双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌

摘要

为建立同步检测检疫性细菌洋葱腐烂病菌和菊基腐病菌的双重PCR方法,根据GenBank上公布的洋葱腐烂病菌ITS序列设计1对特异性引物B3/B6,将设计的引物与已发表的检测菊基腐病菌特异性引物ADE1/ADE2结合,经过条件优化,建立了双重PCR反应体系,并进行特异性和灵敏度验证.应用双重PCR体系能从洋葱腐烂病菌和菊基腐病菌基因组DNA以及人工带菌样品中扩增到特异性条带.结果表明,建立的双重PCR检测方法能同时检测出洋葱腐烂病菌和菊基腐病菌,可应用于口岸进口郁金香种球2种检疫性细菌的检测.%The objective of this study is to establish a duplex PCR technology for simultaneously detecting Burkholde-ria gladioli pv. alliicola and Erwinia chrysanthemi. Based on the 16S-23S rRNA intergenic transcribed spacer se-quence of B. gladioli pv. alliicola in GenBank,the primers B3/B6 were designed. The duplex PCR assay had been developed using the combining primers B3/B6 and ADE1/ADE2 which were specific primers to detect E. chrysanthe-mi. The reaction conditions were optimized and the specificity and sensitivity of the duplex PCR were tested. The ex-pected DNA fragment could be specifically amplified from the genomic DNA of B. gladioli pv. alliicola and E. chry-santhemi. Specificity was confirmed using the duplex PCR assay to detect B. gladioli pv. alliicola and E. chrysanthe-mi in the artificially inoculated tulip leaf samples. The duplex PCR developed in this study can be used in detecting the two pathogens for imported tulip bulbs quarantine.