Agradecemos ao Departamento de Segurança Interna dos EUA para fornecer parte do financiamento.

1. Estirpes bacterianas e de ADN molde Preparação <ol><li> Crescer cepas de E. coli O157: H7, não-O157, e outras espécies patogénicas, tais como Salmonella e Shigella, a 37 ° C durante a noite com agitação. </li><li> Extrair o DNA de culturas de bactérias usando o kit correspondente (ver tabela de materiais) e seguindo as instruções do fabricante. </li><li> Medir a densidade óptica (DO 260) para determinar as concentrações de ADN, utilizando um espectrofotómetro. </li></ol> 2. Primer e Probe de Design para o Ensaio qPCR <ol><li> E. coli O157: H7 são seqüências do GenBank número de acesso AE00517411. Primers e sonda projeto usando o software apropriado para a primeira demão e design sonda para PCR em tempo real (veja a tabela de materiais para mais detalhes). As seqüências de primers e sonda são as seguintes: Z3276-Forward (F) 5&#39;-TATTCCGCGATGCTTGTTTTT-3 &#39; Z3276-Reverse (R) 5&#39;-ATTATCTCACCAGCAAACTGGCGG-3 &#39; Z3276-Probe, FAM-CCGCAATCTTTCC-MGBNFQ </li><li> Reconstituir o pó de iniciador com água, resultando numa concentração de 10 uM como uma solução de trabalho de iniciador. </li><li> Dilui-se as sondas de 10 fiM como soluções de trabalho de sonda. As sondas marcadas variar em potência com lotes. Portanto, titular de cada lote de sondas marcadas antes de usar para um ótimo desempenho qPCR. </li></ol> 3. Definir as condições de ensaio de qPCR <ol><li> Prepare a mistura de reacção, que consiste em 25,0 ul de PCR em tempo real master mix 2x, 200 nM de iniciador de sentido directo, 200 nM de iniciador de sentido inverso e 100 nM de sonda. </li><li> Adicionar 5 uL de ADN da amostra (100 pg) e um volume apropriado de água para atingir um volume final de 50 ul. </li><li> Para controlo nontemplate, utilizar 5 mL de água para substituir amostra de DNA. </li><li> Definir as condições de qPCR como se segue: 95 ° C durante 10 minutos para a activação de TaqMan, seguido por 40 ciclos de 95 ° C durante 10 segundos para desnaturação e 60 &amp;# 176: C durante 1 min para hibridação / extensão. </li></ol> 4. qPCR Teste de Sensibilidade <ol><li> Fazer uma diluição em série de 10 vezes de uma cultura de fase semi-exponencial (DO600 = 0,5, cerca de 1,5 x 10 8 UFC / ml por plaqueamento). </li><li> Adicionar 100 ml de diluições de células para uma placa de 96 poços em triplicado. </li><li> Centrifugar a placa a 2500 xg durante 10 min. </li><li> Adicionar 50 ml de solução de extracção de ADN de cada poço. </li><li> Os sedimentos de células Ressuspender com uma pipeta multi-canal. </li><li> Selar a placa com uma película, ferver a placa num banho de água durante 10 minutos, e centrifugar a 2500 g durante 2 min. </li></ol> 5. Preparação de misturas vivas e mortas celulares para tratamento PMA <ol><li> Cresce de 10 ml de E. coli O157: H7, a 37 ° C até à fase semi-exponencial e dividir a cultura em duas aliquotas. </li><li> Ferver uma alíquota de células durante 10 minutos em um banho de água para as células mortas e deixar a outra aliquota de células vivas. </li><li> Verifique se ore há células vivas da alíquota mortos pelo calor por plaqueamento das células em placas de agar LB. </li><li> Tome 2 ml das células vivas e mortas pelo calor e ajustar a concentração de 8 x 10 6 CFU / ml com meio LB. Criar quatro conjuntos de diluições de células que variam de 8 x 10 0-8 x 10 6 CFU / mL. </li><li> Use os dois primeiros conjuntos de diluições de células para células vivas tratadas com PMA ou não tratada. </li><li> Para a terceira e quarta séries de diluições de células, adicionar 8 x 10 6 células mortas para cada diluição celular (8 x 10 0 - 8 x 10 6) para fazer misturas de células vivas e mortas. </li><li> Trate o terceiro conjunto de diluição com PMA e usar o quarto conjunto de diluição para o controle. </li></ol> 6. Tratamento de células com PMA <ol><li> Dissolve-se PMA em dimetilsulfóxido (ou água) para fazer uma solução estoque de 10 mM e armazenados a -20 ° C no escuro. </li><li> Alíquota 400 mL das células vivas, células mortas, e mistura de células vivas e mortas natrês microtubos separados. </li><li> Adicionar 2,0 ml de 10 mM de PMA a cada alíquota de células para uma concentração final de 50 uM. </li><li> Incubar as amostras à temperatura ambiente durante 5 min no escuro, agitando suavemente algumas vezes, 3 segundos de cada vez. </li><li> Adicionam-se 100 ul de amostras de um placa de 96 poços. </li><li> Selar a placa com uma película óptica e colocar a placa de gelo. </li><li> Definir a placa 20 cm de uma fonte de luz de halogéneo W 650 e expor durante 2 min durante a exposição à luz. </li><li> Centrifugar a placa a 2500 x g durante 10 minutos, rejeitar o sobrenadante suavemente e cuidadosamente drenar a placa sobre uma folha de papel absorvente. </li><li> Pellets de células ressuspender em 50 ml de solução de extração de DNA por pipetagem cima e para baixo vinte vezes com um multi-canal Pipetman. </li><li> Selar a placa com uma película, ferver durante 10 minutos em um banho de água, e centrifugar a 2500 g durante 2 min. O sobrenadante da placa é o ADN a partir de células tratadas com PMA e pronto para qPCR. </li><li> Realize qPCR como descricama acima, utilizando 5 ul de ADN. </li></ol>

<ol>
	<li>Barletta, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+five-colony+pool+analysis+using+multiplex+real-time+PCR+for+detection+of+diarrheagenic+Escherichia+coli.">Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli.</a> J. Clin. Microbiol. 47, 1915-1917 (2009).</li><li>Gannon, V. P., King, R. K., Kim, J. Y., Thomas, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+method+for+detection+of+Shiga-like+toxin-producing+Escherichia+coli+in+ground+beef+using+the+polymerase+chain+reaction.">Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.</a> Appl. Environ. Microbiol. 58, 3809-3815 (1992).</li><li>Cebula, T. A., Payne, W. L., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+identification+of+strains+of+Escherichia+coli+serotype+O157:H7+and+their+Shiga-like+toxin+type+by+mismatch+amplification+mutation+assay-multiplex+PCR.">Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR.</a> J. Clin. 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Os autores não têm nada a revelar.

Um objetivo primário do estudo envolve o uso de uma ORF Z3276, um exclusivo para E. coli O157: H7, como um único biomarcador para o ensaio de qPCR. No momento, a maioria dos ensaios de qPCR são alvo genes de virulência, como stx1, stx2 e eaeA 1,2,5,6 ou genes fenotípicas compartilhados, como uidA 3,4, rfbE e genes Flic 8,9. Ocasionalmente, uma espécie ou estirpe não pode ser definitivamente identificado por gene (s) de virulência, tais como 1e stx stx 2 genes, como os genes estão presentes em diferentes espécies ou estirpes 16 bem. Usando rfbE e fliC para genes alvo, ambos os genes são necessários para ser utilizado para a identificação completa 17. Usando ORF Z3276 como um biomarcador, este ensaio qPCR tem sido demonstrado ser ensaio sensível e específico (Tabela 1). Este ensaio tem sido submetido a testes de inclusão e exclusividade rigorosas, incluindo O157: H7 (n = 367), mais de 100 cepas não-O157, e outras espécies patogênicas, como Salmonella e Shigella. Todos os O157: H7 cepas analisadas foram positivamente detectada, e nenhuma reação cruzada foi encontrada a partir das cepas não-O157 (Tabela 1), indicando que a ORF Z3276 é um biomarcador único e forte para a detecção de E. coli O157: H7. O outro objetivo do estudo é detectar seletivamente as células vivas de células mortas por tratamento PMA antes da extração de DNA. PMA podem penetrar nas células mortas com membrana comprometida e se ligam ao DNA, e portanto inibe a amplificação do DNA de células mortas em PCR 13. Nós modificamos o procedimento PMA-tratamento, e adaptou-a para um formato de placa de 96 poços para o procedimento. A intensidade da direita da exposição à luz é essencial para obter o efeito óptimo de reticulação. Anteriormente, microtubos foram utilizados neste passo 13-15. No entanto, microtubos separados são difíceis de manusear em função expasso Posure, e estes tubos não são absolutamente transparente para obter o melhor cross-gosto. Usando uma placa de 96 poços, que melhorou a eficiência de PMA-tratamento. Estas alterações podem influenciar o ensaio de várias maneiras: eles tornam mais fácil a obtenção de um efeito de reticulação completa e uniforme, em especial com as amostras numerosas; as amostras tratadas podem ser movidos a partir de uma placa para a outra para possibilitar a detecção de um grande número das amostras, e que proporcionam este ensaio PMA-qPCR com potencial de automatização para todo o processo. A limitação deste ensaio PMA-qPCR é que o tratamento com PMA, aumenta o custo do ensaio de PCR ligeiramente e reduziu ligeiramente a sensibilidade do ensaio de PCR.

A PCR é uma técnica comum para a detecção de E. coli O157: H7 a partir de amostras de alimentos e ambientais. A identificação de biomarcadores específicos para E. coli O157: H7 é um dos fatores-chave para a detecção de sucesso em ensaios de PCR. Os biomarcadores comumente utilizados em ensaios de PCR para a detecção de E. coli O157: H7 incluem toxinas Shiga (stx 1 e stx 2) 1,2, 3,4 uidA, eaea 5,6, rfbE 7 e FLIC genes 8,9. Esses biomarcadores podem proporcionar eficiência variável para identificação, no entanto, a maioria dos marcadores não pode ser utilizada como um biomarcador único para a detecção de E. coli O157: H7. Esta inadequação no poder diferenciação dos genes-alvo exige biomarcador (s) mais específico e mais forte para a identificação de E. coli O157: H7, 10. Numerosos sondas para os genes ou grelhas de leitura abertas (ORFs) hipotéticas em E. coliO157: H7 11 foram concebidos para o ensaio de qPCR com base nas pistas de microarrays de genotipagem de DNA de nosso laboratório e através de pesquisa no banco de dados GenBank, do Centro Nacional de Informações sobre Biotecnologia. A sequência de Z3276 ORF, que é um gene fimbrial 11, foi seleccionado para o ensaio de qPCR. Subsequentemente, um ensaio de qPCR foi desenvolvida através de numerosos ensaios sobre os parâmetros de PCR, tais como concentrações dos iniciadores e sondas, bem como de recozimento e de extensão temperaturas (dados não mostrados). Após testes inclusão de incentivo e de exclusividade sobre cepas de referência, tais como E. coli O157: H7, não-O157 e Shigella no qPCR, a ORF Z3276 foi identificado como um marcador genético específico e forte para a identificação de E. coli O157: H7. Então, nós desenvolvemos um novo método de qPCR utilizando este biomarcador único para identificação de E. coli O157: H7. Outro desafio na detecção de E. coli O157: H7 em contaminada foods e meio ambiente é a determinação exata de células vivas a partir de amostras. A presença de ADN estendido de células mortas e a incapacidade para diferenciar viabilidade no ensaio de PCR pode originar leituras de falsos positivos, resultando numa sobreavaliação do número de células vivas na detecção. Consequentemente, isso limita o uso de PCR na detecção precisa de patógenos de origem alimentar 12. Uma nova abordagem para diferenciar ADN de células mortas foi feita através da combinação de propídio monoazide (PMA) de tratamento com um procedimento de PCR nos últimos anos 13-15. PMA é capaz de ir para dentro de células mortas, e intercalam no ADN quando exposto a luz, mas que não pode ir para dentro de células vivas de reagir com o ADN das células vivas. Assim, nas misturas tratadas por PMA de células vivas e mortas, o DNA de células mortas não pode ser amplificado por PCR em 13. Nós combinamos o tratamento PMA com o ensaio qPCR recentemente desenvolvido para detectar seletivamente ao vivo E. coli O157: H7 células. Além disso, fizemos o PMA-qPCR umssay possível para a detecção de alto rendimento.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">AB 7900HT Fast Real-time PCR system</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">2 x Universal master mix</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4304437</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PrepMan Ultra</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4318930</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Primer Express</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="58" rowspan="2" style="height:58px;width:249px;">Probes</td>
			<td rowspan="2" style="width:164px;">ABI</td>
			<td>Top of Form</td>
			<td rowspan="2"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">4316033 Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PMA</td>
			<td style="width:164px;">Biodium</td>
			<td align="right" style="width:171px;">40013</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="87" rowspan="3" style="height:87px;width:249px;">Puregen cell and tissue kit</td>
			<td rowspan="3" style="width:164px;">Qiagene</td>
			<td>Top of Form</td>
			<td rowspan="3"></td>
		</tr>
		<tr height="29">
			<td align="right" height="29" style="height:29px;">158622</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">DU530 spectrophotometer</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Spectrophotometer</td>
			<td style="width:164px;">NanoDrop Technology</td>
			<td style="width:171px;">&#160;ND-1000</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">650 w halogen light source</td>
			<td style="width:164px;">Britek</td>
			<td style="width:171px;">H8061S</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Otptical Adhesive film</td>
			<td>ABI</td>
			<td align="right">4311971</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Optical 96-well reaction plate</td>
			<td>ABI</td>
			<td align="right">4316813</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of live escherichia coli o157:h7 cells by pma-qpcr

Uma estrutura única de leitura aberta (ORF) Z3276 foi identificado como um marcador genético específico para E. coli O157: H7. Um ensaio de qPCR foi desenvolvido para a detecção de E. coli O157: H7, visando ORF Z3276. Com este ensaio, pode-se detectar tão baixo como algumas cópias do genoma de ADN de E. coli O157: H7. A sensibilidade e especificidade do ensaio foram confirmados por testes de validação intensivos com um grande número de E. coli O157: H7 (n = 369) e as estirpes não-O157 (n = 112). Por outro lado, temos procedimento propídio monoazide (PMA) combinado com o protocolo qPCR recentemente desenvolvido para a detecção selectiva de células vivas a partir de células mortas. A amplificação de DNA a partir de células mortas tratadas com PMA foi quase completamente inibida em contraste com amplificação de forma praticamente independente do ADN a partir de células vivas tratadas com PMA. Além disso, o protocolo foi modificado e adaptado para um formato de placa de 96 poços para um manuseamento fácil e consistente de um grande número de amostras. Tseu método deverá ter um impacto sobre microbiológico preciso e monitoramento epidemiológico da segurança alimentar e fonte ambiental.

Detecção de E. coli O157: H7 por qPCR utilizando ORF Z3276 Um dos factores importantes deste ensaio é a sensibilidade e especificidade da sonda Z3276 utilizado no qPCR. Pode detectar tão baixo como algumas cópias do genoma de ADN de E. coli O157: H7, como mostrado na Figura 1A. Das 120 estirpes não-O157 examinados, incluindo os seis maiores estirpes não-O157, STEC O104: H4, estirpes de Salmonella, e as estirpes de Shigella, sem qualquer reactividade cruzada foi encontrado, como mostrado na Tabela 1. Combinação do tratamento com PMA qPCR Incubar E. coli O157: H7 células com PMA (50 mM), durante 5 minutos no escuro e exposição à luz durante 2 min foram seleccionados para as condições de tratamento com PMA. Além disso, modificamos procedimento de tratamento de PMA. Comparado com vários microtubos transparentes utilizadas no passo de reticulação, uma placa de 96 poços foi encontrada para ser mais eficazatingir o efeito óptimo de reticulação e mais conveniente do que o tratamento de um grande número de tubos de amostra durante o processo de exposição à luz. A amplificação de DNA a partir de células vivas e mortas tratadas com PMA em qPCR Os efeitos da inibição mediada por PMA de amplificação de DNA a partir de células mortas neste ensaio qPCR foram mostrados na Figura 1, utilizando o ADN obtido a partir de duas 10 vezes diluições de células vivas ou mortas em série. Os resultados mostram que as curvas geradas a partir de células vivas tratadas com PMA (curva vermelha) e sem PMA (curva azul) parecem quase linear e idênticos uns aos outros. Diferenças de valor de CT foram ligeiras mostrado entre as células vivas tratadas com PMA e sem PMA (Figura 1A), sugerindo que o tratamento com PMA teve pouco efeito sobre a ampliação do ADN das células vivas no qPCR. Mas a 15 - Diferença de valor CT (32.000 vezes) foi exibido entre as células mortas tratadas com PMA e sem PMA, demonstraçãonstrating que o tratamento com PMA suprimiram eficientemente a amplificação de DNA a partir das células mortas (Figura 1B). A detecção de células vivas a partir de misturas de células vivas e mortas na qPCR Dois conjuntos de diluições de células vivas (8 x 10 0-8 x 10 6 UFC) foram tratados com PMA ou sem PMA a detecção de células vivas a partir de misturas de células vivas / mortas. Os resultados de qPCR (figura 2A) demonstraram uma tendência progressiva inversa paralelo de valores de CT em relação com o número de células vivas tratadas (barras a púrpura) para as amostras não tratadas (barras azuis). As células vivas tratadas rendeu um pouco mais elevados do que os valores de CT de células vivas não tratados. Uma tendência progressiva inversa semelhante nos valores do CT foi observada com o número real de células vivas nas misturas live / dead de células tratadas com PMA (barras verdes) bem na Figura 2B. Além disso, esta tendência de queda dos valores do CT foiobservado com o número de células vivas nas misturas, apesar da presença de 106 células mortas. Estes resultados mostraram que os valores de TC de as misturas de células vivas / mortas representado o ADN a partir das células vivas isoladamente, ao passo que a amplificação de DNA a partir das células mortas foi eficientemente suprimidos por tratamento de PMA. Mas, os valores do CT de misturas ao vivo / morto de células tratadas com PMA foram oscilou e não conseguiu reflete o número de células vivas nas misturas da figura 2B (barras amarelas). <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig1highres.jpg" src="/files/ftp_upload/50967/50967fig1.jpg" /> Figura 1. Viver e células mortas tratadas com PMA ou sem PMA em ensaio qPCR. (A) Uma série de 10 vezes diluído em directo E. coli O157: H7 (diluições de células 8 x 10 0-8 x 10 6 UFC) foram tratados com PMA ou sem PMA.Os valores CT representam os valores médios de CT das triplicatas no ensaio qPCR. (B) Comparação da amplificação de DNA de células vivas e células mortas tratadas com PMA e sem PMA no ensaio qPCR comparação q. ΔRn refere-se a fluorescência mudança intensidade. <a href="https://www.jove.com/files/ftp_upload/50967/50967fig1highres.jpg" target="_blank">Clique aqui para ver imagem ampliada.</a> <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig2highres.jpg" src="/files/ftp_upload/50967/50967fig2.jpg" /> Figura 2. Diferenciação de células vivas a partir de misturas de células vivas / mortas por PMA-qPCR Quatro conjuntos de diluições de 10 vezes das culturas de células (8 x 10 0-8 x 10 6 UFC). Foram efectuadas conforme indicado. (A) Primeiro conjunto de diluição de células foi tratado com PMA, enquanto a segunda diluição celular foi ou não tratada antes da extração de DNA.(B) 8 x 10 6 células mortas foram misturados com os terceiro e quarto conjuntos de diluições de células para fazer dois conjuntos de misturas de células vivas / mortas, tal como indicado. Um conjunto das misturas de células foi tratado com PMA e o outro conjunto foi tratado sem PMA antes da extracção do ADN. Cada barra representa os valores médios de TC de uma experiência em triplicado ± DP. <table><tbody><tr><td> Organismo </td><td> O sorotipo </td><td> N º de cepas testadas </td></tr><tr><td rowspan="15"> E. coli </td><td> 2006 espinafre O157: H7 isoladas de surto </td><td> 186 </td></tr><tr><td> 2006 Taco Bell O157: H7 isolados </td><td> 58 </td></tr><tr><td> 2006 Taco John O157: H7 isolados </td><td> 11 </td></tr><tr><td> Coleções DMB tensão de outros O157: H7 surtos </td><td> 112 </td></tr><tr><td> O104: H4 </td><td> 3 </td></tr><tr><td> O26 </td><td> 18 </td></tr><tr><td> O103 </td><td> 13 </td></tr><tr><td> O111 </td><td> 24 </td></tr><tr><td> O121 </td><td> 2 </td></tr><tr><td> O45 </td><td> 5 </td></tr><tr><td> O145 </td><td> 9 </td></tr><tr><td> O113 </td><td> 2 </td></tr><tr><td> O91 </td><td> 1 </td></tr><tr><td> O55 </td><td> 9 </td></tr><tr><td> Outro </td><td> 19 </td></tr><tr><td rowspan="2"> Salmonella </td><td> Typhi </td><td> 1 </td></tr><tr><td> Newport </td><td> 2 </td></tr><tr><td> Shigella dysenteriae </td><td> 6 </td><td> 2 </td></tr><tr><td> Shigella sonnei </td><td> Desconhecido </td><td> 2 </td></tr><tr><td> Shigella flexneri </td><td> 4 </td><td> 1 </td></tr><tr><td> Shigella boydii </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="2"> Total </td><td> 481 </td></tr></tbody></table> Tcapaz 1. estirpes utilizadas para validação de ensaio de PCR em tempo real para identificação de E. coli O157: H7.

Watch this Scientific Journal Video about Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR at JoVE.com

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Um ensaio de qPCR foi desenvolvido para a detecção de Escherichia coli O157: H7 alvo um marcador genético único, Z3276. A qPCR foi combinado com propidium monoazide (PMA) o tratamento para a detecção de células vivas. Este protocolo foi modificado e adaptado a um formato de placa de 96 poços para uma fácil manipulação e consistente de numerosas amostras

Detecção de Vivo<em&gt; Escherichia coli</em&gt; O157: H7 Células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

detection-of-live-escherichia-coli-o157h7-cells-by-pma-qpcr

Research

JoVE Journal

Biology

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

16.5K visualizações.  Food and Drug Administration.  Um ensaio de qPCR foi desenvolvido para a detecção de Escherichia coli O157: H7 alvo um marcador genético único, Z3276. A qPCR foi combinado com propidium monoazide (PMA) o tratamento para a detecção de células vivas. Este protocolo foi modificado e adaptado a um formato de placa de 96 poços para uma fácil manipulação e consistente de numerosas amostras 

Vídeo: Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Um ensaio para medir a atividade do Escherichia coli Induzível Lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Biologia

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening da atividade endoglucanase de fungos em Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeamento bacterianas redes funcionais e vias<em&gt; Escherichia Coli</em&gt; Usando sintéticos matrizes genéticas

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Protocolo Rápido para Preparação de electrocompetentes<em&gt; Escherichia coli</em&gt; E<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Protocolos para implementação de uma<em&gt; Escherichia coli</em&gt; Com base TX-TL Expression System-Free Cell para Biologia Sintética

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

A purificação cromatográfica do não-polipéptidos recombinantes Elastina semelhantes e suas fusões com Peptídeos e Proteínas de<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

As multifacetadas benefícios da proteína Co-expressão em<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Método para a rotulagem transcritos em células individuais Escherichia coli para único-molécula fluorescência In Situ as experiências de hibridação

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