Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum

Abstract

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration’s (DEA) Heroin Signature Program, which seeks to combat rising opioid use.The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.