Molecular Diagnostics: Amplification, Quantification, Contamination Control, and Sequencing
The molecular section covers amplification chemistry, quantification, contamination control, sequencing, and the regulatory status of the test itself.
PCR fundamentals
Cycling comprises denaturation near 95 °C, annealing near 55–65 °C, and extension near 72 °C with Taq polymerase; Mg2+ is a required cofactor, primers define specificity, and dNTPs are the building blocks. Real-time PCR measures product each cycle, and the cycle threshold (Ct) is inversely proportional to starting template.
Quantification
At 100% efficiency, one log10 of copy number equals about 3.32 cycles; an efficiency of 90–100% is acceptable, corresponding to a standard-curve slope near −3.32. Melting curve analysis distinguishes specific product from primer dimers in SYBR green assays, while TaqMan probes provide sequence-specific detection.
Quality and contamination
Use a unidirectional workflow (reagent preparation, extraction, amplification), no-template and negative extraction controls, and uracil-N-glycosylase in assays vulnerable to amplicon carryover. False positives from contamination and false negatives from inhibitors (hemoglobin, heparin) are the dominant failure modes.
Sequencing and variants
Sanger sequencing confirms single variants; NGS provides breadth but lower sensitivity at low variant allele fractions. The limit of detection is the lowest concentration reliably detected, conventionally at 95% probability.
Worked example
Sample A has Ct 22 and sample B has Ct 28 at 100% efficiency. ΔCt = 6, and 6 ÷ 3.32 ≈ 1.8 log10, so sample A contains roughly 63-fold more target than B. That is a quantitative statement, not merely that A is positive.
Traps that cost marks
Reporting a viral load when the internal control failed. Comparing Ct values across different assays or platforms. Calling a result below the limit of detection "zero" rather than "not detected".