Tempo de viabilidade da saliva em meio externo para fins de extração e quantificação de DNA

Abstract

The analysis of genetic material plays a crucial role in forensic investigations, especially in human identification. Saliva has emerged as a viable and promising alternative. In this sense, this dissertation is organized into chapters that refer to articles developed during the master's course. The first chapter presents, through a scope review article published in the Brazilian Journal of Forensic Odontology (RBOL), the state of the art on the topic. Aiming to map the viability time of human saliva in an external environment for DNA extraction and quantification, the review was developed following the Joanna Briggs Institute protocol and was previously registered on the Open Science Framework platform (doi: 10.17605/OSF.IO/PN9ET), with searches conducted in the following databases: PubMed, Web of Science, Scopus, LILACS, Cochrane, and Google Scholar, without restrictions regarding publication period or language. After the initial screening of the 283 identified studies, followed by a full-text reading to confirm eligibility criteria, 6 studies were included in the review. Cigarette butts, dental prostheses, dental compression pads, oral cavity, and FTA cards were described as substrates from which collected saliva samples were obtained. The DNA viability was verified in time frames ranging from 1 day to 11 years. It was found that the sample collection and storage protocol is a factor that can influence the quantity and quality of the examined material; however, viable DNA was observed in analyses conducted a decade after saliva collection, and this was the maximum follow-up time reported in the studies. In the second chapter, a laboratory research article is presented, which investigated the viability time of DNA extracted from human saliva when exposed to the external environment, with the aim of evaluating its potential for human identification. The study involved 10 healthy adult volunteers. Participants chewed a sialogogue for 3 minutes and then expelled their saliva into sterile Falcon tubes. The samples were exposed to the environment and analyzed at five distinct time points: 0, 7, 15, 30, and 60 days. Afterward, respecting the analysis time for each group, the saliva samples were immersed in 800 μL of TRIzol™ Reagent, a solution used for DNA extraction. The samples were then centrifuged and cryopreserved at - 20°C. In the next phase, after extracting the aqueous-phase substances, which contain DNA molecules, these were recovered and transferred to new microtubes, to which alcohol was added to precipitate the DNA molecules after another centrifugation. For tubes containing samples without visible solution, 50 μL of ultrapure water was added, and then 8 μL of each sample was taken and mixed with 10 μL of mastermix and 2 μL of primer for the B-actin gene. Next, the genetic material quantity was assessed using a thermocycler to check for fluorescence emission, real-time PCR (Polymerase Chain Reaction). After collecting the relative results, the data were statistically analyzed using the Jamovi software (version 2.3.21), with a significance level of 5%. The results showed a loss of approximately 20% in the DNA quantity, which remained consistent across all analyses from the 7th day onward. The variations in the DNA values across the different time groups did not show statistically significant differences (ANOVA for repeated measures; p = 0.475). The results indicate that saliva exposed to the external environment for 60 days is still viable for human identification purposes. It can be concluded that the two articles complement each other, as the first article demonstrates what is known about the topic and the gaps in knowledge, such as the lack of a specific saliva collection and storage protocol and the definition of the maximum time limit at which saliva becomes non-viable. The second article addresses this last gap by conducting experimental research that shows saliva is a viable substrate for human identification even after being exposed to the environment for 60 days. This study consolidates saliva as a reliable genetic material for forensic analysis, reinforcing its stability and viability even after long periods of environmental exposure, which expands its potential for application in human identification and in resolving criminal investigations.