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Official websites use. Share sensitive information only on official, secure websites. Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market. Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting HRM analysis using barcoding markers. Humulus lupulus L. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer ITS and rbc L. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation. The melting temperature T m of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed T m differences of 0. An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbc L regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material. The medical properties of Cannabis species have been known for centuries, but the interest on active secondary metabolites as alternative therapies for diverse pathologies has grown in recent years. This potential use has generated a strong impact worldwide concerning public health and the commerce of cannabis products. Indeed, this has led to legislation changes in some countries. Genetic identification of different cannabis strains is critical not only for quality control purposes in a nonregulated growing market, but for forensic purposes and for medical use as well. Its medical use has been focused on the treatment of skin pathologies, seizures, lack of appetite, neurodegenerative diseases, multiple sclerosis, as well as for the management of chemotherapy side effects 3—6 and chronic pain treatment. This could produce cognitive disorders related to day-to-day consumption and especially in cases of long-term cannabis use. This poses a problem since these plant products require a certificate of authentication. Various methods of identification have been developed to authenticate medical plant species such as morphological analysis, chemical profiling, and DNA-based molecular analysis. The study of conserved regions of the genome for identification has allowed broadening the spectrum of analysis in a number of species. This has been applied on both the botanic and forensic fields. These molecular methods can detect differences at the DNA level and they offer numerous advantages over the conventional phenotype-based approaches. The ITS2 region is one of the most used universal barcode for plant species identification. HRM analysis is the method of choice for rapid analysis of sequence variation within PCR amplicons by determining their melting temperature T m. T m differences can be detected by monitoring the fluorescence changes during PCR cycles using real-time PCR differential analysis. Genotypes are then differentiated by their characteristic melting curves, visualized by the loss of fluorescence as the DNA duplex melts 23 HRM analysis allows genotyping of plant, fungus, and animal species. DNA sequence differences, such as single nucleotide polymorphisms and small insertions and deletions Indels , can be detected based on the location of a differential peak and the shape of the melting transition curves. This article describes a case of adulteration of cannabis seeds due to lack of control during the packaging process. Cannabis sativa L. The objective of this study was to authenticate cannabis strains using a real-time PCR and HRM-based molecular method. Using ITS2 and rbcL barcoding HRM, it was possible to detect the presence of different strains in commercial cannabis seed used for medicinal purposes. This method can be very valuable in terms of traceability and authentication of strains in cannabis commercial seeds. Commercial seeds of four different C. Commercial Hops Humulus lupulus L. Plant material was previously powderized before extraction. DNA was quantified using a fluorometer Qubit 2. Genomic DNA integrity was confirmed by agarose gel electrophoresis. Amplicon size and primer specificity were confirmed by 1. The fluorescent data were acquired at the end of each increment step. All experiments were run in duplicate. Melting curve analysis was performed with the Rotor-Gene Q software v. The first step was to calculate triangular similarity matrix based on Euclidean distance and then a multidimensional metric scaling mMDS was performed. This analysis determines the relationships between the profiles at different temperatures for each marker. A k-means clustering was used to detect classes through a set of quantitative variables. DNA extracted from all seeds yielded a specific amplification product for both regions. These sizes are in accordance to previously published sequence data. PCR amplicons were analyzed to determine their specific T m. Results are represented by means of conventional derivative plots. The melting curve is generated by slowly melting the DNA through a range of temperatures in the presence of a dsDNA binding dye. Distinguishable melting curves were obtained for H. T m s resulted to be All cannabis seeds provided by customers yielded different melting curves. A Melting curve profile using ITS region. C Melting curve profile using rbcL marker. The analysis of ITS2 region yielded different outcomes for the four analyzed strains. Two of the strains resulted to have the same genetic profile Fig. A Melting curve profile of three cannabis seeds with same sequence. B Normalized curve profile of three cannabis seeds with same sequence. C Melting curve profile of two cannabis seeds with same sequence and one seed with different sequence. D Normalized curve profile of two cannabis seeds with same sequence and one seed with different sequence. These differences depend on GC content, length of amplified product, and sequence. The undistinguishable seeds resulted to have a consistent Tm of ITS2 results were confirmed through rbcL analysis. Analysis of derivative melt curve resulted in consistent T m s, within the range Three cannabis seeds resulted to have the same rbc L sequence Fig. HRM analysis using the rbc L chloroplast marker. The mMDS analysis showed the relationships between the seeds' profiles at different temperatures for each gene Fig. Four sets of three seeds from different strains were included in the analysis. All seeds from sets 2 and 4 resulted to form a cluster, whereas seeds from sets 1 and 3 appeared to be genetically heterogeneous. A mMDS represent seed samples of the four different packages used in the study. Each point represents a seed for ITS region. B mMDS represent seed samples of the four different packages used in the study. Each point represents a seed for rbc L marker. However, samples from sets 1 and 3 were not able to be assigned to the same cluster due to their genetic sequence difference. These results demonstrated that seeds from sets 2 and 4 appeared to be genetically homogeneous, whereas samples from sets 1 and 3 seemed to be genetically heterogeneous. Results from sets 1 and 3 clearly indicated the presence of a mixture of seeds. We described a case in which 10 samples of seeds from medicinal users and four strains of cannabis seeds were analyzed by HRM coupled with barcoding. This authentication is critical to ensuring the quality of medical cannabis seed. However, up to date there is no standardized method for rapid identification of cannabis. DNA sequencing technology is relatively expensive and time consuming. Thus, Bar-HRM is a better option for a rapid screening of cannabis strains. However, HRM shows some limitations when the genetic variation is extremely small. Four strains were purchased from a specialized store that distributes cannabis seeds for recreational and medicinal use. Molecular authentication showed that cannabis seed are good sources of DNA, providing full and amplifiable genetic material that can be used efficiently for authentication analysis by HRM. Distinction down to genus and—in many cases—species level is possible based on melting temperatures T m of specific PCR products. The ITS2 region was selected as a barcode marker because is widely used for phylogenetic reconstructions at both genus and species levels. The use of a combination of barcode regions is common and recommended for plant identification. To confirm that each package contained seeds of the same strain, we decided to analyze randomly purchased commercial products. In some cases, some cannabis users reported that different phenotypes were observed from seeds purchased as belonging to a single strain. The robustness of such analyzes would also be strengthened by the exploration of markers in organelles and by increasing the universe of samples to be incorporated in the studies. Our results proved that seeds had different genetic sequences. This possible adulteration may be attributed to poor packaging or a mistake during package processing. Each package of each strain contains three seeds each. Surprisingly, it was found that two out of four packages contained mixtures of seeds. We proposed a fast easy low-cost method to distinguish the presence of mixtures in seed packages. This can certainly help confirm the authenticity or at least the homogeneity of the seed contents of a given strain. Although this analysis can be done at any stage of plant development, it was demonstrated that seeds were a good source of amplifiable DNA. The homogeneous packages had the same T m for each of the three seeds of that strain. In contrast, packages containing mixed seeds displayed different T m s. This allowed distinguishing the different genotypes in the same package. In this study, Bar-HRM analysis was proposed to be a fast and accurate technique for authentication testing of cannabis seed of commercial strains. Here, we describe the development of a Bar-HRM method for adulteration testing of cannabis seeds contained in individual packages. These packages correspond to different cannabis strains. This method allowed detecting heterogeneity in two out of four cases in the contents of these packages. This is undoubtedly important at the time of consumption of these plants by medicinal users. In future studies, we will continue to explore the different seed banks commonly used by users of cannabis and incorporating new DNA markers. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this study that could have influenced the results. This study was partially funded by EM-LA The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article. As a library, NLM provides access to scientific literature. Cannabis Cannabinoid Res. Find articles by Jaime Solano. Find articles by Francisco Encina-Montoya. Find articles by Marco Bustos. Find articles by Alejandra Figueroa. Find articles by David Gangitano. Collection date Aug. Copyright , Mary Ann Liebert, Inc. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.