Une méthode hybride d'extraction d'ADN pour l'évaluation qualitative et quantitative des communautés bactériennes de la volaille Échantillons de production
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
L'efficacité des protocoles d'extraction d'ADN peut être très dépend à la fois du type d'échantillon objet de l'enquête et les types d'analyses effectuées en aval. Considérant que l'utilisation de nouvelles techniques d'analyse de la communauté bactérienne (par exemple, microbiomique, métagénomique) est de plus en plus répandue dans les sciences agricoles et environnementales et de nombreux échantillons de l'environnement au sein de ces disciplines peut être physiochimique et microbiologiquement uniques (par exemple, les selles et les échantillons litière / literie de le spectre de la production de volaille), les méthodes d'extraction d'ADN appropriées et efficaces doivent être choisis avec soin. Par conséquent, une méthode d'extraction d'ADN hybride roman semi-automatique a été développé spécifiquement pour une utilisation avec des échantillons de production de volaille de l'environnement. Cette méthode est une combinaison des deux types principaux de: extraction d'ADN enzymatique et mécanique. A deux étapes intense étape d'homogénéisation mécanique (en utilisant cordon de battre spécialement formulé pour enviTal échantillons) ont été ajoutés au début du procédé d'extraction d'ADN de «gold standard» enzymatique des échantillons fécaux pour améliorer l'élimination des bactéries et de l'ADN à partir de la matrice de l'échantillon et d'améliorer la récupération d'éléments à Gram positif communauté bactérienne. Une fois que la portion d'extraction enzymatique du procédé hybride a été lancé, le procédé de purification restant a été automatisé en utilisant un poste de travail robotique pour augmenter et diminuer le débit d'échantillons échantillon erreur de traitement. En comparaison avec les méthodes mécaniques et enzymatiques strictes extraction de l'ADN, cette méthode hybride roman fourni la meilleure performance globale combinée lors de l'examen quantitatif (en utilisant l'ARNr 16S qPCR) et qualitative (en utilisant microbiomique) des estimations des communautés bactériennes totales lors du traitement des excréments de volaille et des échantillons de litière .
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
La méthode d'extraction d'ADN utilisée effectué les estimations totales communautaires bactériennes quantitatifs et qualitatifs pour les deux échantillons de matières fécales et de la litière, supportant l'échantillon analyses nature dépendante des méthodes d'extraction d'ADN vu précédemment 1,3,6. Pour les deux échantillons de matières fécales et de la litière, l'ordre d'exécution des méthodes d'extraction d'ADN était différent pour le quantitatif (mé…
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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