Abstract
High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated.
| Original language | English |
|---|---|
| Journal | APMIS |
| Volume | 123 |
| Issue number | 9 |
| Pages (from-to) | 731-739 |
| ISSN | 0903-4641 |
| DOIs | |
| Publication status | Published - 2015 |
Keywords
- CffDNA
- Low-level DNA
- Mathematical model
- Poisson
- qPCR