Forensic Identification of Cannabis with Plant DNA Barcodes and Cannabinoid Synthesis Genes

<b>Background/Objectives:</b> According to the World Drug Report 2025, cannabis is the most abused drug in the world, being sold in illicit markets in various physical forms ranging from herbal cannabis to cannabis resin and liquid cannabis. Currently, the methods used for cannabis identification are largely based on the morphological features and chemical content of the product. In this respect, identification could be severely impacted if the product is highly fragmented or pulverised. As such, DNA-based molecular techniques offer a viable alternative detection approach. In this study, we have developed a robust DNA testing method for cannabis identification, with high sensitivity and specificity. <b>Methods/Results</b>: Two plant DNA barcode regions, <i>rbcL</i> and <i>matK</i>, were successfully amplified in a cohort of 54 cannabis plant samples. DNA sequences obtained from these samples were blast-searched against GenBank and resulted in returned matched identity of at least 99% compared to their corresponding <i>Cannabis sativa</i> reference sequences. In addition, the amplification of two cannabis-unique markers, the tetrahydrocannabinolic acid synthase (<i>THCAS</i>) and cannabidiolic acid synthase (<i>CBDAS</i>) genes, produced amplicons with expected sizes only in cannabis samples; these amplicons were not detected in those plants closely related to cannabis. Sequence comparison of the majority of samples yielded at least 97% matched identity against <i>C. sativa</i> reference sequences in GenBank. The <i>THCAS</i> and <i>CBDAS</i> markers detected only the cannabis DNA in varying levels of cannabis-hops and cannabis-tobacco DNA mixtures. Lastly, the use of the four markers could effectively differentiate between cannabis and non-cannabis in 27 blinded samples, including 18 actual casework samples. <b>Conclusions:</b> In conclusion, these four genetic markers can be used to discriminate cannabis from other plant species at the genus level, especially in challenging forensic samples lacking morphological features which therefore cannot be determined by traditional detection methods. As such, this method can complement existing techniques to identify a myriad of cannabis samples.