产志贺毒素Ⅱ型变异体大肠杆菌LAMP检测方法的建立  

Development of loop-mediated isothermal amplification for detection of Escherichia coli producing Shiga toxin Ⅱ variant

作　　者：冯瑜菲[1] 朱颖[1] 刘文鑫[1] 江馗语[1] 何丽丽[1] 关玮琨[1] 胡文霞[1] 师东方[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2011年第3期287-291,共5页Chinese Veterinary Science

基　　金：黑龙江省重点攻关项目(GB05B501);东北农业大学创新团队项目(CXZ008-3)

摘　　要：为建立产志贺毒素Ⅱ型变异体(Stx2e)大肠杆菌的环介导恒温扩增(LAMP)检测方法,参照Gen-Bank中12条Stx2e序列和9条Stx2序列,针对Stx2e基因保守区域设计并合成4条引物,分别进行了反应条件优化、特异性试验、敏感性试验及模拟样品检测。结果显示,用所建立的LAMP方法对阳性样品扩增所得产物电泳后呈特征性梯状条带,阴性样品的扩增产物电泳后均无扩增条带。建立的LAMP方法对Stx2e+大肠杆菌的最低检出量为38CFU/管,敏感性高于PCR方法(3.8×103CFU/管)。LAMP和PCR对模拟样品检测结果的符合率为100%。该研究为仔猪水肿病的诊断和Stx2e+大肠杆菌的快速检测提供了新的方法。The objective of this study was to establish a loop-mediated isothermal amplification（LAMP） method for the detection of Stx2e-producing Escherichia coli.According to 12 Stx2e sequences and 9 Stx2 sequences available in GenBank,a set of four primers were designed based on the Stx2e gene sequence.Optimal conditions,specificity and sensitivity tests were performed,respectively.In results,ladder-like products were produced with those Stx2e positive samples by LAMP,while no product was generated with other controls.The LAMP assay had a detection limit equivalent to 38 CFU/tube,which was more sensitive than PCR method（3.8×103 CFU/tube）.The agreement rate between LAMP and PCR was 100% in detecting simulation samples.In conclusion,the developed LAMP assay provides a novel method for the rapid detection of E.coli producing Stx2e and diagnosis of porcine edema disease.

关 键 词：产志贺毒素Ⅱ型变异体 环介导恒温扩增 大肠杆菌 猪水肿病 

分 类 号：S852.612[农业科学—基础兽医学]