胡萝卜软腐欧文氏菌分子检测技术的研究

Molecular Detection of Erwinia carotovora subsp.carotovora,the Main Causal Agent of Bacterial Soft Rot of Crops

胡萝卜软腐欧文氏菌胡萝卜亚种Erwinia carotovorasub sp.carotovora(Ecc)是引起魔芋和马蹄莲软腐病的主要病原细菌.本研究根据细菌L-氨基酸渗透酶同源基因设计URP核苷酸序列特异性引物进行普通PCR、巢式PCR,同时采用细菌16S-23S rDNA间的ITS序列设计引物及TaqMan探针进行实时荧光PCR,对样品中存在的胡萝卜软腐欧文氏菌进行检测,并比较了3种检测方法的特异性和灵敏度.结果表明,应用实时荧光PCR方法检测样品,其中存在的胡萝卜软腐欧文氏菌的最低菌悬液浓度可达10 cfu/mL;巢式PCR灵敏度也可检测到10 cfu/mL;而用普通PCR最低可检测到103cfu/mL.用实时荧光PCR和巢式PCR方法检测病菌,其灵敏度都比常规PCR提高了100倍,且实时荧光PCR方法更快速、简便、安全、准确,不需PCR的后续处理.实时荧光PCR方法适用于种苗、种球等检测领域.

Erwina carotovora subsp, carotovora （ Ecc ） is the main bacterial pathogen leading to bacterial soft rot of Konjac and Zantedesehia. The specific primer sets and TaqMan probe were designed based on the DNA sequence of URP and 16S-23S rDNA ITS. Conventional PCR, Nested-PCR and real-time fluo- rescent PCR were used for detection of Ecc from cell suspension of pure culture and extracts of infected Konjac plant tissues. The results showed that real-time fluorescent PCR and Nested-PCR could achieve higher specific and sensitive detection of Ecc,with threshold 10 cfu/mL compared with conventional PCR （threshold 103 cfu/mL）. However, real-time fluorescent PCR was simple, fast, assure and more accurate. It was suggested that this method was adopted to rapid detection of Ecc for the seed and seedling industry.