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All PCR & qPCR questions

Question 9 of 9 Advanced Practical

Design a dilution and spike-in experiment to detect PCR inhibition in a difficult sample matrix.

Short answer

Use serial sample dilution together with a fixed exogenous spike to separate inhibition from low target concentration, while controlling the recovery of extraction and reverse transcription.

Why

A genuine target dilutes predictably, whereas inhibitory compounds often become less influential as the sample is diluted. A fixed spike added after extraction tests the amplification/RT matrix. A spike added before extraction can also reveal extraction recovery, though its chemistry must resemble the analyte.

How to work it through

  1. Prepare neat, 1:2, 1:5 and 1:10 matrix dilutions alongside water or clean-matrix standards.
  2. Add identical copies of a distinct spike to every diluted aliquot, and separately test endogenous target.
  3. Compare spike Cq/quantities across matrix concentration with the spike-only control.
  4. Include extraction blanks, positive controls, no-template controls and appropriate spike timing.
  5. Predefine acceptance limits for spike recovery and the assay quantitative range.

An example

A neat stool extract suppresses the spike. The spike response normalizes at 1:5 dilution, while dilution-corrected target quantity stabilizes. This supports matrix inhibition, provided recovery and low-copy uncertainty are acceptable.

Where the answer stops holding

Dilution also lowers target molecules and can push samples below the LOD. An RNA spike placed only after RT will not detect RT inhibition. A spike that competes with target in multiplex PCR can itself alter amplification.

A strong answer covers

  • Use serial sample dilution together with a fixed exogenous spike to separate inhibition from low target concentration, while controlling the recovery of extraction and reverse transcription
  • Prepare neat, 1:2, 1:5 and 1:10 matrix dilutions alongside water or clean-matrix standards

Common mistakes

  • Calling an undetected target truly negative without an inhibition control
  • Correcting all Cq values with an assumed universal efficiency