Validation of New Molecular Tests for Microbiological Testing of Clinical Specimens

Access to the most rapid, sensitive, and specific testing is of critical importance to the microbiology laboratory. However, the tests of interest are constantly changing, as new pathogens emerge, as new antimicrobial markers and virulence genes are discovered, and as new technologies are utilized in the detection and characterization of infectious diseases. The tests available from commercial sources may not fit the exact needs of a laboratory, or the tests may not be FDA approved for patient testing. These factors have led many laboratories to develop and validate new molecular assays for their current patient testing needs. The task of developing and validating these tests is considerable, and the laboratory must learn to navigate the field of molecular testing. This article describes the process of assay development and validation of real-time PCR and nucleic acid sequencing assays; included are descriptions of important considerations, such as the PCR workflow, inhibition testing, proper use of controls, and multiplexing.