Os autores estão em dívida com os chefes, chefes e comunidades de Macha para seu unwaveing ​​cooperar durante as coletas de amostras de mosquitos de campo. Dr. Doug Norris e Rebeca Kent ofereceu conhecimentos inestimáveis ​​sobre o protocolo de salting out. Este trabalho foi financiado pela malária Hopkins Johns Research Institute piloto sistema de concessão. Mosquito e padrões de DNA do parasita laboratoriais foram gentilmente doado por MR4 Tipo, American &amp; Culture Collection.

1. Dissecção Preparatória de espécimes do mosquito <ol><li> Mosquito lugar em um corte pequeno de parafilme e montagem em microscópio de dissecção. </li><li> Cobrir a gota de água desionizada, para amaciar o tecido. </li><li> Faça uma incisão na carcaça mosquito precisamente na articulação entre o tórax eo abdômen, assim nicking fora a cabeça eo tórax da seção abdominal. </li><li> Transfira cada seção mosquito dissecado em um tubo de microcentrífuga limpo autoclavado de 1,5 ml prelabeled com detalhes de identificação do mosquito número e adequadamente marcados para indicar seção do corpo (por exemplo, &quot;m&quot; para meados de intestino seção; &quot;ht&quot; para a cabeça e tórax seção). </li><li> Descarte parafilme e limpe cuidadosamente estágio do microscópio com Kimwipe 70% de álcool-umedecidos antes de processar o mosquito seguinte. </li></ol> 2. Extração de DNA a partir de amostras de mosquitos <ol><li> Pipetar 20 ul de água desionizada num tubo de amostra e a ponta da pipeta para moer o uso a secção de mosquito submerso num unsuspensão iForm. </li><li> Adicionar 100 ul de autoclavado 1X PBS / solução de saponina 1% a homogeneizado amostra e misturar por vórtex momentânea suave. </li><li> Incubar à temperatura ambiente durante 20 min. </li><li> Centrifugar a 20.000 xg durante 2 minutos e descartar o sobrenadante. </li><li> Ressuspender o sedimento em 100 ul de PBS 1X. </li><li> Centrifugar novamente a 20000 xg durante 2 min e descartar o sobrenadante. </li><li> Por centrifugação suave (5 segundos), pellet ressuspender em 75 uL de água estéril desionizada e 25 uL de 20% w / v de suspensão de resina Chelex-100, em água desionizada. </li><li> Pierce buraco na tampa do tubo de amostra usando agulha hipodérmica estéril 23G inflamado no bico de Bunsen. </li><li> Suspensão fervura da amostra em banho-maria na cremalheira de flutuação (ou no bloco de aquecimento), durante 10 min. </li><li> Centrifugar a 20.000 xg, durante 1 min e uma solução de transferência de DNA que se seguiu no frasco de armazenamento prelabeled para utilização como molde de PCR em aplicações. </li></ol> 3. Plasmodium falciparum Genotipagem e Anophelesspp. Identificação Molecular <ol><li> Adicionar 2,5 ul do extrato de DNA Chelex em 25 ul de reações de PCR para verificar a qualidade do DNA 11, identificar a espécie Anopheles 10 e ao genótipo P. falciparum 12 meados de intestino e infecções da glândula salivar. </li><li> Para maximizar o rendimento de amplicon, especialmente para P. falciparum, detecção incluem 1.5X albumina de soro bovino (BSA) no ensaio de PCR. A composição de reacção que se segue é recomendada: (2,5 ul template, primers 0,25 uM, 1,5 uM de cloreto de magnésio, 200 uM dNTPs, 1X tampão de PCR, 1,5 X e BSA 1.0u polimerase de ADN Taq, em 25 volumes uL). </li><li> Para verificar a qualidade do DNA em extrato, amplificar região do mitocondrial desidrogenase nicotinamida adenina dinucleotídeo artrópodes (NADH) subunidade 4 (ND4) gene (primers ND4FW [5&#39;-GTD YAT TTA TGA TTR CCT ​​AA-3 &#39;] e ND4RV [5&#39; -CTT CGD CTT CCW ADW CGT TC-3 &#39;]; esperar produto 400pb) 11,13. </li><li> Para diferenciar Anopheles gambiade e sl espécies. irmãos (An. gambiae ss e Uma arabiensis. apenas), amplificar região flanqueando SNPs no espaçador intergênica (IGS) região (primers ONU [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]; esperar 390bp Um produto ss gambiae, 315bp Uma arabiensis) 10,11. </li><li> Para a tipagem P. falciparum antifolato polimorfismos de resistência a drogas, realizar PCR aninhada para amplificar região flanqueadora codões de aminoácidos 108, 51, 59, 16 e 164 no parasita diidrofolato redutase (DHFR), gene: Primers redondos primárias: M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;] e M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] Secundárias primers redondos: M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] com F / [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] Ou F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] com M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). Também amplificar região contendo codões de aminoácidos 436, 437, 540, 581 umnd 613 no P. falciparum dihidropteroato sintetase gene (dhps): Primário redondo primers R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] com R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; Secundárias primers redondas J: [5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] com K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;] Ou K: [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;] com K /, Ou L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] com L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> Amplicon Sujeito 4 ul de alelo-específicos digestões com enzimas de restrição, em 30 ul de reacções seguindo as instruções do fabricante, para detectar drogas antifolato associadas à resistência polimorfismos 12. </li><li> Ul sujeito 5 de PCR amplicon (ou 15 ul de digestão de restrição) por pista de etídio corado com brometo de gel de agarose a 2% para electroforese (100 - 120V) e visualizar as bandas num transiluminador UV. </li></ol>

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O método aqui apresentado Chelex simplificado permite que a extracção de qualidade Anopheles spp e P. falciparum a partir de amostras de DNA de mosquitos passíveis de diversas aplicações de PCR. Esta técnica pode ser utilizada para a identificação molecular de mosquitos vectores da malária e de vigilância de fármaco-resistente P. alelos falciparum em mosquitos para os programas nacionais de controlo da malária. As vantagens da técnica Chelex simplificado incluem a simplicidade, os reagentes menos e, portanto, o custo, a segurança e o tempo de processamento mais curto (37 min) do que os protocolos standard, tais como o método de salting out 10 (2 h 47 min e uma etapa durante a noite, a Tabela 1). As vantagens e os requisitos acima mencionados reagentes mínimos (3 reagentes) em relação ao protocolo padrão atual (10 reagentes, Tabela 1) 11,15, fazer o protocolo simplificado Chelex particularmente amigável para os laboratórios de países endêmicos, onde reagentes uma constantecadeia de fornecimento é muitas vezes difícil de manter. Uma limitação deste método é que, como o protocolo padrão, foi também sujeito a inibidores de PCR conhecidos para ocorrer no tegumento mosquito 16. No entanto, este é prontamente aliviada pela inclusão de BSA nos ensaios. BSA também tem sido empregada com sucesso como um realçador contra os inibidores da amplificação em outras aplicações de PCR 17,18.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Chelex-100, 50-100 mesh seca </td><td> BioRad </td><td> # 143-2832 </td><td></td></tr><tr><td> Saponina </td><td> SIGMA </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td> New England Biolabs </td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

Países endêmicos estão adotando cada vez mais ferramentas moleculares para tipagem eficiente, identificação e vigilância contra os parasitas da malária e mosquitos vetores, como parte integrante de seus programas de controlo 1,2,3,4,5. Para o estabelecimento sustentável dessas abordagens precisos nas operações de pesquisa para fortalecer o controle da malária e os esforços de eliminação, métodos simples e acessíveis, com o reagente parcimoniosa e requisitos de equipamentos são essenciais 6,7,8. Aqui apresentamos uma técnica baseada Chelex simples para extrair parasita da malária e DNA vetor de espécimes coletados no campo de mosquito. Nós identificados morfologicamente 72 Anopheles gambiae sl. Das 156 mosquitos capturados por piretro spray de capturas em quartos de dormir das famílias dentro de uma vizinhança 2.000 km 2, do Instituto Malária em Macha. Após a dissecção de separar a cabeça eo tórax do abdômen para todas as 72 Anopheles gambiae sl. mosquitos, os dois cortes foram colocados individualmente em tubos de microcentrífuga de 1,5 ml e imerso em 20 ul de água desionizada. Utilizando uma ponta de pipeta estéril, cada secção de mosquito foi separadamente homogeneizados a uma suspensão uniforme na água desionizada. Do homogeneizado resultante a partir de cada secção de mosquito, 10 ul foi retida enquanto que o outro 10 ul foram transferidas para um tubo separado ml autoclavado 1.5. As alíquotas separadas foram submetidas à extração de DNA por qualquer Chelex o simplificado ou o padrão de salga a extração protocolo 9,10. O protocolo de salting out é chamado e amplamente utilizado porque emprega altas concentrações de sal em lugar de solventes orgânicos perigosos (tais como fenol e clorofórmio) para o passo de precipitação de proteína durante a extracção de ADN 9. Os extractos foram utilizados como moldes para a amplificação por PCR utilizando iniciadores com alvo mitocondrial artrópode dehydroge nicotinamida-adenina-dinucleótidonase (NADH) subunidade 4 gene (ND4) para verificar a qualidade do DNA 11, um PCR para identificação da espécie Anopheles gambiae 10 irmãos e um PCR para a digitação de infecção por Plasmodium falciparum 12. Comparação com a qualidade do DNA (ND4) PCR mostrou sensibilidade de 93% e especificidade de 82% para a abordagem Chelex relação ao estabelecido protocolo de salting out. Os correspondentes valores de sensibilidade e especificidade foram de 100% e 78%, respectivamente, utilizando-se a identificação da espécie irmão PCR e 92% e 80%, respectivamente, para P. falciparum detecção PCR. Não houve diferenças significativas na percentagem de amostras que deram sinal amplicon com o Chelex ou a regular as trocas salinas protocolo em todas as três aplicações de PCR. A abordagem Chelex necessárias três reagentes simples e 37 min para completar, enquanto o. Salting out protocolo envolveu 10 diferentes reagentes e 2 horas e 47 minutos de tempo &#39;de processamento, incluindo uma etapa durante a noite Nossos resultados mostram ªno método Chelex é comparável à extração salting out existente e pode ser substituído como uma abordagem simples e sustentável de recursos limitados, onde uma cadeia de fornecimento constante de reagente é muitas vezes difícil de manter.

Exemplos de resultados de ensaios de PCR para a qualidade do extracto mosquito DNA (Figura 1), Anopheles arabiensis identificação molecular (Figura 2) e P. falciparum de detecção (Figura 3) mostra que o método de Chelex simplificada dá resultados semelhantes ao padrão de salting out protocolo 10, apesar de muito menos passos (Tabela 1). Com uma qualidade comparável DNA nos respectivos extractos, não é surpreendente que a taxas de positividade de amostra em relação a um. espécies crípticas gambiae, bem como as taxas de infecção do parasita não foram estatisticamente diferentes com base no teste de McNemar qui-quadrado (Figura 3). Sensibilidade (%) foi calculada como TP / (TP + FN) * 100, onde TP denota verdadeiros positivos e falsos negativos FN indica. Especificidade (%) foi determinada como TN / (TN + FP) * 100, onde TN indica verdadeiros negativos e FP denota falsos positivos. A qualidade do ADN (ND4) PCR mostrou sensibilidade de 93% e especificidade de 82% para a abordagem Chelex em comparação com o padrão estabelecido ouro salting out protocolo como. Os correspondentes valores de sensibilidade e especificidade foram de 100% e 78%, respectivamente, utilizando-se a identificação da espécie irmão PCR e 92% e 80%, respectivamente, para P. falciparum detecção PCR. A adição de BSA para misturas de reacção resultou em um aumento geral de positivos de PCR (Figura 3), devido ao alívio da PCR inibidores 14, tanto para o Chelex simplificada e regular salting out protocolo. No entanto, este aumento não foi estatisticamente significativa, exceto para a qualidade do DNA (ND4 PCR) em extratos Chelex (p = 0,039). Específica de alelo de digestão com enzimas de restrição em P. falciparum DHFR (ou outro gene alvo) amplicon permite a genotipagem de infecções da glândula do intestino médio e salivares malária para os alelos de resistência da droga (Figura 4, os dados de glândulas salivares não mostrado). <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> Procedimento simples Chelex </td><td colspan="2"> Padrão Salga fora Procedimento </td></tr><tr><td> Passos </td><td> Reagentes </td><td> Passos </td><td> Reagentes </td></tr><tr><td><ol><li> Adicionar espécime a 1,5 ml tubo de microcentrífuga e homogeneizar em 100 ul de PBS 1X / Saponina solução a 1% (2 min). </li><li> Deixar a temperatura ambiente durante 20 min. </li><li> Centrifugação a 20000 xg durante 2 min e descartar o sobrenadante. </li><li> Adicionar 100 ul de PBS 1X e misture delicadamente por momentaneamente vórtex (3 seg.) </li><li> Centrifugação a 20000 xg durante 2 min e descartar o sobrenadante. </li><li> Adicionar 25 ul de 20% de Chelex w / v em água desionizada e 75 uL de água estéril desionizada. </li><li> Pierce buraco na tampa do tubo de amostra usando quente, agulha hipodérmica estéril 23G e conteúdo do tubo ferva por 10 min. </li><li> Girar o tubo de microcentrífuga de 20000 xg durante 1 min. </li><li> Transferir o sobrenadante para frasco prelabeled estéril e solução de ADN armazenar a -20 ° C até à sua utilização (2,5 ul de reacção de PCR de 25 uL). </li></ol></td><td> PBS Saponina Chelex-100 esferas </td><td><ol><li> Adicionar espécime a 1,5 ml tubo de microcentrífuga e homogeneizar em 100 ul de tampão * Bender com DEPC a 1% (2 min). </li><li> Incubar a 65 ° C durante 1 h. </li><li> Adicionar 15 ul de acetato de potássio a frio 8 M. Misturar suavemente e incubar em gelo durante 45 min. </li><li> Centrifugue a amostra em tubos de microcentrífuga a 20000 xg durante 10 min e de transferência de sobrenadante para um tubo novo ml 1.5. </li><li> Adicionar 250 ul de etanol a 100% e misturar bem invertendo o tubo. </li><li> Incubar amostra à temperatura ambiente durante 5 min. </li><li> Girar as amostras a 20.000 xg durante 15 min. </li><li> Aspirar e descartar o sobrenadante, deixando o sedimento secar completamente no tubo (30 min). </li><li> Ressuspender o granulado em 20 ul de 0,1 X SSC + RNAse (10 ug / ml) durante a noite a 4 ° C. </li><li> Adicionar 80 ul de água tratada com DEPC e armazenar a -20 ° C até à sua utilização. </li></ol></td><td> Dietilpirocarbonato (DEPC) * Bender Buffer: 0,1 M de NaCl Sacarose 0,2 M 0,1 M de Tris-HCL 0,05 M de EDTA, pH 9,1 SDS a 0,5% em água DEPC 8 de acetato de potássio M Etanol 0,1 X salina tampão de citrato de sódio (SSC) RNAse (10 ug / ml) </td></tr><tr><td colspan="2"> Tempo total: 37 min </td><td colspan="2"> Tempo total: 2 horas 47 min, mais durante a noite </td></tr></tbody></table> Tabela 1. Passo a passo de comparação dos reagentes e requisitos de tempo para a Chelex protocolo simples e ficarard salting out método de extração de DNA a partir de amostras de mosquitos. <img alt="Figura 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> Figura 1. ND4 mitocondrial comparação PCR de DNA de qualidade simplificada Chelex &quot;C&quot; e padrão salting out &quot;M&quot; extractos de amostras de campo Anopheles arabiensis. NC, controlo negativo; M1 e C1, C2 e M2, M3 e C3, e C4 e M4 são emparelhados salting out e extractos a partir de uma mesma Chelex. espécimes do mosquito arabiensis. L, 100 bp ADN. <img alt="Figura 2" src="/files/ftp_upload/3281/3281fig2.jpg" /> Figura 2 molecular de PCR para identificação arabiensis Anopheles C1 e M1, M2 e C2, C3 e M3, M4 e C4 denotam amplicon respectiva de salting out &quot;M&quot; e simplificado Chelex &quot;C&quot; extratos de DNA para amostras de mosquitos mesmos;.. AR, Anopheles arabiensiscontrole positivo; L, 100 bp ADN. <img alt="Figura 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> Figura 3. Detecção de positivos sobre Chelex e salga fora extratos de DNA de amostras de campo do mosquito, com ensaios de PCR para a identificação molecular de Anopheles arabiensis (AR) 10, artrópode gene NADH desidrogenase 4 (ND4) teste de DNA de qualidade 11 e P resistência antifolato droga . falciparum DHFR genotipagem 12 (F-M4). Os resultados apresentados para os testes realizados com ou sem BSA na mistura de reacção. O teste de McNemar de qui-quadrado foi usado para determinar se as diferenças entre cento dos PCRs positivas de DNA extraído a partir da Chelex simplificada e o padrão de salting out protocolos foram estatisticamente significativas. <img alt="Figura 4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <strong&gt; Figura 4. Aplicação da simplificado Chelex protocolo em genotipagem P. falciparum em mosquitos (meados de intestino dados mostrados) para os alelos de resistência a drogas. BstN I em digestão amplicon flanqueando codão 108 do P. falciparum gene DHFR 12 mostra os mutantes resistentes cicloguanilo S108T em amostras de mosquitos (M1-M5; mid-intestinal dados apresentados). U, 522 pb digerido amplicon; FCR3, padrão de laboratório P. falciparum clone controle positivo levando S108T; K1, P. falciparum laboratório padrão de controle clone contábil negativo cicloguanilo sensível S108N, L, 100 bp ADN.

Watch this Scientific Journal Video about A Simple Chelex Protocol for DNA Extraction from Anopheles spp. at JoVE.com

Um protocolo Chelex simples para extração de DNA de Anopheles spp. | Imunologia e Infecção | Vídeo

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Uma maneira rápida e acessível para extrair qualidade parasita da malária e de DNA a partir de amostras do mosquito vetor é descrito. Capitalizando propriedades quelantes de resina Chelex, o método simples que permite a determinação do genótipo de parasitas da malária, em fases de glândulas de mosquitos mid-intestinal e de saliva, bem como a identificação molecular da<em&gt; Anopheles</em&gt; Sibling espécies por PCR.

Um Protocolo de Chelex simples para extração de DNA de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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54.7K visualizações.  Malaria Institute at Macha. Uma maneira rápida e acessível para extrair qualidade parasita da malária e de DNA a partir de amostras do mosquito vetor é descrito. Capitalizando propriedades quelantes de resina Chelex, o método simples que permite a determinação do genótipo de parasitas da malária, em fases de glândulas de mosquitos mid-intestinal e de saliva, bem como a identificação molecular da Anopheles Sibling espécies por PCR.

Vídeo: A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

Um Protocolo de Coleta e coloração Hemócitos do mosquito da febre amarela Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Imunologia e Infecção

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

Tecnologia IgY: Extração de anticorpos de gema de ovo de galinha por polietileno glicol (PEG) Precipitação

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Alta e Baixa Screens rendimento com galhas-Nematóides Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Um protocolo simples para agregação plaquetária mediada por<em&gt; Plasmodium falciparum</emEritrócitos&gt; infectados em uma configuração com poucos recursos

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Um protocolo para hepatite C Analisando replicação de vírus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Um protocolo simples e rápido para não enzimática Separar frescos Tecidos Humanos para a Análise de infiltrar Linfócitos

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo de isolamento eficiente para linfócitos B e T a partir de tonsilas palatinas Humanos

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Uma abordagem simples e eficiente para construir vectores de vírus mutante Vaccinia

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

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