The Rapid Identification of Microorganisms

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
  • Salmonella enterica
• to identify the biological agent such as
  • anthrax
  • smallpox
  in a possible terrorist attack so that appropriate measures can be taken quickly.

Methods

1. Culturing

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

2. 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

3. Immunoassays

Use a method that exploits the specificity and sensitivity of the reaction between antigen and antibodies.

Takes 15 minutes or longer.

4. 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)

• 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.

    Discussion of calcium ions as a second messenger.

• 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.

• 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.