Een hybride DNA-extractie methode voor de kwalitatieve en kwantitatieve beoordeling van Bacteriële Gemeenschappen van pluimveeproductie Monsters
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
De doeltreffendheid van DNA-extractie protocollen kunnen zeer afhankelijk zowel van het type monster dat wordt onderzocht en de soorten stroomafwaartse analyses uitgevoerd. Gezien het feit dat het gebruik van nieuwe bacteriële gemeenschap analysetechnieken (bv microbiomics, metagenomica) wordt steeds meer voor in de landbouw- en milieuwetenschappen en veel milieu-monsters binnen deze disciplines kan fysiochemisch en microbiologisch unieke (bijvoorbeeld fecale en strooisel / beddengoed monsters uit zijn de pluimveehouderij spectrum), moeten passende en effectieve DNA-extractie methoden zorgvuldig worden gekozen. Daarom werd een nieuwe semi-automatische hybride DNA extractiemethode speciaal ontwikkeld voor milieu pluimveehouderij monsters. Deze methode is een combinatie van de twee belangrijkste soorten DNA-extractie: mechanische en enzymatische. Een twee-staps intense mechanische homogenisatiestap (met kraal-beating speciaal ontwikkeld voor environmental monsters) werd toegevoegd aan het begin van de "gouden standaard" enzymatische DNA extractiemethode voor fecale monsters naar de verwijdering van bacteriën en DNA uit het monster matrix verhogen en de terugwinning van Gram-positieve bacteriën leden van de gemeenschap. Nadat de enzymatische extractie gedeelte van de hybride methode werd ingeleid, werd het resterende zuiveringswerkwijze geautomatiseerd met een robot werkstation monsterdoorvoer verhogen en monster verwerkingsfout verminderen. In vergelijking met de strenge mechanische en enzymatische DNA-extractie methoden, deze nieuwe hybride methode voorwaarde dat de beste totale gecombineerde prestaties bij het overwegen van kwantitatieve (met behulp van 16S rRNA qPCR) en kwalitatieve (met behulp van microbiomics) een raming van de totale bacteriële gemeenschappen bij het verwerken van gevogelte uitwerpselen en zwerfvuil monsters .
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
De DNA-extractie methode geschiedt de kwantitatieve en kwalitatieve totale bacteriële gemeenschap schattingen voor zowel de fecale en zwerfvuil monsters, het ondersteunen van de analyses van monsters afhankelijke karakter van de DNA-extractie methoden eerder 1,3,6 gezien. Voor zowel de fecale en zwerfvuil monsters, de volgorde van uitvoering van de DNA-extractie methoden was anders voor de kwantitatieve (Mechanische> Hybrid> Enzymatic) en de kwalitatieve (Enzymatic> Hybrid> Mechanical) totale bac…
Divulgations
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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