Gli autori sono in debito con i capi tribù, capi e le comunità di Macha per la loro collaborazione nel corso unwaveing ​​collezioni zanzara campione sul campo. Il dottor Doug e Rebecca Norris Kent offerto competenze di alta qualità sul protocollo salatura fuori. Questo lavoro è stato finanziato dalla Johns Hopkins Malaria Research Institute sistema pilota di sovvenzione. Mosquito e parassiti del DNA standard di laboratorio sono stati gentilmente donati da MR4, tipo American &amp; Culture Collection.

1. Dissezione di preparazione dei campioni Mosquito <ol><li> Zanzara Posto su un piccolo taglio di parafilm e supporto sulla dissezione microscopio. </li><li> Copertina in goccia di acqua deionizzata per ammorbidire i tessuti. </li><li> Incidere la carcassa zanzara proprio in corrispondenza della giunzione tra il torace e l&#39;addome, in modo da intaccare la testa e il torace dalla sezione addominale. </li><li> Trasferire ogni sezione zanzara sezionato in un ambiente pulito in autoclave 1,5 ml tubetto microcentrifuga prelabeled con dettagli zanzare numero ID e che portano l&#39;etichetta per indicare la sezione del corpo (ad esempio, &quot;m&quot; per la metà-gut sezione, &quot;ht&quot; per testa e torace sezione). </li><li> Eliminare parafilm e asciugare con cura tavolino del microscopio con il 70% di alcool-imbevuto Kimwipe prima di elaborare la zanzara successivo. </li></ol> 2. Estrazione del DNA da campioni Mosquito <ol><li> Pipettare 20 ml di acqua deionizzata in provetta e l&#39;uso della punta di pipetta per macinare la sezione zanzara sommerso in una ONUsospensione iForm. </li><li> Aggiungere 100 ml di PBS 1X autoclavato / soluzione di 1% di saponina omogenato del campione e mescolare dolce vortex momentanea. </li><li> Incubare a temperatura ambiente per 20 min. </li><li> Centrifugare a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Risospendere il pellet in 100 ul di PBS 1X. </li><li> Centrifugare nuovamente a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Vortexando gentilmente (5 sec), risospendere il pellet in 75 microlitri di acqua sterile deionizzata e 25 ml di 20% w / v Chelex-100 sospensione resina in acqua deionizzata. </li><li> Pierce foro nel coperchio della provetta sterile con ago ipodermico 23G fiammato in becco Bunsen. </li><li> Ebollizione sospensione del campione in bagnomaria a cremagliera galleggiante (o in blocco di riscaldamento) per 10 min. </li><li> Centrifugare a 20.000 xg per 1 min e soluzione di DNA conseguente trasferimento nella fiala stoccaggio prelabeled per uso come modello in applicazioni PCR. </li></ol> 3. Plasmodium falciparum Genotipizzazione e Anophelesspp. Identificazione molecolare <ol><li> Aggiungere 2,5 ml di estratto DNA Chelex in 25 ul reazioni PCR per verificare la qualità del DNA 11, identificare le specie Anopheles 10 e al genotipo P. falciparum 12 infezioni della ghiandola metà del budello e salivari. </li><li> Per massimizzare la resa amplicone, soprattutto per P. rilevamento falciparum, includono 1.5X albumina di siero bovino (BSA) in PCR. La composizione seguente reazione è raccomandato: (2,5 template pl, 0,25 primers pm, 1,5 mM di cloruro di magnesio, 200 microM dNTPs, 1X PCR Buffer, 1.5X BSA e 1.0U Taq DNA polimerasi, in 25 volumi pl). </li><li> Per verificare la qualità del DNA in estratto, amplificano regione del artropodi deidrogenasi mitocondriale nicotinammide adenin dinucleotide (NADH) subunità 4 (ND4) gene (primer 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;]; aspettiamo prodotto 400BP) 11,13. </li><li> Per differenziare Anopheles gambiae SL. specie sorella (An. gambiae ss e An. arabiensis solo), amplificare affiancano la regione SNPs nel distanziale intergenica (IGS) regione (primer ONU [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]; aspettiamo 390bp prodotto una SS gambiae, 315bp arabiensis An) 10,11. </li><li> Per la tipizzazione P. falciparum antifolato polimorfismi di resistenza ai farmaci, eseguire nested PCR per amplificare regione fiancheggiante codoni amminoacidici 108, 51, 59, 16 e 164 nella parassita diidrofolato reduttasi (DHFR) gene: Primer rotonde primari: M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;] e M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] Secondari primer rotonde: M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] con F / [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] O F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] con M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). Anche amplificare regione contenente codoni amminoacidici 436, 437, 540, 581 And 613 nel P. falciparum diidropteroato sintetasi (DHPS) gene: Primaria tutto primer R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] con R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; Secondari primer rotonde J: [5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] con K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;] O K: [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;] con K /, O L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] con L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> Soggetto 4 amplicone microlitri di digestioni di restrizione allele-specifici enzimi in 30 ul reazioni seguendo le istruzioni del fabbricante, di rilevare antifolato farmaci associate a resistenza polimorfismi 12. </li><li> Soggetto 5 pl di ampliconi PCR (o 15 ml di digestione di restrizione) per pista di bromuro di etidio-colorato gel di agarosio al 2% per elettroforesi (100 - 120V) e visualizzare le bande con transilluminazione UV. </li></ol>

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Nessun conflitto di interessi dichiarati.

Il metodo semplificato Chelex qui presentato permette l&#39;estrazione di qualità Anopheles spp e P. falciparum DNA da campioni di zanzara suscettibili di diverse applicazioni PCR. Questa tecnica può essere utilizzata per l&#39;identificazione molecolare di zanzare vettore malaria e la sorveglianza della farmaco-resistenti P. alleli falciparum in zanzare per i programmi nazionali di controllo della malaria. I vantaggi della tecnica Chelex semplificata includono semplicità, meno reattivi e quindi dei costi, sicurezza e tempi di lavorazione più brevi (37 min) di protocolli standard quali il salting out metodo 10 (2 h 47 min e un passo overnight, Tabella 1). I vantaggi di cui sopra ed i requisiti minimi dei reagenti (3 reagenti) rispetto al protocollo standard attuale (10 reagenti, Tabella 1) 11,15, rendono il protocollo Chelex semplificato particolarmente cordiale ai laboratori dei paesi endemici in cui un reagente costantefiliera è spesso difficile da mantenere. Una limitazione del metodo è che, come il protocollo standard, è stato anche oggetto di inibitori della PCR noti a verificarsi nel tegumento zanzara 16. Tuttavia, questo è facilmente alleviato inclusione di BSA nei saggi. BSA è stata utilizzata con successo come amplificazione enhancer contro inibitori in altre applicazioni PCR 17,18.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Chelex-100, 50-100 mesh a secco </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.

Paesi endemici stanno sempre più adottando strumenti molecolari per la tipizzazione efficiente, identificazione e sorveglianza contro i parassiti della malaria e le zanzare vettore, come parte integrante dei loro programmi di controllo 1,2,3,4,5. Per la creazione sostenibile di questi approcci precisi nelle operazioni di ricerca per rafforzare il controllo della malaria e gli sforzi di eliminazione, metodi semplici e convenienti, con reagente parsimoniosa e alle attrezzature sono essenziali 6,7,8. Qui vi presentiamo una semplice Chelex tecnica basata per l&#39;estrazione parassita della malaria e il DNA da campioni di zanzara vettore raccolti sul campo. Abbiamo identificato morfologicamente 72 Anopheles gambiae sl. Da 156 zanzare catturate dalla piretro spruzzo catture accessorie nelle camere da letto delle famiglie all&#39;interno di un km 2.000 2 pressi dell&#39;Istituto malaria a Macha. Dopo la dissezione, per separare la testa e il torace dall&#39;addome per tutti Anopheles 72 gambiae sl. zanzare, le due sezioni sono state singolarmente messo in tubi microcentrifuga da 1,5 ml e immerso in 20 ml di acqua deionizzata. Usando una pipetta sterile, ogni sezione zanzara stato omogeneizzato separatamente ad una sospensione uniforme in acqua deionizzata. Dell&#39;omogenato derivanti da ciascuna sezione zanzara, 10 microlitri è stata conservata mentre l&#39;altro 10 microlitri è stato trasferito ad una provetta separata autoclavata 1,5 ml. Le aliquote separate sono stati sottoposti ad estrazione del DNA sia dal Chelex semplificata o lo standard salting out estrazione protocollo 9,10. Il protocollo salting out è il cosiddetto e ampiamente utilizzati perché impiega elevate concentrazioni saline in luogo di pericolosi solventi organici (come fenolo e cloroformio) per la fase di precipitazione della proteina durante l&#39;estrazione del DNA 9. Gli estratti sono stati utilizzati come modelli per l&#39;amplificazione PCR utilizzando primers mirati artropodi mitocondriale dehydroge nicotinammide-adenina-dinucleotidenase (NADH) subunità 4 gene (ND4) per verificare la qualità del DNA 11, una PCR per l&#39;identificazione di specie Anopheles gambiae fratelli 10 e un nested PCR per la tipizzazione di infezione da Plasmodium falciparum 12. Confronto con qualità del DNA (ND4) PCR ha mostrato il 93% di sensibilità e specificità 82% per l&#39;approccio Chelex relativo alla stabilita salatura fuori protocollo. Corrispondenti valori di sensibilità e specificità sono state del 100% e 78%, rispettivamente, con fratello identificazione delle specie PCR e il 92% e 80%, rispettivamente, per P. falciparum rilevamento PCR. Non ci sono state differenze significative nella percentuale di campioni che con il segnale amplificato Chelex o il regolare salatura su protocollo in tutte e tre le applicazioni PCR. L&#39;approccio Chelex richiesto tre reagenti semplici e 37 min per completare, mentre la salatura su protocollo ha comportato 10 diversi reagenti e 2 ore e 47 min il tempo di elaborazione &#39;, tra cui un passo durante la notte. I nostri risultati mostrano °al metodo Chelex è paragonabile a quella esistente salting out estrazione e può essere sostituito da un approccio semplice e sostenibile in risorse limitate, dove una costante filiera reagente è spesso difficile da mantenere.

Esempi di risultati di saggi di PCR per la qualità zanzara estratto DNA (Figura 1), arabiensis Anopheles identificazione molecolare (Figura 2) e P. falciparum rilevamento (Figura 3) mostra che il metodo semplificato Chelex produce risultati simili allo standard salting out protocollo 10, nonostante passi molto minori (Tabella 1). Con la qualità del DNA comparabile nei rispettivi estratti non è sorprendente che la positività frequenze di campionamento per quanto riguarda An. specie sorella gambiae così come i tassi di infezione del parassita non erano statisticamente differenti in base a McNemar test Chi-quadrato (Figura 3). Sensibilità (%) è stata calcolata come VP / (VP + FN) * 100, dove TP denota veri positivi e falsi negativi FN indica. Specificità (%) è stato determinato come TN / (TN + FP) * 100, dove TN denota veri negativi e FP denota falsi positivi. La qualità del DNA (ND4) PCR ha mostrato il 93% di sensibilità e specificità 82% per l&#39;approccio Chelex rispetto alla norma stabilita salatura fuori protocollo come l&#39;oro. Corrispondenti valori di sensibilità e specificità sono state del 100% e 78%, rispettivamente, con fratello identificazione delle specie PCR e il 92% e 80%, rispettivamente, per P. falciparum rilevamento PCR. L&#39;aggiunta di BSA a miscele di reazione determinato un generale aumento di PCR positivi (Figura 3) dovute ai rilievi inibitori della PCR 14, sia per il Chelex semplificato e regolare salting out protocollo. Tuttavia, questo aumento non era statisticamente significativo tranne che per qualità del DNA (ND4 PCR) su estratti Chelex (p = 0.039). Allele-specifica digestione con enzimi di restrizione P. falciparum DHFR (o altro gene target) amplicon consente la genotipizzazione di infezioni metà ghiandola salivare intestinale e malaria per alleli di resistenza ai farmaci (Figura 4; ghiandole salivari dati non mostrati). <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> Chelex procedura semplice </td><td colspan="2"> Standard salatura fuori procedura </td></tr><tr><td> Passi </td><td> Reagenti </td><td> Passi </td><td> Reagenti </td></tr><tr><td><ol><li> Aggiungere al campione provetta 1,5 ml microcentrifuga e omogeneizzare in 100 pl di PBS 1X / saponina soluzione 1% (2 min). </li><li> Lascia a temperatura ambiente per 20 min. </li><li> Spin a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Aggiungere 100 ml di PBS 1X e miscelare delicatamente momentaneamente vortex (3 sec). </li><li> Spin a 20.000 xg per 2 minuti ed eliminare il surnatante. </li><li> Aggiungere 25 ml di 20% w / v Chelex in acqua deionizzata e 75 microlitri di acqua sterile deionizzata. </li><li> Pierce foro nel coperchio del tubo campione utilizzando caldo, ago ipodermico sterile 23G e contenuto della provetta bollire per 10 min. </li><li> Spin provetta per microcentrifuga a 20.000 xg per 1 min. </li><li> Trasferire il surnatante in fiala sterile prelabeled e DNA soluzione conservare a -20 ° C fino al momento dell&#39;uso (2,5 microlitri in 25 microlitri di reazione PCR). </li></ol></td><td> PBS Saponina Chelex-100 perle </td><td><ol><li> Aggiungere al campione provetta 1,5 ml microcentrifuga e omogeneizzare in 100 microlitri tampone * Bender con 1% DEPC (2 min). </li><li> Incubare a 65 ° C per 1 ora. </li><li> Aggiungere 15 microlitri fredda 8 M acetato di potassio. Mescolare delicatamente e incubare in ghiaccio per 45 min. </li><li> Spin campione in provette per microcentrifuga a 20.000 xg per 10 min e il trasferimento supernatante in una nuova provetta 1,5 ml. </li><li> Aggiungere 250 microlitri di etanolo al 100% e mescolare bene capovolgendo la provetta. </li><li> Incubare il campione a temperatura ambiente per 5 min. </li><li> Centrifugare i campioni a 20.000 xg per 15 min. </li><li> Aspirare ed eliminare il surnatante, il pellet lasciando asciugare completamente nel tubo (30 ma). </li><li> Risospendere il pellet in 20 ml di 0,1 X. SSC + RNAsi (10 mg / ml) per una notte a 4 ° C. </li><li> Aggiungere 80 microlitri di acqua DEPC e conservare a -20 ° C fino al momento dell&#39;uso. </li></ol></td><td> Dietilpirocarbonato (DEPC) * Bender Buffer: 0,1 M NaCl 0,2 M Saccarosio 0,1 M Tris-HCL 0,05 M EDTA, pH 9,1 0,5% SDS in acqua DEPC 8 M di acetato di potassio Etanolo 0,1 X. Saline di sodio citrato (SSC) tampone RNAsi (10 mg / ml) </td></tr><tr><td colspan="2"> TEMPO TOTALE: 37 min </td><td colspan="2"> TEMPO TOTALE: 2 h 47 min, più durante la notte </td></tr></tbody></table> Tabella 1. Confronto passo per passo dei reagenti e dei tempi necessari per il protocollo Chelex semplice e standard salatura metodo di estrazione del DNA da campioni di zanzara. <img alt="Figura 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> Figura 1. ND4 mitocondriale confronto PCR di DNA di qualità semplificato Chelex &quot;C&quot; e standard salting out &quot;M&quot; su campioni estratti arabiensis campo Anopheles. NC, controllo negativo, M1 e C1, C2 e M2, M3 e C3, C4 e M4 e sono accoppiati salatura ed estratti Chelex da An stessa. arabiensis zanzara campioni. L, 100 bp DNA ladder. <img alt="Figura 2" src="/files/ftp_upload/3281/3281fig2.jpg" /> Figura 2 molecolare PCR per l&#39;identificazione arabiensis Anopheles C1 e M1, M2 e C2, C3 e M3, M4 C4 e denotano rispettivamente da amplicon salting out &quot;M&quot; e semplificato Chelex &quot;C&quot; estratti di DNA per gli esemplari di zanzara stessi,.. AR, Anopheles arabiensiscontrollo positivo, L, 100 bp DNA ladder. <img alt="Figura 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> Figura 3. Rilevamento di positivi su Chelex e salatura su DNA estratto da campioni prelevati sul campo di zanzara, con saggi di PCR per l&#39;identificazione molecolare di Anopheles arabiensis (AR) 10, artropodi gene NADH deidrogenasi 4 (ND4) qualità del DNA test 11, e antifolato P resistenza ai farmaci . falciparum DHFR genotipizzazione 12 (F-M4). Risultati visualizzati per dosaggi con o senza BSA nella miscela di reazione. McNemar chi-square test è stato utilizzato per determinare se le differenze tra cento di PCR positivi da DNA estratto dal Chelex semplificata e lo standard salting out protocolli erano statisticamente significative. <img alt="Figura 4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <strong&gt; Figura 4. Applicazione del protocollo semplificato Chelex genotipizzazione P. infezioni falciparum in zanzare (mid-gut dati illustrati) per gli alleli di resistenza ai farmaci. BstN ho la digestione su amplicone accompagnamento codone 108 del P. falciparum gene DHFR 12 mostra cicloguanile mutanti resistenti S108T in campioni di zanzara (M1-M5; metà gut dati riportati). U, non digerito 522 bp ampliconi, FCR3, standard di laboratorio P. falciparum clone controllo positivo svolgimento S108T, K1, P. falciparum standard di laboratorio di controllo clone negativo portando cicloguanile sensibile S108N, L, 100 bp DNA ladder.

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

Un semplice protocollo Chelex per l'estrazione del DNA da Anopheles spp. | Immunologia e infezione | Video

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Un modo rapido e conveniente per estrarre parassita della malaria qualità e il DNA da campioni di zanzara vettore è descritto. Capitalizzando proprietà chelanti di resina Chelex, il semplice metodo consente genotipizzazione dei parassiti della malaria in fasi zanzara ghiandole mid-intestinali e salivare, nonché identificazione molecolare del<em&gt; Anopheles</em&gt; Sibling specie mediante PCR.

Un protocollo Chelex semplice per l&#39;estrazione del DNA da<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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54.5K Views.  Malaria Institute at Macha. Un modo rapido e conveniente per estrarre parassita della malaria qualità e il DNA da campioni di zanzara vettore è descritto. Capitalizzando proprietà chelanti di resina Chelex, il semplice metodo consente genotipizzazione dei parassiti della malaria in fasi zanzara ghiandole mid-intestinali e salivare, nonché identificazione molecolare del Anopheles Sibling specie mediante PCR.

Video: 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.

Un protocollo per la raccolta e la colorazione ematociti concentrati dal Mosquito febbre gialla 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 ...

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Immunologia e infezioni

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: Estrazione di anticorpi pollo rosso d&#39;uovo con polietilenglicole (PEG) Precipitazioni

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.

Schermi throughput elevato e basso con Nematodi galligeni 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.

Un semplice protocollo per Clumping piastrinica mediata da<em&gt; Plasmodium falciparum</emEritrociti&gt; infette in una risorsa Povero Setting

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

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.

Un semplice e rapido protocollo non-enzimaticamente dissociare freschi tessuti umani per l&#39;analisi di linfociti infiltranti

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

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.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

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

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

Combinando l'analisi di DNA in un'estrazione di greggio virione con l'analisi del RNA dalle foglie infette per scoprire nuovi Virus genomi

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

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

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

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

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

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

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

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

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

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

Una piattaforma di High-throughput per lo Screening della Salmonella spp /Shigella spp.

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

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

Analisi della dinamica del microbiota fecale in topi inclini al lupus utilizzando un metodo di isolamento del DNA semplice ed economico

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...