GENETIC ANALYSIS OF PLANTS IN THE GENUS MITRAGYNA AND THE IDENTIFICATION OF MITRAGYNA SPECIOSA BY REAL-TIME PCR

Abstract

The genus Mitragyna has been used as local traditional medicines. There are four species, M. diversifolia, M. hirsuta, M. rotundifolia and M. speciosa, existing in Thailand. Only M. speciosa, known as kratom, is officially classified as a narcotic plant among the genus Mitragyna. Consumption of M. speciosa is illegal in Thailand. However, this plant is popularly consumed by construction workers for the purpose of work harder. Recently, the pattern of consumption has increasingly changed toward a drink called “kratom cocktail”. The abuse of M. speciosa can affect of the consumer health. Thus, correct identification of M. speciosa is essential for both traditional medicinal and pharmacological studies as well as forensic investigation. This study aimed to develop molecular markers for the accurate identification of M. speciosa. The patterns of interspecific variation were detected in the nucleotide sequences of the ITS2 region in the four Mitragyna species. Based on the nucleotide polymorphism, species-specific primers were designed for the identification and differentiation of M. speciosa from the other closely related species. Moreover, a real-time PCR technique based melting curve analysis was developed for the detection and discrimination of M. speciosa from other Mitragyna species. Based on the distinct melting temperatures of amplification products, the melting curve was highly specific for the detection of M. speciosa DNA with the amplification efficiency value of 95.6%. Additionally, this assay can be used to detection and quantification of M. speciosa DNA in kratom cocktail samples.