洋葱腐烂病菌荧光定量PCR检测体系的建立

摘要

唐菖蒲伯克霍尔德菌洋葱致病型(Burkholderia gladioli pv.alliicola),是重要的植物病原细菌检疫性有害生物.本研究的目的是建立特异、灵敏、快速的TaqMan探针荧光定量PCR方法用于检测洋葱腐烂病菌(B.gladioli pv.alliicola).利用洋葱腐烂病菌保守基因序列设计和筛选特异性引物和TaqMan探针,优化PCR反应体系和反应条件.荧光定量PCR菌液检测灵敏度达到1.02×102 CFU/mL,DNA检测灵敏度达到1.73×10-4 ng/μL,反应时间仅需1h左右,对14种伯克氏菌属Burkholderia)参比菌种的特异性检测结果显示,洋葱腐烂病菌具有明显的扩增曲线生长,表明建立的荧光定量PCR体系具有非常高的特异性.同时对洋葱(Allium cepa)种子进行了模拟带菌检测,结果表明,建立的洋葱腐烂病菌荧光定量PCR检测体系能够检出模拟样品中的洋葱腐烂病菌,验证了检测体系的实用性.本研究建立的洋葱腐烂病菌荧光定量PCR检测体系能够有效用于洋葱腐烂病菌的鉴定,为进出口口岸和基层检验检疫部门提供了一种简便、快速、准确的洋葱腐烂病菌分子检测手段.%Burkholderia gladioli pv. alliicola is an important plant pathogen. This research was to establish the specific, sensitive and fast Taqman Real-time PCR for detection of B. gladioli pv. alliicola. The specific genome region of B. gladioli pv. alliicola was used for the design of Taqman probe and specific primers. The reaction conditions was optimized. Fourteen reference strains were used for the specificity test, the sensitivity of the Real-time PCR was tested as well. The sensitivity of bacterial suspension of Real-time PCR reached 1.02×102 CFU/mL and the sensitivity of DNA reached 1.73×104 ng/μL. Total reaction time took about 1 h or so. Among 14 reference strains belong to the genus of Burkholderia, Real-time PCR showed an extremely high specificity. The simulation test was performed on the onion (Allium cepa) seeds mixed with bacterial suspension of B. gladioli pv. alliicola, the results revealed that the Real-time PCR was practical in the test of B. gladioli pv. alliicola. The Real-time PCR established in this research is useful for the diagnostics of B. gladiolirnpv. alliicola. It provides an easy, fast and accurate method for molecular identification of B. gladioli pv.rnalliicola.