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Advisor(s)
Abstract(s)
A Genética Forense utiliza o DNA como alvo de análise na resolução de casos forenses, contudo
a quantidade presente nas amostras pode variar consideravelmente, o que poderá colocar em causa a
obtenção de perfis genéticos. A quantificação de DNA permite a análise da quantidade e qualidade de
DNA na amostra, de forma a adaptar o restante protocolo. A técnica de qPCR é considerada uma das
mais precisas e sensíveis para quantificação, permitindo acompanhar, em tempo real, o produto
amplificado.
Para a implementação de um ensaio é necessária a sua prévia validação, de forma a testar as suas
capacidades de execução. No Serviço de Genética e Biologia Forenses da Delegação do Sul do Instituto
Nacional de Medicina Legal e Ciências Forenses, I.P., procedeu-se à validação interna de ensaios de
quantificação de DNA, aplicando os kits Quantifiler Trio e Investigator Quantiplex aos equipamentos
QuantStudio5 e CFX Opus 96. A validação foi realizada de forma direta através da comparação de
resultados de 24 amostras problema com o método previamente validado Quantifiler Trio aplicado ao
7500 Real-Time PCR System, e de forma indireta, através da análise da especificidade, sensibilidade,
repetibilidade, reprodutibilidade e capacidade de distinção entre DNA feminino e masculino, através da
análise de amostras controlo.
No âmbito da Genética e Biologia Forenses, os resultados apresentados pelo ensaio Quantifiler
Trio aplicado ao QuantStudio5 permitiram a sua validação interna, demonstrando este um melhor
desempenho, comparativamente aos restantes ensaios estudados. Já os resultados apresentados pelo
ensaio Investigator Quantiplex aplicado ao QuantStudio5 permitiram a sua validação para amostras com
concentração dentro da reta de calibração, enquanto os resultados apresentados pelo ensaio Investigator
Quantiplex aplicado ao CFX Opus 96, apenas permitiram a sua validação para a região-alvo
autossómica. Relativamente ao ensaio Quantifiler Trio aplicado ao CFX Opus 96, será necessária a sua
posterior validação indireta.
Forensic Genetics uses DNA as the main target of analysis in the resolution of forensic cases. However, the amount of DNA present in samples can vary considerably, which may compromise the ability to obtain complete genetic profiles. DNA quantification allows for the analysis of both the quantity and quality of DNA in a sample, enabling the adjustment of the remaining protocol. The qPCR technique is considered one of the most precise and sensitive methods for quantification, allowing realtime monitoring of the amplified product. Before implementing an assay, prior validation is required to test its performance capabilities. At the Forensic Genetics and Biology Service of the Southern Delegation of the National Institute of Legal Medicine and Forensic, I.P., internal validation of DNA quantification assays was conducted using the Quantifiler Trio and Investigator Quantiplex kits on the QuantStudio5 and CFX Opus 96 platforms. The validation was performed directly, by comparing results from casework samples with those obtained using the previously validated Quantifiler Trio assay on the 7500 Real-Time PCR System, and indirectly, through the analysis of specificity, sensitivity, repeatability, reproducibility, and the ability to distinguish between male and female DNA, using control samples. In the context of Forensic Genetics and Biology, the results obtained from the Quantifiler Trio assay on the QuantStudio5 platform allowed for its internal validation, demonstrating superior performance compared to the other assays evaluated. The results from the Investigator Quantiplex assay on the QuantStudio5 supported its validation for samples with concentrations within the calibration curve, while the results from the same assay on the CFX Opus 96 only supported validation for the autosomal target region. As for the Quantifiler Trio assay on the CFX Opus 96, further indirect validation will be required.
Forensic Genetics uses DNA as the main target of analysis in the resolution of forensic cases. However, the amount of DNA present in samples can vary considerably, which may compromise the ability to obtain complete genetic profiles. DNA quantification allows for the analysis of both the quantity and quality of DNA in a sample, enabling the adjustment of the remaining protocol. The qPCR technique is considered one of the most precise and sensitive methods for quantification, allowing realtime monitoring of the amplified product. Before implementing an assay, prior validation is required to test its performance capabilities. At the Forensic Genetics and Biology Service of the Southern Delegation of the National Institute of Legal Medicine and Forensic, I.P., internal validation of DNA quantification assays was conducted using the Quantifiler Trio and Investigator Quantiplex kits on the QuantStudio5 and CFX Opus 96 platforms. The validation was performed directly, by comparing results from casework samples with those obtained using the previously validated Quantifiler Trio assay on the 7500 Real-Time PCR System, and indirectly, through the analysis of specificity, sensitivity, repeatability, reproducibility, and the ability to distinguish between male and female DNA, using control samples. In the context of Forensic Genetics and Biology, the results obtained from the Quantifiler Trio assay on the QuantStudio5 platform allowed for its internal validation, demonstrating superior performance compared to the other assays evaluated. The results from the Investigator Quantiplex assay on the QuantStudio5 supported its validation for samples with concentrations within the calibration curve, while the results from the same assay on the CFX Opus 96 only supported validation for the autosomal target region. As for the Quantifiler Trio assay on the CFX Opus 96, further indirect validation will be required.
Description
Tese de mestrado, Biologia Molecular e Genética, 2025, Universidade de Lisboa, Faculdade de Ciências
Keywords
Quantificação Validação DNA nuclear qPCR Teses de mestrado - 2025