Nested PCR
Related: PCR · qPCR · Broad-Range 16S PCR
1. Principle
Nested PCR uses two successive amplifications: an outer primer pair first, then inner primers targeting within that amplicon — boosting sensitivity and specificity at the cost of higher contamination risk.
2. Step-by-Step Procedure (conceptual)
- Round 1 with outer primers (fewer cycles).
- Transfer diluted product (or use single-tube nested designs) into round 2 with inner primers.
- Detect second-round product.
- Extreme contamination control: separate rooms, unidirectional workflow, UNG/dUTP where applicable.
3. Interpretation
- Ultra-sensitive — interpret low-level positives cautiously.
- Pitfalls: Amplicon carryover false positives; modern labs often replace classical open nested PCR with real-time or closed nested systems.
4. Clinical Use Cases
- Historical / specialized detection of low-burden pathogens (some parasites, mycobacteria research assays).
- Semi-nested variants still appear in reference protocols.
- Prefer qPCR or Digital PCR when validated alternatives exist.
5. Comparison with Other Methods
| Method | Sensitivity | Contamination risk |
|---|---|---|
| Nested PCR | Very high | High if open-tube |
| qPCR | High | Lower (closed) |
| Digital PCR | High for rare targets | Moderate |
6. Mnemonic / Visual Aid
Outer door → Inner door — two locks, two chances to track dirt inside.
Active Recall
- Why is nested PCR contamination-prone?
- What modern methods often replace it?
- What does “semi-nested” mean?