Les auteurs tiennent à remercier le Centre National Laboratory Calicivirus for Disease Control and Prevention (CDC) pour le don de la nature quelques points positifs standards et de contrôle pour NoV, et les laboratoires de l&#39;École de santé publique de Johns Hopkins pour fournir des réactifs.

1. Des échantillons de selles <ol><li> Des échantillons de selles doivent être congelés afin de préserver l&#39;ARN. Pour faire une suspension à 10% fécale, prenez environ 0,1 g de l&#39;échantillon de selles décongelées et compléter à 1 ml avec du PBS. </li><li> Aliquoter dans 200 ul d&#39;éviter les cycles de gel et de dégel. Entreposer les aliquotes à -70 ° C. </li><li> Décongeler et centrifuger à 4000 g aliquotes pendant 10 min avant de l&#39;utiliser dans l&#39;extraction. </li></ol> 2. Préparation de particules de silice pour l&#39;extraction avec de la guanidine et de silice <ol><li> A RT, de suspendre 30 g de dioxyde de silicium et de compléter avec dH 2 O jusqu&#39;à 250 ml. </li><li> Après 24 heures de sédimentation, retirer 215 ml du surnageant par aspiration. Ajouter dH 2 O jusqu&#39;à 250 ml, et de suspendre le culot de silice en secouant. </li><li> Après encore 24 heures, éliminer le surnageant par aspiration, et ajuster le pH de la suspension de silice à pH 2,0, à l&#39;aide d&#39;acide chlorhydrique (HCl). Aliquoter la suspension de silice dans le verre botbouteilles et l&#39;autoclave. </li></ol> 3. Préparation de L6 tampon pour l&#39;extraction avec de la guanidine et de silice <ol><li> A RT, préparer L6 tampon en dissolvant 60 g de thiocyanate de guanidinium (GuSCN) et 1,3 g de Triton X-100 dans 50 ml de Tris 0,1 M de chlorhydrate, pH 6,4, et 11 ml d&#39;une EDTA 0,2 M, pH 8,0 solution. </li></ol> 4. Préparation du Tampon L2 pour l&#39;extraction avec de la guanidine et de silice <ol><li> A RT, préparer un tampon L2 en dissolvant 180 g de thiocyanate de guanidinium (GuSCN) dans 150 ml de 0,1 M Tris chlorhydrate, pH 6,4. </li></ol> 5. Procédure d&#39;extraction <ol><li> Dans chaque extraction d&#39;un échantillon de selles positif NoV, comme contrôle positif, et l&#39;eau traitée au DEPC, comme contrôle négatif, devraient être inclus. </li><li> Dans un tube à centrifuger micro-mélanger le suivant: 1 ml de tampon L6, 20 pl de particules de silice, et ajouter 200 ul de la suspension fécale extrait 10%. Vortexer brièvement et laisser à température ambiante pendant 15 min. </li><li> Centrifuger la suspension à 6000 g pendant 10 s et laver le culot avec 1 ml de tampon L2 deux fois, suivi par deux lavages avec de l&#39;éthanol 70% et un temps avec de l&#39;acétone. Sécher le culot dans un bloc de chauffage à sec à 56 ° C pendant 5 min. </li><li> Hydrater les ARN dans le culot de silice par addition de 50 pl de RNase dH 2 0. Ajouter 1 ul de RNasin, mélanger en retournant le tube et incuber à 56 ° C pendant 15 min. </li><li> Centrifuger pendant 3 min à pleine vitesse, dans une centrifugeuse de table, et de recueillir 40 ul du surnageant contenant l&#39;ARN. </li><li> Quantifier les échantillons extraits d&#39;ARN en utilisant Nanodrop 2000 spectrophotomètre (Thermo Scientific). Puis stocker à -70 ° C jusqu&#39;à son utilisation, jusqu&#39;à 6 mois. (Remarque: Une meilleure façon de préserver intacte l&#39;ARN total est de le convertir en ADNc). </li></ol> 6. Extraction alternative utilisant l&#39;ARN commercial QIAamp Viral RNA Kit Les instructions complètes se trouvent dans le livret provided avec le kit QIAGEN. En bref, la procédure est comme suit: <ol><li> Ul 560 Pipet de tampon AVL contenant les ARN porteur dans un tube de 1,5 ml, et ajouter 140 ul de la suspension de selles de 10%. Mix par impulsions vortex pendant 15 secondes. Incuber à température ambiante pendant 10 min. </li><li> Ajouter 560 ul d&#39;éthanol (96-100%) pour l&#39;échantillon et mélanger par impulsions vortex pendant 15 secondes, puis centrifuger brièvement. </li><li> Appliquer 630 uL de cette solution à une colonne spin placé dans un tube de 2 mL de collecte. Centrifuger 1 min à 6000 g, puis placez la colonne de spin dans un tube de 2 ml propre. </li><li> Laver la colonne contenant de l&#39;ARN liés avec 500 uL de tampon AW1 et centrifuger à 6000 g pendant 1 min. Lieu colonne dans un endroit propre tube collecteur de 2 ml. </li><li> Laver la colonne avec 500 ul de tampon AW2 et centrifuger à pleine vitesse (20.000 g ou 14 000 tpm) pendant 3 min. </li><li> Colonne spin Placer dans un propre de 1,5 ml de micro-tube à centrifuger, ajouter 40 ul eau traitée au DEPC et éluer l&#39;ARN à pleine vitesse. Répéter l&#39;opération surCE pour un final 80 ul d&#39;ARN élues. </li><li> Quantifier les échantillons extraits d&#39;ARN en utilisant Nanodrop 2000 spectrophotomètre (Thermo Scientific). Puis stocker à -70 ° C jusqu&#39;à son utilisation, jusqu&#39;à 6 mois. (Remarque: Une meilleure façon de préserver intacte l&#39;ARN total est de le convertir en ADNc). </li></ol> 7. Détection des norovirus dans l&#39;ARN extrait par une étape en temps réel RT-PCR avec des sondes TaqMan spécifiques Instrument StepOnePlus PCR en temps réel des systèmes (Applied Biosystems). Méthodologie <ol><li> Essuyer et nettoyer toutes les surfaces de travail, les pipettes et des centrifugeuses avec de la RNase, pour supprimer toute contamination éventuelle RNase. </li><li> Déposer le GI et GII NoV de dépistage master mix selon le tableau 1. Primaires et sondes Taqman sont énumérés dans le tableau 2. </li><li> Aliquote de 10 pl de mélange maître approprié à chaque tube de réaction. </li><li> Ajouter 5 ul de l&#39;échantillon non dilué inconnueARN, eau traitée au DEPC comme contrôle négatif, ou IG, respectivement GII contrôle positif ARN, les puits de réaction correspondantes. Tous les échantillons doivent être analysés en double. Les contrôles positifs et les normes de concentration de 300 ug / uL et 500 ug / uL respectivement ont été fournis par National Calicivirus Laboratoire de contrôle des maladies (CDC). </li><li> Centrifuger la plaque de réaction à 6.000 g pendant 10 sec. </li><li> Dans l&#39;expérience les propriétés de l&#39;écran; définir et sélectionner un type d&#39;expérience pour la course. Assurez-vous que les réactifs TaqMan sont affichés comme le type de réactifs, et que lecture pré-PCR et l&#39;amplification sont sélectionnés. </li><li> Exécuter 15 réactions ul en utilisant le profil thermique dans le Tableau 3. </li><li> Analyser les résultats. </li></ol> 8. Génotypage des NoV GI et GII aide conventionnelle RT-PCR et séquençage (Il n&#39;est pas mentionné dans cette vidéo car il s&#39;agit d&#39;une méthode commune et largement utilisé.) Instrument. Applied Biosystems StepOne et StepOnePlus Systèmes Temps réel PCR avec le modèle Quantitec. Méthodologie <ol><li> Avant le génotypage, le génogroupe (GI ou GII) des échantillons sont déterminés par la projection de RT-PCR décrite ci-dessus. GI NoV ARN doit être amplifié avec le mélange GI maître génotypage et le GII NoV ARN avec le mélange GII génotypage maître. </li><li> Déposer le GI et GII NoV génotypage master mix conformément au tableau 4. Primaires GI SKF / SKR et G2 SKF / SKR sont énumérés dans le tableau 5. </li><li> Aliquoter 45 ul du mélange maître approprié de tubes de 0,2 mL de réaction. </li><li> Ajouter 5 pi de eau traitée au DEPC à chaque tube contrôle négatif, et 5 pi de présélection, NoV (GI et / ou GII) à ARN positif du tube de réaction correspondante. </li><li> Exécuter 50 réactions ul en utilisant le profil thermique dans le Tableau 6. </li><li> Envoyer produits PCR CONTAINing au moins 100 ng / pi de la région amplifiée de capside Macrogen Société pour la purification et le séquençage. (9700 Grande Sénèque autoroute. Rockville, MD 20850 et 10 $ par échantillon par séquençage). </li></ol> 9. Les résultats représentatifs <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="Figure 1" /> La figure 1 montre des résultats représentatifs de la Taqman One-Step RT-PCR lorsqu&#39;il est utilisé à l&#39;ARN de dosage extraites à partir d&#39;échantillons de selles d&#39;enfants diarrhéiques. Le cycle seuil (Ct) pour un échantillon positif a été fixé à inférieur ou égal à 37 pour Ct et Ct IG 39 pour GII. Le CT-valeurs pour les contrôles positifs ont été trouvés à moins de 27 et 18 de la jonction ORF1-ORF2 pour GI et GII, respectivement. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="Figure 2" /> Figure 2 </strong&gt; montre une comparaison tête-à-tête de valeurs de Ct de la méthode de silice guanidinium et Qiamp kit ARN viral. Les données des deux essais tombent très près de la ligne d&#39;égalité (= pente 1 et d&#39;interception = 0). Ceci est confirmé par l&#39;analyse de régression et le coefficient de corrélation. Tous les contrôles négatifs ont été évalués et jugés à 0 par les deux méthodes. <table border="1"><tbody><tr><td> Mix Master </td><td> Final Conc </td><td> Volume (pi) </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 Cog R amorce </td><td> 1 uM </td><td> 0,250 </td></tr><tr><td> 50 uM d&#39;amorce F Cog </td><td> 1uM </td><td> 0,250 </td></tr><tr><td> 10 sonde Anneau uM </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 de chaque sonde a été utilisée pour le test GI, tandis que 0.125 pi de chaque sonde a été utilisée pour les tests GII. Tableau 1. Qiagen Quantitect master mix pour le dépistage GI et GII (Trujillo et al., 2006) 7. <table border="1"><tbody><tr><td> Nom </td><td> Geno-groupe </td><td> Utiliser </td><td> Séquence (5 &#39;à 3&#39;) </td></tr><tr><td> Cog 1R </td><td> IG </td><td> Apprêt </td><td> CTT AGA CCG CAT CAT CAT TYA C </td></tr><tr><td> Cog 1F </td><td> IG </td><td> Apprêt </td><td> CGY TGG ATG CGN ATS CAT AG </td></tr><tr><td> Cog 2R </td><td> GII </td><td> Apprêt </td><td> TCG ACG CCA TCT TCA TTC ACA </td></tr><tr><td> Cog 2F </td><td> GII </td><td> Apprêt </td><td> CAR GAR BCN ATG ATS AGR TGG ATG AG </td></tr><tr><td> Bague 1A </td><td> IG </td><td> Sonde </td><td> FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td> 1B Anneau </td><td> IG </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 GGC GAT GAG CCG AAT CT-BHQ-1 </td></tr></tbody></table> Tableau 2. Primer et des oligonucléotides sondes utilisées en temps réel RT-PCR quantitative pour génogroupes I, II (Kageyama et al.,2003) 8. <table border="1"><tbody><tr><td> Étape </td><td> Temp (° C) </td><td> Temps (min) </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> synthèse de l&#39;ADNc </td></tr><tr><td> 2 </td><td> 95 </td><td> 15:00 </td><td> Activation HotStart Taq polymérase </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> Cycles 45X </td></tr></tbody></table> Table3. Le profil thermique pour une étape Taqman en temps réel RT-PCR (Trujillo et al., 2006) 7. <table border="1"><tbody><tr><td> Mix Master </td><td> Final Conc </td><td> Volume (pi) </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> Mélange enzyme </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 SKF uM </td><td> 0,1 uM </td><td> 0,50 </td></tr><tr><td> 10 SKR 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> Tableau 4. Qiagen One-Step RT-PCR Master Mix pour le génotypage GI et GII. <table border="1"><tbody><tr><td> Nom </td><td> Geno-groupe </td><td> Utiliser </td><td> Séquence (5 &#39;à 3&#39;) </td></tr><tr><td> G1SKF </td><td> IG </td><td> Apprêt </td><td> 5&#39;-CTG CCC GAA ATS GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> IG </td><td> Apprêt </td><td> 5&#39;-ACC ACC ACC CAR TTR TAC A - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> Apprêt </td><td> 5&#39;-CNT GGG GCG AGG ATC GCA A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> Apprêt </td><td> 5&#39;-CCR CCN GCA TRH CCR TTR TA CAT-3 </td></tr></tbody></table> Primaires Tableau 5. Utilisées pour l&#39;amplification de la région C de la région de la capside (Kojima et al., 2002) 5. <table border="1"><tbody><tr><td> Étape </td><td> Temp (° C) </td><td> Temps (min) </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> synthèse de l&#39;ADNc </td></tr><tr><td> 2 </td><td> 96 </td><td> 15:00 </td><td> Activation HotStart Taq polymérase </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> Cycles 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> Recuit </td></tr></tbody></table> Tableau 6. Profil thermique pour 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>

Pas de conflits d&#39;intérêt déclarés.

Utilisation de l&#39;économique dans la maison-méthode pour isoler l&#39;acide nucléique à partir d&#39;échantillons de selles, on obtient des résultats égaux avec le commercial QIAmp virale ARN kit de Qiagen, et avec le TaqMan RT-PCR développée dans notre laboratoire, nous permet de détecter un large éventail de NoV génotypes appartenant à la GI et GII génogroupe. Une publication récente du protocole de la région C ont signalé des taux de génotypage de 78% 6. Depuis la diversité des souche...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Isothiocyanate de guanidine </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> BDH Chemicals </td><td> 14530 </td><td></td></tr><tr><td> Diéthylpyrocarbonate </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> Inhibiteur de RNase 2000 unités </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / flacon </td></tr><tr><td> Non-Stick microtubes Rnae-libres </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

Les norovirus (NoV) sont la principale cause des épidémies de gastro-entérite aiguë sporadique dans le monde entier chez les humains de tous âges. Ils sont cause importante d&#39;hospitalisation chez les enfants ayant un impact de santé publique semblable à celle de rotavirus. NoV sont des virus à ARN de grande diversité génétique et il ya un aspect continu de nouvelles souches. Cinq génogroupes sont reconnus; GI et GII avec leurs nombreux génotypes et sous-types étant le plus important pour l&#39;infection humaine. Cependant, le diagnostic de ces deux génotypes reste problématique, ce qui retarde le diagnostic et le traitement. 1, 2, 3 Pour l&#39;extraction d&#39;ARN à partir d&#39;échantillons de selles de la méthode la plus couramment utilisée est le kit RNA viral QIAmp commercial de Qiagen. Ce procédé combine les propriétés de liaison d&#39;une membrane de gel de silice, les tampons qui ARNases contrôle et fournir liaison optimales de l&#39;ARN à la colonne ensemble avec la vitesse de MicroSpin. Cette méthode est simple, rapide et fiable et est carrIed dans quelques étapes qui sont détaillées dans la description fournie par le fabricant. Norovirus est la deuxième à rotavirus est la cause la plus fréquente de la diarrhée. Diagnostic norovirus devrait être disponible dans toutes les études sur la pathogenèse de la diarrhée ainsi que dans les foyers ou les cas de diarrhée individuels. À l&#39;heure actuelle le diagnostic norovirus mais est limitée à seulement quelques centres en raison de l&#39;absence de méthodes simples de diagnostic. Ce diagnostic et le traitement des retards 1, 2, 3. En outre, en raison des coûts de transport et réglementé de tampons corrosifs à l&#39;intérieur et entre les pays l&#39;utilisation de ces kits manufacturés pose des problèmes logistiques. En conséquence, dans ce protocole, nous décrivons une alternative, économique, en interne la méthode qui est basée sur la bôme d&#39;origine et al. La méthode 4 qui utilise les propriétés d&#39;acides nucléiques de liaison de particules de silice ainsi que les propriétés anti-nucléase de thiocyanate de guanidinium . <p class=&quot;Jove_content&quot;&gt; Pour la détection et la genogrouping (GI et GII) des isolats de NoV échantillons de selles, de plusieurs protocoles de RT-PCR en utilisant des objectifs différents ont été développés. Le consensus est que le RT-PCR en utilisant la chimie TaqMan serait la meilleure technique moléculaire pour le diagnostic, car il combine une grande sensibilité, la spécificité et la reproductibilité avec un débit élevé et la facilité d&#39;utilisation. On décrit ici un dosage cible de la région de lecture ouvert trame 1 (ORF1) de jonction-ORF2; la région la plus conservée du génome de NoV et donc le plus approprié pour le diagnostic. Pour l&#39;analyse génétique en outre une RT-PCR conventionnelle qui vise le très variable N-terminal-shell de la protéine majeure de la capside (région C) en utilisant des amorces décrites à l&#39;origine par Kojima et al. 5 est détaillé. Le séquençage du produit de PCR de la PCR conventionnelle permet la différenciation des génotypes appartenant à la GI et GII génogroupes.

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 pour la détection et l&#39;identification génogroupe des isolats de norovirus dans les selles des enfants, qui utilise des amorces et des sondes TaqMan spécifiques au cadre de lecture ouvert 1 (ORF1)-ORF2 région de jonction, la région la plus conservée du génome de Norovirus est décrit. Un non-commerciale, rentable méthode d&#39;extraction d&#39;ARN est détaillé.

Détection et Genogrouping des norovirus dans les selles des enfants en Taqman en une seule étape de 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 vues.  Universidad Peruana Cayetano Heredia. Un One-Step RT-PCR pour la détection et l'identification génogroupe des isolats de norovirus dans les selles des enfants, qui utilise des amorces et des sondes TaqMan spécifiques au cadre de lecture ouvert 1 (ORF1)-ORF2 région de jonction, la région la plus conservée du génome de Norovirus est décrit. Un non-commerciale, rentable méthode d'extraction d'ARN est détaillé.

Vidéo: 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.

Détection MicroRNA dans les tumeurs prostatiques par quantitative PCR en temps réel (qPCR)

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

Recherche

Médecine

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.

Luminescence Cerenkov Imaging (CLI) pour la surveillance de la thérapie du cancer

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.

Implantation de veine cave inférieure interposition de greffe en modèle de souris

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.

Isolement et immortalisation de lignées cellulaires de patients dérivé de la biopsie musculaire pour la modélisation des maladies

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.

Mise en place d&#39;épithéliales humaines Enteroids et Colonoids de tout le tissu et la 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.

Perfusion de canal pancréatique: une méthode efficace et sélective d’administration de médicaments et de 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.

Isolation des hépatocytes de souris primaire pour analyse-synthèse protéine naissante par méthode d’étiquetage non-radioactifs 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.

Détecteur de métaux portatif de dépistage pour l’Ingestion de corps étrangers métalliques chez les enfants

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.

Détection du monoxyde d’azote dans le sang en mesurant Phosphorylation VASP plaquettaire

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.

Évaluation et caractérisation des navires hyaïdis chez les souris

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