Molecular Diagnostics and Emerging Technologies
Core concepts
Understand nucleic acid amplification: PCR (real-time, multiplex), transcription-mediated amplification, and isothermal methods. Know the difference between qualitative (presence/absence) and quantitative (viral load) assays, and the role of sequencing (Sanger, next-generation sequencing) for resistance and outbreak typing.
Assay performance
Know sensitivity, specificity, positive and negative predictive value, and how prevalence affects PPV. Understand contamination control (unidirectional workflow, UNG), internal controls, and the limits of detection. Emerging tools include whole-genome sequencing for outbreak tracing, metagenomic sequencing, and CRISPR-based detection.
Traps
- Confusing analytic sensitivity with clinical sensitivity.
- Forgetting that PPV falls when prevalence is low, even for a highly specific test.
- Assuming a positive PCR always means active infection (contamination, colonization, or residual DNA).
- Overlooking the need for internal controls to detect inhibition.
Worked rule of thumb
A test with 99% specificity in a low-prevalence population (1%) yields a PPV near 50%: of 100 positives, about 50 are false positives. Always interpret a positive result in clinical context and confirm when the pretest probability is low.