首页|出血性大肠埃希菌O157∶H7可视化和实时荧光RPA检测方法的比较

出血性大肠埃希菌O157∶H7可视化和实时荧光RPA检测方法的比较

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目的 以检测出血性大肠埃希菌(EHEC)0157:H7为目的,基于重组酶聚合酶扩增(RPA)技术,分别建立可视化RPA检测方法和实时荧光RPA检测方法.方法 根据出血性大肠埃希菌O157∶H7的rfbE基因设计引物和探针,进行特异性、灵敏度检测,建立实时荧光RPA检测方法.结合SYBR Green Ⅰ在核酸反应中的颜色变化特征,设计可视化RPA检测方法.结果 建立的实时荧光RPA检测方法和可视化RPA检测方法,分别在35 ℃时反应不超过25 min即可检测EHEC O157∶H7.设计的引物和探针仅对EHEC O157∶H7出现特异性结果.两种检测方法的检测限低于10-5 ng/μL.结论 EHEC O157∶H7的可视化RPA检测方法和实时荧光RPA检测方法操作简便,且特异性强、敏感性高,可为EHEC O157∶H7的现场快速检测和实验室检测提供新方法.
Visualization and real-time fluorescence RPA detection methods for enterohemorrhagic Escherichia coli O157∶H7
Objective To detect enterohemorrhagic Escherichia coli(EHEC)O157∶H7,visualization recombinant enzyme polymerase amplification(RPA)detection and real-time fluorescence RPA detection methods were established based on RPA technology.Methods Primers and probes were designed based on the rfbE gene of EHEC O157∶H7,and specificity and sensitivity were determined.A real-time fluorescent RPA method was established.Combined with the color change characteristics of SYBR Green Ⅰ in nucleic acid reactions,a visualization RPA method for the detection of EHEC O157∶H7 was established.Results The established real-time fluorescence RPA detection and visual RPA detection methods both detected EHEC O157∶H7 in 25 min at a reaction temperature of 35 ℃.The designed primers and probes showed specific results only for EHEC O157∶H7.The detection limits of the two methods were lower than 10-5 ng/μL.Conclusion The visual RPA detection and real-time fluorescence RPA detection methods of EHEC O157∶H7 were easy to operate with strong specificity and high sensitivity.This study provides novel methods for the rapid detection of EHEC O157∶H7 in the field and laboratory.

Recombinant enzyme polymerase amplification technologyenterohemorrhagic Escherichia coli O157∶H7visualization RPAchromogenic reactionreal-time fluorescence RPAdetection methodfoodborne pathogenic

徐颖、庄国栋、王丽丽、滕新栋

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青岛国际旅行卫生保健中心,山东青岛 266000

中华人民共和国黄岛海关口岸监管处,山东青岛 266000

重组酶聚合酶扩增技术 出血性大肠埃希菌O157∶H7 可视化RPA 显色反应 实时荧光RPA 检测方法 食源性致病菌

山东省重点研发计划

2021CXCG011306

2024

10.13590/j.cjfh.2024.01.005

中国食品卫生杂志
中国卫生信息与健康医疗大数据学会

中国食品卫生杂志

CSTPCD北大核心
影响因子：1.173
ISSN：1004-8456
年,卷(期)：2024.36(1)
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