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Detection methods :!: Rapid methods, critical microbiological limits (5a)

- - - - Latex agglutination test (identification) - LAT:
         Latex particles coated with antibody (raised in e.g. rabbits)
         Immunochemical reaction between antibody and antigen results in agglutination
      - Enzyme Linked ImmunoSorbent Assay (ELISA)
        ‘Sandwich’-ELISA
      - VIDAS
        1) Antibody coated tip
        2) Salmonella antigen is bound
        3) The immuno complex is detected by anti-Salmonella antibody, conjugated to an enzyme
        4) The bound enzyme catalyses the added substrate
        5) The product is released in the test strip and fluorescence is measured by the VIDAS scanner
      - Immunological methods: Concentration
        Immuno-magnetic concentration
        ● Dynabeads (followed with PCR, ELISA, plating)
        ● PickPen (push-out magnet)
      - VIDAS ® up (Recombinant Phage Protein technology)
        Only use of the recombinant protein from the tail
        ● Most specific part of the phage
        ● Safe technology, no risk of ‘living’ phages
        ● Very specific and sensitive
    - - PCR -Polymerase Chain Reaction: multiplication of DNA sequence
         Identification of microorganisms: multiplication of 16S rDNA gene sequence
        ● 16S part of the ribosome
        ● Most species have subtle differences in sequence (biological signature)
      - Detection of multiplied DNA
        ● Gel analysis: negative charge of DNA allows migration
        ● Microtiter plate detection
         Disadvantage: qualitative
      - RFLP
        16S rDNA universal primers
        Extra step needed to identify microorganism
        ● PCR-Restriction Fragment Length Polymorphism:
        Cutting of 16S rDNA sequence into fragments using a restriction enzyme
        ● DNA fragments are separated on length by gelelectroforesis
        Band pattern specific for each micro-organism
      - Real time PCR: semi quantitative
        ● Monitoring of PCR reaction during amplification
        ● Fluorescent probe (SybrGreen) binds to amplified DNA
        ● Sensitive: 5-500 cfu/ml, depending on matrix
      - Microarray
         DNA-DNA microarray
         DNA amplification
         Immobilization of specific probes to a solid phase: multitarget
         Hybridization with fluorescent labeled DNA
         Identification
      - RNA-sequencing
         Expression of genes
         mRNA -> cDNA
         Sequencing of cDNA
         Absolute abundance of transcripts