**6. VIDAS**

VIDASTM (BioMérieux) is an automated enzyme-linked fluorescent assay (ELFA) method based on the detection of *Salmonella* by using specific antibodies coated on the inner surface of a tip-like disposable pipette which is introduced into the VIDAS system along with the VIDAS *Salmonella* strip containing the boiled *Salmonella* culture.

VIDAS Immuno-concentration *Salmonella* (ICS) is a fully automated method for the concentration of *Salmonella* from foods. It replaces traditional selective enrichment procedures with an automated immunological capture and specific release process (Yeh et al., 2002). The method is based on multistage reaction. The kit contains so called reagent stripes, that is a set of wells with reagents sealed tightly inside, and pipettes, which inner sides are coated with antibodies against specific antigens. The amount of 500 µl of the sample after selective enrichment stage on RVS is introduced to the first well and a strip is placed in the immunoanalyser chamber. Reaction suspension is cyclically pulled up and down by pipettes. A pipet tip-like device, the solid-phase receptacle (SPR) serves as the solid phase as well as the pipet for the assay. The SPR is coated with anti-*Salmonella* antibodies absorbed on the surface. A final enzymatic step releases the captured *Salmonella* into a well. Detection of *Salmonella* antigens is based on enzyme-linked fluorescent immunoassay

performed in the automated VIDAS instrument. ASPR serves as the solid phase as well as the pipet for the assay. The SPR is coated with a cocktail of highly specific monoclonal

Detection of *Salmonella* spp. Presence in Food 407

infecting bacteria. Phages are extremely host-specific. Most bacteria can be infected by particular phages and it is common that a given phage can recognize and infect only one or a few strains or species of bacteria (Hagens & Loessner, 2007). The specificity of these phages is partly mediated by tail-associated proteins that distinctively recognize surface molecules of susceptible bacteria (Kretzer et al., 2007). Bacteriophages or proteins of bacteriophages have been included in various ways in detection methods for pathogens

Although there is a need to perform a collaborative study to further evaluate the methods before it can be concluded that their performances are equal, both the PCR and the ELFAbased assay could provide a rapid and user-friendly screening method for detection of *Salmonella* in food (Uyttendaele et al., 2003; Priego et al., 2009; Kumar et al., 2008; Szabo et

Numerous and diverse alternative methods for microbial analysis of foods, as described above, exist (Bohaychuk et al., 2005; Wu, 2008) . They are currently brought to the market by various suppliers in a variety of formats as a result of recent developments, particularly in the field of biotechnology, microelectronics and related software development. Many of them have been proven to be equivalent to the "golden standard" reference methods with

> **Selective enrichment**

> **Selective enrichment**

**Detection** 

**extraction PCR Detection**

**zation Detection**

**Fixation Hybridi-**

 **Day 1 Day 2 Day 3 Day 4 Day 5** 

**Detection**

**Plating Detection Confirma-**

**tion** 

regard to the performance characteristics of the method.

Fig. 2. Detection of *Salmonella* spp. from food.

(Favrin et al., 2003)

al., 2008)

**ISO** 

**VIDAS SLM** 

**VIDAS SLMX/ UP** 

**Preparation** 

**Non selective pre-enrichment** 

**DNA** 

**PCR** 

**FISH** 

**7. Conclusion** 

antibodies. All of the assay steps are performed automatically by the VIDAS instrument. For the detection of *Salmonella by* VIDAS SLM, the sample is inoculated into lactose broth and incubated for 18 h at 37°C (non-selective pre-enrichment). Subsequently, 0.1 ml of this medium is inoculated into Rappaport–Vassiliadis broth and 1 ml into tetrathionate broth, and then incubated for 8 h at 42°C and 8 h at 37°C, respectively. Then, 1 ml of each broth is inoculated separately into 10 ml of M-broth and incubated at 42°C for 18 h. Finally, 1 ml of each broth is placed in a tube, which is heated for 15 min at 100°C. Following preenrichment, immuno-concentration, and postenrichment of test portions, an aliquot of the boiled test suspension is placed into the reagent strip and is cycled in and out of the SPR for a specific length of time. *Salmonella* antigens, if present, bind to the monoclonal antibodies coating the interior of the SPR. All other unbound material is washed away. Antibodies conjugated with alkaline phosphatase are cycled in and out of the SPR, binding to any *Salmonella* antigen bound to the SPR wall. The final wash step removes unbound conjugate. The substrate, 4-methyl umbelliferyl phosphate, is converted by the enzyme on SPR wall to the fluorescent product, 4-methyl umbelliferone.

The intensity of fluorescence is measured by the optical scanner in VIDAS. The fluorescence intensity is measured twice at 450 nm. The first result is related to the background, the second it the value after incubation of the substrate with enzyme. Based on that, the apparatus calculates the result of the test and interprets it as a positive or negative one. RFV (Relative Fluorescence Value) is calculated as the difference between the sample and background fluorescences. The printed report contains the RFV value of the sample, RFV value of the standard, and test value (TV), which is a quotient of the sample value and standard value. A result was interpreted by the apparatus as positive, if TV ≥ 0.23, while as negative if TV ≤ 0.23. Results are interpreted after the test values and control are compared to thresholds stored in the computer. A positive result requires confirmation with classical culture methods, that is streak plating on two plates with selection growth medium. For confirmation, previously prepared and stored under cold conditions broth culture of the investigated sample is used.

Based on the comparative studies with the standard plate method, it can be concluded that the VIDAS system can be use to get fast results; however, because these results can be false positive then they have to be confirmed by culture method (Yeh et al., 2002; Zadernowska et al., 2010; Walker et al., 2001)

Problems with detection of some *Salmonella* spp. serotypes were observed during detection by the immunoenzymatic method. This may be caused by weak binding of antibodies, which is confirmed by results obtained by other authors. Vitek Immunodiagnostic Assay System (VIDAS, BioMérieux) are currently used in the meat and poultry processing industries (Maciorowski et al., 2006). Several validation studies have been reported that the detection rate of VIDAS systems were comparable to that of culture method (Yeh et al., 2002) and real-time PCR (Uyttendaele et al., 2003) for detecting of *Salmonella* in food.

**VIDAS Salmonella Xpress** (VIDAS SLMX) is most rapid method for the detection of *Salmonella than VIDAS SLM*. The results are obtained as little as 17 hours. The method has been simplified with a single enrichment in buffer peptone water and just one pipeting step. A broad incubation time of 16 to 24 hours simplifies the laboratory workflow, enabling all samples to be processed as they arrive during the day. This test is validated for raw beef and veal meats (including frozen), not flavoured and pasteurized egg products.

**VIDAS UP Salmonella** is a new generation of assay based on **the latest technology**  available for pathogen screening: Phage recombinant protein. Bacteriophages are viruses infecting bacteria. Phages are extremely host-specific. Most bacteria can be infected by particular phages and it is common that a given phage can recognize and infect only one or a few strains or species of bacteria (Hagens & Loessner, 2007). The specificity of these phages is partly mediated by tail-associated proteins that distinctively recognize surface molecules of susceptible bacteria (Kretzer et al., 2007). Bacteriophages or proteins of bacteriophages have been included in various ways in detection methods for pathogens (Favrin et al., 2003)

Although there is a need to perform a collaborative study to further evaluate the methods before it can be concluded that their performances are equal, both the PCR and the ELFAbased assay could provide a rapid and user-friendly screening method for detection of *Salmonella* in food (Uyttendaele et al., 2003; Priego et al., 2009; Kumar et al., 2008; Szabo et al., 2008)
