Development of a Locked Nucleic Acid Real-Time Polymerase Chain Reaction Assay for the Detection of Pinus armandii in Mixed Species Pine Nut Samples Associated with Dysgeusia

Abstract

Recent work has shown that the presence of the species Pinus armandii , even when occurring as species mixtures of pine nuts, is correlated with taste disturbance (dysgeusia), also referred to as "pine mouth". Because of this known possibility of pine nut mixtures, a need was identified for a rapid streamlined assay to detect the presence of this species in the presence of other types of pine nuts. A locked nucleic acid probe was employed in a real-time polymerase chain reaction (RT-PCR) format to detect a single nucleotide polymorphism (SNP) unique to this species. This assay was able to detect P. armandii in homogenates down to ∼1% concentration (the lowest level tested) in the presence of several commonly co-occurring and closely related species of pine and should prove to be a useful tool for the detection of this species in food products.

An analysis of Echinacea chloroplast genomes: Implications for future botanical identification

“…For example if a region that distinguishes important species based on the size of DNA fragments can be identified and validated, this method could be used without sequencing, thus creating a rapid low cost approach to species identification. In the absence of suitable indels, other variable regions in closely related species can be targeted for either PCR, real-time PCR or other sequence based identification methods 40 .…”

“…For example, in 2013 Handy et al . used a large chloroplast dataset to design a species-specific assay to differentiate Pinus armandii , which causes a taste disturbance known as dysgeusia 40 , from other species that do not.…”

A potential trigger for pine mouth: a case of a homozygous phenylthiocarbamide taster

“…This subject was affected by PNS after ingesting pine nuts from Pinus sibirica , which have previously not been associated with PNS [1, 7,8]. However, recent reports of taste disturbances from samples containing nuts from a mixture of Pinus sibirica and Pinus armandii have been reported [2,9,10]. As a study based on a single individual, is it intended to be hypothesis-generating research with limited generalizability.…”

“…Only a few studies of PNS have been reported [3–5], and one study has described the symptoms of pine nut-related dysgeusia in a total of six subjects [6]. A single species of pine nuts ( Pinus armandii ) has previously been associated with this syndrome [1,7,8], although recent studies have reported taste disturbances from samples containing nuts from a mixture of different Pinus species [2,9,10]. However, the syndrome occurs in only a small fraction of individuals who consume pine nuts, and efforts to identify a common trigger or medical cause responsible for the occurrence of PNS have not been successful [4].…”

Development of a Reference Standard Library of Chloroplast Genome Sequences, GenomeTrakrCP

“…After much chemical testing of consumer complaints, the FDA CFSAN could not determine the cause. A DNA-based approach was applied to determine whether or not a species substitution, Pinus armandii Franch., Pinaceae, was causing this effect [10,11]. Traditional DNA barcoding regions (i.e., matK, rbcL, and ITS2) were not useful in identifying these pines [10]; instead a diagnostic region, ycf1, was targeted from 33 Pinus chloroplast genome comparison and was used to differentiate P. armandii from closely related species using a specific real-time polymerase chain reaction assay [11].…”