多酶恒温核酸快速扩增法检测大肠杆菌<b>O157</b>∶<b>H7</b>

王淑娟; 范一灵; 冯震; 蒋波; 宋明辉; 李琼琼; 刘浩; 秦峰; 杨美成

doi:10.19428/j.cnki.sjpm.2022.22048

多酶恒温核酸快速扩增法检测大肠杆菌O157∶H7

**Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157**∶H7

摘要

摘要:
目的采用多酶恒温核酸快速扩增（MIRA）荧光法，结合金属有机骨架免疫磁珠富集功能，开发大肠杆菌O157∶H7快速富集和检测方法。
方法以rfbE基因为靶标，筛选引物、探针及优化反应体系，同时考察该方法的特异性、灵敏度和实际应用情况。
结果检测大肠杆菌O157∶H7菌体的检测限为1.18×10⁵ CFU‧mL^-1，菌体DNA质量浓度检测限为9 pg‧μL^-1，检测过程可在20 min内完成。47株菌（24株目标菌和23株非目标菌）特异性验证结果与实时荧光定量聚合酶链式反应（RT‑PCR）方法一致。
结论该研究开发了一种基于金属有机骨架免疫磁珠富集结合MIRA的检测方法，该法操作简单、反应迅速，适用于食品中大肠杆菌O157∶H7的快速富集和检测。

Abstract:
Objective A rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads.
Methods Using rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated.
Results The detection limit of Escherichia coli O157∶H7 was 1.18×10⁵ CFU‧mL^-1， and the detection limit of DNA concentration was 9 pg‧μL^-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）.
Conclusion A method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.

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多酶恒温核酸快速扩增法检测大肠杆菌O157∶H7

Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7

**Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157**∶H7