Journal of Microbial & Biochemical Technology

Isothermal DNA amplification for Salmonella spp. detection and characterization

Joint Event on 4^th World Congress and Expo on Applied Microbiology & 2^nd International Conference on Food Microbiology

November 29-December 01, 2017 Madrid, Spain

Alejandro Garrido Maestu, Sarah Azinheiro, Pablo Fucinos, Joana Carvalho and Marta Prado

International Iberian Nanotechnology Laboratory, Portugal

Scientific Tracks Abstracts: J Microb Biochem Technol

Abstract:

Statement of the Problem: The genus Salmonella continues to be a major health issue. In Europe, more than 90000 of salmonellosis cases were confirmed in 2015 (1.9% increase respect to 2014), being serovar Enteritidis and Typhimurium the most prevalent ones, representing 45.7% and 15.8%. Conventional culture methods are lengthy and time-consuming, for this reason, the development of new methods that allow early detection of these pathogens is of high interest. Molecular methods have the ability of overcoming the previous limitations, being DNA amplification techniques the most attractive approach. In this sense, in recent years isothermal DNA amplification techniques have gained a lot of interest over other more stablished techniques, such as PCR/ qPCR, as providing several added-on advantages (no need of expensive equipment, many alternatives for product detection, etc.) One of these techniques with increase interest is loop-mediated isothermal amplification (LAMP). Methodology: A selection of the most appropriate genetic targets was done, based on previously published studies, being selected invA for Salmonella spp. detection, STM4497 and safA for Typhimurium and Enteritidis serotyping respectively. Specificity and sensitivity assays were accomplished to ensure correct performance, and finally were implemented as the detection strategy after a simple pre-enrichment and DNA extraction. These new methods were compared against qPCR. Findings: The newly designed assays performed excellent in terms of specificity, but presented lower sensitivity than qPCR. This problem was overcome with a pre-enrichment step. The evaluation of the performance of the assays in spiked food samples (turkey, chicken and egg) indicated that they may be implemented in routine food-testing. Conclusion & Significance: The developed methodology is suitable for routine food analysis, and may be used, either in a sequential mode or for directly assessing the presence of a specific serovar. LAMP represents a valid alternative to conventional DNA amplification techniques, and will allow costs reduction. Recent Publications 1. Garrido Maestu A, Fuciños P, Azinheiro S, Carvalho J and Prado M (2017) Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp. Enteritidis and Typhimurium in food samples. Food Control. 80:297-306. 2. Vial S, Berrahal Y, Prado M and Wenger J (2017) Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates. ACS Sensors. 2(2):251-256. 3. Garrido Maestu, A, Lozano León A, Rodríguez Souto R R, Vieites Maneiro R, Chapela M J and Cabado A G (2016) Presence of pathogenic Vibrio species in fresh mussels harvested in the southern Rias of Galicia (NW Spain). Food control. 59:759-765. 4. Barros Velazquez J, Prado M Espiña, B Fernandez Argüelles, M T Diéguez, L Fuciños et. al. (2015) Chp 14 Detection of foodborne pathogens using nanoparticles. Advantages and trends. Antimicrobial Food Packaging. 183-201. ISBN: 978-0-12-800723-5. 5. Chapela M J, Garrido Maestu A, and Cabado A G (2015) Detection of foodborne pathogens by qPCR: a practical approach for food industry applications. Cogent Food & Agriculture, 1(1):1013771.

Biography :

Alejandro Garrido Maestu is a Research Fellow working at International Iberian Nanotechnology Laboratory, with the expertise in Microbiology and Molecular Biology. At present he is working in a project based on a novel DNA amplification technique (loop-mediated isothermal amplification) for foodborne pathogens detection, combining with microfluidics and gold nanoparticles. This project is expected to have a great impact in the food industry as it will allow fast and accurate detection of the main foodborne pathogens. He has participated in several research projects involving bacterial agents detection, back in Spain and in the University of Florida where he started his Postdoctoral training. He has authored 10 peer-review papers with an h-index of 8, along with 1 book, all focused on food microbiology and bacterial pathogens.