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19.2G: The Rapid Identification of Microorganisms

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    5976
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    The Need

    • diagnosis of infection so that the appropriate treatment (e.g., an antibiotic) can be started.
    • testing of food to ensure that it is not contaminated with infectious organisms like E. coli O157:H7 and Salmonella enterica
    • to identify the biological agent such as anthrax and smallpox in a possible terrorist attack so that appropriate measures can be taken quickly

    Methods

    Culturing

    • The oldest and still most common.
    • For bacteria, spread samples on culture media and examine the resulting colonies for morphology and metabolic traits. For viruses, inoculate cultures of living cells.
    • Disadvantage: it make take several days to learn the results.

    Polymerase Chain Reaction (PCR)

    • Extract DNA from the sample and perform PCR.
    • Advantage: rapid (often less than an hour)
    • Disadvantage: overly sensitive to presence of contaminants

    Immunoassays

    Use a method that exploits the specificity and sensitivity of the reaction between antigen and antibodies. Takes 15 minutes or longer.

    Biosensors (CANARY)

    In the 11 July 2003 issue of Science, a team of scientists at the Lincoln Laboratory in the U. S. reported a new method of rapid identification that exploits living cells. They call their method CANARY (for Cellular Analysis and Notification of Antigen Risks and Yields)

    Their "biosensor" is a clone of B lymphocytes (B cells) that have been genetically engineered to express

    • a B cell receptor for antigen (BCR) selected to interact with an epitope on the suspected agent. The BCR on their clones is surface IgM.
    • aequorin, a protein extracted from the same jellyfish that produces green fluorescent protein.
      • Aequorin emits light when it is exposed to calcium ions (Ca2+).
      • One of the first events (within seconds) when BCRs bind to antigen is a rise in the level of calcium ions in the cytosol.

    Procedure:

    • Prepare the sample.
    • Mix - in separate wells - with B-cell clones each specific for a different suspected agent.
    • Place in a sensitive light detector.
    • If a clone has a BCR for an epitope present in the sample, that clone will emit light within a few seconds.

    Results:

    • highly sensitive: can detect as few as 50 bacteria or 500 virions
    • highly specific: can detect the agent even in the presence of related contaminating agents.
    • fast: time elapsed from sample preparation to signal from the light detector is often less than 5 minutes.

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