New strategies for rapid nucleic acid analysis : Studies and development of biological collecting tools for DNA identification

La criminalistique peut être définie comme l’application de procédés techniques aux investigations judiciaires permettant l’étude scientifique des traces et des indices retrouvés sur les scènes de crime.Depuis la découverte de l’empreinte génétique par Sir Alec Jeffreys en 1984, le monde judiciaire s’est profondément ancré dans l’ère de l’ADN en raison d’évolutions technologiques successives dans le domaine de la biologie moléculaire et ses applications en criminalistique. Le besoin de réponse instantanée est omniprésent dans les esprits. La mise en œuvre de techniques d’analyses simples, sensibles, fiables et permettant d’obtenir des résultats dans les plus brefs délais sont les clés du succès.Au cours des processus techniques, la collecte du matériel biologique, et donc de l’ADN au sein de la trace, constitue une étape incontournable et cruciale qui va conditionner la réussite des analyses. Ce travail de recherche a donc consisté à développer des solutions performantes de prélèvements de matériels biologiques soit en détournant de leur fonction initiale des solutions existantes soit en développant des solutions simples mais innovantes combinant les avantages des solutions existantes. Ces travaux ont permis de donner naissance au micro-écouvillon GendSAG. Les potentialités de GendSAG permettent de proposer une solution alternative aux solutions commerciales de systèmes intégrés d’analyses rapide d’ADN. Cette solution alternative d’analyse rapide et haut débit de l’ADN mise en œuvre dans un laboratoire mobile au plus près de la scène de crime répond non seulement à la grande majorité des avantages des systèmes intégrés mais également à toutes leurs limitations.Forensic sciences can be defined as the used of technical processes to judicial investigations allowing the scientific study of traces and evidences found on crime scenes.Since the discovery of DNA fingerprinting by Sir Alec Jeffreys in 1984, the legal world has become deeply rooted in the DNA by successive technological developments in molecular biology and its applications in forensic. The need for instant response is omnipresent in the minds. The key to success is the implementation of simple, sensitive, reliable analytical techniques that enable results to be achieved in the shortest possible time.During these technical processes, the collection of biological samples, is an unavoidable and a crucial step that will condition the analysis success rate. This study consisted in developing efficient biological collecting solutions either by diverting from their original function the existing solutions or by developing simple but innovative solutions combining the advantages of the existing solutions. This allowed developing the micro-swab GendSAG. The potentialities of GendSAG make it possible to propose an alternative solution to the commercial rapid DNA analysis integrated systems. This rapid, cost effective and high-throughput DNA analysis solution performed in a dedicated mobile laboratory directly into the crime scene enables the large majority of the rapid DNA analysis integrated systems benefits and also all of their limitations

Evaluation of DNA Methylation-Based Age-Prediction Models from Saliva and Buccal Swab Samples Using Pyrosequencing Data

In forensic genetics, the identification of an individual is often carried out by comparing unknown DNA profiles obtained in a case against databases or references. When no match is found, investigators need new tools in order to obtain additional leads. The latest technical advances now make it possible to predict externally visible characteristics. With this objective, predicting the age of an individual through DNA methylation analysis remains one of the last challenges. The prediction models have to account for the specific constraints of this field, including tissue specificity and DNA availability (i.e., low DNA amounts or low-quality DNA). Jung and colleagues have recently produced models from blood, saliva and buccal cells by using a single base extension sequencing method. With the goal of evaluating these models in our own analytical conditions, saliva and buccal cell samples from 115 French individuals between the ages of 0 and 88 years old were collected and analyzed. After having determined the optimal analysis conditions, including the DNA quantity for bisulfite conversion (75 ng), some differences were highlighted in the measured methylation rates between the two studies. Despite these discrepancies, the prediction performance levels remain very similar, our study showing mean absolute errors of 3.5 years, 3.9 years and 3.2 years, respectively, for the saliva, buccal swab and multitissue model, with limitations observed for the oldest and youngest individuals. Furthermore, we propose the use of a prediction interval with an error dispersion and correct prediction rate at ±5 years and ±10 years, respectively

Innovations in Forensic Sciences for Human Identification by DNA in the French Gendarmerie during the Last 10 Years

The IRCGN (Institut de Recherche Criminelle de la Gendarmerie Nationale) is a forensic science institute built by the French Gendarmerie which has the ability to exploit crime scene evidence. Any piece of evidence, anywhere in the world, in any environment, can be examined by IRCGN teams deployed in just a few hours. During the past 10 years, experts specializing in genetics have developed innovative genetic engineering technologies for application in forensic sciences. In this review, we highlight the main innovations and the creation of new tools for human identification, which are fully suited to the French Gendarmerie’s needs. Devices developed by the IRCGN are specific to the Gendarmerie’s purposes

Targeting cell-derived markers to improve the detection of invisible biological traces for the purpose of genetic-based criminal identification

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

A mobile DNA laboratory for forensic science adapted to coronavirusSARS-CoV-2 diagnosis

International audienceThe 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

MicroFLOQ® Direct: A Helpful Tool for the Coronavirus SARS-Cov-2 Rapid Detection without RNA Purification

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Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

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 couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark 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 in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume 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 procedures, 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

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

International audienceThe 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 vo

Outcomes in Newly Diagnosed Atrial Fibrillation and History of Acute Coronary Syndromes: Insights from GARFIELD-AF

BACKGROUND: Many patients with atrial fibrillation have concomitant coronary artery disease with or without acute coronary syndromes and are in need of additional antithrombotic therapy. There are few data on the long-term clinical outcome of atrial fibrillation patients with a history of acute coronary syndrome. This is a 2-year study of atrial fibrillation patients with or without a history of acute coronary syndromes