Polymerase chain reaction and chemometrics detected several Pinus species including Pinus armandii involved in pine nut syndrome

Abstract A dramatic peak in reported cases of pine mouth or pine nut syndrome (PNS) was observed in Europe and in the United States of America in 2008–2012. The PNS symptoms involve a constant bitter and/or metallic taste that appear 1–2 days after ingestion and disappear within 5–14 days. The chemical compound responsible for the symptoms is unknown, but symptoms are related to ingestion of pine nuts from the species Pinus armandii . P. armandii used industrially for non-food purposes has entered the food chain through mislabeling. Consequently, species determination of pine nuts has gained focus in governmental control of food authenticity. In this study, a PCR primer design targeted conserved DNA sequences that span an area of variation between P. armandii and other relevant species. Principal component analysis (PCA) of high-resolution melting curves from PCR amplicons was used to cluster pine species from reference material, and to determine the species of unknown samples. The PCA successfully clustered 2 subspecies/varieties of P. armandii , Pinus bungeana , Pinus massoniana , Pinus pinea , and Pinus wallichiana . Pinus koraiensis / Pinus pumila and Pinus sibirica/Pinus cembra had identical PCR amplicons, respectively, and formed 2 distinct clusters. 12 pine nuts from 4 unknown samples were analyzed. 10 pine nuts clustered together with P. armandii and P. koraiensis / P. pumila . 2 pine nuts were not part of clusters, but probabilities suggested P. armandii , and P. sibirica/P. cembra. These determined species were comparable to external results obtained elsewhere.