Preventing contamination is key 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 critical aspects of preventing erroneous results.
Large quantities of target molecules from previously amplified materials are significant potential sources of contamination. Amplicons may contaminate work surfaces, air spaces, pipettes, and reagents. Scrupulous techniques are one aspect of preventing contamination; another is the physical separation of specific process steps.
Ideally, all molecular work will occur in distinct areas designated for critical aspects of the workflow, with each region having its dedicated equipment, especially pipetting equipment. The three designated areas are:
- Clean area: Master mixes and other reagents are prepared without specimen material. Protection from aerosol contamination is a key consideration in this area. Two ways to address this need are to use a dead air box with ultraviolet (UV) lighting for decontamination or a separate area with controlled airflow.
- Specimen preparation and extraction area: Separated from the area where amplification and detection of the amplified product occur to prevent contamination of samples with amplicons of previously processed specimens.
- Amplification, detection, and identification of the amplified product: Ideally, this area would be separated and enclosed.
To some extent, introducing 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 and a defined workflow.
