Escherichia coli O157:H7 is an important food-borne pathogen and has ability to contaminate food, such as water, meat products, and milk. Moreover, E. coli O157:H7 is the main toxin-producing serotype which can produce Shiga toxin type 1 and type 2 and cause intestinal disease. The strong pathogenicity and lethality of Escherichia coli O157:H7 pose a serious threat to human. This study aims to develop a rapid and visual detection assay of E. coli for rfbE, stx1, and stx2 genes by crossing priming amplification (CPA). The limit of detection of CPA assay for rfbE, stx1, and stx2 genes was 3.20 fg/μl, 320 fg/μl, and 320 fg/μl in genomic DNA, while that of in artificially contaminated food samples was 103 cfu/ml, 105 cfu/ml, and 105 cfu/ml, respectively, which was distinctly higher than that of PCR methods. And the specificity of CPA assay was tested by 22 different bacterial strains and except for E. coli O157:H7 ATCC43895 and other E. coli O157:H7 isolated from eggs, milk, and beef, all strains showed negative results. The visible detection assay was conducted by the addition of calcein in the reaction solutions. The CPA assay showed a successful detection of E. coli O157:H7 (Shiga toxin–producing and non-Shiga toxin–producing) within 60 min under 63 °C with high sensitivity and specificity. These results indicated that the CPA assays with calcein can provide an advanced method to achieve the rapid and visual detection of food-borne E. coli O157:H7.
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This work was supported by the National Key R&D Program of China (2016YFD0400203), YangFan Innovative and Entrepreneurial Research Team Project (2014YT02S029), the National Key Research and Development Program of China (2016YFD04012021), Guangdong Special Support Program (2016TQ03N682), Pearl River S&T Nova Program of Guangzhou (201710010061), the Fundamental Research Funds for the Central Universities (Dr. Zhenbo Xu, D2191310), Collaborative grant with AEIC (KEO-2019-0624-001-1), the 111 Project (B17018), and Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety (KL-2018-05, KL-2018-14, KL-2018-16).
Compliance with Ethical Standards
This is not applicable (humans are not involved).
Conflict of Interest
Zhenbo Xu declares that he has no conflict of interest. Yuting Luo declares that she has no conflict of interest. Thanapop Soteyome declares that he has no conflict of interest. Chii-Wann Lin declares that he has no conflict of interest. Xingyong Xu declares that he has no conflict of interest. Yuzhu Mao declares that she has no conflict of interest. Jianyu Su declares that he has no conflict of interest. Junyan Liu declares that she has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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1.School of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product SafetySouth China University of TechnologyGuangzhouPeople’s Republic of China
2.Department of Microbial Pathogenesis, School of DentistryUniversity of MarylandBaltimoreUSA
3.Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center)GuangzhouChina
4.Home Economics TechnologyRajamangala University of Technology Phra NakhonBangkokThailand
5.Institute of Biomedical EngineeringNational Taiwan UniversityTaipeiChina