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Advisor(s)
Abstract(s)
The Investigator® 26plex QS Amplification Kit, from QIAGEN, provides reliable and rapid DNA profiles while enabling the multiplex amplification of 24 STRs, 2 Quality Sensors and a gender-determining marker, Amelogenin. The Quality Sensors incorporated in this kit provide insight into the sample quality and the PCR's success while alerting to the presence of inhibitors.
Through an extensive internal validation study, this research sought to implement the Investigator® 26plex QS in the laboratory routine of the Forensic Genetic and Biology Service, Central branch (SGBF-C) of the Portuguese National Institute of Legal Medicine and Forensic Sciences. Here we detail the procedures and parameters applied which followed the SWGDAM guidelines, as well as report the results obtained throughout.
Firstly, it was crucial to establish unique analytical criteria that would be the baseline for the interpretation of the results obtained. To accomplish such, analytical, stochastic, heterozygous balance and stutter thresholds were defined. Thereupon, this study intended to gauge the kit’s performance, by evaluating its concordance with normalized methodologies while assessing its sensitivity, specificity, robustness, and precision, besides its efficiency in the presence of degraded and/or inhibited samples. Lastly, in favour of optimizing the laboratory workflow, an assay was carried out to test half volume reactions and direct amplification on reference samples.
In this study a wide range of sample types were analysed, which made it possible to acquire robust data pertaining to the performance of the assay. The laboratory methodology applied comprehended: DNA extraction using Prep-n-Go™ Buffer and PrepFiler Express™ Forensic DNA Extraction Kit, quantification with the Quantifiler™ Trio Quantification Kit, followed by the amplification with the Investigator® 26plex QS. Capillary electrophoresis was performed in the Applied Biosystems™ 3500 Genetic Analyzer, and the electropherograms’ were analysed using the GeneMapper™ ID-X Software v1.6.
Through this work, it was possible to characterize the main advantages of the Investigator® 26plex QS kit, as well as its limitations. The validation data demonstrated that this system produces reliable profiles in the presence of minute DNA quantities and identifies the minor contributor in a mixture up to a 1:50 ratio. The results inferred a high human specificity, robustness, and sensitivity, while producing concordant and reproducible results with optimized protocols for both reference and low copy number DNA samples. Through concordance studies it was further shown that there is a high consistency between this novel amplification kit and those currently implemented in the SGBF-C, thus proving its suitability for the analysis of forensic samples.
Description
Poster apresentado no 30th Congress of the International Society for Forensic(2024), Santiago de Compostela (Espanha)
Keywords
Investigator® 26plex QS Forensic Genetics PCR Internal Validation Analysis Thresholds Forensic Samples STRs