Molecular Diagnostics
Molecular Diagnostics
This domain tests amplification chemistries, interpretation of real-time PCR data, sequencing workflows, and contamination control - the controls that make a molecular result defensible.
Core rules
- PCR cycles: denaturation (~95 °C), annealing (~50–65 °C, primer-dependent), extension (~72 °C for Taq). Specificity comes largely from the primers.
- In real-time PCR, the Ct is the cycle at which fluorescence crosses the threshold. Lower Ct means higher target concentration. A one-cycle difference corresponds to a twofold change at 100% efficiency.
- Amplification efficiency is derived from the standard curve slope: E = 10^(−1/slope) − 1; a slope of −3.32 gives approximately 100% efficiency.
- Sanger sequencing yields one read per amplicon and is suited to known hot spots; NGS provides depth and detects low-level variants but requires bioinformatic filtering.
- Contamination control includes unidirectional workflow, aliquoted reagents, uracil-N-glycosylase, and negative extraction and no-template controls.
Worked example
A patient sample has Ct 30.0; the calibrator at 10^5 copies/mL has Ct 24.0. With a slope of −3.32 (100% efficiency), each cycle is a factor of 2, so six cycles represent a 64-fold difference: 10^5/64 ≈ 1.6 × 10^3 copies/mL. A negative no-template control is required before this value is reported; a signal in the NTC invalidates the run.
Traps that cost marks
- Reporting a result when the internal control failed (possible inhibition).
- Interpreting Ct without the standard curve or efficiency.
- Confusing a detected variant with a confirmed diagnosis in the absence of clinical correlation.
- Overlooking amplicon contamination as the cause of a late-Ct positive.