Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay

Abstract

In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

CelB is a suitable marker for rapid and specific identification of Klebsiella pneumoniae by the loop-mediated isothermal amplification (LAMP) assay

“…LAMP provides an alternative to PCR-based assays; due to its isothermal nature, it may be more suitable for the detection of pathogenic bacteria in the front-line or mobile laboratories in developing countries. LAMP technology was at least 10 times more sensitive than PCR technology [29][30][31]. But in this study, the sensitivity of PCR was same to the LAMP by optimizing the PCR reaction system, which suggested that there was still room for improvement in the sensitivity of PCR.…”

Rapid, specific, and sensitive detection of the ureR_1 gene in Klebsiella pneumoniae by loop-mediated isothermal amplification method

“…The results in Figure 1 suggest that the ureR_1 gene was specific to K. pneumoniae and can be used as a biomarker in clinics. In many studies, the sensitivity of LAMP was reported to be 10-fold higher than that of the PCR method (27,29,34). However, specificity and sensitivity between LAMP and PCR were similar when the ureR_1 gene was used.…”

Improved On-Site Protocol for the DNA-Based Species Identification of <i>Cannabis sativa</i> by Loop-Mediated Isothermal Amplification

“…10) Two loop primers were added to accelerate the LAMP reaction, and a LAMP reagent containing pyrophosphatase was used because its substrate, pyrophosphate, is a byproduct of amplification and shows inhibitory effects. 12,13) The sensitivity and specificity of the optimized LAMP reaction were evaluated. Using purified DNA as the template, 10, Supplementary Table S1.…”