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

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 APPLICATIONS

The 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.