摘要
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0.01ng/μL,目的菌的检测限为10~3CFU/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 10~3 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.
作者
刘婧文
凌莉
黄成栋
王菊芳
蒋丽婷
李志勇
LIU Jing-Wen;LING Li;HUANG Cheng-Dong;WANG Ju-Fang;JIANG Li-Ting;LI Zhi-Yong(Guangzhou Customs Technology Center,Guangzhou 510623,China;Guangdong Provincial Key Laboratory of Import and Export Technical Measures of Animal,Plant and Food,Guangzhou 510623,China;School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China;Guangzhou Institute of Microbiology,Guangzhou 510663,China)
出处
《食品安全质量检测学报》
CAS
2020年第17期6093-6103,共11页
Journal of Food Safety and Quality
基金
国家重点研发计划项目(2017YFC1601203)。
