가금류 생산 샘플에서 세균 공동체의 정성 및 정량 평가를위한 하이브리드 DNA 추출 방법
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
DNA 추출 프로토콜의 효능을 조사되는 시료의 종류와 분석을 수행 하류의 두 종류에 따라 크게 좌우 될 수있다. 새로운 세균 지역 사회 분석 기술 (예를 들어, microbiomics, 메타 지노믹스)의 사용이 분야 내에서 농업과 환경 과학 및 많은 환경 시료에서 더욱 확산되고 있음을 감안하면 물리 화학적 및 미생물 (독특한에서 예를 들어, 배설물과 쓰레기 / 침구 샘플이 될 수 있습니다 가금류 생산 스펙트럼), 적절하고 효과적인 DNA 추출 방법이 신중하게 선택되어야한다. 따라서, 신규 한 반자동 하이브리드 DNA 추출법 환경 가금류 생산 샘플을 사용하기 위해 특별히 개발되었다. 기계적 및 효소 :이 방법은 DNA 추출의 두 가지 주요 유형의 조합이다. 두 단계의 격렬한 기계적 균질화 공정 (비드를 사용하여 고해 구체적 environmen 제형분변 샘플 시료 매트릭스로부터 박테리아를 제거하고 DNA를 향상과 그람 양성 세균 커뮤니티 회원 복구를 향상시키기 위해 탈 샘플)을 "황금 표준"효소 DNA 추출법의 시작 부분에 첨가 하였다. 하이브리드 방식의 효소 추출 부 개시하고 나면, 나머지 정제 공정은 시료 처리량을 증가시키고 시료 처리 오류를 감소시키는 로봇 자동화 된 워크 스테이션을 사용 하였다. 가금류의 배설물과 쓰레기 샘플을 처리 할 때 엄격한 기계 및 효소 DNA 추출 방법에 비해,이 소설 하이브리드 방법은 정량적 고려 전반적으로 최상의 성능을 결합 총 세균 지역 사회의 추정 (microbiomics 사용) (16S rRNA 유전자 qPCR에 사용)과 정성을 제공 .
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
사용 된 DNA 추출법 샘플 이전 1,3,6- 본 DNA 추출법의 종속 특성을 분석지지 모두 분변 샘플 및 깔짚 정량적 총 세균 커뮤니티 추정치들을 달성. 배설물과 쓰레기 샘플 모두를 들어, DNA 추출 방법의 성능의 순서는 정량적 (기계> 하이브리드> 효소)과 정성 (효소> 하이브리드> 기계) 총 세균 지역 사회 추정치 달랐다. 하이브리드 방법은 높은 정량 또는 정성적인 추정치를 생성하지 않는…
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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