Droplet digital PCR for absolute quantification of pathogens

Abstract

The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.