FLEISCHWIRTSCHAFT international_04_2018

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22 Fleischwirtschaft international 4_2018 Hygiene Fig. 1: Rapid agglutination assays show a negative result, when the original color retains; a positive result is indicated by distinct color agglutination. Detection assures food safety State-of-the-art microbiological tests help to reduce the risk of human illness cases – Part 2 Consumption of minimally processed RTE-meats increases the risk of human illness cases since these products generally do not receive any further treatment before consumption. All raw meat can have some level of microbial contamination present. Modern microbiological methods are able to deliver detailed insight of a microbial contamination of meat or a meat product. By Akhilesh K. Verma, A. Prajapati and Pramila Umaraw Conventional or traditional methods for detecting microorganisms in meats are based on the incorporation of samples into a nutrient medium in which microorganisms can multiply, thus providing visual confirmation of their growth. These conventional test methods are simple, easily adaptable, very practical, and generally inexpensive. More and more new technolgies find their way into labs specialized in meat testing. Immunological detection methods These techniques are based on specific body-antibody reactions. Rapid agglutination assays The test is based on visible clumps shown by organisms in presence of specific antibodies. These are rapid and easy to perform. Sensitivity and specificity may vary from organism to organism and the antibodies used which may lack specificity due to non-specific agglutination of some other organisms (CHEES- BROUGH and DONNELLY, 1996). These tests are primarily used for screening of suspected bacterial colonies after culture isolation from selective agar plates. Latex agglutination assay tests are widely used for the rapid detection of Salmonella in which a drop of colony suspension or enrichment broth is mixed with the latex reagent. The latex remains in the homogenous suspension and retains its original color in a negative test. A positive result is indicated by distinct color agglutination against an altered background (Fig. 1). Lateral flow devices Lateral flow devices (LFD) are typically comprised of a simple dipstick made of a porous membrane that contains colored latex beads or colloidal gold particles coated with detection antibodies targeted toward a specific microorganism. The particles are found on the base of the dipstick, which is put in contact with the enrichment medium (POSTHUMA-TRUMPIE et al., 2009). If the target organism is present it will bind with the colored particles. This conjugated cell/ particle moves by capillary action until it finds the immobilized capture antibodies. Upon binding with these, it forms a colored line that is clearly visible in the device window, indicating a positive result (BETTS and BLACKBURN, 2009). LFD also requires previous enrichment. Results are often available within 24 h. False positive results may be observed during the reaction because of denaturation or degradation of the capture antibody and it is likely that the detection antibody or the enzyme-conjugated antibody may also bind the non-specifically to denatured capture antibody. The technique is extremely simple to use and easy to interpret, requires no washing or manipulation, and can be completed within 10 min after culture enrichment (ALDUS et al., 2003). ELISA and ELFA The Enzyme-Linked Immunosorbent Assay (ELISA) is a biochemical technique that combines an immunoassay with an enzymatic assay. An antibody bound to a solid matrix is used to capture the antigen from enrichment cultures and a second antibody conjugated to an enzyme is used for detection. The enzyme is capable of generating a product detectable by a change in color, or in the case of Enzyme-Linked Fluorescence Assay (ELFA) in fluorescence, which allows for indirect measurement using spectrophotometry (or fluorometry for ELFA) of the antigen present in the sample (microorganism or toxin) (COHEN and KERDAHI, 1996; JASSON et al., 2010). The success of an immunoassay depends on the specificity of the antibody. The limit of detection for immunoassays is approximately 10 4 –10 5 CFU/g depending on the type of antibody and its affinity for the corresponding epitope, which means that one or two previous enrichment stages are always required (JASSON et al., 2010). High limits of sensitivity of >10 5 CFU/mL (COX, 1988), cross reactivity and changes to antigens due to acetylation and changing recognition by assay antibodies (KIM and SLAUCH, 1999) are the some disadvantages of ELISA methods. Molecular detection methods These techniques base on specific molecular reactions. DNA based methods The ability of two single stranded DNA-molecules in vitro, under the right conditions, to form double stranded DNA by specific base pairing, i.e. to hybridize, is the basis of all DNA-based detection methods (Fig. 2). While many different methods based on specific base
Fleischwirtschaft international 4_2018 23 Hygiene pairing have been developed (WOL- COTT, 1992), three methods, i.e. colony hybridization (GRUNSTEIN and HOGNESS, 1975), single phase, liquid hybridization assays (CURI- ALE, KLATT and MOZOLA, 1990), and PCR (SAIKI et al., 1988) are in particular relevant for the detection of bacteria in meats. Nucleic acid-based technologies A DNA probe is a short (14–40 bases) single-stranded sequence of nucleotide bases that will bind to specific regions of single-stranded target DNA sequences; the homology between the target and the DNA probe results in stable hybridization. Hybridization is monitored by labeling probes, by attachment to or incorporation into the probe, with compounds that can be detected visually or chemically. Isotopes such as P 32 , enzymes such as alkaline phosphatase or horseradish peroxidase (HRP) and fluorescently labeled compounds such as fluorescein isothiocyanate are often linked to the probe via a chemical linkage. Colony hybridization In the colony hybridization assay, a meat sample or an enrichment culture is spread on a nylon or paper filter and incubated until visible colonies are present. These are processed to destroy the cells, remove cell substances and leave fixed single-stranded DNA for hybridization, usually by treatment with detergents and alkali or by microwave treatment as in the method of DATTA et al. (1987). A radio, enzyme or hapten labeled DNA-probe, which constitutes parts of the target DNA-sequence, is applied to hybridize to the sample DNA. Each signal on the filter corresponds to a positive identification, and upon direct spreading of samples, colony hybridization is a quantitative method. Since oligonucleotides can be synthesized in vivo, short, defined and synthetic oligonucleotides were quickly introduces into food microbiology (HILL et al., 1985) and are now by far preferred as probe molecules. An essential step in colony hybridization is the separation between labeled probe molecules bound to the target DNA and those that bind non-specifically to the filter. This is obtained by stringency washing, which is only possible when the target DNA is fixed to a solid support, i.e. the filter. Isolates of Bacillus cereus from traditional Indian foods were detected by colony hybridization using the PCR-generated phospholipase (PL-1) method. In all, 29 isolates picked up by the probe were confirmed as B. cereus by conventional cultural and biochemical characteristics (RADHIKA et al., 2002). A genetic probe was used to identify and enumerate virulent Yersinia enterocolitica colonies in eleven artificially contaminated foods. Efficiency of enumeration, determined by autoradiography after DNA colony hybridization, ranged from 66% to 100% (average 86%) and was influenced by the number of indigenous bacteria (JAGOW and WE, 1986.) A plasmid containing the cloned listeriolysin gene of Listeria monocytogenes was used as a probe to identify Listeria strains by DNA colony hybridization. Of the 150 Listeria strains and 16 non-Listeria strains examined, the probe hybridized only with L. monocytogenes. The technique was also used to enumerate L. monocytogenes in artificially contaminated foods (DATTA et al., 1993). JONES et al. (2009) examined for detection of Vibrios from postharvest-processed (PHP) oysters and each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus; here was 96% agreement between real-time and DNA colony hybridization. Fluorescent in-situ hybridization Fluorescent in-situ hybridization (FISH) with oligonucleotide probes directed at rRNA is the most common method among molecular techniques not based on PCR. The probes used by FISH tend to be 15–25 nucleotides in length, and are covalently labeled at their 5’ end with fluorescent labels. After hybridization, the specifically stained cells are detected using epifluorescence microscopy (WAGNER et al., 2003). The detection limit of this technique is around 10 4 CFU/g. Following pre-enrichment results can be achieved in 3h. Whole fixed cells can be identified and counted directly by fluorescent in-situ hybridization (FISH), allowing different microbial species in mixed cultures to be distinguished. Detection of specific food contaminants can be achieved by FISH coupled with FCM (FLOW–FISH). For example, rapid and sensitive FLOW–FISH methods have been used for detecting and enumerating Cornybacteria and Lactobacillus brevis in seafood products and lactic acid bacteria used as starter cultures. This technique can provide a rapid Fig. 2: Three DNA-based methods are in particular relevant for the detection of bacteria in meats.
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