玉米细菌性枯萎病菌的环介导恒温扩增(LAMP)检测方法

为了提高口岸和基层实验室检疫和监测玉米细菌性枯萎病菌的准确性和工作效率,利用环介导恒温扩增技术(LAMP),根据内切葡聚糖酶(EGase)基因前导序列,设计2个内引物和2个外引物,对玉米细菌性枯萎病菌进行快速检测。结果表明,使用玉米细菌性枯萎病菌的近缘种或引致相似症状的病原菌菊欧文氏菌玉米致病变种Erwinia chrysanthemi pv.zeae、玉米内州萎蔫病菌Clavibacter michiganensis subsp.nebraskensis、燕麦假单胞菌Pseudomonas avenae、杓兰欧文氏菌Erwinia cypripedii检测其特异性,仅玉米细菌性枯萎病菌有扩增。LAMP检测灵敏度达到2 pg DNA,为普通PCR的100倍;与其它检测方法相比,LAMP方法检测时间短,效率高,不仅降低了设备投入,易于操作,而且具有较高的灵敏度和特异性,适合玉米细菌性枯萎病菌的现场检疫和大规模检测。

Loop-mediated isothermal amplification (LAMP) for detection of Pantoea stewartii subsp. stewartii

In order to improve the accuracy and efficiency of quarantine and monitoring of Pantoea stewartii subsp. stewartii for the port and the basic-level test labs, and establish a rapid detection method, the loop mediated isothermal amplification (LAMP) technology was applied in the laboratory. Based on the endoglucanase (EGase) gene leading sequence, two inner primers and two outer primers were designed through a simple LAMP to detect P. stewartii subsp. stewartii. The results showed that the LAMP primers did not react with suspensions of Erwinia chrysanthemi pv. zeae, Clavibacter michiganensis subsp. nebraskensis, Pseudomonas avenae, and Erwinia cypripedii. The LAMP detection system of P. stewartii subsp. stewartii was designed successfully with the lowest detection limit of 2 pg DNA, 100 times higher than ordinary PCR technology. Compared with other detection methods, the LAMP method for detection of P. stewartii subsp. stewartii in this study was rapid, with higher efficiency and lower equipment investment. The results indicated that LAMP method is easy to conduct, and has high sensitivity and specificity, indicating that it is suitable for the on-site quarantine and large-scale monitoring of P. stewartii subsp. stewartii.