Detection and Identification of Kratom (Mitragyna speciosa) and Marijuana (Cannabis sativa) by a Real‐Time Polymerase Chain Reaction High‐Resolution Melt Duplex Assay^,

Abstract

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used “legal high” opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta‐D‐glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high‐resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/μL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen® PLUS.

Evaluation of Two New Methods for DNA Extraction of “Legal High” Plant Species

“…The PCR HRM results demonstrated that all of the methods were able to extract amplifiable DNA for the plants studied. The melt temperature values obtained in this study with the newly evaluated methods are within or close to the melt temperature ranges determined in previous studies (10,12) with DNA extracted using the Qiagen DNeasy kit. The melt peak shapes were congruent with amplified DNA extracted with the Qiagen kit (10–12).…”

“…Table 1 lists their common and scientific names and family. Table 2 lists the PCR primers used in the PCR HRM assays; additional gene and location details can be found in references (10–12,17). Primer stocks of each primer were prepared at 5 μM with the exception of those for morning glory which were prepared at 10 μM in nuclease‐free water.…”

“…Artemisia absinthium (Wormwood), commonly distilled into absinthe, contains the GABA brain receptors blocker compounds α‐ and β‐thujone making it dangerous to smoke or ingest (11). Mitragyna speciosa (Kratom) induces effects similar to opiates due to the alkaloid mitragynine among other compounds, causing it to be widely abused recreationally as a painkiller and has been considered for classification as a Schedule I drug by the U.S. Drug Enforcement Agency (12). Kratom is a plant that originates in Southeast Asia (8,12).…”

“…Mitragyna speciosa (Kratom) induces effects similar to opiates due to the alkaloid mitragynine among other compounds, causing it to be widely abused recreationally as a painkiller and has been considered for classification as a Schedule I drug by the U.S. Drug Enforcement Agency (12). Kratom is a plant that originates in Southeast Asia (8,12). It can be acquired as a powder, resinous extract, and fresh and dried leaves (8,12).…”

“…Kratom is a plant that originates in Southeast Asia (8,12). It can be acquired as a powder, resinous extract, and fresh and dried leaves (8,12). Papaver somniferum (Opium Poppy) contains opium alkaloids such as morphine and codeine when the seeds are unwashed before distribution and has been known to be brewed into a tea that produces psychoactive and euphoric effects (13,14).…”