Rapid Detection of Food-Borne Escherichia coli O157:H7 with Visual Inspection by Crossing Priming Amplification (CPA)

Xu, Zhenbo; Luo, Yuting; Soteyome, Thanapop; Lin, Chii‐Wann; Xu, Xiaoping; Mao, Yuzhu; Su, Jianyu; Liu, Junyan

doi:10.1007/s12161-019-01651-z

Cited by 18 publications

(15 citation statements)

References 16 publications

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“…Therefore, we established a PMA-CPA method for the detection of E. coli O157:H7 in VBNC cell, as well as its virulence factors. The detection limits of PMA-CPA assay showed consistency with that of CPA assay, no matter in pure bacterial culture or food samples, which had been performed previously (Xu et al, 2020). To the best of our knowledge, this is the first report of a PMA-CPA assay to detect E. coli O157:H7 in VBNC state from food samples.…”

Section: Discussionsupporting

confidence: 78%

“…As mentioned before (Xu et al, 2020), the CPA primers were designed for specific O-antigen rfbE gene and Shiga toxin genes stx1 and stx2 of E. coli via Primer Premier 5. For each of the target genes, a set of primers were designed, including five primers that recognized five distinct regions on corresponding sequences.…”

Section: Design Of Crossing Priming Amplification Primersmentioning

confidence: 99%

“…The mixed samples were centrifuged at 10,000 r/min for 5 min, and the precipitation under the tubes was processed by DNA extraction kit (Dongsheng Biotech, Guangzhou) followed the instruction of the manufacturer, which were prepared as DNA samples for PMA-CPA. The CPA reaction was performed as mentioned before (Xu et al, 2020), using thermostatic equipment or water bath in 26 µl, which contained 20-mM Tris-HCl, 10-mM (NH 4 ) 2 SO 4 , 10-mM KCl, 8.0-mM MgSO 4 , 0.1% Tween 20, 0.7-M betaine (Sigma), 1.4-mM dNTP (each), 8-U Bst DNA polymerase (NEB, United States), a 1.0-µM primer of 2a/1s, a 0.5-µM (each) primer of 2a and 3a, 0.6-µM (each) primer of 4s and 5a, 1-µl mixture 1 https://www.ncbi.nlm.nih.gov/tools/primer-blast/ chromogenic agent (mixture with calcein and Mn 2+ ), and 1µl template DNA, and the volume was made up to 26 µl with nuclease-free water. The mixed chromogenic agent consists of 0.13-mM calcein and 15.6-mM MnCl 2 .4H 2 O.…”

Section: Development Of Propidium Monoazide-crossing Priming Amplificmentioning

confidence: 99%

“…Isothermal amplification is a novel method for DNA amplification at a constant temperature, providing simple, fast, independent of sophisticated instruments and cost-effective techniques to detect biological targets, especially for less well-equipped laboratories, as well as for filed detection. Isothermal technologies mainly include loop-mediated isothermal amplification, rolling circle amplification, single primer isothermal amplification, polymerase spiral reaction, strand displacement amplification, and recombinase polymerase amplification ( Walker et al, 1992 ; Notomi et al, 2000 ; Haible et al, 2006 ; Zhao et al, 2009 , 2010 , 2011 ; Wang et al, 2011 ; Xu Z. et al, 2012 ; Xu et al, 2020 ; Liu et al, 2015 ; Yang et al, 2017 ). Cross priming amplification (CPA) is a novel isothermal method that relies on five primers (2a/1s, 2a, 3a, 4s, and 5a) to amply the target nucleotide sequences ( Xu G. et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

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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

Zhou

Wang

et al. 2020

Front. Microbiol.

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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 10 3 , 10 5 , and 10 5 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 10 3 , 10 5 , and 10 5 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.

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Section: Discussionsupporting

confidence: 78%

Section: Design Of Crossing Priming Amplification Primersmentioning

confidence: 99%

Section: Development Of Propidium Monoazide-crossing Priming Amplificmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Zhou

Wang

et al. 2020

Front. Microbiol.

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“…5). As rapidity was concerned, 10-15, 8-10, 45 and 3-5 min are required for DNA extraction, template DNA loading, LAMP reaction and results determination, respectively [71,72]. In summary, the total time consumption is 66-75 min, for simultaneous detection of 12 samples for 8 different pathogens.…”

Section: Application Of the High Flux Screening Platformmentioning

confidence: 99%

Bioprocessing and integration of a high flux screening systematic platform based on isothermal amplification for the detection on 8 common pathogens

Zhong

Deng²,

Li³

et al. 2020

Bioprocess Biosyst Eng

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During a large variety of common pathogens, E. coli, P. aeruginosa, MRSA, MRCNS, V. parahaemolyticus, L. monocytogenes and Salmonella are the leading pathogens responsible for large number of human infections and diseases. In this study, a high flux screening based on nucleic acid isothermal amplification technique has been developed. For the 8 common pathogens, species-specific targets had been selected and analyzed for their unique specificity. After optimization, separate LAMP reaction assays had been bioprocessed and integrated into one systematic detection platform, including 8 strips (PCR tubes) and 96-well plates. Eight standard strains verified for the accuracy. Application of the established high flux screening platform was used for detection for 48 samples in 4 different 96-well plates, with 2 groups of 2 operators using double-blind procedure. The accuracy of 100% was obtained, with the total time consumption as 66–75 min (for 12 samples detection on 8 different pathogens). As concluded, through the bioprocess of the systematic platform based on LAMP technique, it’s been demonstrated to be capable of simultaneous detection of 8 pathogens, with high sensitivity, specificity, rapidity and convenience.

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Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases

et al. 2021

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Rapid Detection of Food-Borne Escherichia coli O157:H7 with Visual Inspection by Crossing Priming Amplification (CPA)

Cited by 18 publications

References 16 publications

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

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

Bioprocessing and integration of a high flux screening systematic platform based on isothermal amplification for the detection on 8 common pathogens

Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases

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