Defense Date
2022
Document Type
Directed Research Project
First Advisor
Sarah Seashols-Williams, Ph.D.
Second Advisor
Christopher Ehrhardt, Ph.D.
Third Advisor
James Landers, Ph.D.
Abstract
Body fluid identification serves a vital role in contextualizing biological evidence received in forensic laboratories. While several fluids can be presumptively detected using immunological or catalytic tests, these tests are susceptible to false results and have limited sensitivity. True confirmatory testing is still somewhat limited in forensic laboratories. With the advancement of DNA typing technologies, laboratories can develop DNA profiles from trace amounts of sample, increasing the demand for sensitive and accurate body fluid detection. This project proposes using loop-mediated isothermal amplification (LAMP) as a method to rapidly detect microRNAs (miRNAs) that are differentially expressed in various body fluids with high specificity and without the need for specialized equipment. LAMP detection of miRNAs specific to semen and blood were evaluated, testing multiple LAMP template and primer methodologies for their specificity and sensitivity. An assay design utilizing a template DNA with a complementary region for its respective target miRNA was moderately successful. With this design, the target miRNA initiated strand displacement synthesis, prompting exponential amplification. A positive control containing 10^6-10^11 copies of synthetic miRNA could be detected in 45 minutes in both real-time qPCR and gel systems with little to no amplification of a negative control containing the respective template DNA. This window was extremely narrow since amplification of the negative control typically followed within mere minutes. A few methods were evaluated to see if this non-specific amplification could be alleviated by reducing primer and template DNA concentrations, and by adding reagents such as diethylformamide (DEF) and dimethylsulfoxide (DMSO) to improve specificity. The addition of DMSO and DEF slightly improved specificity, but further validation is necessary. With additional optimization, this assay could demonstrate proof of concept for the development of a rapid on-site body fluid identification assay.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
5-6-2022