Table 2.
Comparison of guidelines and documents related to qPCR on acceptance criteria of each items.
| Item | 1) MIQE | 2) Checklist | 4) FDA Guidance | 5) GMO |
|---|---|---|---|---|
| LOD | 95% probability | ≥95% | 95% confidence | ensuring ≤5% false negative results |
| LOQ | – | Item only (Criteria not described) | ≤50 copies of vector/μg genomic DNA | less than 1/10 of the value of the target concentration with CV ≤25% |
| Accuracy (Trueness) | Item only (Criteria not described) | Item only (Criteria not described) | – | RE within ±25% |
| Precision | Item only (Criteria not described) | Item only (Criteria not described) | – | CV ≤25% |
| PCR efficiencya | Item only (Criteria not described) | 90–110% (−3.1 ≥ slope ≥ −3.6) | – | 90–110% −3.1 ≥ slope ≥ −3.6 |
| Correlation coefficient: r2 | Item only (Criteria not described) | 0.99 ≤ r2 ≤ 0.999 | – | r2 ≥ 0.98 |
MIQE, The MIQE Guidelines [89]; Checklist, Checklist for optimization and validation of real-time PCR assays [90]; FDA Guidance, Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for Delayed Adverse Events [91]; GMO, Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing [92]; CV, coefficient of variation; LOD, limit of detection; LOQ, limit of quantification; RE, relative error.
a
PCR efficiency (%) = [10(−1/slope) −1] ×100.