The authors would like to acknowledge Latoya Wiggins and Katelyn Griffin for their assistance in sample acquisition, as well as Laura Lee Rutherford for their assistance in sampling and molecular analyses. We would also like to thank Sarah Owens from Argonne National Lab for microbiomic sample preparation and sequencing. These investigations were supported equally by the Agricultural Research Service, USDA CRIS Projects &#8220;Pathogen Reduction and Processing Parameters in Poultry Processing Systems&#8221; #6612-41420-017-00 and &#8220;Molecular Approaches for the Characterization of Foodborne Pathogens in Poultry&#8221; #6612-32000-059-00.

1. omogeneizzazione meccanica di pollame ambientale campioni di produzione <ol style="margin-left: 40px;"><li> Prima di estrazione, impostare un bagno d&#39;acqua a 95 ° C e lasciare il tempo bagnomaria per raggiungere tale temperatura. </li><li> Pesare 0,33 g di terra o di materiale fecale in una provetta 2 ml di Lisi Matrix E. <ol><li> Non superare 0,33 g di campione nel tubo, perché altrimenti le seguenti soluzioni per superare la capacità del tubo. </li><li> Scongelare i campioni congelati a RT prima della pesata. </li><li> Per analizzare un totale di 1 g di suolo / feci, pesare 3 replicare 0,33 g campioni per ogni campione ambientale individuo. </li><li> Conservare i campioni a -20 ° C all&#39;interno dei tubi conici matrice prima dell&#39;estrazione, se necessario. </li></ol></li><li> Aggiungere 825 microlitri Sodium Phosphate Buffer e 275 ml di soluzione PLS per una provetta. Miscelare utilizzando un vortex per ~ 15 sec e poi centrifugare i campioni a 14000 xg per 5 min. </li><li> Versare il Supernatant e aggiungere 700 ml di tampone ASL. Mescolare con un vortice per 5 sec. <ol><li> Assicurarsi che vi sia spazio di testa (~ 10% del volume totale) disponibili nel tubo conico a questo punto. Se non c&#39;è spazio di testa, i tubi si hanno la tendenza a perdere durante l&#39;omogeneizzazione successivo passo che potrebbe portare a contaminazione incrociata e / o perdita di campione. </li></ol></li><li> Porre i campioni in un FastPrep 24 Instrument, e omogeneizzare i campioni ad una velocità di 6,0 m / sec per 40 sec. </li><li> Centrifugare il campione omogeneizzato a 14000 g per 5 min. Trasferire il surnatante in una sterile 2 ml provetta. </li><li> Per massimizzare il recupero di DNA dal campione, ripetere i punti da 1.4 a 1.6, che combina il surnatante nella stessa provetta sterile, 2 ml. </li></ol> 2. enzimatica Inibizione di inibitori di omogenati campione NOTA: Questo protocollo utilizza il kit QIAamp DNA Sgabello Mini. <ol style="margin-left: 40px;"><li> Incubare lasupernatante in un bagno d&#39;acqua a 95 ° per 5 minuti per massimizzare il recupero del DNA da eventuali cellule rimanenti nel surnatante. <ol><li> Incubare a 70 ° C per i campioni contenenti organismi prevalentemente Gram-negativi. Tuttavia, se sono presenti (che è il caso con pollame campioni fecali) organismi Gram-positivi, incubare a 95 ° C. </li><li> Utilizzare clip di plastica bloccaggio sui tubi microcentrifuga per assicurare che i tubi non &quot;pop&quot; aperto e potenzialmente perdere volume del campione come pressione può accumularsi in questi tubi microcentrifuga sigillati. </li></ol></li><li> Aprire ogni provetta per scaricare la pressione, ri-cap tubi microcentrifuga e miscelare con un vortice per 15 sec. </li><li> Centrifugare il campione a 14000 g per 1 minuto, rimuovere 1,2 ml del surnatante e inserirlo in un nuovo sterile 2 ml provetta. </li><li> Aggiungere 1 scheda Inhibitex per ogni campione, e mescolare con un vortice fino a quando il campione ha un / liquido bianco sporco uniformemente bianco. <ol><li> Evitare touching scheda Inhibitex mentre metterlo nella provetta contenente il campione. Per fare questo, inserire il blister contenente la scheda direttamente sopra la provetta aperta e spingere delicatamente la scheda dal blister e in provetta. </li></ol></li><li> Incubare il campione per 1 min a RT (~ 25 ° C) e centrifugare a 14.000 xg per 5 min. </li><li> Trasferire tutto il liquido sterile 1,5 ml provetta e centrifugare a 14.000 xg per 5 min. <ol><li> Evitare eventuali particelle residue che possono essere pellet sul fondo della provetta al termine della fase 2.5 durante il trasferimento del liquido. </li></ol></li></ol> 3. Automated Purificazione DNA Utilizzando il QIAcube Robotic Workstation NOTA: Il numero di materiali di consumo di plastica, la disposizione degli adattatori rotore campione all&#39;interno della centrifuga, ed i volumi richiesti dei buffer / soluzioni dipendono numBER di campioni che sono in esecuzione. <ol style="margin-left: 40px;"><li> Aggiungi provette di eluizione e tubi di filtro per gli slot all&#39;interno delle schede del rotore. Per ciascun campione, aggiungere 400 microlitri al slot centrale dell&#39;adattatore rotore. Posizionare gli adattatori del rotore nella centrifuga workstation nella disposizione corretta in base al numero di campioni purificato. <ol><li> Assicurarsi che tutti i coperchi provetta siano fissati correttamente all&#39;interno dell&#39;adattatore del rotore dal momento che un fallimento di farlo potrebbe comportare taglio durante una delle fasi di centrifugazione del protocollo di purificazione. </li></ol></li><li> Aggiungere il numero di 1.000 microlitri e 200 microlitri puntali con filtro alla workstation, e riempire le bottiglie tampone in dotazione con il volume di buffer. NOTA: I buffer necessari per questo protocollo di purificazione (AL, AW1, AW2, e AE) sono tutti contenuti all&#39;interno del Kit DNA QIAamp Sgabello Mini. L&#39;utente deve fornire l&#39;etanolo 100% che è necessario per il bu AWffers e come soluzione utilizzata nel processo di purificazione. </li><li> Aggiungere il volume richiesto di soluzione K proteasi fornito in una sterile 1,5 ml provetta e metterlo nella fessura A nella workstation. Inoltre, aggiungere il numero (pari al numero di campioni purificazione) di 2 ml sicuro bloccaggio provettoni microcentrifuga RB alla sezione shaker della workstation. <ol><li> Assicurarsi che i coperchi dei tubi di esempio sono inseriti saldamente nelle apposite fessure sulla workstation, dal momento che un fallimento di farlo si tradurrà in un errore quando la macchina esegue la scansione inizialmente la workstation per assicurarsi che tutte le materie plastiche ei liquidi necessari sono disponibili per la richiesta correre. </li></ol></li><li> Utilizzando il touch screen sulla workstation, selezionare la Stool DNA - Sgabello Umano - patogeni Rilevazione Protocollo, e leggere le schermate successive per assicurare che la workstation è stata caricata correttamente. Una volta che tutti gli schermi di controllo vengono passati, selezionare Avvia per eseguire questo protocollo. <ol><li> Se l&#39;estrazione di DNA da più di 12 campioni, iniziare il processo di omogeneizzazione (Fase 1) per la prossima serie di campioni, dal momento che una corsa di 12 campioni richiede ~ 72 minuti per completare sulla workstation. </li></ol></li><li> Rimuovere i campioni dagli adattatori rotore, li cap, e posto a -20 ° C fino al momento per le successive analisi a valle. <ol><li> A questo punto, combinare le purificazioni 3 replicati per un singolo campione (totale analizzato quantità = 1 g) utilizzando un sistema di centrifugazione / basato evaporazione-. Unire le repliche e le ri-eluire ad un volume finale di 100 ml di tampone Tris-ETDA. </li></ol></li></ol>

<ol>
	<li>Maukonen, J., Simoes, C., Saarela, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+currently+used+commercial+DNA-extraction+methods+give+different+results+of+clostridial+and+actinobacterial+populations+derived+from+human+fecal+samples.">The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples.</a> FEMS microbiology ecology. 79, 697-708 (2012).</li><li>Tang, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+method+for+isolation+of+DNA+from+pig+faeces+and+comparison+of+five+different+methods.">An effective method for isolation of DNA from pig faeces and comparison of five different methods.</a> Journal of microbiological. 75, 432-436 (2008).</li><li>McOrist, A. L., Jackson, M., Bird, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+five+methods+for+extraction+of+bacterial+DNA+from+human+faecal+samples.">A comparison of five methods for extraction of bacterial DNA from human faecal samples.</a> Journal of microbiological. 50, 131-139 (2002).</li><li>Ariefdjohan, M. W., Savaiano, D. A., Nakatsu, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+DNA+extraction+kits+for+PCR-DGGE+analysis+of+human+intestinal+microbial+communities+from+fecal+specimens.">Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens.</a> Nutrition journal. 9, 23 (2010).</li><li>Carrigg, C., Rice, O., Kavanagh, S., Collins, G., O'Flaherty, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+extraction+method+affects+microbial+community+profiles+from+soils+and+sediment.">DNA extraction method affects microbial community profiles from soils and sediment.</a> Applied microbiology and biotechnology. 77, 955-964 (2007).</li><li>Smith, B., Li, N., Andersen, A. S., Slotved, H. C., Krogfelt, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+Bacterial+DNA+Extraction+from+Faecal+Samples:+Comparison+of+Three+Methods.">Optimising Bacterial DNA Extraction from Faecal Samples: Comparison of Three Methods.</a> The Open microbiology journal. 5, 14-17 (2011).</li><li>Claassen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+efficiency+of+five+different+commercial+DNA+extraction+kits+for+extraction+of+DNA+from+faecal+samples.">A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.</a> Journal of microbiological. 94, 103-110 (2013).</li><li>Scupham, A. J., Jones, J. A., Wesley, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+DNA+extraction+methods+for+analysis+of+turkey+cecal+microbiota.">Comparison of DNA extraction methods for analysis of turkey cecal microbiota.</a> Journal of applied microbiology. 102, 401-409 (2007).</li><li>Salonen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+fecal+DNA+extraction+methods+with+phylogenetic+microarray:+effective+recovery+of+bacterial+and+archaeal+DNA+using+mechanical+cell+lysis.">Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.</a> Journal of microbiological methods. 81, 127-134 (2010).</li><li>Peng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+direct+boiling+method+with+commercial+kits+for+extracting+fecal+microbiome+DNA+by+Illumina+sequencing+of+16S+rRNA+tags.">Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags.</a> Journal of microbiological. 95, 455-462 (2013).</li><li>Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z., Forney, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+for+the+extraction+and+purification+of+DNA+from+the+human+microbiome.">Evaluation of methods for the extraction and purification of DNA from the human microbiome.</a> PloS one. 7, 33865 (2012).</li><li>Qu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Metagenomics+Reveals+Host+Specific+Metavirulomes+and+Horizontal+Gene+Transfer+Elements+in+the+Chicken+Cecum+Microbiome.">Comparative Metagenomics Reveals Host Specific Metavirulomes and Horizontal Gene Transfer Elements in the Chicken Cecum Microbiome.</a> PloS one. 3, 2945 (2008).</li><li>Sekelja, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abrupt+temporal+fluctuations+in+the+chicken+fecal+microbiota+are+explained+by+its+gastrointestinal+origin.">Abrupt temporal fluctuations in the chicken fecal microbiota are explained by its gastrointestinal origin.</a> Applied and environmental microbiology. 78, 2941-2948 (2012).</li><li>Oakley, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Poultry-Associated+Microbiome:+Network+Analysis+and+Farm-to-Fork+Characterizations.">The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations.</a> PloS one. 8, 57190 (2013).</li><li>Cook, K. L., Rothrock, M. J., Eiteman, M. A., Lovanh, N., Sistani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+nitrogen+retention+and+microbial+populations+in+poultry+litter+treated+with+chemical,+biological+or+adsorbent+amendments.">Evaluation of nitrogen retention and microbial populations in poultry litter treated with chemical, biological or adsorbent amendments.</a> Journal of environmental management. 92, 1760-1766 (2011).</li><li>Rothrock, M. J., Cook, K. L., Warren, J. G., Eiteman, M. A., Sistani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+mineralization+of+organic+nitrogen+forms+in+poultry+litters.">Microbial mineralization of organic nitrogen forms in poultry litters.</a> Journal of environmental quality. 39, 1848-1857 (2010).</li><li>Rothrock, M. J., Cook, K. L., Warren, J. G., Sistani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+alum+addition+on+microbial+communities+in+poultry+litter.">The effect of alum addition on microbial communities in poultry litter.</a> Poultry science. 87, 1493-1503 (2008).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+QIAamp+DNA+Stool+Mini+Kit+for+ecological+studies+of+gut+microbiota.">Evaluation of QIAamp DNA Stool Mini Kit for ecological studies of gut microbiota.</a> Journal of microbiological. 54, 13-20 (2003).</li><li>Dridi, B., Henry, M., El Khechine, A., Raoult, D., Drancourt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+precalence+of+Methanobrevibacter+smithii+and+Methanosphaera+stadtmanae+detected+in+the+human+gut+using+an+improved+DNA+detection+protocol.">High precalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol.</a> PloS one. 4, 7063 (2009).</li><li>Harms, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+quantification+of+nitrifying+bacteria+in+a+municipal+wastewater+treatment+plant.">Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant.</a> Environmental science and technology. 37, 343-351 (2003).</li><li>Rothrock, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+microvolume+DNA+quantification+methods+for+use+with+volume-sensitive+environmental+DNA+extracts.">Comparison of microvolume DNA quantification methods for use with volume-sensitive environmental DNA extracts.</a> Journal of natural and environmental sciences. 2, 34-38 (2011).</li><li>Navas-Molina, J. A., DeLong Edward, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 531, 371-444 (2013).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-throughput+microbial+community+analysis+on+the+Illumina+HiSeq+and+MiSeq+platforms.">Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms.</a> The ISME journal. 6, 1621-1624 (2012).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+patterns+of+16S+rRNA+diversity+at+a+depth+of+millions+of+sequences+per+sample.">Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 Suppl 1, 4516-4522 (2011).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QIIME+allows+analysis+of+high-throughput+community+sequencing+data.">QIIME allows analysis of high-throughput community sequencing data.</a> Nature methods. 7, 335-336 (2010).</li><li>Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCHIME+improves+sensitivity+and+speed+of+chimera+detection.">UCHIME improves sensitivity and speed of chimera detection.</a> Bioinformatics. 27, 2194-2200 (2011).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Search+and+clustering+orders+of+magnitude+faster+than+BLAST.">Search and clustering orders of magnitude faster than BLAST.</a> Bioinformatics. 26, 2460-2461 (2010).</li><li>DeSantis, T. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Greengenes,+a+chimera-checked+16S+rRNA+gene+database+and+workbench+compatible+with+ARB.">Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.</a> Applied and environmental microbiology. 72, 5069-5072 (2006).</li><li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PyNAST:+a+flexible+tool+for+aligning+sequences+to+a+template+alignment.">PyNAST: a flexible tool for aligning sequences to a template alignment.</a> Bioinformatics. 26, 266-267 (2010).</li><li>Price, M. N., Dehal, P. S., Arkin, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FastTree+2&#8211;approximately+maximum-likelihood+trees+for+large+alignments.">FastTree 2&#8211;approximately maximum-likelihood trees for large alignments.</a> PloS one. 5, 9490 (2010).</li><li>Cook, K. L., Rothrock, M. J., Lovanh, N., Sorrell, J. K., Loughrin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+changes+in+the+microbial+community+in+an+anaerobic+swine+waste+treatment+lagoon.">Spatial and temporal changes in the microbial community in an anaerobic swine waste treatment lagoon.</a> Anaerobe. 16, 74-82 (2010).</li><li>Cook, K. L., Rothrock, M. J., Warren, J. G., Sistani, K. R., Moore, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+alum+treatment+on+the+concentration+of+total+and+ureolytic+microorganisms+in+poultry+litter.">Effect of alum treatment on the concentration of total and ureolytic microorganisms in poultry litter.</a> Journal of environmental quality. 37, 2360-2367 (2008).</li><li>Lovanh, N., Cook, K. L., Rothrock, M. J., Miles, D. M., Sistani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+shifts+in+microbial+population+structure+within+poultry+litter+associated+with+physicochemical+properties.">Spatial shifts in microbial population structure within poultry litter associated with physicochemical properties.</a> Poultry science. 86, 1840-1849 (2007).</li></ol>

The authors have nothing to disclose.

Il metodo di estrazione del DNA usato effettuata totali stime comunità batteriche quantitativi e qualitativi sia per i campioni di feci e di lettiera, sostenendo l&#39;analisi dei campioni natura dipendente di metodi di estrazione del DNA visto in precedenza 1,3,6. Per entrambi i campioni di feci e del disordine, l&#39;ordine di esecuzione dei metodi di estrazione del DNA era diverso per il quantitativo (meccanica&gt; Hybrid&gt; enzimatica) e qualitativa (enzimatica&gt; Hybrid&gt; Meccanica) totali stime del...

When analyzing complex clinical or environmental samples (e.g., feces, soils), there are two main methodologies used for the extraction of DNA. The first is a mechanical disruption of the matrix using an intense bead-beating step, while the second is an enzymatic disruption of the matrix to chemically release bacterial cells and inhibit PCR inhibitors from the matrix simultaneously. Given the different means by which these two types of extraction methods work, it is not surprising that previous studies demonstrated that the appropriate DNA extraction method is both highly sample and analysis dependent. Comparative DNA extraction studies previously showed that some methods are more appropriate for improved DNA quality and quantity from environmental samples1-3, while others demonstrated improvements for community-level analyses such as denaturing gradient gel electrophoresis (DGGE)4-6, terminal restriction fragment length polymorphism (T-RFLP)7, automated ribosomal intergenic spacer analysis (ARISA)8, and phylogenetic microarrays9. Therefore, appropriate DNA extraction methods need to be used, or developed, according to the types of environmental samples and the types of analyses being performed on those samples, especially given the recent advancements in bacterial community analyses. 
Next generation sequencing, in conjunction with more quantitative community assessments (e.g., quantitative PCR (qPCR)), is becoming more prevalent in the environmental and clinical sciences, however, very little research has been performed to determine the effect of DNA extraction methods on these data sets. Most DNA extraction comparison studies dealt with microbiomic community estimates from human or human model samples10,11, not agricultural animal samples. The few poultry-focused next generation sequencing studies dealt with specific metagenomic12,13 or microbiomic14 questions; they did not discuss the effect of DNA extraction method on the resulting microbiomic analyses. Considering the complex nature of environmental samples related to poultry production (e.g., feces, litter/bedding, pasture soil), DNA extraction methods need to be carefully selected. Poultry-related environmental samples are known to contain large numbers of PCR inhibitors and up to 500-fold DNA extract dilutions have been required for PCR and subsequent downstream analysis15-17. Therefore it is essential that DNA extraction methods be optimized for these types of samples in order to not only physically disrupt the matrix, but also to be able to reduce/eliminate the large number of inhibitors that are present. 
The QIAamp DNA Stool Mini Kit, an enzymatic extraction method, has been considered the &#8220;gold standard&#8221; when extracting DNA from difficult gut/fecal samples1,18,19 and has been applied successfully to poultry environmental samples8,14. The enzymatic removal of PCR inhibitors through the use of a proprietary matrix is one of the greatest advantages of using this method for these types of environmental samples, as is the ability to significantly improve throughput (and reduce sample processing error) using automated workstations. One major disadvantage is the lack of a mechanical homogenization step to physically disassociate bacterial cells from the environmental matrix. When testing gut and fecal samples of non-poultry origin, the addition of a bead-beating or mechanical disruption step within a DNA extraction protocol significantly increased extraction efficiency9, DNA yield/quality1,4,5 and significantly improved downstream community analyses in terms of richness, diversity, and coverage5,6,11. These studies compared not only mechanical bead-beating methods to the &#8220;gold standard&#8221; enzymatic method, but some also added the mechanical bead-beating step to the enzymatic protocol to improve results6,9,11. 
According to the results from the above studies, bacterial community analyses (both qualitative and quantitative) could be improved from poultry-related environmental samples through the addition of a mechanical homogenization step to the enzymatic method. Therefore, the goal of this study was twofold: (1) to develop a novel DNA extraction technique that utilizes the most desirable aspects of both the mechanical (powerful homogenization step) and enzymatic (PCR inhibitor removal and automation) extraction methods and (2) compare the quantitative (via qPCR) and qualitative (via microbiomics) bacterial community assessments of this novel method to representative mechanical and enzymatic methods.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysing Matrix E tube</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6914-050</td>
			<td>Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1000 tubes)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Phosphate Solution</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6570-205</td>
			<td>Can be purchased individually, or also contained within the FastDNA Spin Kit for Feces (Cat. No. 116570200)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PLS Buffer</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6570-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buffer ASL (560 ml)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">19082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FastPrep 24 homogenizer</td>
			<td style="width:71px;">MPBio</td>
			<td align="right" style="width:119px;">116004500</td>
			<td>48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA Stool Mini Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">51504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAcube24 (110V)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">9001292</td>
			<td>Preliminary results show that QIAcube HT (Cat. No. 9001793) can be used to improve throughput, but different consumables are required of this machine and more comparative work needs to be done.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter-Tips, 1000 ml (1024)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter-Tips, 200 ml (1024)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990332</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAcube Rotor Adapters (10 x 24)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990394</td>
			<td>For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative.&#160; It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sample Tubes RB (2 ml)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990381</td>
			<td>Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352</td>
		</tr>
	</tbody>
</table>

a hybrid dna extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

L&#39;efficacia di protocolli di estrazione di DNA può essere altamente dipende sia dal tipo di campione indagati ei tipi di analisi effettuate a valle. Considerando che l&#39;uso di nuove tecniche di analisi della comunità batterica (ad esempio, microbiomics, metagenoma) sta diventando sempre più prevalente nelle scienze agrarie e ambientali e molti campioni ambientali all&#39;interno di queste discipline può essere physiochemically e microbiologicamente unico (ad esempio, fecale e campioni lettiera / biancheria da lo spettro della produzione di pollame), metodi di estrazione del DNA appropriati ed efficaci devono essere scelti con cura. Pertanto, un metodo di estrazione del DNA ibrido romanzo semi-automatico è stato sviluppato specificatamente per l&#39;uso con i campioni di produzione di pollame ambientale. Questo metodo è una combinazione dei due tipi principali di estrazione del DNA: meccanica ed enzimatica. A due fasi intense passo omogeneizzazione meccanica (con bead-battendo specificamente formulato per environmencampioni TAL) inserito all&#39;inizio del metodo di estrazione del DNA &quot;standard&quot; enzimatica per campioni fecali per migliorare la rimozione di batteri e DNA dalla matrice del campione e migliorare il recupero di batteri Gram-positivi membri della comunità batteriche. Una volta che è stata avviata la parte di estrazione enzimatica del metodo ibrido, il processo di purificazione rimanente è stato automatizzato utilizzando una stazione di lavoro robotizzata per aumentare il throughput del campione e diminuire errore di elaborazione del campione. Rispetto ai metodi di estrazione del DNA severe meccaniche ed enzimatiche, questo nuovo metodo ibrido fornito le migliori prestazioni complessive combinato quando si considera quantitativa (con 16S rRNA qPCR) e qualitativo (utilizzando microbiomics) le stime del totale comunità batteriche durante l&#39;elaborazione di feci di pollame e campioni di lettiera .

Per questo studio, escrementi fecali freschi e campioni di lettiera sono stati recuperati da una casa commerciale broiler (~ 25.000 uccelli) nel sud-est degli Stati Uniti. I polli da carne (Gallus gallus) erano Cobb-500 attraversa, ed erano 59 giorni di età al momento del campionamento. Freschi campioni di feci e lettiere sono stati recuperati da quattro aree distinte all&#39;interno della casa (vicino a pad di raffreddamento, vicino alle linee waterer / alimentazione, tra le righe waterer / alimentazione, e n...

Watch this Scientific Journal Video about A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples at JoVE.com

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Un ibrido metodo di estrazione del DNA per la valutazione delle comunità batteriche qualitativa e quantitativa di pollame campioni di produzione

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

a-hybrid-dna-extraction-method-for-qualitative-quantitative

Research

JoVE Journal

Biology

14.4K Views.  USDA-Agricultural Research Service. A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities. 

Video: A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purificazione del DNA plasmidi da colonie batteriche Utilizzando il kit Qiagen Miniprep

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Ricerca

Biologia

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

L&#39;uso di Rotorod come un metodo per l&#39;analisi qualitativa del Camminare in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

Estrazione del DNA da 0,22 Filtri mM Sterivex e cloruro di Cesio densità centrifugazione gradiente

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Efficiente Nucleic Acid Estrazione e 16S rRNA sequenziamento del gene per batterica Comunità Caratterizzazione

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Un metodo sezionamento migliorato per Recalcitrante tessuti vegetali Campioni

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Analisi metagenomica quasi di Salmonella da campioni ambientali e alimentari

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Valutazione della produzione di siderofori da isolati di Pseudomonas aeruginosa

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Autore in primo piano: Quantificazione di siderofori e piochelina per il controllo delle infezioni 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Analisi qualitativa e quantitativa della produzione di siderofori da Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...