Digital PCR for Genotype Quantification: A Case Study in a Pasta Production Chain

SIMPLE SUMMARY: Digital polymerase chain reaction (dPCR) is a breakthrough technology able to provide an absolute quantification of the target sequence through the compartmentalization of the sample and independent amplifications of the numerous separate compartments. Such technology has recently found several applications in plant science; however, to the best of our knowledge, it has never been applied until now for the detection and quantification of a specific plant variety along a production chain. As proof of concept, a dPCR assay targeted to the quantification of a durum wheat variety routinely used in an Italian premium pasta production chain has been developed. ABSTRACT: Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized by unprecedented levels of precision. The actual applicability of this analytical technique to quantify the presence of a specific plant genotype, in both raw materials and transformed products, by exploiting a point polymorphism has been evaluated. As proof of concept, an Italian premium pasta production chain was considered and a dPCR assay based on a durum wheat target variety private point mutation was designed and evaluated in supply-chain samples. From the results obtained, the assay can be applied to confirm the presence of a target variety and to quantify it in raw materials and transformed products, such as commercial grain lots and pasta. The performance, costs, and applicability of the assay has been compared to analytical alternatives, namely simple sequence repeats (SSRs) and genotype-by-sequencing based on Diversity Arrays Technology sequencing (DArTseq(TM)).