13-loci STR multiplex system for Brazilian seized samples of marijuana: individualization and origin differentiation

Abstract

It is known that Cannabis in Brazil could either originate from Paraguay or be cultivated in Brazil. While consumer markets in the North and Northeast regions are maintained by national production, the rest of the country is supplied with Cannabis from Paraguay. However, the Brazilian Federal Police (BFP) has exponentially increased the seizure number of Cannabis seeds sent by mail. For this reason, the aim of the study was to assess the 13-loci short tandem repeat (STR) multiplex system proposed by Houston et al. (2015) to evaluate the power of such markers in individualization and origin differentiation of Cannabis sativa samples seized in Brazil by the BFP. To do so, 72 Cannabis samples seized in Brazil by BFP were analyzed. The principal coordinate analysis (PCoA) and probability identity (PI) analysis were computed. Additionally, the Cannabis samples' genotypes were subjected to comparison by Kruskal-Wallis H, followed by a multiple discriminant analysis (MDA). All samples analyzed revealed a distinct genetic profile. PCoA clearly discriminated the seizure sets based on their geographic origin. A combination of seven loci was enough to differentiate samples' genotypes, and the PI for a random sample is approximately one in 50 billion. The Cannabis samples were 100% correct as classified by Kruskal-Wallis H, followed by an MDA. The results of this study demonstrate that the 13-loci STR multiplex system successfully achieved the aim of sample individualization and origin differentiation and suggest that it could be a useful tool to help BFP intelligence in tracing back-trade routes.

Development and Validation of a Novel and Fast Detection Method for Cannabis sativa: A 19-Plex Short Tandem Repeat Typing System

“…The PE values ranged from 0.0711 (9269) to 0.7123 (CS1), with an average value of 0.3218. In this study, CS1 was found to be the most informative maker, and similar results were observed in Houston et al (2016), Fett et al (2019), and Ribeiro et al (2020. In total, the TDP and the CPE of the 19-plex typing system were calculated to exceed 0.999 999 999 999 988 and 0.998 455 889 684 078, respectively, presenting higher efficiency than two multiplex panels containing 13-STR markers (Fett et al, 2019;Ribeiro et al, 2020).…”

“…Based on previous studies and published reports, the STRs of C. sativa were selected as follows: (1) tetranucleotide and trinucleotide repeated STRs were prior to the dinucleotide-repeat STRs. The latter were excluded due to the high stutters and heterozygote imbalances ( Valverde et al, 2014b ); (2) the STRs with high probability of amplification failures were excluded ( Houston et al, 2016 ; Fett et al, 2019 ); and (3) all STRs chosen have been previously nominated using the nomenclature of International Union of Pure and Applied Chemistry (IUPAC) ( Valverde et al, 2014a , b ). In addition, one sex-determining marker and one quality control marker of C. sativa from Pei (2009) were also included in the final assay.…”

“…At present, Houston et al (2016Houston et al ( , 2017 have developed two multiplex panels containing 13-STR markers, according to the International Society of Forensic Genetics (ISFG) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, which are also the most widely used panels for C. sativa. However, these two panels have been evaluated by Fett et al (2019) and Ribeiro et al (2020), which showed locus dropout and relatively low efficiency according to the analysis of population genetics. In addition, although it is very important to distinguish the sex of C. sativa (since differences exist in the THC content of C. sativa plants of different sexes), neither of the two panels could meet the needs.…”

Cannabis Extracts and Their Cytotoxic Effects on Human Erythrocytes, Fibroblasts, and Murine Melanoma

“…In order to identify the seized sample, a DNA extraction followed by the application of a molecular panel, developed for identification and individualization of C. sativa, was performed (Houston et al 2017). The DNeasy® Meri-con® Food Kit was used for the DNA isolation following the protocol described by Fett et al (2019). DNA amplification was performed via polymerase chain reaction (PCR) using the Multiplex PCR Kit (Qiagen, Valencia, CA, USA) according to previously described protocols (Ribeiro et al 2020).…”

Interpol review of controlled substances 2016–2019

“…2019 13-loci STR multiplex system for individualization and origin differentiation of Brazilian seized samples of marijuana [ 716 ]; determination of Cannabinoid concentrations in 531 confiscated cannabis samples [ 717 ]; optimization of protein extraction from cannabis [ 718 ];…”