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. Homogenization מכאני של דוגמאות ייצור עופות הסביבה <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני המיצוי, להגדיר אמבט מים עד 95 ° C ולאפשר זמן אמבט מים כדי להגיע לטמפרטורה זו. </li><li style=";text-align:right;direction:rtl"> לשקול את 0.33 גרם של אדמה או חומר צואתי לתוך צינור lysing מטריקס E 2 מיליליטר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לא יעלה על 0.33 גרם של מדגם בצינור, שכן זה יגרום הפתרונות הבאים למעבר לקיבולת של הצינור. </li><li style=";text-align:right;direction:rtl"> דגימות הפשרה קפוא לRT לפני השקילה. </li><li style=";text-align:right;direction:rtl"> על מנת לנתח את כולל של 1 גרם של אדמה / צואה, שוקל 3 מ- לשכפל 0.33 דגימות g עבור כל דגימה סביבתית בודדת. </li><li style=";text-align:right;direction:rtl"> חנות דגימות ב -20 ° C בתוך צינורות מטריצת חרוטי לפני המיצוי, במידת צורך. </li></ol></li><li style=";text-align:right;direction:rtl"> להוסיף 825 μl סודיום פוספט הצפת וμl 275 של פתרון PLS לצינור מדגם. מערבבים בעזרת מערבולת ל~ 15 שניות ואז צנטריפוגות הדגימות 14,000 XG במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> למזוג supernatant ולהוסיף 700 μl של הצפת ASL. מערבבים בעזרת מערבולת במשך 5 שניות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא שיש אמיץ (10% נפח כולל ~) זמין בצינור חרוטי בשלב זה. אם אין אמיץ, הצינורות יהיו נטייה לדלוף במהלך שלב הומוגניזציה הבא שעלולות להוביל לזיהום צולב ו / או הפסד מדגם. </li></ol></li><li style=";text-align:right;direction:rtl"> מניחים את הדגימות למכשיר 24 FastPrep, וhomogenize הדגימות במהירות של 6.0 מ &#39;/ שנייה למשך 40 שניות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה המדגם הומוגני 14,000 XG במשך 5 דקות. מעביר את supernatant לצינור microcentrifuge 2 מיליליטר סטרילי. </li><li style=";text-align:right;direction:rtl"> על מנת למקסם את התאוששות DNA מן המדגם, חזור על שלבים 1.4-1.6, המשלבים את supernatants לתוך צינור microcentrifuge, 2 מיליליטר סטרילי. </li></ol> 2. אנזימתי עיכוב של המעכבים מhomogenates לדוגמא הערה: פרוטוקול זה משתמש בערכת QIAamp DNA הצואה Mini. <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירהsupernatant באמבט מים C ° 95 במשך 5 דקות על מנת למקסם את התאוששות DNA מכל תאים שנותרו בתוך supernatant. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדגור על 70 מעלות צלזיוס למשך דגימות המכילות אורגניזמים בעיקר גראם שליליים. עם זאת, אם אורגניזמים גראם חיוביים נמצאים (שהוא במקרה עם דגימות צואה עופות), דגירה על 95 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> השתמש בקליפים פלסטיק נעילה על צינורות microcentrifuge כדי להבטיח שהצינורות לא &quot;פופ&quot; פתוחים ואפשרות לאבד נפח דגימה כלחץ יכול להצטבר בצינורות microcentrifuge האטומים אלה. </li></ol></li><li style=";text-align:right;direction:rtl"> פתח כל צינור microcentrifuge כדי לשחרר את הלחץ, צינורות microcentrifuge מחדש כובע ומערבבים בעזרת מערבולת במשך 15 שניות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה מדגם 14,000 XG דקות 1, להסיר 1.2 מיליליטר של supernatant ולמקם אותו לתוך צינור סטרילי חדש 2 מיליליטר microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 1 כרטיסיית InhibitEx מדגם זה, ומערבבים בעזרת מערבולת עד המדגם הופך נוזל אחיד לבן / off-לבן. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הימנע touching כרטיסיית InhibitEx תוך שימה אותו לתוך צינור microcentrifuge המכיל את המדגם. כדי להשיג זאת, הנח את חבילת השלפוחית ​​המכילה את הכרטיסייה ישירות על צינור microcentrifuge הפתוח ודחף בעדינות את הכרטיסייה מחפיסת השלפוחית ​​ולתוך צינור microcentrifuge. </li></ol></li><li style=";text-align:right;direction:rtl"> דגירה המדגם 1 דקות בRT (~ 25 מעלות צלזיוס) וצנטריפוגות ב 14,000 XG במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> להעביר את כל הנוזל לצינור סטרילי 1.5 מיליליטר microcentrifuge ו צנטריפוגות ב 14,000 XG במשך 5 דקות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הימנע מכל חלקיקים שנותרו שאולי pelleted בתחתית צינור microcentrifuge בסוף השלב 2.5 בעת העברת הנוזל. </li></ol></li></ol> 3. אוטומטי DNA טיהור באמצעות QIAcube רובוטית Workstation הערה: מספר מתכלה פלסטיק, ההסדר של מתאמי הרוטור מדגם בתוך צנטריפוגות, וכרכים הנדרשים ממאגרים / הפתרונות תלויים בnumבער של דגימות שמתנהלות. <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף צינורות elution וצינורות מסנן לחריצים המתאימים במתאמי הרוטור. עבור כל דגימה, להוסיף 400 μl לחריץ אמצע מתאם הרוטור. הנח את מתאמי הרוטור בצנטריפוגה תחנת העבודה בהסדר הנכון בהתאם למספר הדגימות מטוהר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא שכל מכסי צינור microcentrifuge מאובטחים כראוי בתוך מתאם הרוטור מאז כישלון לעשות זאת עלולה לגרום לגז באחד משלבי צנטריפוגה של פרוטוקול הטיהור. </li></ol></li><li style=";text-align:right;direction:rtl"> הוסף את המספר הדרוש של 1,000 μl ו -200 μl מסנן-טיפים לתחנת העבודה, ולמלא את בקבוקי חיץ המסופקים עם הנפח הנדרש לאחסון. הערה: כל המאגרים הנדרשים לפרוטוקול טיהור זה (AL, AW1, Aw2, וAE) נמצאים בתוך ערכת QIAamp DNA הצואה Mini. המשתמש צריך לספק אתנול 100% מה שצריך לbu AWffers וכפתרון המשמש בתהליך הטיהור. </li><li style=";text-align:right;direction:rtl"> הוסף את הנפח הנדרש של פתרון K proteinase סיפק לתוך צינור microcentrifuge סטרילי 1.5 מ&quot;ל ומניח אותו לחריץ בתחנת העבודה. כמו כן, להוסיף את המספר הנדרש (שווה למספר של דגימות מטוהרות) של RB צינורות מדגם microcentrifuge בטוח נעילת 2 מיליליטר לסעיף שייקר של תחנת העבודה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא שהמכסים של צינורות המדגם ממוקמים באופן מאובטח לחריצים המתאימים בתחנת העבודה, מאז כישלון לעשות זאת תגרום לשגיאה כאשר המכונה בתחילה סורק את תחנת העבודה כדי לוודא שכל הפלסטיק והנוזלים הדרושים זמינים לבקש לרוץ. </li></ol></li><li style=";text-align:right;direction:rtl"> שימוש במסך המגע בתחנת העבודה, בחר בצואת DNA - צואה אדם - פתוגן איתור פרוטוקול, וקרא במסכים הבאים כדי להבטיח שתחנת העבודה הייתה טעונה בצורה נכונה. ברגע שכל מסכי הבדיקה מועברים, בחר התחל לרוץ בפרוטוקול זה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם חילוץ DNA יותר מ 12 דגימות, להתחיל בתהליך הומוגניזציה (שלב 1) לסדרה הבאה של דגימות, מאז ריצה של 12 דגימות לוקחת ~ 72 דקות כדי להשלים בתחנת העבודה. </li></ol></li><li style=";text-align:right;direction:rtl"> הסר את הדגימות ממתאמי הרוטור, כובעם, ומקום ב -20 ° C עד צורך לניתוחים במורד הזרם שלאחר מכן. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בשלב זה, לשלב את purifications לשכפל 3 למדגם בודד (סכום כולל = 1 גרם ניתחו) באמצעות מערכת צנטריפוגה / מבוסס אידוי. מערבבים את משכפל מחדש elute לנפח סופי של של חיץ טריס-ETDA 100 μl. </li></ol></li></ol>

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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.

שיטת מיצוי DNA משמשת בוצעה הערכות חיידקים כוללת כמותיים ואיכותיות הקהילה לשתי דגימות הצואה והמלטה, התומך במדגם מנתח טבע תלוי בשיטות מיצוי DNA ראה בעבר 1,3,6. עבור שני דגימות צואה והמלטה, סדר ביצועים של שיטות מיצוי DNA היה שונה עבור כמותית (מכאני&gt; היברידי&gt; אנזימתי) ו(...

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

היעילות של פרוטוקולי מיצוי DNA יכולה להיות תלויה במידה רבה על שני הסוג של מדגם נחקר והסוגים של ניתוחים שבוצעו במורד הזרם. בהתחשב בכך שהשימוש בטכניקות חדשות ניתוח קהילת חיידקים (למשל, microbiomics, metagenomics) הופך נפוץ יותר במדעי החקלאות ואיכות הסביבה, ורבים דגימות סביבתיות בתוך דיסציפלינות אלה יכולים להיות physiochemically וmicrobiologically ייחודי (לדוגמא, צואה ודגימות חול / מצעים מ ספקטרום ייצור העופות), שיטות מיצוי DNA מתאימות ויעילות צריכה להיות שנבחרו בקפידה. לכן, שיטת מיצוי DNA ההיברידי אוטומטית-למחצה רומן פותחה במיוחד לשימוש עם דגימות ייצור עופות סביבתיים. שיטה זו היא שילוב של שני סוגים העיקריים של מיצוי DNA: מכאני והאנזימטית. צעד הומוגניזציה מכאנית אינטנסיבי דו-שלבים (באמצעות חרוז הכאה גיבש במיוחד עבור לסביבהדגימות טל) התווספה לתחילתה של השיטה &quot;תקן זהב&quot; האנזימטית מיצוי DNA לדגימות צואה כדי לשפר את ההסרה של חיידקים וDNA ממטריצת המדגם ולשפר את ההתאוששות של חברי קהילת חיידקים גראם-חיוביים. ברגע שחלק החילוץ האנזימטית של השיטה ההיברידית יזם, תהליך הטיהור שנותר היה אוטומטי באמצעות תחנת עבודה רובוטית כדי להגדיל את התפוקה ולהפחית מדגם מדגם שגיאת עיבוד. בהשוואה לשיטות מיצוי DNA המכנית והאנזימטית הקפדניות, השיטה ההיברידית הרומן הזה סיפק את הביצועים הטובים ביותר בשילוב כולל כאשר שוקלים כמותי (באמצעות 16S rRNA qPCR) ואיכותי (באמצעות microbiomics) ערכות של קהילות חיידקים הכוללת עת עיבוד צואת עופות ודגימות חול .

במחקר זה, גללי צואה טריים ודגימות חול שהתאוששו מבית מסחרי פטם (~ 25,000 ציפורים) בדרום-מערב ארה&quot;ב. הפטמים (גאלוס גאלוס) היו קוב-500 חוצים, והם היו בני 59 ימים בזמן הדגימה. דגימות צואה וחול טריות היו התאוששו מארבעה אזורים שונים בתוך הבית (ליד קירור כרית, בקרבת קווי Waterer / ?...

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.

הפקת DNA שיטה היברידית להערכה איכותית וכמותית של הקהילות בקטריאלי מדוגמאות ייצור עופות

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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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.

טיהור פלסמיד דנ&quot;א מ מושבות חיידקים באמצעות Miniprep Qiagen 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 ...

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.

השימוש Rotorod כשיטה לניתוח איכותי של הליכה עכברוש

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.

הפקת DNA של 0.22 מיקרומטר מסננים Sterivex ו כלוריד צסיום צפיפות צנטריפוגה Gradient

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.

הפקת DNA גבוהה מולקולרית משקל הגנום מקרקעות ו משקעים

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.

הפקת DNA מחומר פרפין Embedded עבור ניתוח גנטי epigenetic

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.

הפקת חומצות גרעין יעיל 16S rRNA ג&#39;ין רצף עבור בקטריאלי קהילת אפיון

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.

בידוד סינון של חומצות גרעין: שיטת הפקת DNA פשוטה ומהירה

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).

-Cut ההיברידי: שיטת חתך משופרת עבור דוגמאות רקמות צמח סרבן

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;

ניתוח קוואזי-metagenomic של סלמונלה מזון, סביבתית דגימות

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

הערכה של ייצור סידרופור מבודד 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.

מחבר זרקור: כימות סידרופורים ופיוצ'לין לבקרת זיהומים 

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

ניתוח איכותני וכמותי של ייצור סידרופור מ 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 ...