Development of a similarity discrimination method for cannabis samples using Inter-Simple Sequence Repeat-PCR

In forensic cannabis investigations, it is important not only to prove that seized materials are cannabis or not but also to clarify the relationship between cannabis samples. This study aimed to establish a method for evaluating the similarity of the genetic background of cannabis samples using Inter-Simple Sequence Repeat (ISSR)-PCR analysis, a variant of DNA testing.

Forty-eight cannabis samples from 24 strains were collected. ISSR-PCR was performed using 10 ISSR primers, and the resulting band patterns were used for grouping. After analyzing the characteristics of each primer using a binary classification method, two numerical indicators, the similarity score and total likelihood ratio, were calculated to comprehensively evaluate the similarity between samples and assess their discrimination performance.

Cannabis samples were grouped into band patterns using ISSR-PCR, but no single primer classified all strains. The "similarity score" (0-10), based on primers showing the same band pattern across 10 ISSR-PCRs, averaged 1.69 between different strains, 4.34 between individuals of the identical strain, and 9.48 for the identical individual. The mean "total likelihood ratio" calculated from the positive and negative likelihood ratios for binary classification using 10 primers was 1.28 × 10⁰, 1.11 × 10⁵, and 9.79 × 10⁹, respectively. Each indicator demonstrated high discrimination performance in evaluating genetic similarity.

This study established numerical criteria for estimating the genetic similarity between unknown cannabis samples based on ISSR-PCR results using 10 primers.