Fig. 1

The workflow in ddPCR (the most commonly used platform in parasitology) can be divided into four steps. Step 1: A mixture of primers and DNA-binding dyes (such as EvaGreen^®) or fluorescent probes (containing dye molecules such as FAM™, HEX™ or VIC^®) at their 5′ end and a quencher molecule containing probes at their 3′ end is often mixed with an already prepared supermix (containing premixed amounts of DNA polymerase, deoxynucleoside triphosphates [dNTPs] and salt). Step 2: After sample preparation, microfluidics is used to disperse and subdivide each sample into several thousand nano-sized droplets (partitions), each containing either no template molecules or one or more template molecules of interest. Step 3: The target nucleic acid sequence(s) are subjected to conventional PCR amplification until an endpoint is reached. Step 4: The presence of PCR-positive and PCR-negative reactions is evaluated using a droplet reader (strictly binary). From this, the concentration of the analyte molecule in the sample can be determined by fitting a Poisson distribution to the data. Essentially, the relationship between the proportion of positive reactions and the copies of the target molecule (i.e. ln(1−p), where p is the proportion of positive reactions) is used to determine the copies of the target molecule (with a 95% confidence interval) in the volume of the sample-reaction mixture