Using rDNA ITS2 barcoding to identify kratom (Mitragyna speciosa) from the genus Mitragyna and Neolamarckia cadamba

Abstract

Abstract This study collected 80 samples of suspected kratom plant powder. A polymerase chain reaction sequence analysis was conducted using two sets of DNA barcode primers for plant ribosomal (r)DNA internal transcribed spacers (ITSs), namely, ITS3/ITS4 and ITS‐p3/ITS‐u4. Among the 80 samples, 40 were analyzed using the ITS3/ITS4 primer pair, and then DNA sequences were subjected to a National Center for Biotechnology Information–Basic Local Alignment Search Tool (NCBI–BLAST) comparison. Results showed that 29 samples had a 100% match (364/364) with Mitragyna speciosa (kratom), and 6 samples had a 99.73% match (363/364) with M. speciosa , whereas 5 samples had disordered and unreadable sequences. The 5 unreadable samples and an additional 40 suspected kratom samples were then analyzed using the ITS‐p3/ITS‐u4 primer pair, followed by an NCBI–BLAST comparison. Among these, 32 samples had a 100% match (404/404) with M. speciosa , and 11 samples had a 99.75% match (403/404) with M. speciosa . Among the samples with sequences matching M. speciosa , three distinct types were observed (no variance/404, 287M/404, and 287A/404). One sample had a 99.51% match (404/406) with Neolamarckia cadamba , and another sample had a sequencing length of 305 bp, with 25 positions showing mixed base pairs, indicating a mixture of different species. Analysis of the mixed base pair pattern suggested a possible mixture of M. speciosa and N. cadamba . Actually, M. speciosa and N. cadamba have very similar external morphologies. This indicates that the ITS‐p3/ITS‐u4 primer pair is effective in distinguishing mixtures of M. speciosa and N. cadamba and is thus more suitable than ITS3/ITS4 for identifying and analyzing samples of suspected kratom plant powder.