Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli
O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodolo...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-09-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2020.569105/full |
| _version_ | 1828775506144657408 |
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| author | Wenqu Zhou Kan Wang Wei Hong Caiying Bai Ling Chen Ling Chen Xin Fu Tengyi Huang Junyan Liu |
| author_facet | Wenqu Zhou Kan Wang Wei Hong Caiying Bai Ling Chen Ling Chen Xin Fu Tengyi Huang Junyan Liu |
| author_sort | Wenqu Zhou |
| collection | DOAJ |
| description | O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness. |
| first_indexed | 2024-12-11T15:41:33Z |
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| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-11T15:41:33Z |
| publishDate | 2020-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-9df9d94359224a52897b5d2b77596b9d2022-12-22T00:59:48ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2020-09-011110.3389/fmicb.2020.569105569105Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coliWenqu Zhou0Kan Wang1Wei Hong2Caiying Bai3Ling Chen4Ling Chen5Xin Fu6Tengyi Huang7Junyan Liu8GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, ChinaResearch Center for Translational Medicine, The Second Affiliated Hospital, Shantou University Medical College, Shantou, ChinaGMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, ChinaGuangdong Women and Children Hospital, Guangzhou, ChinaSchool of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou, ChinaResearch Institute for Food Nutrition and Human Health, Guangzhou, ChinaGMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou, ChinaDepartment of Laboratory Medicine, The Second Affiliated Hospital of Shantou University Medical College, Shantou, ChinaDepartment of Civil and Environmental Engineering, University of Maryland, College Park, MD, United StatesO157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.https://www.frontiersin.org/article/10.3389/fmicb.2020.569105/fullpropidium monoazide-crossing priming amplificationviable but non-culturable stateEscherichia coli O157:H7rapid detectionfoodborne pathogens |
| spellingShingle | Wenqu Zhou Kan Wang Wei Hong Caiying Bai Ling Chen Ling Chen Xin Fu Tengyi Huang Junyan Liu Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli Frontiers in Microbiology propidium monoazide-crossing priming amplification viable but non-culturable state Escherichia coli O157:H7 rapid detection foodborne pathogens |
| title | Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli |
| title_full | Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli |
| title_fullStr | Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli |
| title_full_unstemmed | Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli |
| title_short | Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli |
| title_sort | development and application of a simple easy to operate propidium monoazide crossing priming amplification on detection of viable and viable but non culturable cells of o157 escherichia coli |
| topic | propidium monoazide-crossing priming amplification viable but non-culturable state Escherichia coli O157:H7 rapid detection foodborne pathogens |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2020.569105/full |
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