Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples

摘要

European Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect 10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the gLAMP process, which may be easily implemented in other assays where accurate determination of T-t is needed for quantification purposes. (C) 2017 Elsevier Ltd. All rights reserved.