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Development and validation of molecular methodologies for identification of biological traces from crime scenes

Published:October 07, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.10.006

      Abstract

      This study aimed to develop a methodology to identify biological fluids in sexual assault cases through mRNA markers. Biological fluid samples such as blood, saliva, and semen were collected from volunteers and submitted to RT-qPCR reactions with specific primers for the biomarkers HTN3 (saliva), ALAS (blood) andTGM4 (semen). The Melting (Tm) of each biological fluid was analyzed and the result inferred a high specificity capable of differentiating such traces. Biplex systems were generated to improve trace analysis in a single qPCR reaction.

      Keywords

      1. Introduction

      Forensic Genetics emerged in the 1980s and has been improving with the development of technological tools that assist in solving crimes. In addition to techniques for human identification by DNA, knowledge about the identification of biological fluids found at crime scenes has fundamental importance to assist in the elucidation of the crime committed, as it can lead to a possible link between the donor and criminal activity [
      • Lindenbergh A.
      • et al.
      A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.
      ,
      • An J.H.
      • et al.
      Body fluid identification in forensics.
      ,
      • Albani P.P P.P.
      • Fleming R.
      Novel messenger RNAs for body fluid identification.
      ]. For sexual assault, this type of analysis is especially important, as biological fluids are a source of genetic material for possible identification of the aggressor. The analysis of gene expression patterns and the pathophysiological conditions provide information about cell function [
      • Sijen T.
      • Harbison S.
      On the identification of body fluids and tissues: a crucial link in the investigation and solution of crime.
      ], thus, through the use of mRNA as a biomarker, the objective of this study was to develop a methodology for the identification of biological fluids relevant to sexual assault by dissociation curve analysis (MELT).

      2. Materials and methods

      A total of 15 volunteers agreed to participate in the study by donating their biological fluids. After signing and agreeing with the consent form, the sample collection was performed as described below: Blood samples were collected by venipuncture, with a vacuum system, using a tube of EDTA K3 (ethylenediaminetetraacetic tripotassium acid) as an anticoagulant. Saliva sampling in the oral cavity was performed by gently scraping the roof of the mouth, cheeks, and tongue with a cotton stick at the end (sterile swab). The semen sample was collected by masturbation after sexual abstinence for two to five days. The volunteer performed local asepsis with soap and water, and then placed the semen in a sterile bottle. RNA extractions from biological fluid samples were performed with the organic reagent Trizol® (ThermoFisher). After extraction, quantification was determined on a Qubit Fluorometer (ThermoFisher Scientific), using the RNA HS (High Sensitivity) Assay kit. Primers for the PCR reaction were used according to Lindenbergh et al. [
      • Lindenbergh A.
      • et al.
      A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.
      ] and Roeder et al. [
      • Roeder A.M.
      • Haas C.
      Body fluid identification using mRNA profiling.
      ]. The SOLIScript® 1-step SolisGreen kit (SolisBiodyne) was used for amplification and the analysis of the MELT curve was used to characterize the different biological fluids.

      3. Results and discussion

      Biological fluids collected at crime scenes are used to provide evidence in forensic cases because they help to understand the dynamics of the event, which becomes essential for the identification of tissue origin [
      • Liang Q.
      • Sun H.
      • Wu X.
      • Ou X.
      • Gao G.
      • Jin Yi
      • Tong D.
      Development of new mRNA markers for the identification of menstrual blood.
      ]. The most common methods used are not able to identify the tissue type of body fluids, as they are based on old, limited, nonspecific, and subjective techniques, which might generate false results [
      • An J.H.
      • et al.
      Body fluid identification in forensics.
      ]. Thus, more current techniques that analyze gene expression through biomarkers such as mRNA and which allow the identification of tissue origin have become increasingly relevant in forensic genetics. An effective and highly specific analysis occurs through the dissociation curves pattern (MELT), where each biological fluid presents a unique and differentiated profile. In this study, the genes analyzed, HTN3 (saliva), ALAS (blood), and TGM4 (semen) showed high specificity for detection based on the dissociation curve with 90% confidence, the quantification by cycling threshold (Ct) analysis between 14 and 32 cycles and Melt profile (Tm) of 79 °C, 84 °C, and 89 °C respectively. Another important feature in forensic cases concerns the samples collected at crime scenes that may be degraded and/or present in low quantities. Therefore, sensitivity tests were performed in a range of quantification of total RNA between 0.007 and 10 ng and the result was the amplification of the probes even with small traces of RNA in the samples. An assay to identify different biological fluids in a single reaction was standardized for a more efficient and objective biological trace analysis. Reactions with multiple probes were tested and Biplex systems that detect at the same time blood/semen and/or saliva/semen were developed. The observed results (Fig. 1) reveal a system capable of generating a fast, accurate solution, capable of optimizing the forensic work for crime scene trace analysis.
      Fig. 1
      Fig. 1Results by QuantStudio 5 (Thermo Fisher). Amplification plot (A); Melt curve in biplex PCR with mixture samples of blood and semen (B); Melt curve in biplex PCR with mixture samples of saliva and semen (C).

      4. Conclusion

      The current work presents optimizations for trace analysis at a crime scene, mainly in the context of sexual assault with a robust system, a sensitivity capable of detecting low traces of mRNA, and high specificity for the identification of different biological fluids in a single reaction.

      Ethics approval and consent to participate

      This study was approved by the Ethic Committees at the State University of Rio de Janeiro (CAAE 54247521.8.0000.5259), and all participants signing the requirement for informed consent to their participation.

      Funding

      This study was financially supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Brazil (FAPERJ), grant #E-26/E-26/010/100811/2018 – ADT1.

      Conflict of interest

      None.

      Acknowledgments

      Cancer Biology Laboratory (Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro), Histocompatibility and Cryopreservation Laboratory (Polyclinic Piquet Carneiro, State University of Rio de Janeiro) and Genetic Laboratory (Brazilian Arms Institute of Biology).

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