Rapid identification of drug-type and fiber-type cannabis by allele specific duplex PCR

Cannabis is classified into two types: drug-type cannabis, which is abused worldwide, and fiber-type cannabis, which is used for industrial purposes. The two types are a result of differences in the sequences of tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes. In the present study, we aimed to establish a PCR-based method to distinguish between drug-type and fiber-type cannabis by detecting the differences in the sequences of THCAS and CBDAS. We constructed a single-plex PCR targeting active THCAS, and observed drug-type cannabis-specific amplification when using 10pg to 1ng of DNA; however, amplification was also observed in fiber-type cannabis when the DNA content reached 10ng. Similarly, single-plex PCR targeting active CBDAS showed fiber-type cannabis-specific amplification in 100pg of DNA, as well as in >1ng of drug-type cannabis DNA. Therefore, when an allele-specific duplex PCR system was constructed, in which both primer sets were mixed at an appropriate ratio, unintended nonspecific amplification was suppressed and amplicons of different sizes were observed between the drug-type and fiber-type cannabis, using DNA samples in the range of 1pg to 10ng. When the constructed duplex PCR was performed on DNA extracted from various cannabis seed samples, it was possible to distinguish between the drug-type and the fiber-type as well as detect a hybrid-type with both active THCAS and active CBDAS and a special type with neither. The identification method developed in the present study can quickly and accurately distinguish between drug-type and fiber-type cannabis, and is expected to be used for various purposes such as the detection of genetic contamination of industrial hemp as well as forensic examination of cannabis-related cases.