A fluorescence in situ hybridization method using a peptide nucleic acid probe for the identification of Salmonella spp. in a broad spectrum of samples

Abstract

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. Specificity and sensitivity probe matching theoretical estimates were both 100%. The PNA FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species, confirmed the predicted theoretical values of specificity and sensitivity. The PNA FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water and powdered infant formula (PIF).

The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. By performing a previous enrichment step, the PNA FISH method was able to detect 1 CFU per 10 mL of blood (5x109 ± 5x108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10g of PIF (2x107 ± 5x106 CFU/ml after an 8h enrichment step),. The feces and water samples were also enriched according to the corresponding ISO methods, and results showed that the PNA FISH method was able to detect Salmonella immediately after conducting the first enrichment step. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples, taking less than 20 hours to obtain a diagnosis, except for PIF samples where the analysis takes less than 12 hours. This procedure may be used for food processing and municipal waters control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.

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Identifiers

ORCID for Charles W. Keevil: orcid.org/0000-0003-1917-7706

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