Real-time PCR (qPCR)

Principle

• Amplification is monitored during each cycle by measuring fluorescence in a closed tube; no gel is needed.
• SYBR Green: binds any double-stranded DNA; cheap but non-specific, so a melt curve is used to confirm a single product.
• TaqMan (hydrolysis) probe: carries a reporter and quencher; the 5'→3' exonuclease activity of Taq cleaves the bound probe, freeing the reporter to fluoresce.
• Other chemistries: molecular beacons, FRET (hybridisation) probes, Scorpion primers.
• Multiplexing uses probes with different dyes in one tube.

Results

• Ct (Cq): cycle at which fluorescence crosses the threshold. Lower Ct means more starting target.
• At 100% efficiency, a 10-fold difference in starting quantity equals about 3.3 cycles.
• Standard curve: slope of –3.32 = 100% efficiency; acceptable efficiency 90–110% (slope about –3.6 to –3.1) with R² ≥0.98.
• Efficiency = 10^(–1/slope) – 1.
• Absolute quantification uses a standard curve (e.g. viral load in IU/mL); relative quantification uses a reference gene and the 2^–ΔΔCt method.

Quality control and pitfalls

• Include no-template controls, positive controls and internal controls in each run.
• A delayed internal-control Ct suggests inhibition; an invalid IC with a negative target cannot be reported as negative.
• With SYBR Green, primer dimers can give late signals in no-template controls; check the melt curve.
• A passive reference dye (e.g. ROX) normalises well-to-well variation on some instruments.
• Ct values are not directly comparable between different assays or platforms.
• Closed-tube format lowers contamination risk, but good workflow is still required.

Clinical use

• Viral loads (HIV, HBV, HCV, CMV), respiratory pathogen panels, SARS-CoV-2, BCR-ABL1 monitoring in CML, genotyping and SNP detection.