Abstract
Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625–1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28–15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3–14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).
Highlights
Papaver somniferum L., a traditional medicinal plant since the Neolithic age[1], is the source of up to 140 alkaloids[2,3], with morphine, thebaine, codeine, papaverine and noscapine being the most prominent
Consecutive analysis of microsatellites sorted according to the number of repeats (NR) showed a sharp decrease in the number of loci with higher NR (Supplementary Fig. S3)
Most of the remaining candidate loci were excluded during the marker development
Summary
Papaver somniferum L. (opium poppy; Papaveraceae family), a traditional medicinal plant since the Neolithic age[1], is the source of up to 140 alkaloids[2,3], with morphine, thebaine, codeine, papaverine and noscapine being the most prominent. The global production of illicit opium has seen an increasing trend and reached as high as 10 thousand tons in 2017, which is the largest amount recorded over the last two d ecades[6] This alarming situation demonstrates another statistic provided by the United Nations Office on Drugs and Crime (UNODC): 29 million people used opiates in 2017, which is 50% more than in 20166, this shift is partially caused by an improvement in statistical methodology and the retrieval of new information from highly populated countries such as India and Nigeria. An alternative approach based on the assessment of the isotope ratio of chosen elements was proposed and successfully a pplied[11,12] Another way to retrieve more information involves molecular genetic methods, because opium or heroin contains plant (cell) parts and DNA, in limited amounts and degraded[13]. There is no forensically relevant DNA marker system implementing polymorphisms of short tandem repeats (STRs) or single nucleotide polymorphisms (SNPs)
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