实时荧光定量PCR定量检测山核桃干腐病病菌潜伏侵染量方法的建立

Quantifying Botryosphaeria dothidea infection causing canker disease on Carya cathayensis using real-time PCR

由茶麋子葡萄座腔菌Botryosphaeria dothidea引起的山核桃Carya cathayensis干腐病是山核桃栽培过程中的主要病害,该病菌表现明显的"潜伏侵染"特性。研究山核桃树体中干腐病菌的定量检测技术对山核桃干腐病的预测预报及科学防治具有重要的指导意义。根据茶麋子葡萄座腔菌的EF1α基因设计特异性引物,通过普通聚合酶链式反应(PCR)和实时荧光定量PCR(real-time PCR)扩增发现引物EFRT-F1/R1对山核桃干腐病病菌的特异性及扩增效率较高,可稳定扩增出230 bp的目标条带。应用此特异性引物建立的实时荧光定量PCR干腐病病菌检测方法能够定量检测出山核桃植株样品中的病菌含量,灵敏度要比普通PCR高100倍。

Canker disease caused by Botryosphaeria dothidea is the most imp ortant disease that threaten the production of Chinese hickory and has the significant characteristics of 'latent infection'. Therefore, the development of a quantitative detection technique of B. dothidea in hickory plants is the prerequisite for its forecasting and scientific management. In this study, specific primers were respectively designed based on the conserved regions of EF1α gene of B. dothiaea. Results of both real-time PCR and common PCR showed that the specificity and sensitivity of EFRT-F1/R1 was good primer pair. A band of 230 bp could be stably amplified by the primers EFRT-F1/R1. Further study displayed that this real-time PCR technique is more sensitive(more than 100 fold) than the common one. Our molecular detection technique will provide scientific basis for forecast, and management of hickory canker disease.