食品和环境样品中沙门氏菌的准总体基因体分析
Summary
在这里, 我们提出了一个协议, 准备从食物和环境微生物群 DNA 样本, 以协同检测和子类型沙门氏菌通过 quasimetagenomic 测序。结合使用培养富集、免疫磁分离 (IMS) 和多重置换扩增 (MDA), 可有效地将沙门氏菌基因组 DNA 从食物和环境样本中浓缩。
Abstract
准宏基因组测序是指基于测序分析的食品和环境样品的改性微生物群。在本协议中, 微生物菌群的改性是为了集中于目标食源性致病菌污染物的基因组 DNA, 以便在单个工作流程中检测和子类型病原体。在这里, 我们解释并演示了样品制备步骤, 对代表性食品和环境样品中沙门氏菌起因的准宏基因组分析, 包括苜蓿芽、黑胡椒粉、地牛肉、鸡胸肉和环境拭子。样本首先受到沙门氏菌的培养富集的缩短和可调的持续时间 (4–24 h)。然后通过免疫磁分离 (IMS) 选择性地从浓缩培养中捕获沙门氏菌细胞。最后, 进行多位移扩增 (MDA), 以放大来自 IMS 捕获细胞的 DNA。该协议的 DNA 输出可以通过高通量测序平台进行排序。可进行定量 PCR 分析, 以取代沙门氏菌检测的测序, 或在测序前评估沙门氏菌DNA 浓度。
Introduction
宏基因组测序理论允许对食源性病原体进行协同检测和子类型。然而, 食品微生物群的直接测序对病原体分析提出了挑战。首先, 食源性病原体经常存在于食物样本的低水平。大多数商业上可用的快速检测方法仍然需要 8–48 h 培养, 以丰富病原细胞到可检测的1级。其次, 许多食物含有丰富的菌群细胞和/或食物 dna, 使食源性病原体 dna 成为食物宏基因组的一小部分, 并且是通过直接总体基因体测序检测和子类型的难以捉摸的目标。
据报道, 食品微生物群的改性允许大量食源性病原体 DNA 的浓度, 以方便测序检测滋贺毒素产生的大肠杆菌2、3和沙门氏菌起因4。由于改性食品微生物群仍然是不同微生物种类的混合物, 它们的测序被称为准总体基因体分析4。微生物菌群的改性可通过培养富集单独2、3或与免疫磁分离 (IMS) 和多位移扩增 (MDA)4、5相结合进行。IMS 可以通过抗体涂层磁珠选择性地从浓缩培养中捕获病原细胞。MDA 可以通过高效的ɸ29 dna 聚合酶6生成足够数量的基因组 DNA 进行测序。IMS-MDA 允许从临床样本7的独立于文化的病原体检测, 并缩短食品样本4中沙门氏菌的准总体基因体检测和子类型的培养富集。
该方法的总体目标是从食物样本中制备准总体基因体 DNA, 以使沙门氏菌基因组 dna 的靶向浓度和随后的检测和子类型沙门氏菌污染物的测序。与沙门氏菌检测8、9和子类型10的标准方法相比, quasimetagenomic 方法可以大大缩短污染食品和环境样品的周转时间, 从而病原体的分子亚型通过将两个典型的分离分析统一为一个工作流。这种方法特别适用于诸如食源性暴发反应和其他追溯调查, 在这种情况下, 除了病原体检测和快速分析周转, 还需要强健的病原体子类型。
Protocol
1. 样品制备 注: 根据美国农业部食品安全和检验服务部 (USDA-FSIS)11的微生物学实验室指南 (MLG) 和美国食品和细菌分析手册 (BAM), 准备食品样品以进行预浓缩。药物管理局 (FDA)12。 无菌将 25 g 部分的食物样本, 如黑胡椒, 鸡胸肉, 牛肉, 苜蓿芽或环境拭子放入一个无菌实验室搅拌机袋与内置过滤器。无菌用浓缩汤 (拉帕波特-绿, RV) 滋润海?…
Representative Results
在 quasimetagenomic 测序之前, 可通过 fluorospectrometer (表 1) 评估 IMS MDA 产品的总数量和纯度。 浓缩时间 (h) Ct 值 浓度 (ng/ul) 纯度 (260/280) 品牌 A 4 …
Discussion
由于食品和环境样品中沙门氏菌的丰度和均质性存在, 在沙门氏菌检测和子类型的同时, 还需要对其进行培养;因此, 这是该议定书的一个关键步骤。为了确定最佳条件以增加沙门氏菌相对于样本背景菌群的丰度, 可以对不同的富集介质进行特定样品的评估。根据 MLG 和 BAM 的规定, 如 RV 汤和非选择性培养基, 如缓冲的蛋白胨水和乳糖汤, 可用于食品样本中的沙门氏菌浓缩。?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者希望感谢格鲁吉亚大学的马克哈里森和格温, 为这项研究提供了细菌菌株和其他支持。
Materials
| Laboratory blender bag w/filter | VWR | 10048-886 | |
| Buffered peptone water | Oxoid Micorbiology Products | CM0509 | |
| Rappaport Vassiliadis broth | Neogen Acumedia | 7730A | |
| Polysorbate 20 | Millipore Sigma | P9416 | Tween 20 |
| Stomacher blender | Seward | 30010108 | |
| Centrifuge | Fisher Scientific | 75005194 | |
| 50ml Centrifuge tubes | Fisher Scientific | 05-539-6 | |
| Thermal Cycler | Techne Prime | EW-93945-13 | |
| StepOne Real-Time Thermal cycler | Applied Biosystems | 4.76357 | |
| AMPure XP beads | Beckman Coulter | A63881 | PCR purification beads; mix well before use; store at 4C |
| Nextera XT library prep kit | Illumina | FC-131-1024 | Store at -80C |
| MinIon library prep kit | Oxford Nanopore | SQK-LSK108 | Store at -80C |
| NanoDrop | Thermo Scientific | ND-2000 | |
| Dynabead Anti-Salmonella beads | Applied Biosystems | 71002 | Vortex well prior to use |
| Illustra GenomiPhi V2 DNA amplification kit (MDA kit) -Sample buffer -Reaction buffer -Enzyme mix |
GE Healthcare | 25-6600-30 | Store at -80C |
| HulaMixer | Invitrogen | 15920D | |
| DynaMag magnetic rack | Invitrogen | 12321D | |
| TaqMan Universal PCR mastermix | Applied Biosystems | 4304437 | Mix well before use; store at 4C |
| Microfuge | Fisher Scientific | 05-090-100 |
Referências
- Valderrama, W. B., Dudley, E. G., Doores, S., Cutter, C. N. Commercially Available Rapid Methods for Detection of Selected Food-borne Pathogens. Critical Reviews in Food Science and Nutrition. 56 (9), 1519-1531 (2016).
- Leonard, S. R., Mammel, M. K., Lacher, D. W., Elkins, C. A. Application of metagenomic sequencing to food safety: detection of Shiga Toxin-producing Escherichia coli on fresh bagged spinach. Applied and Environmental Microbiology. 81 (23), 8183-8191 (2015).
- Leonard, S. R., Mammel, M. K., Lacher, D. W., Elkins, C. A. Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing. PLoS One. 11 (12), e0167870 (2016).
- Hyeon, J. Y., et al. Quasi-metagenomics and realtime sequencing aided detection and subtyping of Salmonella enterica from food samples. Applied and Environmental Microbiology. , (2017).
- Hyeon, J. Y., Deng, X. Rapid detection of Salmonella in raw chicken breast using real-time PCR combined with immunomagnetic separation and whole genome amplification. Food Microbiology. 63, 111-116 (2017).
- Hosono, S., et al. Unbiased whole-genome amplification directly from clinical samples. Genome Research. 13 (5), 954-964 (2003).
- Seth-Smith, H. M., et al. Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture. Genome Research. 23 (5), 855-866 (2013).
- Jacobson, A. P., Gill, V. S., Irvin, K. A., Wang, H., Hammack, T. S. Evaluation of methods to prepare samples of leafy green vegetables for preenrichment with the Bacteriological Analytical Manual Salmonella culture method. Journal of Food Protection. 75 (2), 400-404 (2012).
- Jacobson, A. P., Hammack, T. S., Andrews, W. H. Evaluation of sample preparation methods for the isolation of Salmonella from alfalfa and mung bean seeds with the Bacteriological Analytical Manual’s Salmonella culture method. Journal of AOAC International. 91 (5), 1083-1089 (2008).
- Deng, X., et al. Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis. Journal of Clinical Microbiology. 53 (1), 212-218 (2015).
- . Microbiology Laboratory Guidebook Available from: https://www.fsis.usda.gov/wps/portal/fsis/topics/science/laboratories-and-procedures/guidebooks-and-methods/microbiology-laboratory-guidebook/microbiology-laboratory-guidebook (2018)
- . Bacteriological Analytical Manual (BAM) Chapter 5: Salmonella Available from: https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm (2018)
- Malorny, B., et al. Diagnostic real-time PCR for detection of Salmonella in food. Applied and Environmental Microbiology. 70 (12), 7046-7052 (2004).
- June, G. A., Sherrod, P. S., Hammack, T. S., Amaguana, R. M., Andrews, W. H. Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella from raw flesh and other highly contaminated foods: precollaborative study. Journal of AOAC International. 78 (2), 375-380 (1995).
- Hammack, T. S., Amaguana, R. M., June, G. A., Sherrod, P. S., Andrews, W. H. Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella spp. from foods with a low microbial load. Journal of Food Protection. 62 (1), 16-21 (1999).
- Wood, D. E., Salzberg, S. L. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biology. 15 (3), R46 (2014).
- Davis, S., Pettengill, J. B., Luo, Y., Payne, J., Shpuntoff, A., Rand, H., Strain, E. CFSAN SNP Pipeline: an automated method for constructing SNP matrices from next-generation sequence data. PeerJ Computer Science. 1 (e20), (2015).
- Zhang, S., et al. Salmonella serotype determination utilizing high-throughput genome sequencing data. Journal of Clinical Microbiology. 53 (5), 1685-1692 (2015).
- Rodrigue, S., et al. Whole genome amplification and de novo assembly of single bacterial cells. PLoS One. 4 (9), e6864 (2009).
- Ramamurthy, T., Ghosh, A., Pazhani, G. P., Shinoda, S. Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria. Front Public Health. 2, 103 (2014).
Citar este artigo
Hyeon, J., Mann, D. A., Townsend, A. M., Deng, X. Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples. J. Vis. Exp. (140), e58612, doi:10.3791/58612 (2018).