Este trabalho é apoiado pelo K-INBRE P20GM103418, da Comissão de soja Kansas (KSC/PSU 1663), ACS PRF 56629-UNI7 e PSU polímero química inicialização, só para SS. Agradecemos a cinegrafista da Universidade, Sr. Jacob Anselmi, pelo seu excelente trabalho com o vídeo. Agradecemos também o Sr. Roger Heckert e Sra. Katha Heckert pelo seu generoso apoio para a investigação.

1. síntese e Functionalization de multi paramétrico Magneto-fluorescente Nanosensors (MFnS).
<ol><li>Síntese de óxido de ferro superparamagnético nanopartículas (IONPs) <ol><li>para se preparar para a síntese IONP, preparar as seguintes 3 soluções: solução 1: FeCl 3 (0,70 g) e FeCl 2 H 2 O (2 mL), 2 solução: NH 4 OH (2,0 mL, 13,4 M) em H 2 O (15 mL) e a solução 3: ácido poliacrílico (0,855 g) em H 2 O (5 mL).</li>
		<li>Adicionar 90 µ l de ácido clorídrico de 2 M (HCl) à solução 1 e então misture com 2 solução enquanto num Vortex em 875 rpm. Em seguida, adicione solução 3. Continuar num Vortex de 60 min.</li>
		<li>Centrifugar por 20 min no 1.620 g. x de centrifugação o sobrenadante por 20 min em 2.880 g. x</li>
		<li>Purificar o sobrenadante final através de diálise. Adicionar o sobrenadante para um saco de diálise (MWCO 6 &#8722; 8 K) e coloque-o em um béquer contendo tamponado fosfato salino (PBS) (pH = 7,4) e barra de rotação. Deixe de molho por 12h, substituindo com buffer fresco a cada 2-3 h.</li>
	</ol></li> <li>Conjugação de anticorpos de alvos de superfície de IONPs <ol><li>preparar as seguintes quatro soluções: solução a: 5 mg de 1-etil - 3-(3-dimetilaminopropil) carbodiimida cloridrato (CED), em 250 µ l MES [2-(N- tampão de ácido etanesulfónico de Morpholinos)] (0,1 M, pH = 6,8), solução b: 3 mg de N-Hidroxisuccinimida (NHS) em 250 µ l de tampão MES (0,1 M, pH = 6,8), solução c: 5 µ g de IgG1, o mAb de Escherichia coli, em 225 µ l de tampão fosfato salino (PBS) (pH 7,4) e a solução d: 4 mL de IONP (5,0 mmol) em 1 mL de PBS (pH 7,4).</li>
		<li>Então imediatamente adicionar 2-(N-Morpholinos) ácido etanesulfónico (MES) do buffer para a solução A, adicionar solução-A solução d em pequenos incrementos, no prazo de 15 s, invertendo a solução após cada adição para a mistura.</li>
		<li>Imediatamente começam a adicionar solução B a solução D em pequenos incrementos ao longo de um período de três minutos. Mix, invertendo-se a solução após cada adição.</li>
		<li>Adicionar solução C solução em incrementos de 5 µ l, invertendo a solução após cada adição para a mistura.</li>
		<li>Permitir a reação continuar para 3h em temperatura ambiente e em seguida, continuar a incubação a 4 ° C durante a noite.</li>
		<li>Purificar as IONPs Ab-conjugado resultantes através de coluna magnética usando PBS (pH = 7,4, final [Fe] = 3,5 mmol) para remover quaisquer anticorpos unconjugated.
		<ol><li>Lavar a coluna magnética com PBS (1 mL). Unir a coluna para a placa magnética e adicionar a solução IONP.</li>
			<li>Lavar a coluna magnética com PBS (1 mL). Remova a coluna magnética da placa magnética. Adicionar PBS para a coluna e recolher a solução. Loja a 4 ° C</li></ol></li></ol></li> <li>encapsulamento de 1,1 fluorescente &#39; - Dioctadecyl - 3,3,3 &#39;, 3 &#39;-corante Tetramethylindocarbocyanine perclorato (DiI) para o revestimento (PAA) o ácido poliacrílico os conjugado anticorpo IONPs usando um método de difusão de solventes.
	<ol><li>De 4 mL dos IONPs, Adicionar gota a gota 2.0 μL de tintura de DiI (2 mmol) em 100 µ l de DMSO com mistura contínua a 1100 rpm.</li>
		<li>Dialize a solução resultante (MWCO 6-8 KDa, 12 h) contra PBS, criando uma solução final de MFnS com uma concentração final de ferro [Fe] = 2 mmol.</li>
	</ol></li> <li>Caracterização de MFnS <ol><li>para análise espectrofotométrica, usar um leitor de placas para detectar encapsulado DiI tintura com emissão de fluorescência em 595 nM.</li>
		Difusão dinâmica da luz <ol><li>de <li>coloca uma amostra da solução de MFnS em um zetasizer para determinar o tamanho médio e carga de superfície da MFnS. 
			Nota: Aqui, o tamanho médio e carga de superfície de MFnS funcionais foram encontrados para ser 77.09 nm e-22.3 mV, respectivamente.</li>
		</ol></li></ol></li></ol> 2. Bactérias Culturing e preparação de solução estoque <ol><li>bacteriana cultivo <ol><li>hidratar uma pelota liofilizada (Escherichia coli O157: H7) em 1 mL de caldo nutriente e em seguida, adicionar um adicional 5 mL de caldo de carne.</li>
		<li>Aplicam-se 100 µ l desta solução para uma placa de ágar e raia utilizando uma ansa de inoculação estéril, seguida de incubação a 37 ° C por 24 h.</li>
		<li>Após 24 h, selecione uma colônia isolada da placa de ágar e adicioná-lo a um 15 mL de caldo de cultura. Incubar a 37 ° C, durante 4-6 h, enquanto monitora o valor de densidade óptica (absorvância a 600 nm).</li>
		<li>Uma vez um valor de densidade óptica de 0.1 obtido, pare de cultivo e realizar diluições em série.</li>
	</ol></li> <li>Diluições em série <ol><li>Adicionar 900 µ l de caldo nutriente para oito tubos estéreis microcentrifuga.</li>
		<li>Adicione 100 µ l da suspensão bacteriana ao primeiro tubo (diluição 10 -1).</li>
		<li>Levar 100 µ l do primeiro tubo e adicioná-lo para o segundo, para obter uma diluição de 10 -2. Continue este padrão para os tubos restantes, terminando com uma diluição final de 10 -8.</li>
		<li>Transferir 100 µ l de cada tubo para separar as placas de ágar e incubar por 24 h a 37 ° C.</li>
		<li>No dia seguinte, contar as unidades (CFUs) formadoras em cada prato. Selecione a placa com CFUs ~ 100. Usar as correspondentes diluições para novas experiências (100 µ l = CFUs ~ 100). 
		Nota: Aqui era a diluição de 10 -6 que resultou em 100 ~ CFUs.</li>
	</ol></li></ol> 3. Rápida deteção de E. coli O157: H7 usando MFnS <ol><li>Spike várias soluções de PBS (1. X, pH 7,4, 300 µ l) com a quantidade crescente do estoque de 10 -6 bacteriana, resultando em CFU varia de 1-100. Adicionar uma quantidade consistente de MFnS (100 µ l) para essas soluções.</li>
	<li>Criar uma solução de linha de base que contém somente PBS (1. X, pH 7,4, 300 µ l) e MFnS (100 µ l).</li>
	<li>Incubar soluções por 30 min a 37 ° C e então deixe-os arrefecer à temperatura ambiente.</li>
	<li>Transferência de soluções individuais para o relaxometer magnético (Tesla 0,47) e gravar alterações nos tempos de relaxamento (T2) em relação as CFUs em cada solução.
	<ol><li>Para gravar as alterações em valores de T2, Comece medindo o valor de T2 da solução de linha de base que contém somente PBS e MFnS.
		Relaxometer <ol><li>lugar a solução em magnética. Abra o respectivo software, selecione o &#34; relaxamento T2 &#34; definição e pressione &#34; medida. &#34;</li></ol></li> <li>seguindo a coleção de linha de base T2, medir os valores de T2 das soluções adicionais que foram cravados com vários concentrações de bactérias. 
		Nota: A mudança em T2 é equivalente à linha de base de T2 subtraída a T2 cravado.</li>
	</ol></li> <li>Remover amostras da relaxometer magnética e centrifugar os tubos a 2880 x g durante 10 min.</li>
	<li>Decante o sobrenadante e ressuspender pelotas bacterianas em 100 µ l de PBS (1. X, pH 7,4).</li>
	<li>Adicionar 80 µ l de cada ressuspensão de uma placa de 96 poços e intensidades de fluorescência recorde em 595 nM. 
	Nota: Teste pode ser repetido usando solventes diferentes, incluindo a água do lago, leite e outros, conforme descrito na seção de resultados</li></ol>

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A aplicação demonstrada esta nanotecnologia ainda não está aprovada pelo FDA.

Este protocolo foi projetado para produzir MFnS totalmente funcionais como simplesmente possível. No entanto, existem muitos pontos-chave em que a alteração do protocolo pode ser útil, dependendo do objetivo do final do usuário. Por exemplo, o uso de anticorpos diferentes permitiria segmentação dos muitos outros patógenos. Além disso, este protocolo não é limitado ao uso de anticorpos como alvo moléculas. Qualquer molécula que tem afinidade de ligação específica para pató...

A ocorrência persistente de contaminação bacteriana em ambos produzidos comercialmente alimentos e fontes de água criou uma necessidade de plataformas de diagnósticos cada vez mais rápidas e específicas. 1 , 2 alguns dos contaminantes bacterianos mais comuns responsáveis pela contaminação de alimentos e água são dos gêneros Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus e Escherichia. 3 , 4 a contaminação bacteriana por esses patógenos frequentemente resulta em sintomas como diarreia, cólera, gastroenterite e febre. 4 contaminação das fontes de água, muitas vezes tem efeitos adversos e drásticos em comunidades sem acesso a água filtrada suficientemente, e contaminação de alimentos tem levado a um grande número de doenças e esforços de recuperação do produto. 5 , 6
A fim de reduzir a ocorrência de doenças causadas por contaminação bacteriana, tem havido uma série de esforços para desenvolver métodos pelos quais água e alimentos podem ser eficientemente verificados antes da venda ou consumo. 3 técnicas como a PCR,1,7,8,9,10 ELISA,11,(mediada por loop de amplificação isotérmica de12 LÂMPADA),13,14 entre outros,15,16,17,18,19,20,21, 22,23,24 recentemente tem sido usado para detecção de vários patógenos. Comparado ao tradicional bacteriana, métodos de cultivo, estas técnicas são muito mais eficientes em relação à especificidade e tempo. No entanto, estas técnicas ainda lutam com falsos positivos e negativos, procedimentos complexos e custo. 1 , 3 , 25 ... é por essa razão que Multiparamétrico magneto-fluorescente nanosensors (MFnS) propõem-se como um método alternativo para a deteção bacteriana.
Estes nanosensors com exclusividade par juntos relaxação magnética e modalidades fluorescentes, permitindo uma plataforma dual-deteção rápida e exata. Usando Escherichia coli O157: H7 como um contaminante de amostra, é demonstrada a capacidade de MFnS para detectar tão pouco quanto 1 CFU em poucos minutos. Específicas do patógeno anticorpos são usados para aumentar a especificidade, e a combinação de modalidades magnéticas e fluorescentes permite a detecção e quantificação de contaminantes bacterianos em ambas as gamas de alta e baixa --contaminação. 16 no caso de contaminação bacteriana, o nanosensors vai enxame à volta as bactérias devido as habilidades alvos dos anticorpos específicos do patógeno. A ligação entre a nanosensors magnética e bactérias limita a interação entre o núcleo de ferro magnético e os prótons de água circundante. Isto causa um aumento nos tempos de relaxamento T2, como registrado por uma relaxometer magnética. Como a concentração de bactérias na solução aumenta, os nanosensors se dispersar com o aumento do número de bactérias, resultando em valores mais baixos de T2. Por outro lado, a emissão de fluorescência aumentará em proporção com a concentração de bactérias, devido ao aumento do número de nanosensors diretamente ligado ao patógeno. Centrifugação das amostras e o isolamento da pelota bacteriana, só irão conservar as nanopartículas diretamente conectadas com as bactérias, removendo qualquer nanosensors flutuante e correlacionando diretamente a emissão de fluorescência com o número de bactérias presentes na solução. Uma representação esquemática deste mecanismo é representada na Figura 1.
Esta plataforma de MFnS foi projetada com rastreio de point-of-care em mente, resultando em características de baixo custo e portátil. MFnS são estáveis à temperatura ambiente e só são necessários em concentrações muito baixas para detecção precisa de bactérias contaminantes. Além disso, após a síntese, o uso da MFnS é simples e não requer o uso de profissionais treinados no campo. Por último, esta plataforma de diagnóstica permite direcionamento altamente personalizável, proporcionando um meio pelo qual este uma plataforma pode ser usada para detectar agentes patogénicos de todos os tipos, em muitas configurações diferentes.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
			<td></td>
		</tr>
	</tbody>
</table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrhagic Escherichia coli O157: H7 tem sido associada a ambos de origem hídrica e doenças transmitidas por alimentos e continua a ser uma ameaça, apesar dos métodos de seleção de alimentos e de água usada atualmente. Enquanto os métodos de detecção bacteriana convencional, tais como a reação em cadeia da polimerase (PCR) e ensaios imunoenzimático (ELISA) especificamente podem detectar contaminantes patogênicos, eles exigem preparação da amostra extensa e longos períodos de espera. Além disso, estas práticas exigem instrumentos de laboratório sofisticado e configurações e devem ser executadas por profissionais treinados. Neste documento, propõe-se um protocolo para uma técnica de diagnóstica mais simples que apresenta uma combinação única de parâmetros magnéticos e fluorescentes em uma plataforma baseada em nanopartículas. O nanosensors de magneto-fluorescente Multiparamétrico proposto (MFnS) pode detectar a contaminação de Escherichia coli O157: H7 com tão pouco quanto 1 unidade formadoras presente em solução dentro de menos de 1 h. Além disso, a capacidade de MFnS permanecem altamente funcional em meios complexos tais como leite e água do lago foi verificada. Ensaios de especificidade adicionais também foram utilizados para demonstrar a capacidade de MFnS para detectar somente as bactérias do alvo específico, mesmo na presença de espécies bacterianas semelhantes. O emparelhamento de modalidades magnéticas e fluorescentes permite a detecção e quantificação de contaminação do patógeno em uma ampla gama de concentrações, exibindo seu alto desempenho em ambos deteção de contaminação e tarde-estágio inicial. A eficácia, acessibilidade e portabilidade dos MFnS torná-los um candidato ideal para o rastreio de point-of-care para bactérias contaminantes em uma ampla gama de configurações, de reservatórios aquáticos para alimentos embalados comercialmente.

O mecanismo de ação MFnS é representado na Figura 1. O agrupamento de MFnS em torno da superfície de bactérias contaminantes interfere com as interações entre os magnetic cores dos MFnS e os núcleos de hidrogênio circundante. Como resultado deste relaxamento de clustering, magnético valores aumentam. À medida que aumenta a concentração de bactérias contaminantes, reduz a clusterização e a alteração nos valores de T2 diminui. Portanto, a adição de uma modalidade fluorescen...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

O objetivo geral do presente protocolo é para sintetizar nanosensors funcional para o portátil, econômica, e rápida detecção de direcionado especificamente bactérias patogênicas através de uma combinação de relaxação magnética e as modalidades de emissão de fluorescência.

Patógeno de origem alimentar rastreio usando Nanosensor Magneto-fluorescente: Rápida detecção de Escherichia Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

7.6K visualizações.  Pittsburg State University. O objetivo geral do presente protocolo é para sintetizar nanosensors funcional para o portátil, econômica, e rápida detecção de direcionado especificamente bactérias patogênicas através de uma combinação de relaxação magnética e as modalidades de emissão de fluorescência.

Vídeo: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de Salmonella On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Imunologia e Infecção

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Triagem rápida de HIV transcriptase reversa e integrase Inibidores

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isothermal amplificação (lâmpada), Ensaios mediada por loop para detecção específica da espécie os de<em&gt; Eimeria</em&gt; Que infectam Galinhas

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rápida Detecção Molecular e diferenciação de Influenza vírus A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Criação de um denso Transposon inserção biblioteca usando bacteriana conjugação em entérobactérienne cepas tais como Escherichia Coli ou Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extração rápida, segura e simples Manual cabeceira de ácidos nucleicos para a detecção de vírus em amostras de sangue total

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Uma plataforma de alto rendimento para o rastreio de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Patógeno de origem alimentar rastreio usando Nanosensor Magneto-fluorescente: Rápida detecção de Escherichia Coli O157: H7

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