利用环介导等温扩增方法快速检测瓜类细菌性果斑病菌和番茄细菌性溃疡病菌

Rapid detection of Acidovorax avenae subsp. citrulli and Clavibacter michiganensis subsp. michiganensis with loop-mediated isothermal amplification

为建立简单、快速和灵敏地检测瓜类细菌性果斑病菌Acidovorax avenae subsp. citrulli(Aac)和番茄细菌性溃疡病菌Clavibacter michiganensis subsp. michiganensis(Cmm)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,以Aac的ugpB基因和Cmm的micA基因为靶标,分别设计、合成和筛选特异性引物,摸索和优化各项反应条件和反应体系,成功建立了以钙黄绿素颜色为指示且只需要金属浴恒温反应30～60 min的LAMP扩增体系。特异性分析表明该LAMP方法可以快速检出5株不同的Aac菌株和2株不同的Cmm菌株,其它对照菌株如燕麦嗜酸菌燕麦亚种A. avenae subsp. avenae、丁香假单胞菌Pseudomonas syringae、水稻黄单胞菌水稻致病变种Xanthomonas oryzae pv. oryzae和茄科雷尔氏菌Ralstonia solanacearum则呈现阴性反应;引物比目前报道的LAMP引物有更高的DNA样品检测灵敏度,Aac和Cmm的灵敏度分别为1.72×10~2fg/μL和1.26×10~2fg/μL。研究结果表明,所设计的Aac和Cmm引物特异性好、灵敏度高,更有利于从源头上控制这2种检疫性细菌病害的流行和传播。

To establish a simple,sensitive and rapid loop-mediated isothermal amplification (LAMP) method for the detection ofAcidovorax avenae subsp,citrulli (Aac)causing melon bacterial fruit blotch and Clavibacter michiganensis subsp,michiganensis (Cmm)causing tomato bacterial canker disease, ugpB gene of Aac and micA gene of Cmm were used as the target for design of LAMP primers.Following primers screening and optimization of reaction systems and conditions,the LAMP method was finally established based on 30-60min incubation by using calcein as an indicator.It showed a good prospect for application due to the reduction in both the reaction time and cost.The results of specificity analysis indicated a positive reaction for the five strains of Aac and two strains of Cmm and a negative reaction for the reference strains A.avenae subsp,avenae,Pseudomonas syringae,Xanthomonas oryzae pv.oryzae,Ralstonia solanacearum,etc.In addition,the sensitivity of LAMP was 1.72×10^2 fg/μL for Aac and 1.26×10^2 fg/μL for Cmm,respectively,which were higher than those of the previous reports.In general,the LAMP system is highly sensitive and specific and helpful for control of the epidemics and spread of the two bacterial diseases.