The authors thank Dr. Yoav Barak from the Chemistry Faculty, Weizmann Institute, for support and advice. This study was supported by the Israel Science Foundation (grant # 913/12), and by the Minerva Foundation with funding from the Federal German Ministry for Education and Research.

<ol>
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The authors have nothing to disclose.

Este trabalho descreve métodos de extração e detecção para quantificar tanto endotoxinas e DNA presente em amostras de aerossóis recolhidas em filtros. Os métodos requerem rotinas precisas e pode ser executada facilmente, desde que o experimentalista adere a alguns pontos essenciais e importantes discutidos aqui. Para o passo de detecção de endotoxina, note que o lisado solução é bastante viscosa e tende a produzir bolhas mediante pipetagem. É difícil de remover ti bolhas, e eles levam a alterações nos valores de microplacas-reader. Portanto, é importante para o primeiro lugar a amostra na placa e apenas depois de se assegurar que todas as bolhas desapareceu, continuar com a adição da solução de lisado. Uma técnica útil para prevenir a formação de bolhas no presente passo consiste em trabalhar-se lentamente com a pipeta, e não para pressionar todo o caminho durante a drenagem do lisado para os poços. Como reacção começar imediatamente uma vez que o lisado está a ser adicionado à endotoXin extrair, que é essencial para iniciar a leitura o mais cedo possível. Para encurtar o passo lisado disso, uma pipeta de múltiplos canais pode ser usado. Note-se que em alguns kits e protocolos existe uma etapa de pré-incubação adicional das amostras a 37 ° C durante 10 min, após o que é colocado na placa de micro titter. 29 Só após esta etapa, o lisado pode ser adicionado. Um ponto importante para a extracção da amostra ambiental de endotoxinas é a presença de inibidores das amostras (por exemplo, na amostra de ar de alta poluição). Neste caso, a diluição torna-se crítico, e pode evitar falsa reforço, tipicamente observado para a amostra não diluída. 38 Da mesma forma, existem vários pontos de importância para garantir o sucesso e confiabilidade da detecção q-PCR. 1) Como o volume das soluções é muito pequena, se eles não são colocados no fundo do poço, o teor de água na gota de ADN pode ser reduzida por evaporação while dispor as amostras na placa. Para evitar este problema, colocar a amostra de ADN no fundo do poço, arrefece-se a placa, colocando-o em gelo ou outra plataforma de arrefecimento, e preparar-se tudo com antecedência para acelerar este passo, tanto quanto possível. 2) Quando se prepara a mistura de reacção de Q-PCR (Tabela 4), ​​cobrir o tubo com folha de alumínio para evitar a foto-degradação. 3) O presente experimento descreve a preparação de uma reacção de 10 ul. No entanto, em alguns instrumentos Q-PCR, o volume de reacção é de 20 uL e, em seguida, o volume de cada reagente é multiplicado em conformidade. 4) As condições de ciclos térmicos pode alterar óptimas para diferentes iniciadores e misturas de polimerase 39. Por exemplo., A temperatura de recozimento deve ser fixado em torno da temperatura de fusão dos iniciadores, e é determinado empiricamente como a temperatura à qual ocorre a amplificação mais eficiente. Por esta razão, é também importante para preparar uma curva de calibração para cada analisado Q-PCRplaca, para assegurar que a quantificação é preciso. As curvas de calibração, bem como filtros-padrão cravado também servir como um controlo positivo, para assegurar que não ocorre inibição. Para evitar a inibição, é recomendado o uso de kits de extracção destinadas a eliminar os inibidores a partir de amostras ambientais. 5) O número de ciclos térmicos nesta experiência foi definido como o número máximo, de 45, a fim de aumentar a sensibilidade como as concentrações de ADN a partir dos extractos de filtro foram muito baixos. 6) Certifique-se de que os ciclos para Threshold (Cτ) valores para todas as amostras de DNA estão dentro do alcance dinâmico da curva de calibração. Também garantir que qualquer amplificação de uma amostra de controlo negativo é, pelo menos, oito ciclos maior do que a das amostras de DNA. 7) Se estiver trabalhando com o reagente Verde SYBR, certifique-se de que não existem vários picos de temperatura de fusão por reacção. Para o teste turbidimétrico, dois métodos de quantificação de endotoxinas estão disponíveis: o método de cinética ea endpoint método cromogénico. Na abordagem cinética, como realizado no presente protocolo, o tempo necessário para a amostra atingir a absorvância máxima é medida. Aqui, um intervalo de reacção mais curto indica uma concentração mais elevada de endotoxinas na amostra. No método cromogénico ponto de extremidade, a absorção de luz é medida após um tempo de incubação definido para determinar a concentração de endotoxinas. Ambos os métodos exigem uma curva de calibração padrão para quantificação 40. Embora a precisão e exactidão da endotoxina extracção acima descrito é muito alta, a eficiência de extracção 14 pode ser melhorada. É possível que um tipo diferente de filtro ou a adição de um detergente (tal como o polissorbato 20) para o procedimento de extracção pode melhorar o rendimento de endotoxina extraída a partir do filtro. A eficiência da extracção de Endotoxina pode também ser influenciado por diferentes partículas ensaiadas em paralelo sobre o filtro. 33 Para o nosso sistema experimental, olimite do método de detecção é estimada como sendo de 0,001 m -3 UE 14. Técnicas para quantificar o extracto de ADN que pode servir como uma alternativa à Q-PCR incluem abordagem de clonagem por PCR e gota digitais (DD-PCR). As vantagens de Q-PCR em relação à abordagem de clonagem para a identificação de espécies são a sua maior sensibilidade e que requer uma concentração de DNA mais baixa para o passo de amplificação inicial. Além disso, ao contrário de Q-PCR, o método de clonagem, o que é trabalhoso e demorado, não é quantitativa. Um método alternativo para a quantificação do extracto de ADN é DD-PCR. 19 A vantagem desta técnica é que ele não necessita de uma curva de calibração, tal como a detecção baseia-se numa única estirpe de ADN em cada gotícula. No entanto, para as amostras ambientais, nenhum método fiável existe até agora para de detecção de gota de DNA extraído a partir de filtros. A eficiência com que o DNA é extraído FRom o filtro, bem como a exactidão e precisão das medições de ADN, pode ser influenciado por factores como o tipo de filtro, microrganismos alvo, e instrumentação. 34,41 Por conseguinte, deve ser definida antes da análise de amostras. Em nossa configuração experimental, o limite de detecção pode chegar até 3,5 genoma m -3. 14 Para concluir, o método descrito aqui é aplicável para a identificação e quantificação de espécies biológicas amostragem de ar, tanto de fontes naturais e antropogênicas. uso preciso do ensaio apropriado permite investigações valiosas de questões ambientais complexas a serem realizadas.

Amostragem de ar em filtros é uma ferramenta básica na investigação aerossóis atmosférica. 1 Os filtros são amostrados o ponto de partida para várias propriedades químicas, físicas e biológicas das caracterizações partículas ambiente recolhida. 2-11 A vantagem desta abordagem é que várias análises podem ser realizado fora de linha na mesma amostra. Compilando os dados de todas as diferentes análises permite ao pesquisador obter uma boa compreensão das características das partículas e auxiliares colhidos em resolver questões complexas nas ciências atmosféricas. 12,13 Por exemplo, aparelhos de ar amostras marinhas e fluviais tomada durante o mesmo período pode ser comparado com respeito à toxicidade de partículas amostrado e composição biológica. 14 para este estudo, lipopolissacáridos (LPS), componentes de células, as paredes bacterianas gram-negativas, também conhecidos como endotoxinas, foram extraídos a partir de filtros amostrados em terra e em um site para o interior, e foram avaliados usando olimulus amebócito lisado (LAL) de teste. Em paralelo, uma avaliação do conteúdo genómico bacteriano (bactérias totais, bactérias gram negativas, e cianobactérias) foi realizada na mesma amostra, utilizando a reacção em cadeia da polimerase quantitativa (Q-PCR). O teste LAL baseia-se em medições de turvação formada após a adição de um extracto aquoso de amebócitos do caranguejo-ferradura, Limulus polyphemus, a uma solução aquosa que contém as endotoxinas. Quanto maior for a concentração de endotoxinas na amostra, a turbidez mais rapidamente se desenvolve. 15 A análise Q-PCR baseia-se num sinal de fluorescência emitido como um fragmento de ADN específico é amplificado. 16 por monitorização em tempo real do sinal durante a fase exponencial da PCR reacção e de calibragem com uma curva padrão, a quantidade inicial de ADN pode ser quantificada. A combinação destas duas análises em conjunto com os outros, como detalhado em outra parte, 14 pode fornecer uma boa estimativa dos níveis de endotoxina e aquantidade de bactérias nas amostras de origem. O objectivo do método aqui apresentado consiste em obter uma análise precisa e altamente fiável do conteúdo de endotoxina e de ADN de bio-aerossóis extraídos de filtros. Embora os métodos para a amostragem das características físico-químicas e inorgânicos dos aerossóis são bem conhecidos e, mais recentemente, têm sido desenvolvidos métodos para investigar a sua componente de matéria orgânica, 17 houve uma pesquisa escassa no componente biológico de aerossóis. 18 A razão para a corrente de método é preencher esta lacuna, apresentando em pormenor um método robusto para extrair, analisar e identificar a fração biológica de aerossóis 14. O método detalhado aqui é esperado para encontrar uma ampla utilização em projectos de investigação biológicos interiores e exteriores de aerossol que envolvem a análise de filtro 20-24.

<table border="0" cellpadding="0" cellspacing="0" width="631">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Filter sampling</td>
			<td style="width:123px;"></td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">HiVol 3000 - High Air Volume Sampler</td>
			<td style="width:123px;">Ecotech</td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Quartz Microfiber Filters</td>
			<td style="width:123px;">Whatman</td>
			<td style="width:103px;">1851-865</td>
			<td style="width:220px;">203mm X 254mm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">ELF - Laboratory Chamber Furnaces</td>
			<td style="width:123px;">Carbolite</td>
			<td style="width:103px;">ELF 11series</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Aluminum foil</td>
			<td style="width:123px;">Opal</td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:187px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:220px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Endotoxin</td>
			<td style="width:123px;"></td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Ethanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">16368&#160;</td>
			<td style="width:220px;">Laboratory Reagent, 96%</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Airstream Class II Biological Safety Cabinet AC-4E1</td>
			<td style="width:123px;">ESCO</td>
			<td style="width:103px;">10011712</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Pyrotell -T</td>
			<td style="width:123px;">Associates of Cape Cod, Inc.</td>
			<td style="width:103px;">T0051</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Control Standard Endotoxin</td>
			<td style="width:123px;">Associates of Cape Cod, Inc.</td>
			<td style="width:103px;">E0005-1</td>
			<td style="width:220px;">Escherichia coli&#160;O113:H10, 0.5 &#181;g/vial 1 Pack</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">LAL Reagent Water</td>
			<td style="width:123px;">Associates of Cape Cod, Inc.</td>
			<td style="width:103px;">W0051</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">10 mL sterile syringe with Luer-Lok Tip</td>
			<td style="width:123px;">Becton-Dickinson &#38; Co.</td>
			<td style="width:103px;">309605</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">BD Precisionglide syringe needle</td>
			<td style="width:123px;">Becton-Dickinson &#38; Co.</td>
			<td style="width:103px;">305129</td>
			<td style="width:220px;">Sterile&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Parafilm-M sealing tape</td>
			<td style="width:123px;">Parafilm</td>
			<td style="width:103px;">P7543</td>
			<td style="width:220px;">Sigma catalog number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Microtubes</td>
			<td style="width:123px;">Axigen</td>
			<td style="width:103px;">MCT-200-C</td>
			<td style="width:220px;">2ml, pyrogen free</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">1.12 cm diameter Cork Borer</td>
			<td style="width:123px;">Boekel Scientific</td>
			<td style="width:103px;">1601 BD Series - Steel</td>
			<td style="width:220px;">Part of a cork borer set containing borers with various diameters.&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">50 mm Petri Dish</td>
			<td style="width:123px;">Miniplast Ein-Shemer</td>
			<td style="width:103px;">72050-01</td>
			<td style="width:220px;">Aseptic</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Vortex Genie 2&#160;</td>
			<td style="width:123px;">Scientific Industries, inc.</td>
			<td style="width:103px;">SI-0297</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Microcentrifuge 5415 D</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:103px;">22621408</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:187px;">TC MicroWell 96 F SI w/lid</td>
			<td style="width:123px;">Nunc</td>
			<td style="width:103px;">167008</td>
			<td style="width:220px;">Flat bottom wells (with lid (individually wrapped)), sterile, pyrogen free</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:187px;">Synergy HT Multi-Detection Microplate Reader</td>
			<td style="width:123px;">Biotek</td>
			<td style="width:103px;">7091000</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:187px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:220px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">DNA</td>
			<td style="width:123px;"></td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA away</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">7010</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:187px;">Standard DNA of the microbial species of interest</td>
			<td style="width:123px;">ATCC or other culture collection</td>
			<td style="width:103px;"></td>
			<td style="width:220px;">Either the appropriate microbial strain for DNA extraction or the extracted DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Neubauer-improved</td>
			<td style="width:123px;">Marienfeld</td>
			<td style="width:103px;">640030</td>
			<td style="width:220px;">hemocytometer</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">TE buffer, Low EDTA</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:103px;">12090-015</td>
			<td style="width:220px;">10 mM Tris-HCl (pH 8.0)&#160;0.1 mM EDTA&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Nuclease-free PCR-grade water&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">3315959001</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">PCR primers</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;"></td>
			<td style="width:220px;">Targets the microbial species of interest</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Dual-Labeled Probes</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;"></td>
			<td style="width:220px;">Targets the microbial species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Screw cap tubes</td>
			<td style="width:123px;">Axigen</td>
			<td style="width:103px;">ST-200-SS</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">PowerSoil DNA extraction kit&#160;</td>
			<td style="width:123px;">Mo Bio Laboratories</td>
			<td style="width:103px;">12888-100</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Glass beads, acid-washed 425-600 Microns</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">G8772-100G</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Glass beads, acid-washed &#60;106 microns</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">G4649-100G</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">PowerSoil Solution C1</td>
			<td style="width:123px;">Mo Bio Laboratories</td>
			<td style="width:103px;">12888-100-1</td>
			<td style="width:220px;">Cell lysis buffer , Power soil Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Magic Touch ice bucket</td>
			<td style="width:123px;">Bel-Art</td>
			<td style="width:103px;">18848-4001</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Mini-Beadbeater-16</td>
			<td style="width:123px;">BioSpec</td>
			<td style="width:103px;">607EUR</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4376600</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Fast SYBR Green Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4385612</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4370048</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:187px;">MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4346906</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">MicroAmp Splash-Free 96-Well Base</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4312063</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">MicroAmp Optical Adhesive Film</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4311971</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Centrifuge 5810 R</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:103px;">5811 000.010</td>
			<td style="width:220px;">Rotor A-4-62 with MTP buckets&#160;</td>
		</tr>
	</tbody>
</table>

air-sampled filter analysis for endotoxins and dna content

Aerosol Research Outdoor comumente utiliza partículas amostradas em filtros. Este procedimento permite que várias caracterizações das partículas recolhidas para ser realizado em paralelo. O objectivo do método aqui apresentado consiste em obter uma análise precisa e altamente fiável do conteúdo de endotoxina e de ADN de bio-aerossóis extraídos de filtros. A extracção de moléculas orgânicas de elevado peso molecular, tais como lipopolissacáridos, de filtros amostrados envolve a agitação da amostra, num meio à base de água livre de pirogénio. A subsequente análise baseia-se numa reacção enzimática que pode ser detectada utilizando uma medição turbidimétrica. Como resultado do elevado teor orgânico nos filtros da amostra, a extracção de ADN a partir de amostras é realizada usando um kit de extracção de DNA comercial que foi originalmente concebido para solos e modificados para melhorar o rendimento de ADN. A detecção e quantificação de espécies microbianas específicas usando Reacti cadeia da polimerase quantitativaem análise (Q-PCR) são descritos e comparados com outros métodos disponíveis.

É comum para estudar aerossóis atmosféricos usando &quot;off-line&quot; análises de filtros amostra (ver Figura 2). Analisa 32 química do material incluem orgânico (por exemplo, proteínas, as moléculas de hidrocarboneto, sacáridos) e inorgânico (por exemplo, metais, sais) conteúdo amostrado . análises biológicas incluem conteúdo viáveis ​​e não viáveis ​​microrganismo, identificação de espécie utilizando uma abordagem de ADN ou microscopia, bem como a quantificação baseado no ADN. <img alt="Figura 2" src="/files/ftp_upload/53444/53444fig2.jpg" /> Figura 2: Filer gráfico de exemplo de fluxo de análise Filtro de pós-recolha de ar (A), a preparação do filtro para análises a jusante (B), e análise dos componentes da amostra (C).. E = &quot;1&quot;&gt; Para a extracção de endotoxina, filtros de sub-amostras (1,12 cm 2) são agitados em 1 ml PFW durante 60 min à temperatura ambiente. As amostras são então centrifugadas durante 10 min a 375 x g. Diferentes velocidades de centrifugação pode resultar em diferentes eficiências de extracção, tal como indicado na Figura 3A. Um teste preliminar para eficiência de extração devem ser conduzidos para o filtro específico escolhido e protocolo realizado. Na Figura 3B, as eficiências de extracção mais elevados são obtidos a partir spiking limpo em comparação com filtros de quartzo amostrados, e ambas essas eficiências são mais baixos do que a conseguida através da detecção directa da solução padrão. Como discutido noutro lugar, a natureza dos aerossóis ambiente amostrados no filtro também pode afectar a eficiência da extracção endotoxina 14,33. g3.jpg &quot;/&gt; Figura 3: Endotoxina eficiência da extracção O efeito da velocidade de centrifugação (A) e filtro de carga (B) na eficiência de extracção endotoxina.. As barras de erro representam o desvio padrão. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53444/53444fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> A reacção para a detecção de endotoxina envolve uma proporção de 1: 1 de reagente LAL para o padrão de endotoxinas, as endotoxinas extraiu-amostra, ou os espaços em branco. Recomenda-se usar triplicados para a análise da amostra. No entanto, para curvas padrão, duplicatas são suficientes. Quando as leituras de absorção (a 405 nm) são concluídas, a densidade óptica resultante (OD) é analisada depois exportá-lo em um programa de planilha de análise de dados para análise posterior dos dados. <p class="jove_content" fo:keep-together.within-page = &quot;1&quot;&gt; A extração de DNA a partir do filtro de amostragem ar é executada diretamente a partir de porções de filtros de sub-amostradas. a extracção do ADN é realizada utilizando um kit de isolamento de amostras de solo de ADN comercial, com algumas modificações para aumentar o rendimento de ADN. Assim, contas de vidro são adicionadas para agitar os filtros em um grânulo-batedor para melhorar a libertação de ADN dentro da solução. Para evitar um aquecimento da amostra (que pode levar à desnaturação do ADN), esta etapa é dividido em cinco intervalos de 1 min, com o tubo arrefecido em gelo entre os intervalos. A extracção pode, então, prosseguir de acordo com o procedimento do fabricante. Tal como na extracção endotoxina, é importante para executar uma análise preliminar da eficiência de extracção de ADN sob as condições experimentais específicas. Isto é feito pela adição de uma quantidade conhecida de uma solução padrão celular do organismo para uma peça cortada a partir do filtro, seguindo o protocolo de extracção de DNA acima descrito. <sup&gt; 34 A concentração do microrganismo alvo extraído dos aerossóis da amostra é determinada usando Q-PCR. Aqui, a técnica de hidrólise da sonda é utilizada, o que tem a vantagem de especificidade elevada, por causa da sonda adicional ligada ao ADN molde. 35 O método de verde SYBR também pode ser aplicado para o efeito. As curvas de calibração são obtidos a partir do ADN extraído a partir dos organismos escolhidos de interesse, utilizando o método de extracção descrito acima. Uma associação mista dos compostos de reacção, com excepção da amostra de ADN, padrão ou controlo, é preparada e mantida no escuro e em gelo até serem necessárias. Em seguida, uma alíquota da piscina mista é adicionada a cada poço de uma placa de micropoços óptico 96. O padrão de ADN, a amostra ou controlo é adicionado após a mistura da reacção de acordo com o arranjo de placas. Depois de todos os reagentes terem sido inseridos na placa, que é marconduzido com uma película adesiva óptica e centrifugadas para recolher todas as gotículas no fundo dos poços. A placa é que inserida no instrumento Q-PCR para a amplificação de ciclo termal e detecção de sinal. Os dados consistem em valores de saída de PCR Ct, que são definidas como o número do ciclo em que a quantidade de ADN amplificado alcança o nível de limiar. Utilizando os valores curva de calibração, pode ser obtida a concentração de ADN alvo (ver Figura 4). 36 As concentrações de células do microrganismo pode ser calculada a partir dos valores do Ct de a eficiência da extracção em comparação com uma contagem de hemocitómetro da solução padrão organismo. 37 <img alt="Figura 4" src="/files/ftp_upload/53444/53444fig4.jpg" /> Figura 4: Lote de amplificação de PCR quantitativa em (A) e Calibrat.curva de ion que varia entre 2 x 10 -4 -2 x 10 2 (B) realizada para DNA bacteriano gram negativa representativa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53444/53444fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Air-sampled Filter Analysis for Endotoxins and DNA Content at JoVE.com

Air-sampled Filter Analysis for Endotoxins and DNA Content

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Análise Filtro amostrado-Air para endotoxinas e conteúdo de DNA

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

air-sampled-filter-analysis-for-endotoxins-and-dna-content

Nanyang Technological University

Research

JoVE Journal

Environment

9.6K visualizações.  Weizmann Institute of Science. Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Vídeo: Air-sampled Filter Analysis for Endotoxins and DNA Content

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

Análise do conteúdo de ácidos graxos e composição em Microalgas

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Ambiente

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Uso de Chironomidae (Diptera) Pupal Exuviae como um Bioavaliação Protocolo Rápido para massas de água de superfície flutuante

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Modificação e Aplicação de uma Folha Blower-vac para Campo Amostragem de Artrópodes

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

O uso de um cartucho de filtro para Amostras de filtração de água e extração de DNA Ambiental

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Protocolo para Microplásticos Amostragem na superfície do mar e análise de amostras

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Extração de Pesticidas Organoclorados de Pelotização de Plástico e Análise de Tipo de Plástico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

Sequenciamento de DNA Nanopore para análise de solo Metagenomic

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Coleta e extração de ar ocupacional amostras para análise de DNA fúngico

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Quantificação de proteínas solúveis vegetais e carboidratos digeríveis conteúdo, usando o milho (Zea mays) como um Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Combining Eye-tracking Data with an Analysis of Video Content from Free-viewing a Video of a Walk in an Urban Park Environment

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. Eye-tracking informatics are known to connect to a range of feelings, health conditions, mental states and actions. But because vision is the result of constant eye-movements, teasing out what is important from what is noise is complex and data intensive. Furthermore, a significant challenge is controlling for what people look at compared to what is presented to them.
The following presents a methodology for combining and analyzing eye-tracking on a video of a natural and complex scene with a machine learning technique for analyzing the content of the video. In the protocol we focus on analyzing data from filmed videos, how a video can be best used to record participants&#39; eye-tracking data, and importantly how the content of the video can be analyzed and combined with the eye-tracking data. We present a brief summary of the results and a discussion of the potential of the method for further studies in complex environments.

The objective of the protocol is to detail how to collect video data for use in the laboratory; how to record eye-tracking data of participants looking at the data and how to efficiently analyze the content of the videos that they were looking at using a machine learning technique.

Combinando dados de rastreamento de olhos com uma análise de conteúdo de vídeo de livre-visualizando um vídeo de uma caminhada em um ambiente de parque urbano

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. ...