摘要
用PBS和种子研磨后的普通病原提取液稀释番茄溃疡病菌纯菌液,并以梯度纯菌液和带菌种子提取液作为模板进行直接PCR和免疫捕捉PCR,比较2种方法在纯菌液和带菌种子提取液中的检测灵敏度,找到一种高特异性、高灵敏度、简便快捷的方法用于检测番茄种子携带的番茄溃疡病菌。结果显示在纯菌液中直接PCR灵敏度为104cfu/mL,免疫捕捉PCR为102cfu/mL;在带菌种子提取液中直接PCR灵敏度为106cfu/mL,免疫捕捉PCR为104cfu/mL;免疫捕捉PCR比直接PCR灵敏度高100倍,尤其在实际种子检测中优势更明显,而且检出时间短,重复性好。
Clavibacter michiganensis subsp.michiganensis culture were 10-fold diluted in PBS,then draw some dilution as template for direct-PCR and immunocapture-PCR.Seed extract samples were prepared using previously tested noninfected commercial tomato seeds;uncontaminated seed extracts were spiked with Clavibacter michiganensis subsp.michiganensis as described above.Bacterial suspensions,tomato seed extracts spiked with Clavibacter michiganensis subsp.michiganensis were detected by immunocapture-PCR.These results were compared with direct-PCR to find a high specificity,high sensitivity,high sensitivity,simple and fast method for detection of Clavibacter michiganensis subsp.michiganensis in tomato seeds.The direct-PCR provided positive results for 10^4cfu/mL in bacterial suspensions and 10^6cfu/mL in seed extracts respectively.As a new method,Immunocaptre-PCR provided positive results for10^2cfu/mL in bacterial suspensions and 10^4cfu/mL in seed extract respectively,The sensitivity is 100-fold than direct-PCR.Immunocapture-PCR provided the better results regarding sensitivity and specificity for Clavibacter michiganensis subsp.michiganensis detection,the sensitivity and specificity indicated that the immunocapture-PCR developed in our study could be used for routine seed born diseases detection.
出处
《植物检疫》
北大核心
2010年第4期12-16,共5页
Plant Quarantine
基金
甘肃省科技厅中小企业创新基金计划(项目编号:0802NCCA028)
国家质监总局项目(项目编号:2007IK259
2009IK276)
