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Challenges for Implementation: Separation of Key Activities

Prevention of contamination is a very key consideration when introducing molecular methods into the routine diagnostic laboratory setting. Because amplification methods are so sensitive, the incidental introduction of even a few copies of exogenous nucleic acid can lead to false positive results. Both physical design and process controls are key aspects of preventing erroneous results.

Significant potential sources of contamination are the large quantities of target molecules from previously amplified materials. Amplicons may contaminate work surfaces, air space, pipettes, and reagents. Scrupulous technique is one aspect of preventing contamination; another is the physical separation of specific steps of the process.

Ideally, all molecular work will take place in distinct areas designated for key aspects of the workflow, with each area having its own dedicated equipment (especially pipetting equipment). The three designated areas are:

  1. Clean area: Where master mixes and other reagents are prepared in the absence of any specimen material. In this area, protection from aerosol contamination is a key consideration. Use of a dead air box with ultraviolet (UV) lighting for decontamination, or a separated area with controlled airflow, are two ways to address this need.
  2. Specimen preparation and extraction area: Separated from the area where amplification and detection of amplified product occurs, in order to prevent contamination of samples with amplicons of previously processed specimens.
  3. Amplification, detection, and identification of the amplified product: Ideally, this area would be both separated and enclosed.

To some extent, the introduction of platforms that utilize automated specimen processing equipment and/or closed amplification and detection systems mitigates stringent separation and space requirements. Good practice, however, would always include designated spaces for each activity, as well as a defined workflow.