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.
डीएनए निष्कर्षण प्रोटोकॉल की प्रभावकारिता जांच की जा रही नमूना के प्रकार और प्रदर्शन किया बहाव के विश्लेषण के प्रकार दोनों पर अत्यधिक निर्भर हो सकता है। नई बैक्टीरियल समुदाय विश्लेषण तकनीक (जैसे, microbiomics, metagenomics) के उपयोग से इन विषयों के कृषि और पर्यावरण विज्ञान और कई पर्यावरण के नमूनों में अधिक प्रचलित होता जा रहा है यह देखते हुए कि physiochemically और microbiologically (अद्वितीय से जैसे, मल और कूड़े / बिस्तर नमूने किया जा सकता है पोल्ट्री उत्पादन स्पेक्ट्रम), उपयुक्त और प्रभावी डीएनए निष्कर्षण तरीकों सावधानी से चुना जाना चाहिए। इसलिए, एक उपन्यास अर्द्ध स्वचालित संकर डीएनए निष्कर्षण विधि पर्यावरण पोल्ट्री उत्पादन के नमूनों के साथ उपयोग के लिए विशेष रूप से विकसित किया गया था। यांत्रिक और enzymatic: इस विधि में डीएनए निष्कर्षण के दो प्रमुख प्रकार का एक संयोजन है। एक दो कदम तीव्र यांत्रिक homogenization के कदम (का उपयोग कर मनका पिटाई विशेष रूप से पर्यावरणीय के लिए तैयारमल के नमूने नमूना मैट्रिक्स से बैक्टीरिया के हटाने और डीएनए को बढ़ाने और ग्राम पॉजिटिव बैक्टीरिया समुदाय के सदस्यों की वसूली में सुधार करने के लिए ताल नमूने) "सोने के मानक 'एंजाइमी डीएनए निष्कर्षण विधि की शुरुआत करने के लिए जोड़ा गया है। संकर विधि के enzymatic निकासी भाग शुरू किया गया था एक बार, शेष शुद्धिकरण की प्रक्रिया नमूना throughput बढ़ाने और नमूना प्रसंस्करण त्रुटि को कम करने के लिए एक रोबोट कार्य केंद्र का उपयोग कर स्वचालित किया गया था। पोल्ट्री मल और कूड़े के नमूने प्रसंस्करण जब सख्त यांत्रिक और enzymatic डीएनए निष्कर्षण तरीकों की तुलना में, इस उपन्यास संकर विधि मात्रात्मक जब विचार सबसे अच्छा समग्र संयुक्त प्रदर्शन कुल बैक्टीरियल समुदायों का अनुमान (microbiomics का प्रयोग करके) (-16 rRNA qPCR का उपयोग) और गुणात्मक प्रदान की ।
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.
इस्तेमाल किया डीएनए निष्कर्षण विधि नमूना पहले से 1,3,6 देखा डीएनए निष्कर्षण तरीकों के आश्रित प्रकृति का विश्लेषण करती है समर्थन कर रहा है, दोनों मल और कूड़े के नमूने के लिए मात्रात्मक और गुणात्मक कु?…
The authors have nothing to disclose.
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.
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 |