Defense Date
2024
Document Type
Directed Research Project
First Advisor
Dr. Tracey Dawson Green
Abstract
ABSTRACT
With the recent passing of the Farm Bill, industrial hemp was removed from the definition of “marijuana”. Hemp is defined as cannabis with a THC level less than 0.3% dry weight. Consequently, there has been an influx of commercially available hemp derived CBD products on the market. To reduce costs, increase potency and bypass regulatory issues, there has been an increase in synthetic CBD products on the market. Due to the varying pathways for CBD synthesis, unintentional production of numerous illegal and hazardous synthetic cannabinoids can be produced. Synthetic cannabinoids have longer-lasting effects, and their use may result in rapid heart rate, vomiting, agitation, confusion, hallucinations, kidney failure, and even death. Specifically, Synthetic edible CBD products have been linked to negative health effects in users causing a growing public health concern. With that, there is a growing need to test the authenticity of those products. Historically, gas chromatography-mass spectrometry (GC-MS) has been widely used for qualitative analysis and authentication of CBD. However, due to various sample types and matrices such as gummies and chocolates, obtaining a chemical profile has proven to be challenging, thus creating difficulties in developing a fast and efficient protocol for obtaining chemical profiles. Additionally, with the sticky nature of those matrices, they can damage the instrumentation. Thus, the goal of this project was to develop a straightforward, fast, and reliable qPCR-based method that will allow for the determination of naturally derived CBD in edibles and other commercially available products.
As the information regarding this topic is limited, this research began with developing a reliable positive control to serve as a genetic standard to compare test samples. The positive control was developed from cannabis plant material received from NIDA containing varying levels of CBD and THC for determination of naturally derived CBD. In addition to the determination of a positive control, thirteen STR primer sets’ performance was evaluated for the reliable and consistent detection of cannabis DNA. Out of the 13 primer sets tested, primer sets 1528, 9043, D02 and 9269 were determined to be the most reliable to carry forward to qPCR. The best performing positive controls and four primer sets were then employed in qPCR testing of edible samples. In this study, 11 unique edible samples (gummies and chocolates) with varying levels of cannabinoids (THC, CBD, CBN and CBG) were tested. Each sample was extracted with four commercially available kits, DNeasy® Plant Mini Kit, DNA IQ™ System, DNeasy® mericon Food Kit and SureFood® PREP Advanced. These samples were then tested at two DNA input levels (maximum input and ~1:2 dilution) with each of the four primer sets to evaluate the best performing reaction combination for cannabis DNA detection from commercial edibles. The qPCR data revealed that there was no notable difference in whether samples were amplified or crossed threshold between the maximum DNA input and ~1:2 dilution input. However, in evaluation of the same qPCR data STR primer 1528 was shown to be the best performing primer set, and the best performing extraction kit was the DNeasy® Plant Mini Kit with cannabis DNA detected in 25% of samples. This study has shown that the qPCR detection of natural cannabis in commercially available CBD products is possible. Future testing is required to optimize DNA extraction methods and to assess the repeatability of these results.
Keywords: hemp, edibles, cannabis, THC, CBD, cannabinoids, qPCR
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
8-5-2024
Included in
Forensic Science and Technology Commons, Molecular Genetics Commons, Other Genetics and Genomics Commons