Molecular Contamination and Amplification Product Inactivation

Laboratory strategies for diagnosing and monitoring infections now routinely include nucleic acid amplification techniques (NAATs), in particular variations of the polymerase chain reaction. These molecular approaches represent a paradigm shift and can often carry diagnostic advantages over traditional culture-based methodologies. Nevertheless, molecular infectious disease testing is not without its caveats and challenges. Notably, the high sensitivity and target amplification nature of these tests can represent a double-edged sword, as it creates the theoretical potential for false-positive results due to carryover contamination from specimen-to-specimen or amplicon-to-specimen. Clinical laboratories that conduct such assays must be acutely aware of this risk, given the potential risk for patient harm that can be caused by spurious detections. The current chapter reviews the phenomenon of carryover contamination in the molecular environment, as well as specific strategies that can be implemented to mitigate the risk as part of a molecular quality assurance program. These measures include both incorporation of biochemical techniques for amplification product inactivation—such that generated amplicons are no longer able to serve as detectable targets for future reactions—and more general practices in assay design and laboratory workflow. Special consideration is given to carryover contamination in the context quantitative assays, multiplex/syndromic molecular platforms, and broad-range PCR technologies.