Die Autoren danken den Chefs, Häuptlinge und Gemeinden Macha für ihre unwaveing ​​Zusammenarbeit während mosquito Feldprobe Sammlungen. Dr. Doug Norris und Rebekka Kent angeboten unschätzbares Fachwissen auf dem Aussalzen Protokoll. Diese Arbeit wurde von der Johns Hopkins Malaria Research Institute Pilot-Grant-System finanziert. Mosquito und Parasiten DNA-Labor Standards wurden freundlicherweise von MR4, American Type Culture Collection &amp; gespendet.

Ein. Vorbereitende Präparation Mosquito Proben <ol><li> Ort Mücke auf einem kleinen Schneiden von Parafilm und Halterung am Binokular. </li><li> Cover in Tropfen entionisiertes Wasser, um Gewebe zu erweichen. </li><li> Einzuschneiden die Mücke Karkasse genau an der Verbindungsstelle zwischen dem Thorax und Abdomen, damit Nicking den Kopf und Thorax von der abdominalen Abschnitt. </li><li> Übertragen Sie jede seziert mosquito Abschnitt in einem sauberen autoklaviert 1,5 ml Mikrozentrifugenröhrchen mit Moskito-ID-Nummer Details und entsprechend gekennzeichnet, um Körper Abschnitt (zB &quot;m&quot; für Mitte-gut Abschnitt; &quot;ht&quot; für Kopf und Thorax-Bereich) bezeichnen prelabeled. </li><li> Entsorgen Parafilm und wischen Mikroskoptisch mit 70% Alkohol angefeuchtet Kimwipe vor der Verarbeitung der nächsten Mücke. </li></ol> 2. DNA Extraktion aus Mosquito Proben <ol><li> Je 20 ul entionisiertem Wasser in Probenröhrchen und Pipettenspitze, die untergetaucht mosquito Abschnitt in eine un schleifeniform Suspension. </li><li> Fügen Sie 100 ul autoklaviertem 1X PBS / 1% Saponin Lösung Probenhomogenat und durch vorsichtiges momentanen Vortexen. </li><li> Inkubieren bei Raumtemperatur für 20 min. </li><li> Zentrifuge bei 20.000 xg für 2 min und Überstand verwerfen. </li><li> Pellet in 100 ul 1X PBS. </li><li> Zentrifugieren bei 20.000 xg für 2 min und Überstand verwerfen. </li><li> Durch leichtes Vortexen (5 sec), Pellet in 75 ul sterilem entionisiertem Wasser und 25 ul 20% w / v Chelex-100 Harzsuspension in deionisiertem Wasser. </li><li> Pierce Loch im Deckel der Probenröhrchen mit sterilen 23G Injektionsnadel in Bunsenbrenner geflammt. </li><li> Boil Probensuspension im Wasserbad auf schwimmenden Zahnstange (oder im Heizblock) für 10 min. </li><li> Zentrifugieren bei 20.000 × g für 1 Min. und Übertragung anschließenden DNA-Lösung in prelabeled Lagervials zur Verwendung als Matrize in PCR-Anwendungen. </li></ol> 3. Plasmodium falciparum Genotypisierung und Anophelesspp. Molecular Identification <ol><li> Fügen Sie 2,5 ul des Chelex DNA-Extrakt in 25 ul PCR Reaktionen auf DNA-Qualität 11 kontrollieren, zu identifizieren Anopheles Arten 10 und dem Genotyp P. falciparum 12 Mitte Darm und Speicheldrüsen Infektionen. </li><li> Um Amplikon Ertrag, vor allem für P. zu maximieren falciparum Detektion umfassen 1.5X Rinderserumalbumin (BSA) in dem PCR-Assay. Die folgende Reaktion Zusammensetzung wird empfohlen: (2,5 ul Vorlage, 0,25 pM Primer, 1,5 uM Magnesiumchlorid, 200 uM dNTPs, 1X PCR-Puffer, 1,5 X BSA und 1.0U Taq DNA-Polymerase, in 25 ul Volumen). </li><li> Um DNA-Qualität im Extrakt zu überprüfen, zu verstärken Region der Arthropoden mitochondrialen Nicotinamidadenindinucleotid Dehydrogenase (NADH)-Untereinheit 4 (ND4) Gen (Primer ND4FW [5&#39;-GTD YAT TTA TGA TTR CCT ​​AA-3 &#39;] und ND4RV [5&#39; -CTT CGD CTT CCW ADW CGT TC-3 &#39;]; erwarten Produkt 400bp) 11,13. </li><li> Um Anopheles gambia differenzierene sl. Geschwisterart (An. gambiae ss und An. arabiensis only), verstärken flankierenden SNPs in der intergenen Spacer (IGS) Region (Primer UN [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]; erwarten Produkt 390bp Ein gambiae ss, 315bp Ein arabiensis) 10,11. </li><li> Für die Typisierung P. falciparum Antifolat Arzneimittelresistenz Polymorphismen, führen verschachtelten PCR zur ​​Region flankieren Aminosäurecodons 108, 51, 59, 16 und 164 in der Parasit Dihydrofolatreduktase (DHFR)-Gen amplifizieren: Primäre Runde Primer: M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;] und M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] Sekundäre Runde Primer: M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] mit F / [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] Oder F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] mit M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). Auch verstärken enthaltenden Bereich Aminosäurecodons 436, 437, 540, 581 And 613 in der P. falciparum Dihydropteroat Synthetase (DHPS)-Gen: Primäre Runde Primer R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] mit R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; Sekundäre Runde Primer J: [5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] mit K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;] Oder K: [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;] mit K /, Oder L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] mit L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> Gegenstand 4 ul Amplikon zu allelspezifischen Restriktionsenzym Aufschlüsse in 30 ul Reaktionen nach den Anweisungen des Herstellers zum Aufspüren Antifolat Medikamentenresistenz-assoziierten Polymorphismen 12. </li><li> Betreff 5 ul PCR Amplikon (oder 15 ul Restriktionsverdau) pro Spur von Ethidiumbromid-gefärbten 2% Agarosegel-Elektrophorese (100 - 120V) und visualisieren Banden unter UV Durchleuchtung. </li></ol>

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Keine Interessenskonflikte erklärt.

Die vereinfachte Chelex hier vorgestellte Methode ermöglicht die Extraktion der Qualität Anopheles spp und P. falciparum DNA aus Moskito-Proben zugänglich diverse PCR-Anwendungen. Diese Technik kann für die molekulare Identifizierung von Malariavektorkontrollschulung Mücken und Überwachung der Arzneimittel-resistente P. eingesetzt werden falciparum Allele in Mücken National Malaria Control Programs. Die Vorteile des vereinfachten Chelex Technik gehören Einfachheit, weniger Reagenzien und damit Kosten, Sicherheit und kürzere Bearbeitungszeiten (37 min) als Standard-Protokolle wie das Aussalzen Verfahren 10 (2 hr 47 min und einer Nacht Schritt, Tabelle 1). Die oben genannten Vorteile und minimale Reagenz Anforderungen (3 Reagenzien) auf den aktuellen Standard-Protokoll (10 Reagenzien, Tabelle 1) 11,15 gegenüber, machen die vereinfachte Chelex Protokoll besonders freundlich zu endemischen Land Laboratorien, in denen eine konstante ReagenzSupply Chain ist es oft schwierig zu pflegen. Eine Beschränkung des Verfahrens ist, dass wie die Standard-Protokoll, auch war Gegenstand PCR-Inhibitoren bekannt, in der Mücke Integument 16 auftreten. Jedoch ist dies ohne weiteres durch Einschluß von BSA in den Assays erleichtert. BSA wurde auch erfolgreich als Verstärkung Enhancer gegen Inhibitoren in anderen PCR-Anwendungen 17,18 beschäftigt.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Chelex-100, 50-100 trockenen mesh </td><td> BioRad </td><td> # 143-2832 </td><td></td></tr><tr><td> Saponin </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.

Endemischen Ländern übernehmen zunehmend molekulare Werkzeuge für die effiziente Typisierung, Identifizierung und Überwachung gegen Malariaparasiten und Vektor Mücken, als integralen Bestandteil ihrer Bekämpfungsprogramme 1,2,3,4,5. Für eine nachhaltige Etablierung dieser genauen Ansätze in Operations Research zur Malaria-Kontrolle und Beseitigung Bemühungen, einfache und kostengünstige Methoden, mit sparsamen Reagenz und Anforderungen an die Ausrüstung zu stärken sind wesentliche 6,7,8. Hier präsentieren wir Ihnen eine einfache Chelex-basierte Technik zur Gewinnung von Malaria-Parasiten und Vektor-DNA aus dem Feld gesammelt mosquito Proben. Wir morphologisch identifizierten 72 Anopheles gambiae sl. Von 156 Moskitos durch Pyrethrum Spray fängt in den Schlafräumen der Haushalte innerhalb eines 2.000 km 2 Nähe des Malaria Institut Macha erfasst. Nach Dissektion, die Kopf und Thorax aus dem Bauch zu trennen für alle 72 Anopheles gambiae sl. Mücken, wurden die beiden Abschnitte einzeln in 1,5 ml Mikrozentrifugenröhrchen platziert und untergetaucht in 20 ul entionisiertem Wasser. Verwendung einer sterilen Pipettenspitze wurde jeder Abschnitt separat Mücken zu einer gleichmäßigen Suspension in entionisiertem Wasser homogenisiert. Der anschließenden Homogenat aus jeder Mücke Abschnitt wurde 10 ul beibehalten, während die anderen 10 ul einer separaten autoklavierten 1,5 ml Röhrchen überführt wurde. Die separaten Aliquots wurden DNA-Extraktion entweder durch die vereinfachte Chelex oder die Standard Aussalzen Extraktionsprotokoll 9,10 unterzogen. Das Protokoll ist Aussalzen sogenannte und weit verbreitet, weil sie hohe Salzkonzentrationen verwendet anstelle von gefährlichen organischen Lösungsmitteln (wie Phenol und Chloroform) für das Protein Fällungsschritt während der DNA-Extraktion 9. Extrakte wurden als Matrizen für die PCR-Amplifikation unter Verwendung von Primern verwendet Targeting Arthropoden mitochondrialen Nicotinamidadenindinucleotid Dehydrogenasenase (NADH) Untereinheit 4-Gen (ND4) an DNA-Qualität 11, eine PCR zur ​​Identifizierung von Anopheles gambiae Geschwisterart 10 und eine verschachtelte PCR zur ​​Typisierung von Plasmodium falciparum-Infektion 12 zu überprüfen. Vergleich mit DNA-Qualität (ND4) PCR zeigten 93% Sensitivität und 82% Spezifität für die Chelex Ansatz gegenüber der etablierten Aussalzen Protokoll. Entsprechende Werte von Sensitivität und Spezifität waren 100% bzw. 78%, jeweils unter Verwendung Geschwisterart Identifizierung PCR und 92% und 80%, jeweils für P. falciparum Nachweis PCR. Es gab keine signifikanten Unterschiede in der Anteil an Proben, bei Amplikon Signals mit der oder den regulären Chelex Aussalzen Protokoll über alle drei PCR-Anwendungen. Die Chelex Ansatz erforderlich drei einfachen Reagenzien und 37 min in Anspruch, während das Aussalzen Protokoll brachte 10 verschiedene Reagenzien und 2 h und 47 min &#39;Bearbeitungszeit, einschließlich einer Übernachtung Schritt. Unsere Ergebnisse zeigen, tham Chelex Verfahren ist vergleichbar mit der vorhandenen Aussalzen Extraktion und kann als eine einfache und nachhaltige Ansatz mit beschränkten Ressourcen wo eine konstante Reagenz Lieferkette ist oft schwierig zu warten substituiert sein.

Beispiele für Ergebnisse von PCR-Assays für Moskito DNA-Extrakt Qualität (Abbildung 1), Anopheles arabiensis molekulare Identifizierung (Abbildung 2) und P. falciparum-Erkennung (Abbildung 3) zeigen, dass die vereinfachte Chelex Verfahren zu ähnlichen Ergebnissen liefert der Standard Aussalzen Protokoll 10, obwohl viel weniger Schritte (Tabelle 1). Bei vergleichbaren DNA-Qualität in den jeweiligen Extrakten ist es nicht verwunderlich, dass die Probe Positivität Preise in Bezug auf An. gambiae Geschwister Arten sowie Parasiten Infektionsraten waren statistisch nicht unterschiedlich basierend auf McNemar-Chi-Quadrat-Test (Abbildung 3). Sensitivität (%) wurde berechnet als TP / (TP + FN) * 100, wo TP bezeichnet wahren Positiven und FN bezeichnet falsche Negative. Spezifität (%) wurde als TN / bestimmt (TN + FP) * 100, wo TN bezeichnet wahre Negative und FP bezeichnet Fehlalarme. Die DNA-Qualität (ND4) PCR zeigten 93% Sensitivität und 82% Spezifität für die Chelex Ansatz im Vergleich zu den etablierten Aussalzen Protokoll als Goldstandard. Entsprechende Werte von Sensitivität und Spezifität waren 100% bzw. 78%, jeweils unter Verwendung Geschwisterart Identifizierung PCR und 92% und 80%, jeweils für P. falciparum Nachweis PCR. Der Zusatz von BSA an Reaktionsgemische ergab eine allgemeine Erhöhung der PCR Positives (3) infolge Entlastung der PCR-Inhibitoren 14, sowohl für die vereinfachte Chelex und regelmäßige Aussalzen Protokoll. Allerdings war dieser Anstieg statistisch nicht signifikant mit Ausnahme von DNA-Qualität (ND4 PCR) auf Chelex Extrakte (p = 0,039). Allel-spezifische Restriktionsenzymverdau auf P. falciparum DHFR (oder andere Target-Gen) Amplikon ermöglicht die Genotypisierung von Mitte Darm und Speicheldrüse Malariainfektionen für Arzneimittelresistenz Allele (Fig. 4; Speicheldrüse Daten nicht gezeigt). <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> Einfache Chelex Procedure </td><td colspan="2"> Standard Aussalzen Procedure </td></tr><tr><td> Treppe </td><td> Reagenzien </td><td> Treppe </td><td> Reagenzien </td></tr><tr><td><ol><li> Fügen Probe 1,5 ml Mikrozentrifugenröhrchen und homogenisieren in 100 ul 1X PBS / 1% Saponin-Lösung (2 min). </li><li> Hinterlegen bei Raumtemperatur für 20 min. </li><li> Spin bei 20.000 xg für 2 min und Überstand verwerfen. </li><li> Fügen Sie 100 ul 1X PBS und vorsichtig mischen durch kurzes Vortexen (3 sec). </li><li> Spin bei 20.000 xg für 2 min und Überstand verwerfen. </li><li> Fügen Sie 25 ul 20% w / v Chelex in entionisiertem Wasser und 75 ul sterilem entionisiertem Wasser. </li><li> Pierce Loch im Deckel des Probenröhrchens mit heißer, steriler 23G Injektionsnadel und Tubeninhalt Sieden für 10 min. </li><li> Spin Mikrozentrifugenröhrchen bei 20000 × g für 1 min. </li><li> Überstand in sterile prelabeled Fläschchen und store DNA-Lösung bei -20 ° C bis zur Verwendung (2,5 ul in 25 ul PCR-Reaktion). </li></ol></td><td> PBS Saponin Chelex-100 Perlen </td><td><ol><li> Hinzufügen Probe zu 1,5 ml Mikrozentrifugenröhrchen geben und homogenisieren in 100 ul Puffer Bender * mit 1% DEPC (2 min). </li><li> Inkubieren bei 65 ° C für 1 Stunde. </li><li> Fügen Sie 15 ul kalten 8 M Kaliumacetat. Vorsichtig mischen und auf Eis inkubieren für 45 min. </li><li> Spin Probe in Mikrozentrifugenröhrchen bei 20.000 xg für 10 min und Transfer Überstand in ein neues 1,5 ml-Tube. </li><li> Fügen Sie 250 ul 100% Ethanol und gut mischen durch Invertieren des Röhrchens. </li><li> Inkubieren Probe bei RT 5 min. </li><li> Spin Proben bei 20.000 × g für 15 min. </li><li> Ansaugen und Überstand verwerfen, so dass das Pellet vollständig trocknen im Rohr (30 min). </li><li> Pellet in 20 ul 0,1 X SSC + RNAse (10 ug / ml) über Nacht bei 4 ° C. </li><li> Fügen Sie 80 ul DEPC Wasser und bei -20 ° C bis zur Verwendung. </li></ol></td><td> Diethylpyrocarbonat (DEPC) * Bender Buffer: 0,1 M NaCl 0,2 M Sucrose 0,1 M Tris-HCL 0,05 M EDTA, pH 9,1 0,5% SDS in DEPC Wasser 8 M Kaliumacetat Ethanol 0,1 X Saline Natriumcitrat (SSC)-Puffer RNAse (10 ug / ml) </td></tr><tr><td colspan="2"> TOTAL TIME: 37 min </td><td colspan="2"> TOTAL TIME: 2 Std. 47 min, plus Nacht </td></tr></tbody></table> Tabelle 1. Schritt Vergleich der Reagenzien und Zeitbedarf für einfache Chelex Protokoll Schritt und stehenard Aussalzen Methode zur DNA-Extraktion aus Moskito-Proben. <img alt="Abbildung 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> Abbildung 1. ND4 mitochondrialen PCR Vergleich der DNA-Qualität von vereinfachten Chelex &quot;C&quot; und Standard Aussalzen &quot;M&quot;-Extrakten auf Anopheles arabiensis Feldproben. NC, Negativkontrolle; M1 und C1, M2 und C2, C3 und M3 und M4 und C4 sind gepaart Aussalzen und Chelex Extrakte von gleichen An. arabiensis mosquito Proben. L, 100 bp DNA-Leiter. <img alt="Abbildung 2" src="/files/ftp_upload/3281/3281fig2.jpg" /> Abbildung 2 Molecular Identifizierung PCR für Anopheles arabiensis C1 und M1, M2 und C2, C3 und M3 und M4 bedeuten C4 jeweiligen Amplikon aus Aussalzen &quot;M&quot; und vereinfacht Chelex &quot;C&quot; DNA Extrakte zur gleichen Moskito Proben;.. AR, Anopheles arabiensispositive Kontrolle; L, 100 bp DNA-Leiter. <img alt="Abbildung 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> Abbildung 3. Erkennung von Positive auf Chelex und Aussalzen DNA-Extrakte für Moskito Feldproben mit PCR-Assays für die molekulare Identifizierung von Anopheles arabiensis (AR) 10, Arthropoden NADH-Dehydrogenase-Gen 4 (ND4) DNA Qualitätstest 11 und Antifolat drug resistance P . falciparum DHFR Genotypisierung 12 (F-M4). Ergebnisse bei Versuchen mit oder ohne BSA in der Reaktionsmischung ausgeführt dargestellt. McNemar-Chi-Quadrat-Test wurde verwendet, um festzustellen, ob die Unterschiede zwischen Prozent positive PCRs von DNA aus der vereinfachten Chelex extrahiert und die Standard Aussalzen Protokolle waren statistisch signifikant. <img alt="Abbildung 4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <strong&gt; Abbildung 4. Anwendung des vereinfachten Chelex Protokoll Genotypisierung P. falciparum-Infektionen bei Mücken (mid-gut-Daten gezeigt) für Resistenz-Allele. BSTN I Verdauung auf Amplikon flankierenden Codon 108 des P. falciparum DHFR-Gen 12 zeigt Cycloguanil-resistente Mutanten in Moskito S108T Proben (M1-M5; mittleres Darm Daten gezeigt). U, unverdaute 522 bp Amplikon; FCR3, Labor-Standard P. falciparum positive Kontrolle Klon trägt S108T; K1, P. falciparum Laborstandard Klon negative Kontrolle Durchführung Cycloguanil-sensitive S108N; L, 100 bp DNA-Leiter.

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

Ein einfaches Chelex-Protokoll zur DNA-Extraktion aus Anopheles spp. | Immunologie und Infektion | Video

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Eine schnelle und kostengünstige Möglichkeit, die Qualität Malariaparasiten und Vektor-DNA aus mosquito Proben zu extrahieren beschrieben. Aufbauend auf chelatisierenden Eigenschaften der Chelex Harz, ermöglicht die einfache Methode Genotypisierung von Malaria-Parasiten in Mücken Mitte Darm und Speicheldrüsen Phasen sowie molekulare Identifizierung der<em&gt; Anopheles</em&gt; Geschwister Spezies durch PCR.

A Simple Chelex Protokoll zur DNA-Extraktion aus<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 Aufrufe.  Malaria Institute at Macha. Eine schnelle und kostengünstige Möglichkeit, die Qualität Malariaparasiten und Vektor-DNA aus mosquito Proben zu extrahieren beschrieben. Aufbauend auf chelatisierenden Eigenschaften der Chelex Harz, ermöglicht die einfache Methode Genotypisierung von Malaria-Parasiten in Mücken Mitte Darm und Speicheldrüsen Phasen sowie molekulare Identifizierung der Anopheles Geschwister Spezies durch PCR.

Serie: 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.

Ein Protokoll für die Erfassung und Färbung Blutzellen aus dem Gelbfiebermücke 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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Immunologie und Infektion

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.

IgY-Technologie: Extraction of Chicken Antikörper aus Eidotter durch Polyethylenglykol (PEG) Niederschlag

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.

High-und Low Throughput-Screens mit Wurzelknoten-Nematoden 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.

Ein einfaches Protokoll für Thrombozyten-vermittelte Verklumpung<em&gt; Plasmodium falciparum</em&gt;-Infizierten Erythrozyten in einer Ressource Schlechte Einstellung

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.

Eine einfache und schnelle Protokoll zum Nicht enzymatisch Dissociate Frische menschlichen Geweben zur Analyse der infiltrierenden Lymphozyten

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.

Eine einfache und effiziente Methode zu konstruieren Mutant Vacciniavirusvektoren

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.

Kombination von Analyse von DNA in eine grobe Virion-Extraktion mit der Analyse der RNA aus den infizierten Blättern, entdecken neue Virus-Genom

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).

Schnelle, sichere und einfache manuelle am Krankenbett Nukleinsäure-Extraktion für den Nachweis des Virus in Vollblut-Proben

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.

Eine High-Throughput-Plattform für das Screening von 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.

Analyse der Dynamik fäkaler Mikrobiota in Lupus-anfälligen Mäusen mit einer einfachen, kostengünstigen DNA-Isolierungsmethode

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