Gli autori desiderano ringraziare il Centro Nazionale Calicivirus Laboratory for Disease Control and Prevention (CDC) per la gentile dono di un positivi standard e di controllo per NoV e laboratori della School of Public Health presso la Johns Hopkins per la fornitura dei reagenti.

1. I campioni di feci <ol><li> Campioni di feci devono essere congelati e conservati per conservare l&#39;RNA. Per effettuare una sospensione al 10% nelle feci, prendere circa 0,1 g di campione scongelato e completare a 1 ml con PBS. </li><li> Aliquotare in 200 microlitri per evitare ripetuti congelamenti e scongelamenti. Conservare le aliquote a -70 ° C. </li><li> Sciogliere e centrifugare le aliquote a 4000 g per 10 min prima dell&#39;uso in estrazione. </li></ol> 2. Preparazione di particelle di silice per estrazione con guanidina &amp; Silica <ol><li> A RT, sospendere 30 g di biossido di silicio e completare con dH 2 O fino a 250 mL. </li><li> Dopo 24 ore di sedimentazione, rimuovere 215 mL del supernatante mediante aspirazione. Aggiungi dH 2 O fino a 250 mL, e sospendere la silice pellet agitando. </li><li> Dopo altre 24 ore, rimuovere il supernatante mediante aspirazione, e regolare il pH della sospensione di silice a pH 2,0, usando acido cloridrico (HCl). Aliquotare la sospensione di silice in bot vetrostoli e autoclave. </li></ol> 3. Preparazione del L6 tampone per estrazione con guanidina &amp; Silica <ol><li> A RT, preparare L6 tampone sciogliendo 60 g di guanidinio tiocianato (GuSCN) e 1,3 g di Triton X-100 in 50 mL di 0,1 M Tris cloridrato, pH 6,4, e 11 mL di una EDTA 0,2 M, pH 8,0 soluzione. </li></ol> 4. Preparazione del tampone L2 per l&#39;estrazione con guanidina &amp; Silica <ol><li> A RT, preparare tampone L2 sciogliendo 180 g di guanidinio tiocianato (GuSCN) in 150 mL di 0,1 M Tris cloridrato, pH 6,4. </li></ol> 5. Procedura di estrazione <ol><li> In ogni estrazione positivo campione di feci NoV, come controllo positivo, e DEPC acqua trattata, come controllo negativo, dovrebbero essere inclusi. </li><li> In una micro-centrifuga tubo miscelare il seguente: 1 ml di tampone L6, 20 pl di particelle di silice, e aggiungere 200 microlitri della sospensione al 10% di estratto fecale. Vortex brevemente e lasciare a temperatura ambiente per 15 min. </li><li> Centrifugare la sospensione a 6000 g per 10 s e lavare il pellet con 1 ml di tampone L2 due volte, seguito da due lavaggi con 70% etanolo e una volta con acetone. Essiccare il pellet in un blocco di riscaldamento a secco a 56 ° C per 5 min. </li><li> Idratare i RNA della silice pellet aggiungendo 50 pl di RNase-free dH 2 0. Aggiungere 1 ml di RNasin, mescolare capovolgendo la provetta e incubare a 56 ° C per 15 min. </li><li> Centrifugare per 3 min a velocità piena, in una centrifuga da tavolo, e raccogliere 40 pl del supernatante contenente l&#39;RNA. </li><li> Quantificare i campioni di RNA estratti utilizzando NanoDrop 2000 spettrofotometro (Thermo Scientific). Poi conservare a -70 ° C fino al momento dell&#39;uso, fino a 6 mesi. (Nota: Un modo migliore per preservare intatto l&#39;RNA totale sta convertendo in cDNA). </li></ol> 6. Estrazione alternativa usando l&#39;RNA Commercial QIAamp Viral RNA Kit Le istruzioni complete si trovano nella prov librettoite con il kit QIAGEN. Brevemente la procedura è la seguente: <ol><li> 560 microlitri di tampone Pipettare AVL contenente l&#39;RNA carrier in una provetta da 1,5 ml, e aggiungere 140 microlitri della sospensione fecale al 10%. Mix di impulsi vortex per 15 sec. Incubare a temperatura ambiente per 10 min. </li><li> Aggiungere 560 pl di etanolo (96-100%) al campione e miscelare impulsi vortex per 15 secondi, quindi centrifugare brevemente. </li><li> Applicare 630 pl di questa soluzione ad una colonna di rotazione posto in una provetta di raccolta 2 mL. Centrifugare per 1 min a 6.000 g; quindi inserire la Micro Spin Column in un pulito 2 Tubo mL. </li><li> Lavare la colonna contenente RNA associati con 500 microlitri di tampone AW1 e centrifugare a 6000 g per 1 min. Colonna Mettere in una provetta di raccolta clean 2 mL. </li><li> Lavare la colonna con 500 microlitri di tampone AW2 e centrifugare a piena velocità (20.000 o 14.000 rpm g) per 3 min. </li><li> Colonnine posto in un ambiente pulito 1,5 mL micro-tubo di centrifuga, aggiungere 40 microlitri DEPC trattata con acqua e eluire l&#39;RNA a piena velocità. Ripetere ilce per un finale 80 pl di RNA eluiti. </li><li> Quantificare i campioni di RNA estratti utilizzando NanoDrop 2000 spettrofotometro (Thermo Scientific). Poi conservare a -70 ° C fino al momento dell&#39;uso, fino a 6 mesi. (Nota: Un modo migliore per preservare intatto l&#39;RNA totale sta convertendo in cDNA). </li></ol> 7. Rilevazione di Norovirus in RNA estratto da One-step Real Time RT-PCR, con specifiche sonde TaqMan Strumento StepOnePlus Real Time PCR Systems (Applied Biosystems). Metodologia <ol><li> Pulire e pulire tutte le superfici di lavoro, pipette, centrifughe e con RNasi via per rimuovere qualsiasi contaminazione potenziale RNasi. </li><li> Preparare la GI e GII NoV Master Mix di screening secondo la Tabella 1. Primer e sonde Taqman sono elencati nella Tabella 2. </li><li> Aliquotare 10 pl di master mix appropriato per ciascun tubo di reazione. </li><li> Aggiungere 5 microlitri di non diluito campione sconosciutoRNA, acqua trattata con DEPC come controllo negativo, o GI rispettivamente GII RNA positivo di controllo, ai pozzetti di reazione corrispondenti. Tutti i campioni devono essere eseguiti in duplicato. I controlli positivi e gli standard con concentrazione di 300 ug / ul e 500 mg / pl, rispettivamente, sono stati forniti dalla National Laboratory Calicivirus Center for Disease Control and Prevention (CDC). </li><li> Centrifugare la piastra di reazione a 6.000 g per 10 sec. </li><li> Nella schermata esperimento proprietà; definire e selezionare un tipo di esperimento per la corsa. Assicurarsi reagenti TaqMan sono visualizzati come il tipo di reagenti, e che pre-amplificazione PCR di lettura e sono selezionati. </li><li> Eseguire reazioni 15 pl usando il profilo termico in Tabella 3. </li><li> Analizzare i risultati. </li></ol> 8. Genotipizzazione di NoV GI e GII con i convenzionali RT-PCR e il sequenziamento (Non è menzionato in questo video perché è un metodo comune e largamente usato.) Strumento. Applied Biosystems StepOne e StepOnePlus sistemi PCR in tempo reale con il modello Quantitec. Metodologia <ol><li> Prima genotipizzazione, il genogruppo (GI o GII) dei campioni sono determinati dalla proiezione RT-PCR descritto sopra. GI NoV RNA deve essere amplificato con il mix master genotipizzazione GI e GII NoV RNA con il mix GII genotipizzazione master. </li><li> Preparare la GI e GII NoV genotipizzazione Master Mix secondo la Tabella 4. Primers GI SKF / SKR e G2 SKF / SKR sono elencati nella Tabella 5. </li><li> Aliquotare 45 microlitri della master mix appropriato per provette 0,2 ml di reazione. </li><li> Aggiungere 5 microlitri di acqua trattata con DEPC ad ogni provetta di controllo negativo, e 5 pl di pre-screening, NoV (GI e / o GII) RNA positivi per il tubo di reazione corrispondente. </li><li> Eseguire reazioni 50 pl usando il profilo termico in Tabella 6. </li><li> Invia PCR prodotti conteniNing almeno 100 ng / pl della regione amplificata capside a Macrogen Società per la purificazione e sequenziamento. (9700 Hwy grande Seneca. Rockville, MD 20850 e $ 10 per ogni campione di sequenziamento). </li></ol> 9. Risultati rappresentativi <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="Figura 1" /> La figura 1 mostra i risultati rappresentativi del Taqman One-Step RT-PCR quando viene utilizzato per test RNA estratto da campioni di feci di bambini diarroiche. Il ciclo di soglia (Ct) per un campione positivo è stato fissato a minore o uguale a 37 Ct per GI e Ct 39 per GII. I valori Ct per i controlli positivi sono risultate meno di 27 e 18 del ORF1-ORF2 bivio per GI e GII, rispettivamente. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="Figura 2" /> Figura 2 </strong&gt; mostra un testa a testa di confronto tra i valori di Ct nel metodo di silice guanidinio e QiAmp kit di RNA virale. I dati di entrambi i test cadono molto vicino alla linea di uguaglianza (= pendenza 1 e intercetta = 0). Ciò è confermato dalla analisi di regressione e il coefficiente di correlazione. Tutti i controlli negativi sono stati valutati e trovati a 0 con entrambi i metodi. <table border="1"><tbody><tr><td> Master Mix </td><td> Finale Conc </td><td> Volume (pl) </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 micron Cog R fondo </td><td> 1 pM </td><td> 0,250 </td></tr><tr><td> 50 micron Cog fondo F </td><td> 1pM </td><td> 0,250 </td></tr><tr><td> 10 sonda Anello pM </td><td> 1 pM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10,00 </td></tr></tbody></table> * 0,250 pl di ciascuna sonda è stato utilizzato per il test GI, mentre 0,125 pl di ciascuna sonda è stato utilizzato per il test GII. Tabella 1. Qiagen Quantitect master mix per lo screening GI e GII (Trujillo et al., 2006) 7. <table border="1"><tbody><tr><td> Nome </td><td> Geno-group </td><td> Utilizzare </td><td> Sequenza (5 &#39;a 3&#39;) </td></tr><tr><td> Cog 1R </td><td> GI </td><td> Primer </td><td> CTT AGA CGC CAT CAT CAT TYA C </td></tr><tr><td> Cog 1F </td><td> GI </td><td> Primer </td><td> CGY TGG ATG CGN TTY CAT GA </td></tr><tr><td> Cog 2R </td><td> GII </td><td> Primer </td><td> TCG ACG TCT CCA TCA TTC ACA </td></tr><tr><td> Cog 2F </td><td> GII </td><td> Primer </td><td> CAR GAR BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td> Anello 1A </td><td> GI </td><td> Sonda </td><td> FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td> Anello 1B </td><td> GI </td><td> Sonda </td><td> FAM-AGA TCG CGG TCT CCT GTC CA-BHQ-1 </td></tr><tr><td> Ring2-TP </td><td> GII </td><td> Sonda </td><td> FAM-TGG GAG GGC GAT CGC AAT CT-BHQ-1 </td></tr></tbody></table> Tabella 2. Oligonucleotidi primer e sonde utilizzate per quantitativa in tempo reale RT-PCR per genogruppi I, II (Kageyama et al.,2003) 8. <table border="1"><tbody><tr><td> Passo </td><td> Temp (° C) </td><td> Tempo (min) </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> sintesi del cDNA </td></tr><tr><td> 2 </td><td> 95 </td><td> 15:00 </td><td> HotStart Taq polimerasi attivazione </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> Cicli di 45X </td></tr></tbody></table> Tavolo3. Profilo termico per One-step Taqman Real time RT-PCR (Trujillo et al., 2006) 7. <table border="1"><tbody><tr><td> Master Mix </td><td> Finale Conc </td><td> Volume (pl) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29,50 </td></tr><tr><td> 5X Qiagen RT-PCR Buffer </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> Enzyme Mix </td><td></td><td> 2,00 </td></tr><tr><td> 40 U / pl RNAsin </td><td> 20 U / pl </td><td> 0,50 </td></tr><tr><td> 10 pM SKF </td><td> 0,1 pM </td><td> 0,50 </td></tr><tr><td> 10 pM SKR </td><td> 0,1 pM </td><td> 0,50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45,00 </td></tr></tbody></table> Tabella 4. Qiagen One-Step RT-PCR master mix per la genotipizzazione GI e GII. <table border="1"><tbody><tr><td> Nome </td><td> Geno-group </td><td> Utilizzare </td><td> Sequenza (5 &#39;a 3&#39;) </td></tr><tr><td> G1SKF </td><td> GI </td><td> Primer </td><td> 5&#39;-CTG CCC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> GI </td><td> Primer </td><td> 5&#39;-CCA ACC CAR CCA TTR TAC A - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> Primer </td><td> 5&#39;-GGG CNT AGG GCG ATC GCA A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> Primer </td><td> 5&#39;-CCR CCN GCA TRH CCR TTR TA CAT-3 </td></tr></tbody></table> Primer Tabella 5. Usato per l&#39;amplificazione della regione C della regione capsidica (Kojima et al., 2002). 5 <table border="1"><tbody><tr><td> Passo </td><td> Temp (° C) </td><td> Tempo (min) </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> sintesi del cDNA </td></tr><tr><td> 2 </td><td> 96 </td><td> 15:00 </td><td> HotStart Taq polimerasi attivazione </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> Cicli di 40X </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> Ricottura </td></tr></tbody></table> Tabella 6. Profilo termico per 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>

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Utilizzando la economico in casa metodo per isolare acido nucleico da campioni di feci, si ottengono risultati uguali come con il commerciale Viral QIAmp RNA kit da Qiagen, e insieme con la RT-PCR TaqMan sviluppato nel nostro laboratorio si può rilevare una vasta gamma di NoV genotipi appartenenti al GI e GII genogruppo. Una recente pubblicazione della regione C protocollo riportato tassi di genotipizzazione del 78% 6. Dal momento che la diversità dei ceppi contemporanei nov è andato aumentando negli anni ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Guanidina isotiocianato </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> Silice </td><td> Sigma-Aldrich </td><td> S5631 </td><td></td></tr><tr><td> Triton X 100 </td><td> Prodotti chimici BDH </td><td> 14530 </td><td></td></tr><tr><td> Dietilpirocarbonato </td><td> Sigma-Aldrich </td><td> D-5758 </td><td></td></tr><tr><td> QIAamp RNA virale Mini Kit (250) </td><td> QIAGEN </td><td> 52906 </td><td></td></tr><tr><td> QuantiTec sonda 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> Inibitore della RNasi 2000 unità </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / flaconcino </td></tr><tr><td> Non-Stick Rnae senza provette per microcentrifuga </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> Ultrapura 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

Norovirus (NoVs) sono la principale causa di sporadici focolai di gastroenterite acuta in tutto il mondo negli esseri umani di tutte le età. Essi sono causa importante di ospedalizzazione nei bambini con un impatto sulla salute pubblica simile a quella del Rotavirus. NoVs sono virus a RNA di grande diversità genetica e vi è una continua comparsa di nuovi ceppi. Cinque genogruppi sono riconosciuti, GI e GII con i loro numerosi genotipi e sottotipi che sono i più importanti per l&#39;infezione umana. Tuttavia, la diagnosi di questi due genotipi resta problematica, ritardando diagnosi e trattamento. 1, 2, 3 Per l&#39;estrazione di RNA da campioni di feci il metodo più comunemente usato è il kit QIAmp Viral RNA commerciale da Qiagen. Questo metodo combina le proprietà di legame di una membrana di gel di silice, che RNasi buffer di controllo e fornire vincolanti ottimali degli RNA alla colonna insieme alla velocità di MicroSpin. Questo metodo è semplice, veloce ed affidabile ed è carried in alcuni passaggi che sono dettagliati nella descrizione fornita dal produttore. Norovirus è secondo solo al rotavirus come la causa più comune di diarrea. La diagnosi Norovirus dovrebbero essere disponibili in tutti gli studi sulla patogenesi della diarrea come pure in caso di focolai o di casi di diarrea individuali. Attualmente tuttavia la diagnosi norovirus è limitata a pochi centri a causa della mancanza di semplici metodi di diagnosi. Questa diagnosi ritardi e trattamento 1, 2, 3. Inoltre, a causa dei costi di trasporto e regolamentato di buffer corrosivi all&#39;interno e tra i paesi l&#39;uso di questi kit manufatti pone problemi logistici. Come risultato, in questo protocollo descriviamo alternativa, economico, internamente metodo che si basa sulla originale Boom et al. 4 metodo che utilizza le proprietà di legame di acido nucleico di particelle di silice insieme alle proprietà anti-nucleasi tiocianato di guanidinio . <p class=&gt; &quot;Jove_content&quot; Per l&#39;individuazione e la genogrouping (GI e GII) NoVs di isolati da campioni di feci, diversi RT-PCR che utilizzano protocolli diversi target sono stati sviluppati. Il consenso è che una RT-PCR TaqMan utilizzando la chimica sarebbe la migliore tecnica molecolare per la diagnosi, perché unisce alta sensibilità, specificità e riproducibilità con elevata produttività e facilità d&#39;uso. Qui si descrive un saggio di targeting la lettura aperta telaio 1 (ORF1)-ORF2 regione di giunzione, la regione più conservata del genoma NoV e quindi più adatto per la diagnosi. Per ulteriori analisi genetica un convenzionale RT-PCR che gli obiettivi del altamente variabile N-terminale-shell dal principale proteina del capside (Regione C) utilizzando primers originariamente descritti da Kojima et al. 5 è dettagliato. Il sequenziamento del prodotto di PCR dal convenzionale PCR consente la differenziazione di genotipi appartenenti al GI e genogruppi GII.

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

Un One-Step RT-PCR per il rilevamento e l&#39;identificazione genogruppo di isolati Norovirus da feci bambini, che utilizza primer e sonde TaqMan specifiche al telaio di lettura aperta 1 (ORF1)-ORF2 regione di giunzione, la regione più conservata del genoma Norovirus è descritto. Un non-commerciale e conveniente metodo di estrazione dell&#39;RNA è dettagliato.

Rilevamento e Genogrouping di Norovirus dalle feci dei bambini By 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 Views.  Universidad Peruana Cayetano Heredia. Un One-Step RT-PCR per il rilevamento e l'identificazione genogruppo di isolati Norovirus da feci bambini, che utilizza primer e sonde TaqMan specifiche al telaio di lettura aperta 1 (ORF1)-ORF2 regione di giunzione, la regione più conservata del genoma Norovirus è descritto. Un non-commerciale e conveniente metodo di estrazione dell'RNA è dettagliato.

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

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.

Rilevamento MicroRNA in tumori della prostata dal 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 ...

Ricerca

Medicina

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.

Luminescenza Imaging Cerenkov (CLI) per il monitoraggio Cancer Therapy

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.

L&#39;impianto di Vena cava inferiore Interposizione Graft in modello murino

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.

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia 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.

Istituzione di umano epiteliali Enteroids e Colonoids dal tessuto intero e biopsia

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.

Infusione del dotto pancreatico: un metodo efficace e selettivo di somministrazione di farmaci e virus

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.

Isolamento degli epatociti primari del topo per l'analisi di sintesi della proteina nascente dal metodo di etichettatura Non radioattivo L-azidohomoalanine

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.

Metal Detector palmare Screening per ingestione di corpo estraneo metallico nei bambini

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.

Rilevazione basata su piastrine di ossido nitrico nel sangue misurando VASP fosforilazione

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.

Valutazione e caratterizzazione delle navi ialoidi nei topi

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