苹果树腐烂病菌qPCR检测方法的建立

摘要

Universal primers ITS, EF-1 alpha and beta tubulin genes were used to amplify the pathogen strain Vmm41. The PCR product sequences were compared with that of GenBank, and the specific primer pair VE1-F/VE1-R was selected. The target fragment was ligated to the vector pUC19, and used as standard sample. A real-time quantitative PCR (qPCR) detection system was established by optimizing the annealing temperature and primer concentration. The specificity, sensitivity and reliability of the system were verified. The results showed that the detection limit of the qPCR system reached 26.4 fg/μL. Fungal species commonly found in apple orchard such as Botryosphaeria dothidea, Alternaria spp. , Fusarium proliferatum, Trichoderma spp. , and Penicillium spp. were amplified with the qPCR system, and no PCR product could be found. The system was used to detect the apple leaves inoculated with V. mali. The results showed that the copy numbers of pathogen on leaves without lesion could be detected by the qPCR system. The qPCR system established in the present study can be used for rapid detection of V. mali,providing new method for the research on infection and expansion, and early warning of AVC.%利用ITS、β-微管蛋白和EF-1a基因序列的通用引物来扩增苹果树腐烂病菌菌株Vmm41,产物序列,与GenBank中的序列对比,设计并筛选出特异性引物对VE1-F/VE1-R.将目的片段连接到载体pUC19上制成标准品,通过优化退火温度和引物浓度,建立了实时定量PCR检测体系,并对该体系的特异性、灵敏度和可靠性进行了验证.结果表明,该体系可检测到26.4 fg/μL的模板浓度,并可从苹果轮纹病菌、链格孢菌、层出镰刀菌、木霉菌和青霉菌等苹果园常见真菌中特异性的检测出苹果树腐烂病菌.用该体系对人工接种腐烂病菌的苹果叶片进行检测验证,结果表明,利用该检测体系可检测出叶片未发病部分的腐烂病菌数量.本研究建立的定量PCR检测体系可用于苹果树腐烂病菌的快速检测,为苹果树腐烂病侵染规律及预警研究提供新方法.