多堆柄锈菌潜育期侵染量实时荧光定量PCR检测体系的建立

Establishment of quantitative real-time PCR detection system for latent infection of southern corn rust pathogen Puccinia polysora

为快速及时诊断并定量监测玉米内多堆柄锈菌Puccinia polysora潜育期的侵染量,根据多堆柄锈菌的ITS序列和玉米Actin2基因序列,分别设计多堆柄锈菌特异性引物PpoF/PpoR和TaqMan探针PpoP以及玉米特异性引物ZmF/ZmR和TaqMan探针ZmP,对引物和探针的特异性和灵敏度进行测定,并用这2对引物和探针建立多堆柄锈菌潜育期的实时荧光定量PCR检测体系,定量监测接种后玉米叶片中多堆柄锈菌DNA量随时间的变化。结果表明,多堆柄锈菌和玉米的特异性引物和探针对各自靶标片段均具有良好的特异性,灵敏度分别为10^(-3) ng/μL和10^(-2) ng/μL;在接种后24 h,利用所建实时荧光定量PCR检测体系即可在玉米叶片中检测到多堆柄锈菌,且潜育期内多堆柄锈菌的侵染量随时间呈指数增长。表明所建实时荧光定量PCR检测体系可用于多堆柄锈菌潜育期侵染量的定量检测和监测。

To rapidly and timely diagnose and quantify southern corn rust pathogen Puccinia polysora in infected leaves during latent period,P.polysora-specific primer pair PpoF/PpoR and TaqMan probe PpoP,Z.mays-specific primer pair ZmF/ZmR and TaqMan probe ZmP were designed respectively according to the ITS sequence of P.polysora and the Actin2 gene sequence of corn.Then specificity and sensitivity of the designed primer pairs were confirmed.A quantitative real-time PCR(qPCR)detection system was established to detect the DNA content of P.polysora in infected corn leaves during latent period and the DNA content of corn leaves as a reference.The dynamics of P.polysora in corn leaves during the latent period were monitored by using the qPCR assay.The results showed that the primers had high specificity and sensitivity.The sensitivities for PpoF/PpoR and ZmF/ZmR were 10^(-3) ng/μL and 10^(-2) ng/μL,respectively.Puccinia polysora could be detected as early as 24 h after inoculation and its content increased exponentially before the symptom appeared.The results indicated that the qPCR detection system could be used to quantitatively detect and monitor the latent infection of P.polysora.