Loop-Mediated Isothermal Amplification-Based Workflow for the Detection and Serotyping of Salmonella spp. in Environmental Poultry Flock Samples

Abstract

Salmonella spp. is one of the most important foodborne pathogens worldwide. Given the fact that poultry and poultry products are the main source of human infection, Salmonella control in these farms is of utmost importance. To better control this pathogen in farms, boot swabs are used to sample farm environments but the analysis of these swabs is mainly based on culture-dependent methods. In the present study, a novel loop-mediated isothermal amplification (LAMP) method was developed for the rapid screening of Salmonella spp. in boot swab samples from broiler flock environments. Four different DNA extraction protocols were evaluated in depth, including a simple thermal lysis, a chelex-based protocol and two thermal lysis protocols followed by the purification of magnetic beads made of silica (“glass milk”) in order to determine the most suitable alternative for potential on-site, farm analyses. The methodology evaluation included a blind interlaboratory assay and as a proof-of-concept, a naked-eye colorimetric assay was also included. Following the final methodology, it was possible to reach an LoD50 of 1.8 CFU/25 g of the samples, with a high relative sensitivity (95.7%), specificity (100%) and accuracy (96.6%) along with Cohen’s kappa of concordance with respect to the ISO standard 6579-1:2017 of 0.9, with an RLOD of 1.3. In addition to this, due to the relevance of certain serotypes with the genus Salmonella spp., a serotype LAMP panel for the specific identification of S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar and S. Virchow was also developed. Even though some degree of cross-reactivity among the primers developed was observed, all the serotypes could be accurately identified based on their melt curve analysis profile. Taken together, in the present study, a rapid Salmonella spp. screening method, suitable for farm applications, was developed, along with a serotyping panel that could be used in a laboratory setup for the identification of the most relevant serotypes of the genus, taking advantage of real-time amplification followed by melt curve analysis.