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Detection and Identification of Kratom ( Mitragyna speciosa ) and Marijuana ( Cannabis sativa ) by a Real‐Time Polymerase Chain Reaction High‐Resolution Melt Duplex Assay ,
Author(s) -
Cowan Ashley F.,
Elkins Kelly M.
Publication year - 2020
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/1556-4029.14167
Subject(s) - agarose gel electrophoresis , sybr green i , chemistry , chromatography , amplicon , microbiology and biotechnology , polymerase chain reaction , melting curve analysis , dna , biology , biochemistry , gene
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.