**4. Molecular methods**

378 Salmonella – A Dangerous Foodborne Pathogen

typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate (ABTS or 3,3',5,5'-tetramethylbenzidine) to produce a visible signal (colorimetric or fluorescent product) due to the enzymatic cleavage of the substrate. Colorimetric equipment is used to measure the signal indicating colorimetric equipment

ELISAs are highly specific, sensitive, rapid, easy to perform, and scalable, allowing laboratories to easily adopt the technology for routine microbiological testing. The ELISA reactivity however, is influenced by various components of the enrichment medium and incubation conditions used. With most ELISA methods, negative results can be obtained within 24 h after an overnight incubation in selective broth. Positive results may still require further cultural isolation and serological and biochemical confirmation depending of

Currently, there are numerous ELISA plate based assay systems for the detection on *Salmonella: Salmonella* ELISA (BIO ART SA), TRANSIA® PLATE *Salmonella* Gold (BioControl), and RIDASCREEN® *Salmonella* ELISA (R-Biopharm AG). Some of these tests have the advantage of being able to process numerous samples at once in 96 well microtitre plates, and some such as the Tecra™ *Salmonella* Visual Immunoassay (3M), provide a visual indication of detection without the use of colorimetric equipment. In addition ELISA systems have been automated to facilitate routine laboratory testing such as the EIAFoss (Foss Electronics) and the VitekImmuno Diagnostic Assay System (VIDAS) (BioMerieux). For example, the VIDAS®SLM assay (BioMérieux), is intended for use with the VIDAS as an automated qualitative enzyme-linked fluorescent immunoassay (ELFA) for the detection of *Salmonella* in food and food ingredients. The VIDAS instrument performs all of the assay steps automatically. In contrast to the manual manipulation required for microtitre plate based systems, a pipette tip-like disposable unit (a solid phase receptacle or SPR) serves as the solid phase as well as a pipetter during the process. The SPR is coated with polyclonal anti-*Salmonella* antibodies and reagents for the assay are sealed in reagent strips. An aliquot of the enrichment broth is placed into the reagent strip and the sample and reagents are sequentially cycled in and out of the SPR for a specific length of time until the instrument

Nevertheless, ELISA methods are not without disadvantages, some of which include high limits of sensitivity of >105 cfu/mL (Cox 1988) variable cell surface antigen production (Peplow et al. 1999); cross reactivity (Westerman et al. 1997), and changes to antigens due to acetylation and changing recognition by assay antibodies (Kim and Slauch, 1999). Newer ELISA-like techniques utilize fluorogenic, electrochemiluminescent, and real-time PCR reporters to create quantifiable signals. However, given that the general principles in these

Lateral flow immunoassays typically use a sandwich type ELISA and the majority use polyclonal antibody as a capture antibody and a monoclonal antibody as the detection antibody. The antibodies are fixed on a hydrophobic polyvinylidine difluoride-based membrane. A drop of an enrichment sample is placed in a reaction window and travels by capillary action across the membrane to react with the antibodies and provide a colour change. Results are often available within 24 hours. False positive results may be observed

assays are largely similar, they are often grouped in the same category as ELISAs.

indicating the presence of target antigen in the sample.

regulatory requirements.

detects fluorescence.

**3.3 Lateral flow immunoassays** 
