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

Cowan, Ashley F.; Elkins, Kelly M.

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 degrees C and 77.61 +/- 0.46 degrees C, respectively, using SYBR (R) Green I; 80.18 +/- 0.27 degrees C and 80.59 +/- 0.08 degrees C, respectively, using Radiant((TM)) Green; and 82.19 +/- 0.04 degrees C and 82.62 +/- 0.13 degrees C, respectively, using the LCGreen (R) PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/mu L. 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 degrees C and 85.58 +/- 0.23 degrees C, respectively, using LCGreen (R) PLUS.