VALIDATION OF A MULTIPLEX TaqMan qPCR SYSTEM FOR DETECTION OF Salmonella SPP.

SARITA REYES; MILAGROS SAID ADAMO; RAMIRO POMA; HECTOR ANTONIO CRISTÓBAL

Buenos Aires

Congreso; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2021

Salmonella enterica is one of the major bacterial agents that cause foodborne infections in humans all over the world. Traditional Salmonella detection methods are based on cultures using selective media and characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming. The TaqMan system was designed as a multiplex reaction to simultaneously detect three molecular markers of Salmonella spp. The target sequences for Salmonella spp. were one housekeeping gene and two genes that encode for virulence factors. The validation assays for multiplex TaqMan system was carried out by real time PCR using different collection strains. The strains were: Salmonella Choleraesuis (ATCC 4931), Salmonella Typhi (CECT 4594), Salmonella Typhimurium LT2 (ATCC 15277), Salmonella enterica subsp arizonae (CECT 4395, 4396), Shigella disentereae (CECT 584), Shigella sonnei (CECT 457), Escherichia coli (50365 NCTC), Escherichia coli O157:H (NCTC 12900), Serratia marcescens (ATCC 14041, 13880), Vibrio campbellii (ATCC 25920), Enterobacter sakazakii (ATCC 29544), Yersinia enterocolitica (CECT 500), Listeria monocytogenes (CECT 4032), Staphylococcus aureus (ATCC 9144). Additionally, a total of 141 Salmonella spp clinical strains were isolated during the 2018 outbreak in Salta-Argentina, 16 Salmonella spp. and 12 Escherichia coli strains isolated from Laboratory food hygiene inspection and control laboratory from Spain, were tested. The limit of detection (LoD) was determined using the standard curve of Salmonella Typhimurium ATCC 14028. The viable counts (CFU/mL) of cell suspension was determined by plating 100 μL of each dilution on S-S Agar (Britania) in triplicate and were incubated at 37°C for 24 h. The detection system TaqMan qPCR multiplex has 100% inclusivity and exclusivity, all Salmonella strains used were accurately detected. The method did not report any false-positive results. After standardization, the efficiency of the TaqMan qPCR reaction was> 98%, with a dynamic range of 7 orders (R2= 0.98) for all molecular markers. The cut off assumed values of > 43.67 (Ct) for the three genes. The sensitivity of TaqMan qPCR systems was 7x102 CFU/mL. We have described a multiplex TaqMan qPCR method for Salmonella spp. that is simple, sensitive and enables the quick and precise detection of the most prevalent serotypes of Salmonella in human clinical samples.