双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌

Duplex PCR detection of Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi

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Gen Bank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 Burkholderia gladioli pv.alliicola and Erwinia chrysanthemi.Based on the 16S-23S rRNA intergenic transcribed spacer sequence 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.chrysanthemi.The reaction conditions were optimized and the specificity and sensitivity of the duplex PCR were tested.The expected DNA fragment could be specifically amplified from the genomic DNA of B.gladioli pv.alliicola and E.chrysanthemi.Specificity was confirmed using the duplex PCR assay to detect B.gladioli pv.alliicola and E.chrysanthemi 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.