Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Abstract

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.

Evaluation of Two New Methods for DNA Extraction of “Legal High” Plant Species

“…The amplifiability of the extracted DNA was evaluated with published PCR HRM assays (10–12) and the melt temperature results are shown in Table 4. All of the DNA extracted with both kits was amplifiable.…”

“…PCR HRM for the A. absinthium and D. stramonium duplex was conducted in a Rotor‐Gene Q (Qiagen, Germantown, MD) as follows: initial hold at 95°C for 2 min; 40 cycles at 95°C for 20 sec, 62°C for 20 sec with touchdown every 0.5°C for 10 cycles, and 72°C for 20 sec; final hold at 45°C for 1 min; and a melt ramp from 60 to 95°C, rising 0.3°C every 4 sec (11). The temperature program for the I. purpurea (10) and P. somniferum assays was as follows: initial hold at 95°C for 2 min; 40 cycles at 95°C for 20 sec, 55°C for 20 sec, and 72°C for 20 sec; a final hold at 45°C for 1 min; and a melt ramp from 60 to 95°C, rising by 0.3°C every 5 sec.…”

“…Plant material for extraction was collected as described in references (10–12) and includes I. purpurea , D. stramonium , P. rhoeas, Salvia officinalis, Humulus lupulus, Cannabis sativa, P. somniferum , A. absinthium , and M. speciosa . Table 1 lists their common and scientific names and family.…”

“…Real‐time polymerase chain reactions coupled with high‐resolution melt (PCR HRM) have been developed to detect and identify several legal high plant species (10–12). This technique indirectly examines the melt characteristics of double‐stranded DNA based on length, GC‐content, and base composition.…”

“…Datura stramonium (Jimson Weed) is recreationally smoked or ingested for the psychoactive and antimuscarinic components scopolamine and atropine (10). Artemisia absinthium (Wormwood), commonly distilled into absinthe, contains the GABA brain receptors blocker compounds α‐ and β‐thujone making it dangerous to smoke or ingest (11). Mitragyna speciosa (Kratom) induces effects similar to opiates due to the alkaloid mitragynine among other compounds, causing it to be widely abused recreationally as a painkiller and has been considered for classification as a Schedule I drug by the U.S. Drug Enforcement Agency (12).…”