Evaluation and implementation of commercial antibodies for improved nanoparticle-based immunomagnetic separation and rea
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ORIGINAL ARTICLE
Evaluation and implementation of commercial antibodies for improved nanoparticle-based immunomagnetic separation and real-time PCR for faster detection of Listeria monocytogenes Alejandro Garrido-Maestu1 • Sarah Azinheiro1 • Joana Carvalho1 Begon˜a Espin˜a1 • Marta Prado1
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Revised: 6 April 2020 / Accepted: 15 April 2020 Ó Association of Food Scientists & Technologists (India) 2020
Abstract L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surfacevolume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.
& Alejandro Garrido-Maestu [email protected] 1
Department of Life Sciences, Nano4Food – Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre Jose´ Veiga s/n, 4715-330 Braga, Portugal
Keywords Rapid methods Magnetic nanoparticles IMSqPCR hly Listeria monocytogenes
Introduction Listeria monocytogenes is one of the most serious foodborne pathogens due to its high mortality rate and ubiquity (Zilelidou et al. 2016). Data from EU surveillance of human listeriosis, mainly focused on severe, invasive forms of the disease, indicates that it affects several risk groups including elderly, immunocompromised people as well as pregnant women and infants, causing high hospitalization and mortality, particularly among the elderly. Invasive listeriosis has shown a significant increasing trend since EU surveillance was initiated in 2008 and continued this trend in the last surveilled 5 years (2013–2017) (EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control) 2018). Currently, official methods for the detection of bacterial foodborne pathogens are based on classical microbiology, which require several days for bacterial isolation and identification. P
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