Author ORCID Identifier
https://orcid.org/0009-0000-9747-9625
Defense Date
2024
Document Type
Directed Research Project
First Advisor
Tracey Dawson Green, Ph.D
Abstract
Due to the destructive nature of mass casualty incidents (MCIs) or mass fatality events (MFEs), tooth samples are often one of the only viable sources for Disaster Victim Identification (DVI) because of the incredible integrity of the enamel layer surrounding the tooth. However, the resilient structure of the tooth requires harsh chemicals and a labor-intensive sample preparation protocol to ensure successful STR profile development. Considering this difficulty, one of the promising resolutions is the Applied Biosystems RapidHIT™ ID ID platform, which is a fully automated system that is designed to perform cell lysis, DNA isolation, polymerase chain reaction (PCR) amplification, electrophoretic separation, and data analysis from buccal swabs from saliva or blood sample swabs. To accommodate the need for rapid results in MCIs, this study evaluates the performance and feasibility of the RapidHIT™ ID system for use in DVI by identifying the limit of sensitivity of the instruments and optimizing the tooth sample preparation protocols that were previously published. The sensitivity and performance of the updated instrument and software were experimentally evaluated using buccal swab samples from four different donors with total DNA inputs of 5, 7, 10, 20, 50, and 65 ng. Each swab was processed by the RapidHIT™ ID System v2.0 using RapidINTEL™ Plus sample cartridge and the RapidLINK™ Software v2.0 following the general protocol with a lysis volume of 500uL. The resulting STR profiles were compared to the corresponding reference STR profiles to confirm accuracy and concordance to determine the lowest amount of input required to obtain a profile of adequate quality. Furthermore, to optimize the tooth sample preparation protocol for the RapidLINK™ Software v2.0, 40 tooth samples collected from 13 donors were grouped into eight subsets of different combinations of tooth preparation steps. The first group included all steps from the previously developed, and the remaining seven groups systematically omitted specific steps to evaluate their ability to produce reliable STR profiles. After narrowing down to the four best contenders, five more tooth samples were added to each group to be reevaluated to strengthen the confidence in the findings of this paper. Overall, the limit of detection of the RapidHIT™ ID System v2.0 using RapidINTEL™ Plus sample cartridge is determined to be ~0.226ng with the recommended DNA input of at least 20 ng. From all buccal swab samples with known DNA input, the on-board quantitation marker indicated that only ~1.13% of the DNA from the sample chamber is amplified. For the tooth samples, 100% of all alleles were detected and concordant to the reference DNA profiles. Generally, groups that did not involve the fragmentation step yielded higher quality STR profiles compared to those that included the fragmentation step. Based on the time-consumption and qualitative metrics evaluated from each group, the group with the overall highest success in developing STR profiles is group 8, which includes deionized water soak and rinse and surface impurity removal using swabs and enamel thinning steps with no fragmentation or pulverization. However, when encountering tooth samples that do not fit the sample chamber of the RapidINTEL™ Plus cartridge (~ 6mm), it is recommended that the sample preparation protocol from group 7 be implemented as it performed best amongst groups with the fragmentation step. The results from this paper support the idea that using the optimized protocols, the RapidHIT™ID system could be adapted for DVI to rapidly produce STR profiles from tooth samples with minimal intervention from forensic analysts.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
12-12-2024
Included in
Biotechnology Commons, Forensic Science and Technology Commons, Molecular Genetics Commons