PCR-reverse dot blot of the nucleotide signature sequences of matK for the identification of Mitragyna speciosa, a narcotic species

Four species of Mitragyna plants exist in Thailand, including Mitragyna speciosa, M. rotundifolia, M. hirsuta, and M. diversifolia. M. speciosa, or “Kratom” in Thai, is the only narcotic species that has been prohibited by law in Thailand. It causes addiction and sometimes fatalities when overdosed. The accurate detection of this species is needed in forensic justice for legal proceedings. In this study, we aim to develop an assay based on DNA barcoding for the detection of M. speciosa in forensic specimens. DNA barcodes of the matK, rbcL, psbA-trnH and ITS2 regions of the four Mitragyna species were established. The matK nucleotide signature site was selected as a target for the discrimination of M. speciosa, a narcotic species, from non-narcotics by using PCR-reverse dot blot (RDB) hybridization assay. This RDB assay is highly specific and selective for M. speciosa, costs little and is easily performed without advanced analytical equipment. This method is an alternative to DNA sequencing for the detection of M. speciosa in suspected samples. Using PCR-RDB to detect the polymorphisms of matK holds promise as a routine method to test for the presence of M. speciosa in forensic specimens.