Droplet digital polymerase chain reaction (ddPCR) as a novel method for absolute quantification of major...

In this paper, we present for the first time a new tool, based on Droplet Digital™ Polymerase Chain Reaction (ddPCR), for absolute quantification of key genera of gastrointestinal (GI) nematode parasites of grazing livestock. Four combinations of primers/probe sets targeting the internal transcribed spacer region 2 (ITS2) of the ribosomal RNA gene array were designed using the Primer3 software, following in silico analysis of nucleotide sequences from nematodes of interest downloaded from common databases. The amplified regions include both a universal region for detection of any strongylid gastrointestinal parasite and three different genus specific regions, making it possible to differentiate between the most important GI nematodes of sheep in Sweden: Haemonchus, Teladorsagia and Trichostrongylus. Analysis of samples containing serial dilutions and different mixtures of genomic DNA extracted from different species of adult worms proved useful in assessment of different threshold settings with the QuantaSoft software. Analysis of template DNA from these worms indicated that ddPCR is a viable choice for detection and absolute quantification of the different genera and also in samples with multiple species. Interpretation of the ddPCR results was straightforward and choice of analytical approach had little influence on the final results. Thus, the results obtained in the different analytical approaches seemed to be robust and the concentrations determined were uniform. Furthermore, the linear range of the Haemonchus ddPCR assay was similar to that of real-time PCR (qPCR). Taken together, our data confirm the suitability of ddPCR for detection and absolute quantification of three major sheep pathogens when tested on larval cultures from pooled ovine faeces. The results also indicate that ddPCR can be a useful complement to applications based on conventional egg counting methods such as the faecal egg reduction test (FECRT).