4. What are the causes of abnormal amplification curves and their solutions?
2026-03-24 11:18
A standard real-time fluorescent PCR amplification curve includes baseline phase, exponential amplification phase, linear amplification phase and plateau phase, showing a typical S-shape. Analyze irregular curves according to specific conditions:
- Sloped rising curve: Malfunction or loose heated lid leading to inaccurate temperature control and liquid evaporation. Solution: Replace the heated lid or fasten it tightly;
- Discontinuous curve: Baseline end exceeds Ct value, usually caused by high template DNA concentration (CT<15) with baseline still set at 3-15 cycles (including partial amplification signals and suppressing the curve). Solution: Shorten the baseline end to 4 cycles before the Ct value and reanalyze the data;
- Linear amplification curve: Partial degradation of probes in some samples. Solution: Replace reagents for retesting;
- Plateau phase decline: Loose reaction tube cap. Solution: Check and fasten the PCR reaction tube cap tightly after covering.
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