Development of a Novel Phagomagnetic-Assisted Isothermal DNA Amplification System for Endpoint Electrochemical Detection of Listeria monocytogenes
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Published:2023-04-07
Issue:4
Volume:13
Page:464
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ISSN:2079-6374
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Container-title:Biosensors
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language:en
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Short-container-title:Biosensors
Author:
Maciel Cláudia1, Silva Nádia F. D.2ORCID, Teixeira Paula1ORCID, Magalhães Júlia M. C. S.2ORCID
Affiliation:
1. Laboratório Associado, Escola Superior de Biotecnologia, CBQF—Centro de Biotecnologia e Química Fina, Universidade Católica Portuguesa, Rua Diogo Botelho 1327, 4169-005 Porto, Portugal 2. REQUIMTE/LAQV, Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, 4200-465 Porto, Portugal
Abstract
The hitherto implemented Listeria monocytogenes detection techniques are cumbersome or require expensive non-portable instrumentation, hindering their transposition into on-time surveillance systems. The current work proposes a novel integrated system resorting to loop-mediated isothermal amplification (LAMP), assisted by a bacteriophage P100–magnetic platform, coupled to an endpoint electrochemical technique, towards L. monocytogenes expeditious detection. Molybdophosphate-based optimization of the bacterial phagomagnetic separation protocol allowed the determination of the optimal parameters for its execution (pH 7, 25 °C, 32 µg of magnetic particles; 60.6% of specific capture efficiency). The novel LAMP method targeting prfA was highly specific, accomplishing 100% inclusivity (for 61 L. monocytogenes strains) and 100% exclusivity (towards 42 non-target Gram-positive and Gram-negative bacteria). As a proof-of-concept, the developed scheme was successfully validated in pasteurized milk spiked with L. monocytogenes. The phagomagnetic-based approach succeeded in the selective bacterial capture and ensuing lysis, triggering Listeria DNA leakage, which was efficiently LAMP amplified. Methylene blue-based electrochemical detection of LAMP amplicons was accomplished in 20 min with remarkable analytical sensitivity (1 CFU mL−1). Hence, the combined system presented an outstanding performance and robustness, providing a 2.5 h-swift, portable, cost-efficient detection scheme for decentralized on-field application.
Funder
European Union (FEDER funds through the NORTE2020—Programa Operacional Regional do Norte Fundação para a Ciência e a Tecnologia Portuguese national funds
Subject
Clinical Biochemistry,General Medicine,Analytical Chemistry,Biotechnology,Instrumentation,Biomedical Engineering,Engineering (miscellaneous)
Reference83 articles.
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