Rapid Detection of Salmonella in Chicken Meat Using Immunomagnetic Separation, CHROMagar, ELISA and Real-time Polymerase Chain Reaction (RT-PCR)

Abstract

The main objective of this study was to standardize and compare rapid methods for the detection of Salmonella in meat samples using Immuno-Magnetic Separation (IMS) followed by culturing in CHROMagar Plus media, Enzyme-Linked Immunosorbent Assay (ELISA) and Real-Time Polymerase Chain Reaction (RT-PCR). Ten-fold dilutions of bacterial suspension (S. typhymurium, ATCC13311) were prepared from the original concentration of 1.6 x 10⁶cfu/ml. Chicken wing samples of 25 g each negative for Salmonella were spiked with six different concentrations of bacteria ranging from 10⁶ to 10¹. These samples were incubated in buffered peptone water for 4 h as pre-enrichment step and were tested repeatedly. The IMS technique involved the use of paramagnetic polystyrene microscopic beads coated with purified antibodies against Salmonella. The CHROMagar Plus media containing chromomeric substrate facilitated detection of Salmonella species from other flora. The Assurance EIA Salmonella Kit with polyclonal antibodies directed against Salmonella facilitated easy and rapid detection. In the RT-PCR primers targeting invA gene was used which amplified a 378 bp fragment. Comparing to conventional culture method (4 days), CHROMagar plate culture following IMS showed light mauve to mauve-colored colonies of Salmonella in 23 h with high sensitivity (99%) at 1.6 cfu/ml. IMS-ELISA combination also showed high sensitivity (75%) at 1.6 x 10³ cfu/ml in 8 h and minimized cross-reactivity with many Enterobacteraceae. The combination of IMS with RT-PCR took less than 7 h and was even more sensitive (100%) at 1.6 cfu/ml. Sensitivities of IMS-RT-PCR and IMS-CHROMagar were higher compared to IMS-ELISA. IMS-CHROMagar was easier to perform and detects only living Salmonella. These methods will be highly suitable for routine detection and may significantly assist the processing industry in avoiding costly recalls and the timely investigation of outbreaks.