水稻细菌性谷枯病菌的实时荧光PCR检测技术研究

Real-Time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

水稻细菌性谷枯病菌Burkholderia glumae于2007年被列为我国进境植物检疫性有害生物,国内急需建立针对该菌切实可行的检测技术,以有效控制它在我国的传播。采用实时荧光PCR(real-time fluorescence PCR)和经典PCR技术进行水稻细菌性谷枯病菌检测。研究结果表明,供试所有谷枯病菌都能产生139bp左右的特异性片段,非谷枯菌株均无特异性片段产生。两种检测方法的灵敏度比较发现,常规PCR技术在病菌浓度为104CFU/mL时即可检测到,实时荧光PCR技术在病菌浓度为102CFU/mL时即可检测到,后者比前者的灵敏度高100倍。将模拟带菌种子与灭菌种子按1:100混合,实时荧光PCR技术可以检测到该菌的存在。

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effeetiye detection methods for the pathogen in order to prevent further spread of this disease. The real-time PCR method was compared with classical PCR to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR method, while others showed negative PCR result. Comparing the sensitivity of these two detecting methods, about 104 CFU/mL of the bacterial suspension were detected by general PCR and below 102 CFU/mL could only be detected by the real-time fluorescence PCR method. B. glumae can be detected when the healthy seeds and inoculated seeds are mixed with a proportion of 1 ： 100.