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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.

The use of biological agents as potential weapons has been a concern of security agencies for many years. Security agencies require alternative field protocols for handling forensic samples that could be contaminated with biological weapons. In this study, manual and automated DNA extractions were compared for the ability to remove biological agents and for their effectiveness and consistency when samples were contaminated with bacteria, spores or toxins. Purified DNA was evaluated for the absence of the agents, and for the effects of the process on the isolated human DNA. Results demonstrated that incubation of samples in a cell lysis solution eliminated bacteria and toxins, but an additional 0.22 µm filtration step was necessary to successfully remove bacterial spores. Blood and buccal swab samples exposed to some bacteria showed DNA loss and/or degradation. The automated extraction procedure would be preferable over the manual protocol to isolate human DNA contaminated with biological weapons.

The use of biological agents as potential weapons has been a concern of security agencies for many years. Security agencies require alternative field protocols for handling forensic samples that could be contaminated with biological weapons. In this study, manual and automated DNA extractions were compared for the ability to remove biological agents and for their effectiveness and consistency when samples were contaminated with bacteria, spores or toxins. Purified DNA was evaluated for the absence of the agents, and for the effects of the process on the isolated human DNA. Results demonstrated that incubation of samples in a cell lysis solution eliminated bacteria and toxins, but an additional 0.22 µm filtration step was necessary to successfully remove bacterial spores. Blood and buccal swab samples exposed to some bacteria showed DNA loss and/or degradation. The automated extraction procedure would be preferable over the manual protocol to isolate human DNA contaminated with biological weapons.

Starter cultures of different bacteria strains like lactic acid producing bacteria, Staphylococcus and Kocuria are used when making salami. Starter cultures give the sausage specific flavours and improve the quality and ripening of the final product. Probiotic strains can also be added during the production of salami. Studies have shown that probiotics are good for health and are therefore added to food, such as fermented sausages. In order to work as a probiotic strain, the bacteria have to survive during the production process, storage and through the whole human gastrointestinal tract. The aim of this study was to trace the probiotic strains Lactobacillus casei and Lactobacillus paracasei in salami samples to see if they had survived the production process. Methods used were DNA extraction, PCR, colony PCR and gel electrophoresis. Out of 100 samples in duplicate run in PCR, probiotics were found in only 3 of them. To see if screening of probiotics directly from plates was possible, a colony PCR was done. Colony PCR was made on colonies of two different strains of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus sakei. From each bacteria strain, 5 colonies were analysed. Result showed that colony PCR, to screen for probiotic is a possible method.

The use of biological agents as potential weapons has been a concern of security agencies for many years. Security agencies require alternative field protocols for handling forensic samples that could be contaminated with biological weapons. In this study, manual and automated DNA extractions were compared for the ability to remove biological agents and for their effectiveness and consistency when samples were contaminated with bacteria, spores or toxins. Purified DNA was evaluated for the absence of the agents, and for the effects of the process on the isolated human DNA. Results demonstrated that incubation of samples in a cell lysis solution eliminated bacteria and toxins, but an additional 0.22 µm filtration step was necessary to successfully remove bacterial spores. Blood and buccal swab samples exposed to some bacteria showed DNA loss and/or degradation. The automated extraction procedure would be preferable over the manual protocol to isolate human DNA contaminated with biological weapons.

Orientador: Maria Inês de Moura Campos Pardini / Resumo: Este trabalho visou a comparação de 5 diferentes métodos de extração de DNA, a partir de amostras de materiais de arquivo (tecido incluído em parafina, lâmina de hemograma corada e não corada com Leishman, lâmina de mielograma, gotas de sangue em Guthrie card) e fontes escassas (células bucais, 1 e 3 bulbos capilares, 2 mL de urina), para avaliar a facilidade de aplicação dos mesmos e possibilidade de amplificação desse DNA pela técnica de PCR. Os métodos incluíram digestão por proteinase K, seguida e não seguida por purificação com fenol/clorofórmio, utilização de Chelex 100 Ò, utilização de InstaGeneÒ e fervura em água estéril. O DNA obtido, foi testado por PCR, para a amplificação de três fragmentos gênicos: de Brainderived neutrophic factor (764 pb), de Fator V Leiden (220 pb) e de Abelson (106 pb), sendo que, a amplificação para o primeiro, eliminava a necessidade dos demais. Conforme o tamanho do fragmento gênico estudado, a fonte potencial de DNA e o método de extração utilizado, os resultados caracterizaram o melhor caminho para padronização dos procedimentos técnicos a serem incluídos e apresentados no manual de Procedimentos Operacionais Padrão do Laboratório de Biologia Molecular do Hemocentro - HC - UNESP - Botucatu. / Abstract: The present work aimed to compare five different methods of DNA extraction of archieved materials samples (paraffin-embedded tissues, periferic blood smear - stained or non-stained with Leshman, aspired bone marrow smears and blood guts in Guthrie card) and rare sources (oral cells, 1 and 3 capilar bulbs, 2 mL urine), to avaliate the aplication facility and the amplification possibility one for PCR. The methods included proteinase K digestion - followed or non by phenol/chloroform purification, Chelex 100Ò (BioRad), InstaGeneÒ (BioRad) and boilling in sterile water. The DNA obteined, was tested for amplification of 3 genic fragments: from Brainderived neutrophic factor gene (764 bp), Factor V Leiden gene (220 bp) and Abelson gene (106 bp). According to the genic fragment lenght studed, the DNA potential source and the extraction method used, the results characterized better guidelines for padronization of the techniques procedures for to Good Manufacturing Practices from Molecular Biology Laboratory from Blood Center - Medicine School - UNESP - Botucatu . / Mestre

Made available in DSpace on 2015-07-22T20:50:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-11-24 / Tecidos fixados em formalina e incluídos em parafina permitem investigações retrospectivas valiosas para estudos moleculares, especialmente em estudos genéticos, nos casos em que o DNA não se encontra disponível em amostras congeladas e/ou frescas. Entretanto, de acordo com alguns autores, é difícil obter um DNA de boa qualidade, uma vez que o processo de fixação resulta na fragmentação dos ácidos nucleicos. O objetivo deste trabalho foi avaliar o DNA extraído de tecidos parafinados, após 1 e 5 anos de armazenamento, através de 3 métodos de extração. Para isso, foram utilizados o gene da β-actina (136pb), a fim de detectar a viabilidade e fragmentação do DNA extraído, e da amelogenina (X: 212pb e Y: 218pb) para diferenciação do sexo do indivíduo e viabilidade na utilização de primers com comprimento maior. O estudo envolveu 12 casos de autópsia recentes, onde amostras normais de fígado (n=10), baço (n=10) e cérebro (n=10) foram coletadas em duplicata, de modo que um grupo seguiu para o processo de fixação e inclusão em parafina, e outro grupo seguiu para o congelamento. Além disso, foram utilizados os mesmos tipos de tecidos, normais (n=10 cada), oriundos de 13 casos de autópsia armazenados por 1 ano e 15 casos armazenados por 5 anos. Após a remoção da parafina, as amostras foram submetidas às extrações com kit comercial (QIAGEN QIAamp Mini), Salting-Out e fenol-clorofórmio. O DNA extraído foi quantificado no aparelho Nanodrop® e ajustado para PCR (10ng/μl). Os produtos de PCR foram visualizados em gel de agarose a 1%. As amostras de baço e fígado apresentaram maior rendimento em relação à extração de DNA quando comparado ao cérebro, em todos os tempos. Todas as amostras arquivadas apresentaram boas condições de extração de DNA, porém deve-se levar em consideração o processo de fixação e inclusão dos tecidos, que podem comprometer a qualidade do DNA. A extração pelo fenol rendeu maior quantidade de DNA e grau de pureza em relação aos outros métodos estudados, porém o kit comercial mostrou melhores resultados quanto à amplificação do DNA obtido. Houve amplificação do gene da amelogenina em todas as amostras utilizadas, porém recomenda-se a utilização de primers menores para uma completa análise do fragmento a ser estudado. / Formalin fixed and paraffin embedded tissues provide valuable retrospective investigations for molecular studies, especially for genetic studies, when the DNA is not available in fresh and/or frozen samples. However, according to some authors, is difficult to obtain a DNA of good quality, since the fixation process results in nucleic acids fragmentation. The aim of this study was to evaluate the DNA extracted from paraffin embedded tissues, after 1 and 5 years of storage, by 3 methods of extraction. For this, the gene of β-actin (136pb) were used, to detect the viability and DNA fragmentation, and the gene of amelogenin (X: 212pb e Y: 218pb) for sexual differentiation and viability in primers with greater length. The study involved 12 recent autopsy cases, where samples of liver (n=10), spleen (n=10) and brain (n=10) were collected in duplicate, which one group followed to the process of fixation and inclusion and the other group followed to freezing. Moreover, the same kind of tissues, normal (n=10 each), from 13 autopsy cases archived for 1 year and 15 cases archived for 5 years were used. After paraffin remove, the samples were submitted to DNA extraction with commercial kit (QIAGEN QIAamp Mini), Salting-Out and phenol-chlorophorm. The DNA extracted was quantified in Nanodrop® and adjusted for PCR (10ng/μl). The PCR products were visualized in agarose gel 1%. The samples of spleen and liver showed more yield in DNA extraction than the brain samples, in all the times. All the samples archived showed good extraction conditions, however should take in consideration the fixation and embedded process, which could compromise the DNA quality. The extraction by phenol-chloroform yielded more DNA quantity and purity than the other methods. However, the commercial kit extraction showed better results in DNA amplification. The primer of the gene of amelogenin was amplified in all utilized samples, however recommends the utilization of smaller primers for a complete analyze of the fragment studied. / TEDE / BV UNIFESP: Teses e dissertações

The use of biological agents as potential weapons has been a concern of security agencies for many years. Security agencies require alternative field protocols for handling forensic samples that could be contaminated with biological weapons. In this study, manual and automated DNA extractions were compared for the ability to remove biological agents and for their effectiveness and consistency when samples were contaminated with bacteria, spores or toxins. Purified DNA was evaluated for the absence of the agents, and for the effects of the process on the isolated human DNA. Results demonstrated that incubation of samples in a cell lysis solution eliminated bacteria and toxins, but an additional 0.22 µm filtration step was necessary to successfully remove bacterial spores. Blood and buccal swab samples exposed to some bacteria showed DNA loss and/or degradation. The automated extraction procedure would be preferable over the manual protocol to isolate human DNA contaminated with biological weapons.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / Ichthyophonus hoferi is a highly pathogenic histozoic parasite that has low host specificity capable of producing mass mortalities of epizootic proportions in marine commercial fish populations. Currently in Southern Africa, I. hoferi has been reported from flathead mullet (Mugil cephalus) from the Kowie lagoon and from multiple species on exhibit at the Two Oceans Aquarium. Since epizootiologists rely on accurate assessments of prevalence to establish patterns of morbidity and mortality within populations, using the most accurate diagnostic techniques for accurate assessments of infection is imperative. Currently, several diagnostic techniques have been employed to detect I. hoferi in infected fish hosts. These include macroscopic examination of tissues, microscopic examinations of wet-mount squash preparations of tissue, histological examination of tissue sections, in vitro culture of tissue explants, the polymerase chain reaction (PCR) using I. hoferi-specific primers and real-time quantitative PCR (qPCR) using I. hoferi-specific primers and a hydrolysis probe.