网状分枝扩增技术检测产志贺样毒素大肠埃希菌

Detection of shiga toxin in shiga toxin-producing Echerichia coli by ramification amplification method

目的 通过检测产志贺毒素大肠埃希菌中的志贺样毒素,确定网状分枝扩增技术的灵敏度和特异性,从而进一步探讨该方法用于检测食品和临床标本中0157:H7和STEC的可行性。方法 用网状分枝扩增技术检测合成的志贺样毒素2的基因和临床分离的菌株,确定该方法的灵敏度和特异性,与聚合酶链反应进行比较。结果 网状分枝扩增技术最少能检测10个志贺样毒素DNA分子,与PCR灵敏度一样。E.coli0157:H7和志贺痢疾杆菌志贺样毒素阳性,而非致病性大肠埃希菌为阴性。用网状分枝扩增技术与PCR对分离的菌株进行检测,结果也一致。结论 网状分枝扩增技术具有高度的灵敏度和特异性,操作简便,而且在等温条件下扩增核酸,因此可替代PCR检测食品和临床标本中的产志贺样毒素大肠埃希菌。

Objective To determine the sensitivity and specificity of ramification amplification method for detecting Shiga toxin and to determine the feasibility in detecting E. coli 0157: H7 and other shiga toxin-producing E. coli isolated from food and clinical specimens. Methods The sensitivity and specificity of RAM were compared with those of PCR by detecting synthetic Shiga toxin DNA target and isolates from food and clinical specimens. Results The lowest number of targets detected by RAM assay was 10 molecules. Several clinical isolates of E. coli 0157 :H7, Shigella dysenteriae and nonpathogenic E. coli were further tested. The results showed that E. coli 0157: H7 and Shigella dysenteriae were positive for shiga toxin gene,while nonpathogenic E. coli were negative. The results of RAM and PCR by detecting isolates were same. Conclusion RAM assay could be an alternative to PCR to detect STEC in food product and clinical specimens because of its high sensitivity and specificity , simplicity and isothermal amplification.