Development of a novel phagomagnetic-assisted isothermal DNA amplification system for endpoint electrochemical detection of Listeria monocytogenes
| Autor: | Cláudia Maciel, Nádia F. D. Silva, Paula Teixeira, Júlia M. C. S. Magalhães |
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| Přispěvatelé: | Veritati - Repositório Institucional da Universidade Católica Portuguesa |
| Jazyk: | angličtina |
| Rok vydání: | 2023 |
| Předmět: |
prfA
Loop-mediated isothermal DNA amplification Clinical Biochemistry Listeria monocytogenes bacteriophage P100 magnetic capture milk analysis loop-mediated isothermal DNA amplification electrochemical detection Biomedical Engineering Milk analysis General Medicine Bacteriophage P100 Analytical Chemistry Magnetic capture Electrochemical detection Instrumentation Engineering (miscellaneous) Biotechnology |
| Zdroj: | Biosensors; Volume 13; Issue 4; Pages: 464 |
| ISSN: | 2079-6374 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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