Die Autoren danken der National Laboratory Calicivirus Center for Disease Control and Prevention (CDC) für die freundliche Spende von einer Standard-und Kontrolle Positives für Nov danken und Laboratorien der School of Public Health an der Johns Hopkins für die Bereitstellung der Reagenzien.

1. Stuhlproben <ol><li> Stuhlproben sollten eingefroren gelagert, um die RNA zu bewahren. Um eine 10% fäkal Federung machen, dauert ca. 0,1 g aufgetauten Stuhlprobe und füllen auf 1 ml mit PBS. </li><li> Aliquotieren in 200 ul zu vermeiden, wiederholtes Einfrieren und Auftauen. Lagern Sie die Aliquots bei -70 ° C. </li><li> Auftauen und zentrifugieren Aliquots bei 4.000 g für 10 min vor der Verwendung in Extraktion. </li></ol> 2. Vorbereitung von Silica-Partikeln für die Extraktion mit Guanidin &amp; Silica <ol><li> Bei RT auszusetzen, 30 g Siliciumdioxid und komplett mit dH 2 O bis 250 mL. </li><li> Nach 24 Stunden der Sedimentation, zu entfernen 215 ml des Überstands durch Sog. Fügen Sie dH 2 O bis zu 250 ml, sowie die Aussetzung der Kieselsäure Pellet durch Schütteln. </li><li> Nach weiteren 24 Stunden wurde der Überstand entfernt durch Absaugen und der pH-Wert der Kieselsäure-Suspension auf pH 2,0 unter Verwendung von Salzsäure (HCl). Aliquot der Kieselsäuresuspension in Glas BotFlaschen und Autoklaven. </li></ol> 3. Vorbereitung der L6 Puffer für die Extraktion mit Guanidin &amp; Silica <ol><li> Bei RT-Puffer vorzubereiten L6 durch Lösen von 60 g Guanidiniumthiocyanat (GuSCN) und 1,3 g Triton X-100 in 50 ml 0,1 M Tris-Hydrochlorid, pH 6,4 und 11 ml einer 0,2 M EDTA, pH 8,0 Lösung. </li></ol> 4. Vorbereitung der L2-Puffer für die Extraktion mit Guanidin &amp; Silica <ol><li> Bei RT Herstellung L2-Puffer durch Auflösen von 180 g Guanidiniumthiocyanat (GuSCN) in 150 ml 0,1 M Tris-Hydrochlorid, pH 6,4. </li></ol> 5. Extraktionsverfahren <ol><li> In jeder Extraktion ein NoV positive Stuhlprobe, als positive Kontrolle und DEPC-behandeltem Wasser, als negative Kontrolle, einbezogen werden sollten. </li><li> In einem Mikro-Zentrifugenröhrchen das folgende; 1 ml Puffer L6, 20 uL von Silica-Partikel, und fügen Sie 200 ul der 10% fäkal-Extrakt Suspension. Vortex kurz verlassen und bei RT für 15 min. </li><li> Zentrifugieren Sie die Suspension bei 6000 g für 10 s und Waschen des Pellets mit 1 ml Puffer L2 zweimal, gefolgt von zwei Waschungen mit 70% Ethanol und einmal mit Aceton. Trocknen Sie das Pellet in einem trockenen Heizblock bei 56 ° C für 5 min. </li><li> Hydrat die RNA in der Silica-Pellet durch Zugabe von 50 ul RNase-freies dH 2 0. Fügen Sie 1 ul RNasin, mischen durch Invertieren des Röhrchens und inkubieren bei 56 ° C für 15 min. </li><li> Zentrifuge für 3 min bei voller Geschwindigkeit in einer Tischzentrifuge, und sammeln Sie 40 ul des Überstands mit der RNA. </li><li> Quantifizierung der extrahierten RNA-Proben mit Nanodrop 2000 Spektrophotometer (Thermo Scientific). Dann bei -70 ° C gelagert bis zur Verwendung, bis zu 6 Monaten. (Hinweis: Eine bessere Möglichkeit, Gesamt-RNA intakt bewahren wandelt diese in cDNA). </li></ol> 6. Alternative Extraktion mit dem QIAamp Viral RNA Handelsregister RNA Kit Vollständige Anweisungen finden Sie in der Broschüre PROV gefundenBedieneroberfläche mit dem QIAGEN-Kit. Kurz das Verfahren ist wie folgt: <ol><li> Jeweils 560 ul Puffer AVL mit den Träger-RNA in ein 1,5 ml Röhrchen, und fügen Sie 140 ul der 10% der Suspension. Mix von Puls-Vortexen für 15 Sekunden. Inkubation bei RT für 10 min. </li><li> Fügen Sie 560 ul Ethanol (96-100%) zur Probe und mischen Sie durch Puls-Vortexen für 15 Sekunden, dann kurz zentrifugieren. </li><li> Tragen Sie 630 ul dieser Lösung zu einem Spin-Säule in ein 2 ml Collection Tube platziert. Säule für 1 min bei 6.000 g; platzieren Sie dann den Spin-Säule in ein sauberes 2 ml Tube. </li><li> Die Säule wird mit gebundenen RNA mit 500 ul Puffer AW1 und Zentrifuge bei 6.000 g für 1 min. Platz Säule in ein sauberes 2-ml-Collection-Tube. </li><li> Die Säule wird mit 500 ul Puffer AW2 und Zentrifuge bei voller Geschwindigkeit (20.000 g oder 14.000 rpm) für 3 min. </li><li> Platz Spin-Säule in ein sauberes 1,5 ml Mikro-Zentrifugenröhrchen, dann werden 40 ul DEPC-behandeltem Wasser eluieren und die RNA mit voller Geschwindigkeit. Wiederholen Sie aufce für eine abschließende 80 ul eluierte RNA. </li><li> Quantifizierung der extrahierten RNA-Proben mit Nanodrop 2000 Spektrophotometer (Thermo Scientific). Dann bei -70 ° C gelagert bis zur Verwendung, bis zu 6 Monaten. (Hinweis: Eine bessere Möglichkeit, Gesamt-RNA intakt bewahren wandelt diese in cDNA). </li></ol> 7. Nachweis von Noroviren in extrahierten RNA durch Ein-Schritt-Real-Time RT-PCR mit spezifischen TaqMan-Sonden Instrument StepOnePlus Real Time PCR Systems (Applied Biosystems). Methodik <ol><li> Wischen und Reinigen Sie alle Arbeitsflächen, Pipetten und Zentrifugen mit RNase AWAY, um mögliche RNase-Kontaminationen zu entfernen. </li><li> Pipettieren Die Nov GI und GII Screening Mastermixen nach Tabelle 1. Primern und TaqMan-Sonden sind in Tabelle 2 aufgeführt. </li><li> 10 ul Aliquot von geeigneten Master-Mix zu jedem Reaktionsgefäß. </li><li> 5 l der unbekannten Probe unverdünntRNA, DEPC-behandeltem Wasser als Negativkontrolle oder GI bzw. GII positive Kontroll-RNA, für die entsprechenden Reaktionsvertiefungen. Alle Proben sollten in doppelt durchgeführt werden. Positive Kontrollen und Standards mit Konzentration von 300 ug / ul und 500 ug / ul wurden jeweils von National Laboratory Calicivirus Center for Disease Control and Prevention (CDC) zur Verfügung gestellt. </li><li> Zentrifugieren Sie die Reaktionsplatte bei 6.000 g für 10 sek. </li><li> In dem Experiment Eigenschaften Bildschirm; definieren und wählen Sie eine Art von Experiment für den Lauf. Stellen Sie sicher, TaqMan-Reagenzien als Reagenzien-Typ, und dieser Pre-PCR-Amplifikation und Lese sind ausgewählt werden angezeigt. </li><li> Ausführen 15 ul Reaktionen unter Verwendung des thermischen Profils in Tabelle 3. </li><li> Analysieren Sie die Ergebnisse. </li></ol> 8. Genotypisierung von NOV GI und GII mit herkömmlichen RT-PCR und Sequenzierung (Es ist noch nicht in diesem Video erwähnt, da sie eine häufige und weit verbreitete Methode ist.) Instrument. Applied Biosystems StepOne StepOnePlus und Real Time PCR-Systeme mit dem Quantitec Vorlage. Methodik <ol><li> Vor der Genotypisierung, sind die Genogruppe (GI oder GII) der Proben durch das Screening-RT-PCR oben beschrieben bestimmt. GI NoV RNA muss mit dem GI Genotypisierung Master-Mix und der GII NoV RNA mit dem GII Genotypisierung Master-Mix verstärkt werden. </li><li> Pipettieren Die Nov GI und GII Genotypisierung Mastermixen nach Tabelle 4. Grundierungen GI SKF / SKR und G2 SKF / SKR sind in Tabelle 5 aufgeführt. </li><li> 45 ul Aliquot des entsprechenden Master-Mix auf 0,2 ml Reaktionsgefäße. </li><li> 5 l der DEPC-behandeltem Wasser auf jede negative Kontrolle Rohr, und 5 ul sorgfältig geprüften, Nov (GI und / oder GII) RNA positiv auf die entsprechende Reaktionsrohr. </li><li> Ausführen 50 ul Reaktionen unter Verwendung des thermischen Profils in Tabelle 6. </li><li> Senden Sie PCR-Produkte ContaiNing mindestens 100 ng / ul des amplifizierten Kapsid Region um Unternehmen für die Reinigung und Sequenzierung Macrogen. (9700 Große Seneca Hwy. Rockville, MD 20850 und $ 10 pro Probe pro Sequenzierung). </li></ol> 9. Repräsentative Ergebnisse <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="1" /> Abbildung 1 zeigt repräsentative Ergebnisse aus der TaqMan-One-Step RT-PCR-Assay-RNA, wenn sie aus Stuhlproben von Kindern extrahiert Durchfallerkrankungen eingesetzt. Der Schwellenwert (Ct) für eine positive Probe wurde bei weniger oder gleich 37 Ct für GI und GII für 39 Ct eingestellt. Die CT-Werte für die positiven Kontrollen wurde festgestellt, dass weniger als 27 und 18 des ORF1 ORF2-Knotenpunkt für GI und GII, beziehungsweise. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung anzuzeigen</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="2" /> 2 </strong&gt; zeigt ein Kopf-an-Kopf-Vergleich von CT-Werte von Silica in Guanidinium-Methode und QIAmp Viral RNA Kit. Daten der beiden Tests fallen sehr nahe an der Linie Gleichheit (Steigung = 1 und Achsenabschnitt = 0). Dies wird durch die Regressionsanalyse und der Korrelationskoeffizient bestätigt. Alle negativen Kontrollen wurden ausgewertet und festgestellt, 0 sein mit beiden Methoden. <table border="1"><tbody><tr><td> Master Mix </td><td> Endkonzentration </td><td> Volume (ul) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 2,875 </td></tr><tr><td> QuantiTect RT-PCR Master Mix </td><td> 1x </td><td> 6,250 </td></tr><tr><td> RT-Mix </td><td> 1x </td><td> 0,125 </td></tr><tr><td> 50 uM Primer Cog R </td><td> 1 uM </td><td> 0,250 </td></tr><tr><td> 50 uM Primer Cog F </td><td> 1uM </td><td> 0,250 </td></tr><tr><td> 10 uM Ringsonde </td><td> 1 uM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10,00 </td></tr></tbody></table> * 0,250 ul jeder Sonde wurde für GI-Tests verwendet, während 0,125 ul jeder Sonde für GII Tests verwendet wurde. Tabelle 1. Qiagen QuantiTect Master-Mix für das Screening GI und GII (Trujillo et al., 2006). 7 <table border="1"><tbody><tr><td> Name </td><td> Geno-Gruppe </td><td> Verwenden </td><td> Sequenz (5 &#39;nach 3&#39;) </td></tr><tr><td> Cog 1R </td><td> GI </td><td> Grundierung </td><td> CTT CGC AGA Katze Katze Katze TYA C </td></tr><tr><td> Cog 1F </td><td> GI </td><td> Grundierung </td><td> CGY TGG ATG CGN TTY CAT GA </td></tr><tr><td> Cog 2R </td><td> GII </td><td> Grundierung </td><td> TCG ACG CCA TCT TCA TTC ACA </td></tr><tr><td> Cog 2F </td><td> GII </td><td> Grundierung </td><td> Auto gar BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td> Ring 1A </td><td> GI </td><td> Sonde </td><td> FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td> Ring 1B </td><td> GI </td><td> Sonde </td><td> FAM-AGA TCG CGG TCT CCT GTC CA-BHQ-1 </td></tr><tr><td> Ring2-TP </td><td> GII </td><td> Sonde </td><td> FAM-TGG GAG GGC GAT CGC AAT-CT-BHQ-1 </td></tr></tbody></table> Tabelle 2. Primer und Sonden für quantitative Echtzeit-RT-PCR für Genogruppen I, II (Kageyama et al. Verwendet,2003). 8 <table border="1"><tbody><tr><td> Schritt </td><td> Temp (° C) </td><td> Zeit (min) </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> cDNA-Synthese </td></tr><tr><td> 2 </td><td> 95 </td><td> 15.00 </td><td> HotStart Taq-Polymerase Aktivierung </td></tr><tr><td> 3 </td><td> 95 </td><td> 00.15 </td><td></td></tr><tr><td></td><td> 60 </td><td> 01.00 </td><td> 45X-Zyklen </td></tr></tbody></table> Tabelle3. Thermal-Profil für Ein-Schritt-TaqMan real time RT-PCR (Trujillo et al., 2006). 7 <table border="1"><tbody><tr><td> Master Mix </td><td> Endkonzentration </td><td> Volume (ul) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29,50 </td></tr><tr><td> D5 Qiagen RT-PCR-Puffer </td><td> 1x </td><td> 10,00 </td></tr><tr><td> 10 mM dNTP-Mix </td><td> 0,4 mM </td><td> 2,00 </td></tr><tr><td> Enzym-Mix </td><td></td><td> 2,00 </td></tr><tr><td> 40 U / ul RNAsin </td><td> 20 U / ul </td><td> 0,50 </td></tr><tr><td> 10 uM SKF </td><td> 0,1 uM </td><td> 0,50 </td></tr><tr><td> SKR 10 uM </td><td> 0,1 uM </td><td> 0,50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45,00 </td></tr></tbody></table> Tabelle 4. Qiagen One-Step RT-PCR Master-Mix für die Genotypisierung GI und GII. <table border="1"><tbody><tr><td> Name </td><td> Geno-Gruppe </td><td> Verwenden </td><td> Sequenz (5 &#39;nach 3&#39;) </td></tr><tr><td> G1SKF </td><td> GI </td><td> Grundierung </td><td> 5&#39;-CTG CCC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> GI </td><td> Grundierung </td><td> 5&#39;-CCA CCA ACC CAR TTR TAC A - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> Grundierung </td><td> 5&#39;-CNT GGG AGG GCA GCG ATC A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> Grundierung </td><td> 5&#39;-GCA CCN CCR CCR TRH TTR TA CAT-3 </td></tr></tbody></table> Tabelle 5. Primer für die Amplifikation der Region C der Capsid Region (Kojima et al., 2002). 5 <table border="1"><tbody><tr><td> Schritt </td><td> Temp (° C) </td><td> Zeit (min) </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> cDNA-Synthese </td></tr><tr><td> 2 </td><td> 96 </td><td> 15.00 </td><td> HotStart Taq-Polymerase Aktivierung </td></tr><tr><td> 3 </td><td> 94 </td><td> 00.030 </td><td></td></tr><tr><td></td><td> 52 </td><td> 01.00 </td><td> 40X-Zyklen </td></tr><tr><td></td><td> 72 </td><td> 00.30 </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10.00 </td><td> Glühen </td></tr></tbody></table> Tabelle 6. Thermische Profil der TaqMan-One-Step RT-PCR.

<ol>
	<li>Medici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+Norovirus+infections+in+sporadic+cases+of+viral+gastroenteritis+among+children+in+Northern+Italy.">Molecular epidemiology of Norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy.</a> L. Medical Virology. 78, (2006).</li><li>Vidal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+recombinant+Norovirus+causing+outbreaks+of+gastroenteritis+in+Santiago,+Chile.">Novel recombinant Norovirus causing outbreaks of gastroenteritis in Santiago, Chile.</a> J. Clinica Microbiology. 4, (2006).</li><li>Xavier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+caliciviruses+associated+with+acute+infantile+gastroenteritis+in+Salvador,+an+urban+center+in+Northeast+Brazil.">Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil.</a> Braz. J. Med. Biol. Res. 42, (2009).</li><li>Boom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J. Clin. Microbiol. 28, 495-503 (1990).</li><li>Kojima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genogroup-specific+PCR+primers+for+detection+of+Norwalk-like+viruses.">Genogroup-specific PCR primers for detection of Norwalk-like viruses.</a> J. Virol. Methods. 100, 107-114 (2002).</li><li>Mattison, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+comparison+of+two+norovirus+ORF2-based+genotyping+protocols.">Multicenter comparison of two norovirus ORF2-based genotyping protocols.</a> J. Clin. Microbiol. 47, 3927-3932 (2009).</li><li>Trujillo, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+TaqMan+real-time+reverse+transcription-PCR+for+rapid+detection,+quantification,+and+typing+of+norovirus.">Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus.</a> J. Clin. Microbiol. 44, 1405-1412 (2006).</li><li>Kageyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broadly+reactive+and+highly+sensitive+assay+for+Norwalk-like+viruses+on+real-time+quantitative+RT-PCR.">A broadly reactive and highly sensitive assay for Norwalk-like viruses on real-time quantitative RT-PCR.</a> J. Clin. Microbiol. 41, 1548-1557 (2003).</li></ol>

Keine Interessenskonflikte erklärt.

Mit dem wirtschaftlichen Inhouse-Verfahren zur Isolierung von Nukleinsäure aus Stuhlproben, erhalten wir gleiche Ergebnisse wie mit dem kommerziellen QIAmp Viral RNA Kit von Qiagen, und zusammen mit der TaqMan RT-PCR in unserem Labor entwickelt können wir erkennen ein breites Spektrum von NOV Genotypen aus der GI und GII Genogruppe. Eine aktuelle Publikation der Region C-Protokoll berichtet Genotypisierung Raten von 78% 6. Da die Vielfalt der zeitgenössischen NoV Stämme hat sich in den vergangenen Jahren ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Guanidinisothiocyanat </td><td> Sigma-Adrich </td><td> G9277 </td><td></td></tr><tr><td> Tris-HCl </td><td> Sigma-Aldrich </td><td> T5941 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E5134 </td><td></td></tr><tr><td> Kieselerde </td><td> Sigma-Aldrich </td><td> S5631 </td><td></td></tr><tr><td> Triton X-100 </td><td> BDH Chemicals </td><td> 14530 </td><td></td></tr><tr><td> Diethylpyrocarbonat </td><td> Sigma-Aldrich </td><td> D-5758 </td><td></td></tr><tr><td> QIAamp Viral RNA Mini Kit (250) </td><td> QIAGEN </td><td> 52906 </td><td></td></tr><tr><td> QuantiTec Probe RT-PCR-Kit (200) </td><td> QIAGEN </td><td> 204443 </td><td></td></tr><tr><td> Qiagen One Step RT-PCR Kit (200) </td><td> QIAGEN </td><td> 210212 </td><td></td></tr><tr><td> RNase-Inhibitor 2000 Einheiten </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / Fläschchen </td></tr><tr><td> Non-Stick-freie Rnae Mikrozentrifugenröhrchen </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> UltraPure Agarose 1000 </td><td> Invitrogen </td><td> 16550-100 </td><td></td></tr></tbody></table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

Noroviren (NoVs) sind die führende Ursache von Ausbrüchen der sporadischen akuten Gastroenteritis weltweit bei Menschen aller Altersgruppen. Sie sind wichtige Ursachen für Hospitalisierungen bei Kindern mit einer gesundheitlichen Auswirkungen ähnlich der von Rotavirus. NoVs sind RNA-Viren von großer genetischer Vielfalt und es gibt eine kontinuierliche Auftreten neuer Stämme. Fünf Genogruppen anerkannt werden; GI und GII mit ihren vielen Genotypen und Subtypen das wichtigste für eine Infektion beim Menschen. Allerdings ist die Diagnose dieser beiden Genotypen problematisch, Verzögern Diagnose und Behandlung. 1, 2, 3 Für die RNA-Extraktion aus Stuhlproben der am häufigsten verwendete Methode ist die QIAmp Viral RNA kommerziellen Kit von Qiagen. Dieses Verfahren kombiniert die Bindungseigenschaften eines Silicagelmembran, Puffer, dass die Kontrolle RNasen und eine optimale Bindung der RNA an der Säule zusammen mit der Geschwindigkeit des MicroSpin. Diese Methode ist einfach, schnell und zuverlässig und ist Carrin wenigen Schritten, die in der Beschreibung des Herstellers detailliert sind IED. Norovirus ist nur noch von Rotavirus als die häufigste Ursache von Durchfall. Norovirus-Diagnostik sollte in allen Studien über die Pathogenese von Durchfall als auch in Ausbrüchen oder einzelne Durchfall Fällen zur Verfügung. Derzeit jedoch Norovirus Diagnose ist auf nur wenigen Zentren aufgrund des Fehlens von einfachen Methoden der Diagnose beschränkt. Dies verzögert Diagnose und Behandlung 1, 2, 3. Darüber hinaus bedienen sich aufgrund der hohen Kosten und geregelten Transport von korrosiven Puffer innerhalb und zwischen den Ländern dieser gefertigten Bausätze wirft logistische Probleme. Als Ergebnis werden in diesem Protokoll beschreiben wir eine Alternative, wirtschaftlichen, Hausmethode die auf der ursprünglichen basiert Boom et al. Verfahren 4, die die Nukleinsäure-bindenden Eigenschaften von Siliciumdioxid-Teilchen verwendet zusammen mit den Anti-Nuklease Eigenschaften Guanidiniumthiocyanat . <p class=&quot;Jove_content&quot;&gt; Für die Erkennung und genogrouping (GI und GII) von NoVs Isolate aus Stuhlproben, mehrere RT-PCR-Protokolle, die unterschiedliche Ziele entwickelt worden. Der Konsens ist, dass eine RT-PCR unter Verwendung von TaqMan-Chemie wäre die beste Methode zur molekularen Diagnose sein, weil es hohe Sensitivität, Spezifität und Reproduzierbarkeit mit hohem Durchsatz und einfache Bedienbarkeit vereint. Hier beschreiben wir einen Assay zur Förderung der offenen Leserahmen 1 (ORF1)-ORF2 Übergangsbereich, die am stärksten konservierte Region des Genoms und daher NoV am besten geeignet für die Diagnose. Für weitere genetische Analyse eine konventionelle RT-PCR, die die hoch variablen N-terminalen-Schale-Ziele aus der Haupt-Protein des Kapsids (Bereich C) unter Verwendung der Primer ursprünglich von Kojima et al. 5 beschrieben. Die Sequenzierung des PCR-Produkt aus der herkömmlichen PCR ermöglicht die Unterscheidung von Genotypen aus der GI und GII Genogruppen.

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

Ein Ein-Schritt-RT-PCR-Assay zum Nachweis und zur Genogruppe Identifizierung von Norovirus Isolate von Kindern der Stuhl, die Primer und TaqMan-Sonden spezifisch für die offenen Leserahmen 1 (ORF1)-ORF2 Übergangsregion, die am stärksten konservierte Region des Norovirus Genom verwendet beschrieben. Eine nicht-kommerzielle, kostengünstige RNA-Extraktion-Methode wird ausführlich beschrieben.

Erkennung und Genogrouping von Noroviren aus Kinder-Hocker mit der TaqMan One-Step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

17.1K Aufrufe.  Universidad Peruana Cayetano Heredia. Ein Ein-Schritt-RT-PCR-Assay zum Nachweis und zur Genogruppe Identifizierung von Norovirus Isolate von Kindern der Stuhl, die Primer und TaqMan-Sonden spezifisch für die offenen Leserahmen 1 (ORF1)-ORF2 Übergangsregion, die am stärksten konservierte Region des Norovirus Genom verwendet beschrieben. Eine nicht-kommerzielle, kostengünstige RNA-Extraktion-Methode wird ausführlich beschrieben.

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MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNA-Detektion in Prostatatumoren durch quantitative real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Forschung

Medizin

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Cerenkov Lumineszenz Imaging (CLI) for Cancer Therapy Überwachung

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Die Implantation von Vena Cava Interposition Graft in Maus-Modell

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolierung und Immortalisierung von Patienten stammende Zelllinien von Muskelbiopsie for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Gründung des Menschen Epithelial Enteroids und Colonoids von Whole Gewebe und Biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Pankreasgang-Infusion: Eine effektive und selektive Methode der Medikamenten- und Virusabgabe

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Isolierung der primäre Maustaste Hepatozyten für entstehende Protein Synthese Analyse von nicht-radioaktiven L-Azidohomoalanine Kennzeichnung Methode

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Handheld-Metalldetektor-Screening für metallische Fremdkörper Verschlucken bei Kindern

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Thrombozyten-basierte Erkennung von Stickoxid im Blut durch die Messung VASP Phosphorylierung

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

Bewertung und Charakterisierung von Hyaloidgefäßen bei Mäusen

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...