Abstract
At a crime scene, investigators are faced with a multitude of traces. Among them, biological traces are of primary interest for the rapid genetic-based identification of individuals. “Touch ...DNA” consists of invisible biological traces left by the simple contact of a person’s skin with objects. To date, these traces remain undetectable with the current methods available in the field. This study proposes a proof-of-concept for the original detection of touch DNA by targeting cell-derived fragments in addition to DNA. More specifically, adhesive-structure proteins (laminin, keratin) as well as carbohydrate patterns (mannose, galactose) have been detected with keratinocyte cells derived from a skin and fingermark touch-DNA model over two months in outdoor conditions. Better still, this combinatory detection strategy is compatible with DNA profiling. This proof-of-concept work paves the way for the optimization of tools that can detect touch DNA, which remains a real challenge in helping investigators and the delivery of justice.
Abstract Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person’s skin’s contact with an object or another person. Many factors influence ...touch DNA transfer, including the “destination” substrate’s surface. The latter’s physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates’ physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates’ intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.
The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The ...traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.
The Forensic Science Institute of the French “Gendarmerie Nationale” (IRCGN™) developed in 2015 an ISO 17025 certified mobile DNA laboratory for genetic analyses. This Mobil’DNA laboratory is a fully ...autonomous and adaptable mobile laboratory to perform genetic analyses in the context of crime scenes, terrorism attacks or disasters. To support the hospital task force in Paris during the peak of the COVID-19 epidemic, we adapted this mobile genetic laboratory to perform high-throughput molecular screening for coronavirus SARS-CoV-2 by real-time PCR. We describe the adaptation of this Mobil’DNA lab to assist in Coronavirus SARS-CoV-2 diagnosis.
Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal ...events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca2+ flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7–6.7), whereas anti-TLR3-mediated Ca2+ flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15–40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling.
Le développement d’une méthode de prélèvement de l’ADN sur le terrain a été l’opportunité pour la Gendarmerie de s’inscrire dans une démarche innovante débouchant sur un brevet et un partenariat pour ...la production industrielle de l’invention conçue par un gendarme. Cette réalisation a été le prélude à d’autres projets.
The Forensic Science Institute of the French "Gendarmerie Nationale" (IRCGN™) developed in 2015 an ISO 17025 certified mobile DNA laboratory for genetic analyses. This Mobil'DNA laboratory is a fully ...autonomous and adaptable mobile laboratory to perform genetic analyses in the context of crime scenes, terrorism attacks or disasters.To support the hospital taskforce in Paris during the peak of the COVID-19 epidemic, we adapted this mobile genetic laboratory to perform high-throughput molecular screening for coronavirus SARS-CoV-2 by real-time PCR. We describe the adaptation of this Mobil'DNA lab to assist in Coronavirus SARS-CoV-2 diagnosis.
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