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Comparison of different proteases and direct cell lysis methods used for the recovery of exogenous DNA from fingernail evidenceIzzo, Caitlin Rose 02 November 2017 (has links)
Fingernail samples are analyzed in forensic casework to determine the source of the nail and/or to recover a foreign profile from beneath the nail. When extracting from a fingernail sample, it is possible to recover deoxyribonucleic acid (DNA) of the nail donor from within the nail and from the surface of the nail; similarly, foreign DNA may also be present on and recovered from the nail surface. When attempting to recover the latter, fingernail samples present particular problems. Often, the foreign component is masked by the greater mass of donor DNA present within and on the nail sample. This masking effect is exacerbated by the use of proteinase K (PK) in DNA extractions, as PK, with an average of 200 cut sites per keratin molecule, is capable of breaking open the keratin matrix of the nail and exposing the nail DNA intercalated in the matrix. Directly extracting nail clippings, in contrast to swabbing or scraping, would further introduce nail DNA when using proteinase K. The present study explores whether utilizing other proteases (ZyGEM, Acrosolv, and Factor Xa) with fewer cut sites than PK or direct lysis methods (IGEPAL® CA-630 and MAWI iSWABTM-ID) would minimize recovery of nail
DNA from within the nail and thus mitigate the masking effect often seen with fingernail samples.
The endogenous DNA extraction efficiency of each suggested method was compared with the manufacturer’s standard QIAGEN QIAamp® DNA Investigator extraction protocol for hand-washed and/or laboratory-cleaned nails. The extraction results from the hand-washed nails demonstrate variability both within samples from the same donor and between donors. In contrast to previously published literature, a comparison of the results between the hand-washed and cleaned nails suggests that much of the endogenous DNA recovered from fingernail samples is derived from DNA on the surface rather than from within the nail.
QIAamp® extraction with the inclusion of dithiothreitol (DTT) recovered significantly more DNA (0.845 ± 0.651 nanograms of DNA per milligram of nail [ng DNA/mg nail]; p = 0.0045) than the same protocol without DTT (0.278 ± 0.253 ng DNA/mg nail). IGEPAL® recovered the least endogenous DNA (0.005 ± 0.012 ng DNA/mg nail) from the nail. The ZyGEM extraction recovered the second lowest amount (0.163 ± 0.161 ng DNA/mg nail) and both the Acrosolv (0.546 ± 0.607 ng DNA/mg nail) and MAWI’s iSWABTM-ID (0.681 ± 0.780 ng DNA/mg nail) methods recovered more DNA than the QIAamp® protocol without DTT. An assessment of the electropherograms resulting from cleaned fingernails across all extraction methods for one donor showed that both IGEPAL® and MAWI failed to recover a complete profile, whereas the remaining methods were able to recover complete profiles of the nail donor.
An assessment of donor variability found variations in terms of endogenous nail DNA recovery.
Fingernails were also spiked with blood, saliva, or semen to assess the recovery of foreign DNA. The extractions of the spiked nail samples demonstrate variability across all samples, owing, to some degree, to inconsistencies of sample preparation. IGEPAL®’s inability to recover complete foreign profiles suggests that the method may not be viable for extraction of fingernail samples. Conversely, the ZyGEM, Acrosolv, and MAWI extraction methods demonstrate potential as alternative extraction methods for fingernail samples and would benefit from additional experimentation.
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Évaluation de l’exposition populationnelle aux métaux et métalloïdes issus de l’extraction minière artisanale d’or au MaliDiarra, Zeïnaba 04 1900 (has links)
L'exploitation artisanale de l'or peut libérer des éléments métalliques dans l'environnement, ce qui peut entraîner des expositions professionnelles et environnementales. Cette étude pilote visait à évaluer l'exposition aux métaux et métalloïdes chez les travailleurs d'une mine d'or artisanale, les habitants d'un village minier et les habitants d'un village non minier au Mali par une biosurveillance multiéléments. Un échantillonnage par convenance de 315 personnes a été réalisé, réparti de manière égale dans chaque localité (105 par site) et stratifié par groupe populationnel (hommes adultes, femmes adultes et personnes âgées de moins de 18 ans). Des échantillons d'ongles d'orteils et d'ongles de doigts ont été prélevés sur chaque participant de ces différents groupes et 21 éléments (argent, aluminium, arsenic, baryum, béryllium, cadmium, cobalt, chrome, cuivre, fer, gallium, lithium, manganèse, nickel, plomb, sélénium, strontium, thallium, uranium, vanadium et zinc) ont été quantifiés dans ces matrices par spectrométrie de masse avec plasma à couplage inductif (ICP-MS). Les résultats ont montré que les concentrations de 12 éléments dans les ongles des doigts ou des orteils (en particulier l’arsenic, le cobalt et le cuivre à la fois dans les ongles des doigts et des orteils) étaient plus élevées dans le groupe des travailleurs de la mine par rapport aux groupes des villages miniers et non miniers. Dans le groupe des travailleurs de la mine en particulier, les concentrations d’arsenic dans les ongles des mains et des pieds étaient significativement plus élevées chez les hommes. Les concentrations d’arsenic dans les deux matrices étaient également significativement plus élevées chez les fumeurs par rapport aux non-fumeurs. Les concentrations de nickel, strontium et zinc dans les ongles des doigts et des orteils étaient statistiquement plus élevés chez les femmes que chez les hommes. La plupart des métaux présentaient également une forte corrélation positive dans l’ensemble. Au vu des résultats, il serait pertinent d'étendre cette étude en incluant une évaluation de l'exposition par biosurveillance humaine avec d'autres matrices biologiques telles que le sang et l'urine, couplée à des évaluations environnementales pour mieux identifier d'autres sources potentielles d'exposition tout en tenant compte des facteurs de confusion potentiels et pour comparer les résultats obtenus aux normes internationales.
Mots-clés : Exploitation minière artisanale et à petite échelle de l'or, biosurveillance humaine, ongles des orteils, ongles des doigts. / Artisanal gold mining can release metallic elements in the environment that can result in occupational and environmental exposures. This pilot study aimed to assess exposure to metals and metalloids in artisanal gold mine workers, inhabitants of a mining village and inhabitants of a non-mining village in Mali by multi-element biomonitoring. A convenience sampling of 315 individuals was performed equally distributed in each location group (105 per location) and stratified by populational group (adult males, adult females, and people <18 years). Toenail and fingernail samples were collected from each participant of these different groups and twenty-one elements (silver, aluminum, arsenic, barium, beryllium, cadmium, cobalt, chromium, copper, iron, gallium, lithium, manganese, nickel, lead, selenium, strontium, thallium, uranium, vanadium, and zinc) were quantified in those matrices by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that concentrations of 12 elements in fingernails and/or toenails (in particular arsenic, cobalt, and copper both in toenails and fingernails) were higher in the mine worker group compared to the mining and non-mining village groups. In the mine worker group specifically, arsenic concentrations in both fingernails and toenails were significantly higher in males. Arsenic concentrations in both matrices were also significantly higher in smokers compared to non-smokers. Nickel, strontium and zinc concentrations in both fingernails and toenails were statistically higher in females than in males. Most metals also had a strong positive correlation overall. In view of the results, it would be relevant to extend this study by including a human biomonitoring exposure assessment with other biological matrices, such as blood and urine, coupled with environmental assessments to better identify other potential sources of exposure while accounting for potential confounders and to compare the results obtained to international standards.
Keywords: Artisanal and small-scale gold mining, human biomonitoring, toenails, fingernails
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