真菌 DNA 分析用职业空气标本的收集与提取
Summary
确定环境中的真菌多样性是在职业健康研究中用来确定健康危害的一种方法。该协议描述了从职业空气样本中提取的 DNA, 用于对真菌的区域进行扩增和测序。这种方法可以检测到许多真菌物种, 这种方法可被传统的评估方式忽视。
Abstract
在职业环境中识别真菌暴露的传统方法, 如以文化和显微镜为基础的方法, 有若干限制, 导致许多物种被排除在外。过去两年来, 该领域的进展促使职业健康研究人员转向以分子为基础的方法来识别真菌危害。这些方法导致了在室内和职业环境中发现的许多物种没有使用传统方法检测到。本议定书详细介绍了通过基因组 DNA 提取、扩增、测序以及真菌内转录间隔 (其) 区域的分类学鉴定来确定空气样品中真菌多样性的方法。它的测序结果是检测到许多真菌物种, 它们要么没有被发现, 要么难以用文化或显微镜识别到物种水平。虽然这些方法不提供真菌负担的定量措施, 但它们提供了一种新的危险识别方法, 可用于确定一个职业环境中的总体物种丰富度和多样性。
Introduction
室内和职业环境中的真菌暴露可能导致呼吸道疾病, 包括过敏性敏感和哮喘1。真菌危害的鉴定对于评估风险和防止工人暴露是很重要的。这些真菌危害可能是室内污染、室外空气侵入或环境干扰造成的, 导致将真菌材料输送到工人所在的地区2。评价真菌暴露的方法包括可行的培养基取样和真菌孢子的显微鉴定。这些方法有几个局限性, 往往忽略了许多真菌物种, 可能导致整个真菌负担3。以文化为基础的方法只能区分那些可以在营养培养基上培养的可行的真菌有机体。通过显微镜识别真菌孢子到物种水平可以混淆的孢子分享相似的形态。这两种方法都高度依赖于 mycologists 来分析和鉴定真菌种类, 其中许多残留不明。
为了改进现有的职业危害识别和暴露评估方法, 许多研究人员转而采用分子技术。以测序为基础的评估室内和职业环境中微生物多样性的方法表明, 与显微学和可行文化等方法相比, 所遇到的真菌种类更广泛3,4 ,5。本文介绍了职业环境的空气取样和基因组 DNA 的提取, 以确定潜在的真菌危害。危险识别是通过序列化核核糖体内部转录间隔或其在真菌中高度可变的区域来完成的, 并且通常用于区分真菌种类6,7, 8,9。在职业环境中发现的许多物种, 例如属于 Basidiomycota 的一些物种, 在可行的文化中是无法辨认的, 很难在显微镜下加以区分。这些真菌已被观察到在室内和职业环境中的高相对丰度评估的排序真菌其区域3,4,10。它的测序为在室内和职业环境中遇到的真菌多样性提供了更多的知识。
此处描述的协议详细介绍了用于收集、提取和放大真菌的区域从 bioaerosols 进行序列分析的方法。该方法利用国家职业安全与健康研究所 (NIOSH) 两级旋风气溶胶取样器收集空气中的微粒。该取样器是为了收集 bioaerosols 和分离可吸入 (≤4µm 空气动力学直径) 和不可吸入 (> 4 µm 空气动力学直径) 粒子, 它允许在室内环境中识别真菌有机体最可能由工作人员11吸入。其他空气取样器, 包括旋风取样机, 可在市场上提供, 可以在可吸入范围内 (< 4 µm) 中收集粒子, 方法是使用滤镜12,13。相比之下, NIOSH 两级旋风气溶胶取样器将基于其空气动力学直径的真菌分离成可一次性的聚丙烯管, 可以在下游应用中立即处理14。
本议定书详细介绍了提取基因组 DNA 和放大真菌的过程。所提出的提取方法专门用于从真菌和细菌中提取基因组 DNA, 因为许多商业试剂盒靶向哺乳动物细胞、细菌或特定的酵母15。本研究中使用的引物根据其1及其2区域4、5的总体覆盖率选择。这些区域的排序可以比较许多已存入的序列, 包括序列其1区域、2区域或其1及其2区域的顺序。用这些方法在室内环境中收集的空气样品的真菌多样性, 揭示了大量的序列放置在门 Ascomycota 和 Basidiomycota 以及其他序列属于较不占优势的真菌门, 如Zygomycota。使用这种方法确定的真菌序列的广泛多样性不会使用传统的危险识别方法, 如种植或显微镜来捕获。真菌的测序其区域提供了一种增强的方法来识别真菌的危害, 并允许更好地了解室内和职业真菌暴露。
Protocol
Representative Results
Discussion
使用基于排序的方法确定职业环境中的真菌多样性, 改善了真菌危害识别和暴露评估。使用这种方法, 可以检测到许多额外的真菌物种, 通常没有使用文化或显微镜的评估方法检测到。本文介绍了一种从职业和室内环境中取样 bioaerosols 的方法, 并从空气样本中提取基因组 DNA 进行扩增和测序。用这些方法测定真菌的多样性是高度依赖于: (1) 成功和完全提取的基因组 DNA 从空气样本, (2) 扩增的 gDNA 与引…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作部分是由 NIOSH 与 NIEHS (AES12007001-1-0-6) 之间的机构间协议支持的。
Materials
| NIOSH BC251 bioaerosol cyclone sampler | NIOSH | BC251 | The NIOSH sampler is not yet commercially available. Please contact William Lindsley, PhD (wlindsley@cdc.gov) for information on obtaining the NIOSH sampler |
| Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps | Fisher Scientific | 02-681-373 | 1.5 mL polypropylene microcentrifuge tubes for air sampling; screw top threading must match the threading of the NIOSH sampler |
| Falcon 15 mL Conical Centrifuge Tubes | Corning | 352096 | 15 mL polypropylene tubes for air sampling |
| Clean Room Vinyl Tape, Easy-Remove, 1/4" Width | McMaster-Carr | 76505A1 | sealing tape |
| Filter Cassette, Clear Styrene, 37 mm | SKC Inc. | 225-3LF | 3-piece sampling cassette (no filter). Contains: cassette base, extension cowl, cassette cap and inlet/outlet plugs |
| PTFE hydrophobic fluoropore membrane filters, 3.0 µm, 37 mm | EMD Millipore | FSLW03700 | Contains: 37 mm, 3.0 µm PTFE filters and support pads |
| Fisherbrand filter forceps | Fisher Scientific | 09-753-50 | filter forceps |
| Model 502 Precision PanaPress | PanaVise | 502 | pneumatic cassette press is constructed from this precision arbor press |
| Scotch Super 33+ vinyl electrical tape | McMaster-Carr | 76455A21 | 19 mm tape |
| Multi-purpose Calibration Jar, Large | SKC Inc. | 225-112 | calibration jar |
| Universal PCXR4 Sample Pump | SKC Inc. | 224-PCXR4 | sampling pump |
| Mass Flowmeter 4140 | TSI Inc. | 4140 | flow meter |
| Roche High Pure PCR Template Kit | Roche Diagnostics | 11796828001 | Kit used for genomic DNA extraction. Contains: Lysis buffer, Binding buffer, Proteinase K, Inhibitor removal buffer, Wash buffer, Elution buffer, Glass fiber filter tubes and 2 ml collection tubes |
| Fisherbrand 2 mL Reinforced Polypropylene Screw Cap Tubes with Caps | Fisher Scientific | 15340162 | 2 mL reinforced tubes for bead homogenization |
| Glass beads, acid washed, 212-300 µm | Sigma-Aldrich | G1277 | glass beads |
| Fisher Scientific Bead Mill 24 Homogenizer | Fisher Scientific | 15-340-163 | bead homogenizer |
| CelLytic B Cell Lysis Reagent, 10X | Sigma-Aldrich | C8740 | lysis reagent |
| Platinum Taq polymerase | Invitrogen | 10966-018 | Contains: Platinum Taq polymerase, 10X PCR buffer (no MgCl2), 50 mM MgCl2, KB Extender |
| dNTP Mix | Invitrogen | 18427-088 | 10 mM dNTP mix |
| QIAquick PCR Purification Kit | Qiagen | 28106 | Kit used to purify fungal amplicons. Contains: Buffer PB (binding buffer), Buffer PE (washing buffer), Buffer EB (elution buffer), pH Indicator dye (optional), and GelPilot loading dye |
| Owl EasyCast Mini Gel Electrophoresis System | Thermo Fisher | B1 or B2 | |
| TrackIt 1 KB Plus DNA Ladder | Thermo Fisher | 10488-085 | DNA ladder |
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