洋葱腐烂病菌PCR检测方法的建立

Development of PCR detection for Burkholderia gladioli pv.allicola

为了快速准确检测洋葱腐烂病菌(Burkholderia gladioli pv. alliicola,简称Bga),根据GenBank中Bga与相关种的序列差异设计特异引物BG5/BG7和探针Bga-P,建立了Bga常规PCR和荧光PCR检测方法。测试结果表明,引物和探针对供试的11株Bga菌株表现为阳性反应,其他64株供试菌株和空白对照均为阴性。常规PCR和荧光PCR方法的检测灵敏度分别为24 pg和240 fg菌体DNA。美国、法国、意大利等国进境的80批次洋葱种子样品的检测结果显示,这两种方法的阳性检出率分别为7.5%和12.5%。选取阳性样品进行病菌分离,成功从2批次法国进境洋葱种子中分离到目的菌。本文建立的洋葱腐烂病菌PCR检测方法可为口岸检测部门提供更高效、灵敏和特异的检测手段。

In order to rapidly and accurately detect Burkholderia gladioli pv.allicola(Bga),the specific primer BG5/BG7 and probe Bga-P were designed according to the sequence difference between Bga and related strains in GenBank.The detection methods of Bga by conventional PCR and fluorescent PCR were established.The conventional PCR with primer BG5/BG7 and the real time PCR with probe Bga-P could amplify 11 strains of Bga,but no target band or positive amplification for other 64 strains of relat-ed species.These two methods could detect as low as 24 pg and 240 fg cell DNA of Bga,respectively.80 batches of onion seed samples imported from the United States,France and Italy showed positive de-tection rates of 7.5%and 12.5%for these two methods,respectively.Positive samples were selected for pathogen isolation,and the Bga were successfully isolated from 2 batches of onion seeds imported from France.These PCR detection method for Burkholderia gladioli pv.allicola can provide a more efficient,specific and sensitive detection method for port inspection departments.