PCR combined with lateral flow immunochromatographic assay to differentiate the narcotic Mitragyna speciosa from related species and detect it in forensic evidence

Plants in the genus Mitragyna (Rubiaceae) are used in traditional medicine because of their broad therapeutic activity. Four Mitragyna species, M. speciosa (Roxb.) Korth. (MS), M. rotundifolia (Roxb.) Kuntze (MR), M. diversifolia (Wall. ex G. Don) Havil. (MD), and M. hirsuta Havil. (MH), occur in Thailand. M. speciosa, commonly known as 'Kratom' in Thai, is the only narcotic species for which buying, selling, importing or possessing has been prohibited by law in Thailand and some other countries. Mitragynine and 7-hydroxymitragynine, the major psychoactive compounds, are important in the treatment of opioid withdrawal. However, this species is used in traditional medicine to relieve pain and inflammation. Consequently, a rapid and easy technique for differentiating M. speciosa from closely related species is needed for routine forensic analysis. In this study, polymerase chain reaction coupled with lateral flow immunochromatographic assay (PCR-LFA) based on matK was developed for the detection of M. speciosa in forensic specimens. Duplex primers (MS-F-FAM, Ctrl-F-DIG and Ctrl-R-Biotin) were designed based on species-specific nucleotide indels observed exclusively in the matK sequences of M. speciosa. Positive results for M. speciosa are indicated by the clear presence of three black lines on the lateral flow cassette. Forensic samples were investigated, and the three black test lines indicating M. speciosa were observed for seven of eight specimens. PCR-LFA has been proven to be fast, easy and efficient for detecting the narcotic M. speciosa and could be developed as a rapid forensic diagnostic technique for other plants.