Implementation of droplet digital PCR for the specific quantification of nucleic acids (CAD-Infect)

Absolute nucleic acid quantification in highly complex samples

Alongside climate change, emerging and multi-resistant pathogens are the greatest challenge of the 21st century. In terms of sustainable health research, strategies must therefore be developed that make customised therapeutic agents available in the shortest possible time, depending on societal needs.

ddPCR machine surrounded by boxes with written examples for application
Schematic representation of applications of ddPCR

An essential component of lead qualification is the determination of mutation frequencies. Mutagenicity studies usually require a lot of manpower and need large quantities of the test substances, which are not available at early stages of development. Quantitative PCR methods with very low detection limits (such as ddPCR) could solve this problem in the future, as they can reliably detect specific mutations even in relatively small culture volumes.

Compared to conventional methods, droplet-based nucleic acid quantification leads to increased selectivity, precision and reproducibility through sample partitioning and thus to very reliable measurements of the target sequence. Furthermore, partitioning leads to a considerable reduction in the detection limit with simultaneous lower sensitivity to interfering amplification inhibitors and background DNA.

  • Hight throughput ddPCR system QX ONE (Source: Leibniz-HKI)
  • Droplet readout (Source: Leibniz-HKI)

In summary, ddPCR is a universal method with versatile application perspectives in biomedical research and development. An ideal ratio of throughput, precision and operating costs make it currently the most powerful routine procedure on the market. A conversion of many established laboratory procedures to ddPCR methods is therefore strongly implied in the future.

Funding

REACT-EU -Funded as part of the reaction of the Union to the COVID-19 pandemic.