IMS USING IN‐HOUSE MONOCLONAL ANTIBODY‐COATED MAGNETIC BEADS ASSOCIATED TO PCR ASSAY FOR DETECTION OF SALMONELLA TYPHIMURIUM IN RAW MEATS

Abstract

ABSTRACT An immunomagnetic separation (IMS) technique and a PCR assay were developed for use in detection of Salmonella Typhimurium in meat samples. To prevent false‐negative results, an internal amplification control was developed. The polymerase chain reaction (PCR) using primers specific for an omp gene sequence of Salmonella spp has shown 100% sensitivity and specificity and a detection limit of 104 cfu/mL. The IMS‐PCR methods using PCR immediately after IMS and using 6 h postenrichment in brain heart infusion between IMS and PCR resulted in detection limits of 103 cfu/mL and 1–10 cfu/mL, respectively. The lowest level of S. Typhimurium that could be detected by the IMS‐PCR method in the presence of natural microbiota from inoculated meat samples was 1–10 cfu/25 g. When samples were analyzed using enrichment protocols without IMS, several false‐negative results were obtained.PRACTICAL APPLICATIONSThe immunomagnetic separation‐polymerase chain reaction (IMS‐PCR) method developed enabled a rapid and sensitive detection of Salmonella Typhimurium in inoculated meat samples. Monoclonal antibody (Mab)‐coated magnetic beads prepared in‐house were efficient in concentrating and separating the bacteria from the food matrix, thus improving detection limit and avoiding false‐negatives. The internal amplification control (IAC), now mandatory in PCR assays, using the same primers of the target DNA further prevented false‐negative results. Therefore, the IMS‐PCR method developed in this study could be used in the future by the Brazilian food industry as a substitute for the expensive imported kits for Salmonella detection in foods. We are now developing a panel of Mabs against conserved antigens of Salmonella for use in the IMS‐PCR method in order to extend its applicability for detection of other serovars.