Ph. Eur. 2.6.21. Nucleic acid amplification techniques

This chapter sets general requirements, to be applied when NAT is specified in a monograph or agreed with regulators (e.g., plasma pool testing, virus safety). NAT encompasses target amplification and signal amplification.

NAT methods offer very high sensitivity and specificity, enabling detection of low-level microbial/viral nucleic acids in biological materials (e.g., plasma pools) when properly designed, controlled, and validated; EDQM provides reference standards and validation guidance for such uses.

Core implementation elements

• Contamination prevention: Segregated areas (master-mix, pre-PCR, closed amplification, open post-PCR), controlled flows, and decontamination procedures.

• Run controls (per assay set):

  • Negative control (matrix proven target-free).

  • Positive control (defined copy number, near the cut-off).

  • Internal control (same nucleic-acid type as the sample; ideally spiked before extraction to monitor extraction→detection and inhibition).

  • Appropriate calibration standards (traceable to reference material) to define quantitation thresholds in quantitative assays.

  • Result confirmation: Because of high sensitivity and contamination risk, repeat the entire procedure to confirm positives (preferably on a new aliquot), especially for borderline or unexpected positives, confirmation should be performed on a separate aliquot.

Validation

• Qualitative assays: Define the positive cut-off point (copies/volume detected in 95% of runs) and demonstrate specificity & robustness. Validation must include determination of the limit of detection (LOD) distinct from the positivity cut-off, and demonstration that inhibition controls reliably detect interference.

• Quantitative assays: Establish accuracy, precision, specificity, quantitation limit, linearity, range, and robustness; use suitable standards/materials. Participation in external quality assessment programs is recommended as part of ongoing assay performance verification.