实时荧光PCR检测瓜炭疽病菌

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39.6pg的核酸量就可以被检测到,检测灵敏度达到1.584pg/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。

Detection of Colletotrichum orbiculare by the real-time PCR

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.