下一代 16S rRNA 扩增子测序的蜱菌群鉴定
Summary
在这里, 我们提出了下一代测序协议 16S rRNA 测序, 使识别和鉴定微生物群落在载体内。该方法包括 DNA 的提取、扩增和条形码样品的 PCR、序列化和生物信息学, 将序列数据与系统进化信息相匹配。
Abstract
近几十年来, 媒介传播的疾病以惊人的速度重新出现和扩大, 在全世界造成相当大的发病率和死亡率。大多数这些疾病缺乏有效和广泛使用的疫苗, 这就有必要制定新的疾病缓解战略。为此, 一个有希望的疾病控制途径是针对媒介微生物群, 即居住在载体中的微生物群落。载体菌群在病原体动力学中起着举足轻重的作用, 微生物群的操控导致了少数媒介传播疾病的媒介丰度或病原体传播。然而, 将这些发现转化为疾病控制的应用需要对媒介微生物生态学的透彻理解, 这在历史上受此领域技术不足的限制。下一代测序方法的出现使不同微生物群落的快速、高度平行测序得以实现。以高度保守的 16S rRNA 基因为靶, 促进了在不同生态环境和实验条件下, 在载体内的微生物的刻画。这项技术包括扩增 16S rRNA 基因, 通过 PCR 样品条形码, 将样品加载到流细胞进行测序, 以及生物信息学方法将序列数据与系统进化信息相匹配。对于大量复制, 通常可以通过这种方法实现物种或属类级别的识别, 从而规避传统培养、显微学或组织学染色中低检测、分辨和输出的挑战。技术。因此, 该方法非常适合于不同条件下的载体微生物的表征, 但目前尚不能提供微生物功能、载体内位置或抗生素治疗反应的信息。总体而言, 16S 的下一代测序技术是更好地了解媒介微生物在疾病动力学中的身份和作用的有力手段。
Introduction
近几十年来, 媒介传播疾病的死灰复燃和传播对全球人类和野生动物的健康构成严重威胁。对大多数这些疾病缺乏有效的疫苗, 控制努力受到媒介和媒介宿主相互作用的复杂生物学特性的阻碍。了解在病原体传播媒介中微生物相互作用的作用, 可以为规避这些挑战的新策略的发展提供帮助。特别是, 与载体相关的微生物 commensals、共生和病原体之间的相互作用, 称为微生物, 可能对病原体传播有重要的影响。现在压倒性的证据支持这一断言, 举例说明向量菌群与疟疾、Zika 病毒和莱姆病1、2、3等疾病的能力之间的联系。然而, 将这些发现转化为疾病控制策略需要对向量微生物的结构、功能和起源有更详细的了解。在不同的生态学和实验条件下, 载体微生物群落的识别和表征是这一领域前进的重要途径。
通过利用西部黑腿蜱、 Ixodes pacificus、莱姆病病原体螺旋体螺旋体的载体, 在这里提供了一种鉴定病原体微生物的方法。虽然蜱比任何其他节肢动物更有人类病原体的种类, 但对蜱微生物4的生物学和群落生态学的了解相对较少。很明显, 蜱港有多种病毒, 细菌, 真菌和原生动物, 包括 commensals, endosymbionts 和瞬态微生物居民5,4。先前的工作表明, Ixodes微生物与地理, 物种, 性别, 生命阶段, 和血膳食来源6,7,8的强烈变化。然而, 这种变化所依据的机制仍不得而知, 并对这些微生物群落的起源和组装进行更详细的调查。蜱可以通过垂直传播获得微生物, 与寄主接触, 通过气孔、嘴巴和肛门孔9吸收环境。了解蜱菌群最初形成和发展的因素, 特别是垂直和环境传播的相对贡献, 对于了解蜱的自然形态和变化非常重要。微生物多样性以及这些群落在病原体传播过程中的相互作用, 并可能应用于疾病或媒介控制。
强大的分子技术, 如下一代测序, 现在已经存在, 以确定微生物群落, 并可用于表征向量微生物在不同的环境或实验条件。在这些高通量测序方法出现之前, 微生物的鉴定主要依靠显微学和文化。显微术是一种快速、简便的技术, 其识别微生物的形态学方法固有的主观性和粗糙性, 且灵敏度低, 检测10。基于培养基的方法广泛用于微生物鉴定, 可用于测定微生物对药物治疗的敏感性11。然而, 这种方法也有低灵敏度, 因为它已经估计, 2% 的环境微生物可以培养在实验室设置12。组织学染色方法也被用来检测和本地化的特定微生物的载体, 使调查的不同分类群分布在蜱, 并研究假说的微生物相互作用。然而, 在选择合适的污渍之前, 必须事先了解微生物身份, 使这种方法不适合于微生物的鉴定和鉴定。此外, 组织学染色是一个高度时间密集, 费力的过程, 并没有很好的规模大样本大小。传统的分子方法, 如桑格测序同样有限的敏感性和检测不同的微生物群落。
下一代测序允许从大量样品中快速识别微生物。标准标记基因和参考数据库的存在进一步促进了分类分辨率的提高, 通常是属或物种水平。小亚基核糖体 rna 经常被用于达到这个目标, 16S rRNA 是最普遍的由于存在保守和可变区域在基因之内, 允许创造普遍引物以唯一的 amplicons 为每个细菌种类13,14。本报告详细介绍了通过 16S rRNA 下一代测序在蜱微生物群中识别分类的程序。特别是, 本议定书强调了为测序准备样品所涉及的步骤。提供了关于排序和生物信息学步骤的更广义的详细信息, 因为目前有各种测序平台和分析程序, 每个都有广泛的现有文档。这种下一代测序方法的总体可行性是通过将其应用于一个关键疾病载体内微生物群落组装的研究中得到证明的。
Protocol
1. 蜱收集和表面灭菌 通过在蜱相关的栖息地上拖动1米2白色布来收集刻度, 去除附着在寄主物种上的蜱, 或在实验室15、16中饲养蜱。使用细钳操纵蜱和储存在-80 摄氏度。 将蜱在各自的 PCR 管和去除表面污染物由涡流连续十五年代与 500 ul 过氧化氢 (H2O2), 70% 乙醇和 ddH2O。 将刻度放在新的 PCR 管中, 让?…
Representative Results
从三个单独的卵子离合器和两个环境暴露期, 0 和2周的土壤中, 对微生物序列进行了总共42个刻度。每个治疗组, 被认为是一个单一的离合器和曝光时间, 包含6-8 复制蜱样本。这些经过处理的蜱提取物加载到下一代排序器上, 产生了12885713配对端读取传递滤波器。此运行中包含的是从抽取步骤中产生的3个负控制项, 总共生成211214个读取 (包括在上一个计数中)。表 2…
Discussion
16S rRNA 的下一代测序已成为微生物鉴定的标准方法, 并使研究载体微生物如何影响病原体的传播。这里概述的协议详细介绍了利用此方法来调查莱姆病的一个载体pacificus的微生物群落组装。然而, 它可以很容易地用于研究其他蜱种或节肢动物的载体物种。
事实上, 微生物分析的 16S rRNA 测序法已广泛应用于研究微生物, 包括蚊子, psyllids, 和采蝇蝇29,</s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到国家科学基金会赠款的支持, 犍为县 (DEB #1427772, 1745411, 1750037)。
Materials
| Item | Name of Material/Equipment | Company | Catalog # |
| 1 | DNeasy Blood & Tissue Kit | Qiagen | 69504 |
| 2 | Qubit 4 Fluorometer | ThermoFisher Scientific | Q3326 |
| 3 | NanoDrop 8000 Spectrophotometer | ThermoFisher Scientific | ND-8000-GL |
| 4 | 2x KAPA HiFi HotStart ReadyMix | Kapa Biosystems | KK2501 |
| 5 | AMPure XP beads | Agen Court | A63880 |
| 6 | Magnetic Rack | ThermoFisher Scientific | MR02 |
| 6 | TE buffer | Teknova | T0223 |
| 7 | Nextera Index Kit | Illumina | FC-121-1011 |
| 8 | KAPA Library Quantification Kit | Roche | KK4824 |
| 9 | MiSeq System | Illumina | SY-410-1003 |
| 10 | MiSeq Reagent Kit v3 | Illumina | MS-102-3001 |
| 11 | 10 mM Tris-HCl with 0.1% Tween 20 | Teknova | T7724 |
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Cite This Article
Couper, L., Swei, A. Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing. J. Vis. Exp. (138), e58239, doi:10.3791/58239 (2018).