Validation of a peptide nucleic acid fluorescence in situ hybridization for the specific detection of salmonella species in food matrices

Abstract

Salmonella is a Gram-negative flagellated rod-shaped bacterium that is one of the most important etiological agents in bacterial foodborne diseases [1,2]. Despite human salmonellosis generally presenting as a self-limiting episode of enterocolitis, the infection can degenerate into chronic and debilitating conditions [2]. To diagnose a Salmonella infection, standard cultural methods are routinely used, which implies bacterial identification by biochemical and serological tests, to confirm the suspect colonies grown on the selective agar [3]. However, this methodology is time-consuming and takes too long to deliver the results [4]. Due to these limitations, more rapid techniques for detection have been developed [5-7]. For that, in this study, we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA FISH) method for the specific detection of Salmonella spp. The method was based on a new PNA probe, SalPNA1692, coupled with a novel blocker probe in a 1:1 ratio. The method was optimized for the detection of Salmonella in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using Buffered Peptone water as a pre-enrichment for 24 h followed by 16 h of selective enrichment in RambaQuick broth. For validation in food samples, fresh ground beef was artificially contaminated with two ranges of inoculum: a low level (0.22 CFU/25 g) and a high level (210 CFU/25 g). For both levels of contamination, the confirmed positives were the same comparing the PNA-FISH method and the reference method (ISO 6579-1: 2017). The new PNA-FISH method presented a specificity of 100 % and is a faster time-to-result method, making it a good candidate for routine application in food safety laboratories.