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

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.