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>
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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.

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	<tbody>
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			<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 ...

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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. 