Molecular Diagnostics and Emerging Technologies
Molecular Diagnostics tests nucleic acid extraction, amplification, detection, contamination control, and result interpretation. The exam focuses on PCR principles, Ct values, controls, and the limits of molecular tests.
Core rules
- NAAT detects nucleic acid, not viable organisms. A positive result can persist after organism death; a negative result does not exclude infection if the sample is poor or inhibitors are present.
- Real-time PCR measures fluorescence each cycle. Ct is the cycle at which signal crosses threshold; lower Ct means more target nucleic acid.
- Extraction removes inhibitors and concentrates target. Inhibition causes false-negative or delayed Ct; internal controls detect it.
- Contamination causes false positives. Use unidirectional workflow, separate pre- and post-amplification areas, aliquoted reagents, and negative controls.
- Sequencing and genotyping identify resistance mutations and variants. Assay limits of detection and coverage must match the clinical question.
Worked example
Two samples run on the same real-time PCR assay: sample A Ct 22, sample B Ct 35. Interpretation: sample A has a higher target load than sample B, assuming equivalent efficiency and valid controls. Reasoning: fewer amplification cycles are needed to reach threshold when starting target is abundant. A Ct of 35 near the assay limit should be interpreted cautiously and correlated with clinical findings.
Traps
A negative PCR does not rule out infection if the specimen was collected poorly or contains inhibitors. Contamination from amplicons can produce false positives. Do not compare Ct values across different assays without validation. Do not report a genotype without confirming the assay’s target coverage.