实时荧光重组酶聚合酶扩增检测大肠埃希氏菌O157

实时荧光重组酶聚合酶扩增检测大肠埃希氏菌O157
目的  建立实时荧光重组酶聚合酶扩增(real-time recombinase polymerase amplification, real-time RPA)检测大肠埃希氏菌O157的分析方法。方法  根据大肠埃希氏菌O157的rfbE基因(S83460.1), 设计特异性引物和exo探针, 分别进行引物探针筛选和特异性、灵敏度以及稳定性探究, 并对人工污染样品和实际样品进行检测, 验证本研究方法的实用性。结果  本方法在37 ℃恒温20 min内可完成对大肠埃希氏菌O157的定性检测, 目的DNA的检测限为0.01 ng/μL, 目的菌的检测限为103 CFU/mL。在稳定性实验中, 选择从100~0.001 ng/μL的10倍梯度稀释的目的DNA进行每个浓度8个平行的测试, 0.01 ng/μL及以上浓度的DNA的8个平行均可稳定检出。对于大肠埃希氏菌O157的初始污染量为4 CFU/ 25 g的牛奶和鸡肉人工污染样品, 增菌9 h后, 可检出其中的目的菌。用本方法和国标方法GB 4789.36-2016同时检测20份实际样品, 结果一致。结论  该方法快速、简便, 可作为一种常温下大肠埃希氏菌O157的快速检测手段, 应用于基层实验室或现场检测。

Objective  To establish a method for the detection of Escherichia coli O157 by real-time recombinase polymerase amplification (real-time RPA). Methods  According to the rfbE gene of Escherichia coli O157 (S83460.1), specific primers and exo probes were designed to conduct primer probe screening and specificity, sensitivity and stability exploration, respectively, and artificial contaminated samples and actual samples were detected to verify the practicability of this research method. Results  The qualitative detection of Escherichia coli O157 could be completed within 20 min at 37 ℃. The detection limit of Escherichia coli O157 DNA was 0.01 ng/μL and the detection limit of this bacterial was 103 CFU/mL. In stability study, the assay was performed at a 10 fold gradient dilution of target DNA from 100 ng/μL to 0.001 ng/μL in 8 parallels. Among the 8 parallels, the DNA concentration of 0.01 ng/μL and above could be detected steadily. The pathogen could be detected after 9 h of incubation in artificially contaminated milk and chicken samples with an initial Escherichia coli O157 concentration of 4 CFU/25 g. This method and GB 4789.36-2016 were used to simultaneously test 20 actual samples, and the results were consistent. Conclusion  The proposed method is fast, simple, and practical for quick screening of Escherichia coli O157 at room temperature in basic lab or on-site inspection.

标题：实时荧光重组酶聚合酶扩增检测大肠埃希氏菌O157
英文标题：Detection of Escherichia coli O157 by real-time fluorescence recombinase polymerase amplification

作者：
刘婧文 广州海关技术中心；广东省动植物与食品进出口技术措施研究重点实验室；华南理工大学生物科学与工程学院
凌莉 广州海关技术中心；广东省动植物与食品进出口技术措施研究重点实验室
黄成栋 广州海关技术中心；广东省动植物与食品进出口技术措施研究重点实验室
王菊芳 华南理工大学生物科学与工程学院
蒋丽婷 广州市微生物研究所
李志勇 广州海关技术中心；广东省动植物与食品进出口技术措施研究重点实验室

中文关键词:大肠埃希氏菌O157,实时荧光重组酶聚合酶扩增,常温扩增,
英文关键词:Escherichia coli O157,real-time recombinase polymerase amplification,isothermal amplification,

发表日期：2020-04-21

2025-2-27 20:26 上传 实时荧光重组酶聚合酶扩增检测大肠埃希氏菌O157.pdf

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