改良RNA提取法应用于RT-PCR检测O157︰H7活菌  

Detection of viable Escherichia coli O157: H7 by reverse-transcriptase polymerase chain reaction with improved RNA extraction

作　　者：黄偌颖[1] 张娟胜 吕红亮[1] 阿娜尔古丽.伊敏 罗影殊[1] 王国庆[1] 

机构地区：[1]四川大学华西公共卫生学院(华西第四医院),四川成都610041

出　　处：《现代预防医学》2014年第9期1658-1661,共4页Modern Preventive Medicine

摘　　要：目的将改良的RNA提取法应用于RT-PCR,建立一种快速灵敏检测肠出血性大肠杆菌O157:H7活菌的方法。方法以O157︰H7 rfbE基因为靶基因设计引物,样品中加入较大浓度的非目标菌后,用TRIzol试剂提取细菌总RNA后,gDNA Eraser去除其中污染的DNA,通过RT-PCR检测O157︰H7 rfbE的mRNA。结果 RT-PCR法能有效检测样品中的O157︰H7活菌,死菌不被检测。未加入非目标菌(志贺菌)检测O157︰H7检出限为3.4×108cfu/ml,加入非目标菌共沉淀提取RNA,检出限下降至3.4×105cfu/ml,检测灵敏度提高了1 000倍。结论改良RNA提取法应用于RT-PCR能够快速灵敏检测O157︰H7活菌,在突发公共卫生事件的应急处理方面有良好的应用前景。Objective To establish a rapid and sensitive reverse-transcriptase polymerase chain reaction with an improved proce- dure for RNA extraction to detect viable enterohemorrhagic Eschrichia coli O157：H7. Methods Specific primers were designed to target at Eschrichia coli O157：H7 ribE gene. High concentration of non-target bacterial cells were added to the sample, and then bacterial total RNA was extracted by using of TRIzol reagent, contaminated DNA was removed by using gDNA Eraser, mRNA of E- HEC O157：H7 rfbE which indicated the presence of viable EHEC O157：H7 was tested via RT-PCR. Results The specific RT-PCR could efficiently detect only viable EHEC O157：H7, dead cells showed negative results. The low limit of the RT-PCR de- tection of Eschrichia coli O157：H7 was reduced from 3.4 x lO%fu/ml to 3.4 x lOScfu/ml, which meant a 1 O00-fold more sensitive. Conclusion The established RT-PCR detection based on the improved procedure can effectively and sensitively detect live bacteria of Eschrichia coli O 157： H7, it has good prospect in rapid detection of emergent events of public health.

关 键 词：肠出血性大肠杆菌0157 H7 RNA 逆转录聚合酶链反应 

分 类 号：R115[医药卫生—公共卫生与预防医学]