taq Man probes were designed according to glyceraldehyde-3-phosphate dehydrogenase(GAPDH )gene and gluta-mine synthase(GS )gene sequences in C .orbiculare ,and the reaction conditions were optimized.C .orbiculare could be detected from other strains in melon and other similar strains by real-time PCR.The sensitivity test indi-cated that 39.6 pg DNA could be detected in 25 μL reaction system.The real-time PCR sensitivity could reach to 1.584 pg/μL,100 times more sensitive than ordinary PCR.The primers and Taqman probe were shown to be spe-cific by detecting infected plants sampled in the field and unknown samples.