Food safety, especially for value-added and high-value products, has always been a critical issue. Due to rising market demand and limited production because of a loss of available cultivated land, consumers often purchase food items which have been adulterated with other products. Therefore, species identification of plant-based food products is critical for to be able to trace the composition of their food, which has implications on allergy and disease information for the consumer.
Pistachio (Pistacia vera L.) has high economical value and is ranked fifth in total level of global production. Pistachios have been consumed in human diets since prehistoric times. Pistachio nuts are rich in lipids, protein, potassium, phenols, lutein . As a result of the mineral and vitamin content of pistachios, it is recommended to consume them for their health benefits to aid against diseases such as cancer . As our understanding of the nutritional properties of pistachios increases, the amount of pistachio nuts consumed increases. Pistachios are used in several diverse ways, such as direct consumption of the nuts, or as a flavouring in bakery and deli products. Ground nuts are also used to produce pistachio paste. The health properties and multiple usages make pistachios an expensive nut, meaning that pistachio products are susceptible to adulteration. Green pea (Pisum sativum L.) is the most common and widely used adulterant of pistachio products due to its similar colour and texture when grounded. Moreover, green pea is six to seven times cheaper than pistachio in the market. It is not easily possible for consumers to identify green pea adulteration in a pistachio product, especially grounded products. It is also difficult for researchers to detect green pea adulteration of pistachio products in the lab, for example by using spectroscopic methods [3, 4].
Because of these issues, a rapid, robust and cost-effective method is needed to detect green pea adulteration of pistachio products. Identification of the adulterants at the level of DNA is more dependable since DNA is a stable molecule and exists in all tissues. Although a novel polymerase chain reaction (PCR)-based method targeting the trnL-trnF intergenic spacer with capillary electrophoresis (CE) was developed and successfully applied to detect green pea at the ratio of 5% , a more sensitive, faster and simpler method, which does not require post-PCR processing and electrophoresis, is still missing. A novel DNA hybridisation-based method DNAFoil was described in the literature  which is practical and easy to in field usage product which offers 30 min in field analysis time for detection of the target organism. However, it has some drawbacks compared to in lab techniques such as at least 1000 mg of sample required, gives only one (±) answer is provided and being a single use product. Since it is a commercial product with limited production, it is difficult to reach every researcher or consumer.
High resolution melting (HRM) is a DNA-based method that distinguishes DNA variants from one another by single nucleotide polymorphisms (SNP) and insertions and deletions. The differences between amplicons generate different melting patterns and distinguish between genotypes or species, which makes the method extremely sensitive. Since it is an add-on step implemented to the end of the PCR reaction, it does not require extra laboratory duties or extensive bioinformatics knowledge. It is also not necessary to sequence the PCR products or hybridisation . Moreover, since HRM is a closed-tube method, there is no contamination risk compared with other post-PCR methods.
HRM has previously been successfully applied to various food products. In a previous study, the method was applied to detect bovine, ovine and caprine adulterants in Greek Protected Designation of Origin (PDO) feta cheese . HRM accurately identified the presence of bovine milk, which is not allowed in feta cheese, at a concentration of 0.1%. HRM has also been used to identify another PDO food product that contains a type of Turkish apricot (Prunus armeniaca cv. Şalak) , where a simple sequence repeat (SSR)-HRM assay identified the PDO apricot from other closely related cultivars. In another study, microsatellite-based HRM analysis was applied to verify grapevine and olive cultivars .
HRM is also used to identify the components of medicinal plants and products. Liquorice is a traditional herbal medicine which is susceptible to substitution and adulteration. Researchers applied SSR-HRM to identify true liquorice from another species . Results discovered that SSR markers generated distinct melting profiles for different liquorice species. DNA barcoding regions can also be used as HRM markers. Researchers used the internal transcribed spacer-2 (ITS2) barcode region coupled with HRM (Bar-HRM), to differentiate Psammosilene tunicoides, which is an important herb using in traditional Chinese medicine, from other ingredients . This novel approach both detected the adulterant Silene viscidula and quantified the most common admixture.
Another successful usage of HRM is to differentiate edible plants from poisonous plants for food safety. Poisonous Urobotrya siamensis Hiepko can cause death when consumed due to misidentification by consumers. It has young leaves that are similar to vegetables such as Melientha suavis Pierre and Sauropus androgynus (L.) Merr. Bar-HRM can successfully identify seedlings of those species at amounts of 0.001 ng of DNA .
There are many studies that demonstrate how HRM is useful for genotyping [14–17], identifying medicinal plants [12, 18, 19] and identifying species of fish [20–22].
Herein, we developed and evaluated a novel HRM-based method using specific chloroplastidial primers for the rapid, dependable, closed-tube and minimum sample handling detection of green pea adulteration of pistachio. HRM was proven to be capable of detecting the presence of green pea down to concentrations of 0.1% in pistachio.