qPCR
Also called: real-time PCR · quantitative PCR
Related: PCR · RT-PCR · Multiplex PCR · Digital PCR · Nucleic Acid Quantification
1. Principle
Real-time PCR monitors amplification each cycle via fluorescent reporters (intercalating dyes or sequence-specific probes), yielding a cycle threshold (Ct/Cq) that reflects starting template amount.
2. Step-by-Step Procedure (conceptual)
- Extract NA (DNA Extraction / RNA Extraction ± RT).
- Assemble master mix with primers ± TaqMan/Molecular Beacon/FRET probes or SYBR-type dye.
- Run thermocycler with real-time optics; include positive, negative, and internal control.
- Set thresholds; read Ct; optional melt-curve (dye assays) for specificity.
- Interpret qualitatively (detected/not) or quantitatively with standard curve / digital calibrators.
3. Interpretation
- Lower Ct ≈ more target (semi-quantitative; assay-dependent).
- Undetected: Below LOD — not proof of absence.
- Pitfalls: Probe mismatch from variants; inhibition (use internal control); high Ct near LOD → confirm/repeat; colonization still possible.
4. Clinical Use Cases
- Viral load / qualitative ID (SARS-CoV-2, influenza, HSV CSF, etc.).
- Bacterial/resistance markers (mecA, vanA, toxin genes) on validated panels.
- Ortho/transplant viral monitoring.
5. Comparison with Other Methods
| Method | Pros | Cons |
|---|---|---|
| qPCR | Fast, closed-tube, quantitative | Limited plex in many formats |
| Endpoint PCR + gel | Simple | Contamination risk, not quantitative |
| Digital PCR | Absolute counts, rare variants | Cost, lower throughput |
| Isothermal NAAT | Simple hardware | Multiplex/quant limits vary |
6. Mnemonic / Visual Aid
Ct down, load up — remember it’s assay-specific, not a universal viral-load unit.
Inventor / History Link
- Builds on Kary Mullis PCR + fluorescence chemistry
Active Recall
- Probe vs dye chemistry — which needs melt curves more often for specificity?
- What does an internal control failure suggest?
- Why can’t you compare Ct across different kit brands blindly?