هجين طريقة استخراج الحمض النووي للتقييم النوعي والكمي للمجتمعات البكتيرية من عينات الدواجن الإنتاج
Summary
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.
Abstract
فعالية بروتوكولات استخراج الحمض النووي يمكن أن تعتمد بشكل كبير على كل من نوع العينة التي يجري التحقيق فيها وأنواع من التحليلات المصب تنفيذها. يمكن النظر إلى أن استخدام تقنيات تحليل المجتمع البكتيرية الجديدة (على سبيل المثال، microbiomics، metagenomics) أصبحت أكثر انتشارا في مجال العلوم الزراعية والبيئية والعينات البيئية العديدة ضمن هذه التخصصات تكون physiochemically وفريدة من نوعها الميكروبيولوجية (على سبيل المثال، البراز وعينات القمامة / الفراش من الدواجن الطيف الإنتاج)، بحاجة الى طرق استخراج الحمض النووي المناسبة والفعالة ليتم اختياره بعناية. لذلك، تم تطوير DNA هجين طريقة استخراج رواية شبه الآلي خصيصا للاستخدام مع عينات إنتاج الدواجن البيئي. هذا الأسلوب هو مزيج من اثنين من أنواع رئيسية من استخراج الحمض النووي: الميكانيكية والأنزيمية. ومكثفة خطوة التجانس الميكانيكية من خطوتين (باستخدام حبة الضرب وضعت خصيصا للبالبيءهتمت إضافة عينات التل) إلى بداية "المعيار الذهبي" الأنزيمية طريقة استخراج الحمض النووي لعينات البراز لتعزيز إزالة البكتيريا وDNA من عينة مصفوفة وتحسين الانتعاش من أعضاء غرام إيجابي المجتمع البكتيري. مرة واحدة وقد بدأ الجزء استخراج الأنزيمي من الأسلوب الهجين، تم أتمتة عملية تنقية المتبقية باستخدام محطة عمل الروبوتية لزيادة الإنتاجية وتقليل العينة خطأ معالجة العينة. وبالمقارنة مع طرق استخراج الحمض النووي صارمة الميكانيكية والأنزيمية، وقدم هذه الطريقة الهجينة الرواية على أفضل أداء مجتمعة عموما عند النظر في الكمية (باستخدام 16S الرنا الريباسي QPCR) والنوعي (باستخدام microbiomics) تقديرات من إجمالي المجتمعات البكتيرية عند معالجة البراز الدواجن والعينات القمامة .
Introduction
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 “gold standard” 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 “gold standard” 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.
Protocol
Representative Results
Discussion
طريقة استخراج الحمض النووي المستخدمة تنفذ إجمالي تقديرات المجتمع البكتيرية الكمية والنوعية لكل من عينات برازية والقمامة، ودعم تحاليل العينة طبيعة تعتمد أساليب الاستخراج DNA كما كان في السابق 1،3،6. لكل من عينات برازية والقمامة، وكان ترتيب أداء أساليب الاستخراج…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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 “Pathogen Reduction and Processing Parameters in Poultry Processing Systems” #6612-41420-017-00 and “Molecular Approaches for the Characterization of Foodborne Pathogens in Poultry” #6612-32000-059-00.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Lysing Matrix E tube | MPBio | 6914-050 | Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1000 tubes) |
| Sodium Phosphate Solution | MPBio | 6570-205 | Can be purchased individually, or also contained within the FastDNA Spin Kit for Feces (Cat. No. 116570200) |
| PLS Buffer | MPBio | 6570-201 | |
| Buffer ASL (560 ml) | Qiagen | 19082 | |
| FastPrep 24 homogenizer | MPBio | 116004500 | 48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step |
| QIAamp DNA Stool Mini Kit | Qiagen | 51504 | |
| QIAcube24 (110V) | Qiagen | 9001292 | 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. |
| Filter-Tips, 1000 ml (1024) | Qiagen | 990352 | |
| Filter-Tips, 200 ml (1024) | Qiagen | 990332 | |
| QIAcube Rotor Adapters (10 x 24) | Qiagen | 990394 | For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative. It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690 |
| Sample Tubes RB (2 ml) | Qiagen | 990381 | Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352 |
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