**3. Immunological based methods**

#### **3.1 Rapid agglutination assays**

Several rapid latex agglutination assay tests are widely used for the rapid detection of *Salmonella*. These assays however, are primarily used as a confirmation screen for presumptive *Salmonella* colonies after culture isolation from selective agar plates, with further confirmation and identification work carried out on those organisms giving a positive latex reaction. An aliquot of a colony suspension or enrichment broth is simply mixed with the latex reagent and after a few minutes rotation, the results are clearly visible. If the test is negative, the latex remains in smooth suspension and retains its original colour. A positive result is indicated by distinct colour agglutination against an altered background. By reducing the number of samples requiring further confirmatory testing, these tests save time and resources and allow negative results to be reported at least 24 hours earlier than by conventional culture methods. However, depending on the antibodies used they may lack specificity due to non-specific agglutination of some organisms (Cheesbrough and Donnelly, 1996). Some commercial kits include Remel Wellcolex Colour tests for the presumptive identification of *Salmonella* serogroups A, B, C, D, E, and G, and the Vi antigen using just two reagents. Similar tests include Oxoid Salmonella latex test, Microgen Salmonella Latex test, and Denka-Seiken, among others.

#### **3.2 Enzyme-linked immunosorbent assay (ELISA)**

Enzyme-linked immunosorbent assay (ELISA) also known as an enzyme immunoassay (EIA), is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. In the context of *Salmonella* detection, a sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtitre plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "Sandwich" ELISA). After the antigen is immobilized, a detection antibody linked to an enzyme such as Horse Radish Peroxidase (HRP) is added, forming a complex with the antigen. Between each step, the plate is

*Salmonella* Detection Methods for Food and Food Ingredients 379

during the reaction because of denaturation or degradation of the capture antibody and it is likely that detection antibody or enzyme-conjugated antibody may also bind non-specifically to denatured capture antibody. Commercially available lateral flow immunoassays for the detection of *Salmonella* include: DuPont™ Lateral Flow System *Salmonella*, Singlepath *Salmonella* (Merck), Reveal® *Salmonella* lateral flow (Neogen), VIP Gold (BioControl), and RapidChek® SELECT (SDIX). Recently, serotype specific lateral flow immunoassays for the detection of *S.* Enteritidis have also been introduced to serve the egg and poultry industry such as RapidChek® SELECT *S.* Enteritids (SDIX) and Reveal *S.*  Enteritidis (Neogen). In general, these types of immunoassays are ideally suited where a low testing throughput is expected. The implementation of these tests is beneficial in that

Over the past 15 years there has been an important evolution in molecular approaches for the rapid detection of food borne pathogens rather than relying on their biochemical and phenotypic characteristics. Foremost among these tools is the Polymerase Chain Reaction (PCR), a technique based on the specific amplification of a short target DNA sequence (Mullis et al. 1986). Briefly, extracted DNA is first subjected to heat denaturation into single stranded DNA. Next, specific short DNA fragments (primers) are annealed to the single DNA strands, followed by extension of the primers complementary to the single stranded DNA with the aid of a thermostable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium *Thermus aquaticus* (Chien et al. 1976). Each new double-stranded DNA is then a targeted during a new thermal cycle and thus the exponential amplification of the specific DNA sequence is achieved. The amplified product is then separated by gel electrophoresis and visualized by staining with fluorescent ethidium bromide. This type of conventional or endpoint PCR, although sensitive and specific under optimized conditions, is time consuming and labour intensive due to postamplification steps, not sensitive enough to measure the accumulated DNA copies accurately, and can only provide a qualitative result. Nevertheless, PCR techniques have expedited the process of pathogen detection and in some cases, replaced traditional methods for bacterial identification, characterization, and enumeration in foods (McKillip

The development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time PCR as the method of choice for detection of *Salmonella* (Espy et al. 2006). This method combines amplification and detection stages of the process so that nucleic acid amplification is monitored and recorded continuously hence eliminating the need for post-amplification steps such as gel electrophoresis. The detection of PCR products is accomplished via the generation of a fluorescent signal by any of the commercially available chemistries for realtime PCR: TaqMan® (Applied Biosystems®), Molecular Beacons, Scorpions®, and SYBR®

The simplest approach involves the use of the intercalating fluorescent dye SYBR® Green. This fluorogenic dye exhibits little fluorescence when in solution, but emits a strong

they require low technical expertise, and minimal capital expenditure.

**4. Molecular methods** 

and Drake 2004).

**4.2 Real-time PCR** 

Green (Molecular Probes), among others.

**4.1 Polymerase chain reaction (PCR)** 

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 indicating the presence of target antigen in the sample.

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 regulatory requirements.

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 detects fluorescence.

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

#### **3.3 Lateral flow immunoassays**

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 during the reaction because of denaturation or degradation of the capture antibody and it is likely that detection antibody or enzyme-conjugated antibody may also bind non-specifically to denatured capture antibody. Commercially available lateral flow immunoassays for the detection of *Salmonella* include: DuPont™ Lateral Flow System *Salmonella*, Singlepath *Salmonella* (Merck), Reveal® *Salmonella* lateral flow (Neogen), VIP Gold (BioControl), and RapidChek® SELECT (SDIX). Recently, serotype specific lateral flow immunoassays for the detection of *S.* Enteritidis have also been introduced to serve the egg and poultry industry such as RapidChek® SELECT *S.* Enteritids (SDIX) and Reveal *S.*  Enteritidis (Neogen). In general, these types of immunoassays are ideally suited where a low testing throughput is expected. The implementation of these tests is beneficial in that they require low technical expertise, and minimal capital expenditure.
