内毒素和DNA含量的空气采样滤波器分析
Summary
Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.
Abstract
室外气溶胶的研究通常使用的过滤器采样的颗粒物。此过程使得能够并行执行所收集的颗粒的各种特征。这里提出的方法的目的是获得从过滤器中提取的生物气溶胶的内毒素和DNA含量的高度准确和可靠的分析。高的分子量的有机分子，如脂多糖，从采样滤波器的提取包括摇动在一个无热原的水性介质中的样品。随后的分析是基于可使用浊度测量来检测酶反应。作为上采样滤波器的高有机含量的结果是，使用该最初设计用于土壤和修改，以提高DNA产量市售的DNA提取试剂盒进行DNA的样品的提取。的检测和使用定量聚合酶链reacti特定微生物物种的量化上（Q-PCR）分析中描述并与其他可用的方法进行对比。
Introduction
在过滤器上空气采样是在大气气溶胶研究的基本工具。1所述的取样过滤器可用于各种化学，物理和所收集的环境粒子的生物表征的起点。2-11这种方法的优点是，各种分析可离线对同一样品执行的。编译来自所有不同分析的数据使研究者获得的收集的颗粒和助剂的特性很好地理解在解决在大气科学的复杂问题。12,13例如，在相同的拍摄海洋和内陆空气采样期间可相对于所采样的颗粒的毒性和生物组合物。14本研究进行比较，脂多糖（LPS），对革兰氏阴性细菌细胞壁，也被称为内毒素组分，从过滤器中提取的采样在岸和在内陆部位，并使用该评价鲎变形细胞溶解物（LAL）测试。并行地，细菌含量的基因组的评估（总菌，革兰氏阴性，和蓝藻）是使用定量聚合酶链反应（Q-PCR）对同一样品执行的。在LAL试验是基于形成的加成从鲎， 美洲鲎阿米巴细胞的含水提取物之后，向含有内毒素的水溶液浊度的测量。样品在较高的内毒素浓度，更快浊度开发15的Q-PCR分析是基于发射作为特定的DNA片段被扩增荧光信号16由信号的实时监测在PCR指数期反应和校准用标准曲线，初始的DNA量进行量化。这两种的组合与他人一起分析，如别处详述，14可提供的内毒素和水平的良好估计样品中的源的细菌量。
这里提出的方法的目的是获得从过滤器中提取的生物气溶胶的内毒素和DNA含量的高度准确和可靠的分析。而用于采样气溶胶的物理和无机化学特性的方法是，最近，已开发了探讨其有机质成分众所周知，17一直存在很少研究气溶胶的生物成分上18为当前的理方法是通过在详细介绍，用于提取，分析和确定空气气溶胶的生物分数一个可靠的方法来解决这个间隙14中
这里详述的方法可望找到在涉及过滤分析生物的室内和室外的气溶胶研究项目广泛使用。20-24
Protocol
注：在材料部分显示的所有材料，并在该协议中使用仪器的详细清单。 1.空气采样过滤器上过滤器的研制对于高流量采样，使用20.3点¯x25.4厘米2过滤器。选择，具体类型的过滤器是最适合的研究需要，以及在滤波器的截止大小，如果适用。1在此，使用石英微纤维过滤器。 用于有机和生物化合物采样预烘焙滤波器破坏有机残基。到预先烘?…
Representative Results
它是常见使用“离线”采样滤波器的分析来研究大气气溶胶（参见图2）。取样物质包括有机（ 例如蛋白质，烃类分子，糖类）和无机（ 例如金属，盐）含量的32化学分析。生物分析包括存活和非存活的微生物含量，使用DNA的方法或显微镜物种鉴定，以及基于DNA的定量。 <img alt="图2" src="/files/f…
Discussion
这部作品描述了收集器的气溶胶样品中既定量的内毒素和DNA提取存在和检测方法。该方法要求精确的例程和只要实验者附着在这里讨论了一些基本的和重要的点可以容易地进行。
用于内毒素检测步骤，请注意，裂解物溶液是相当粘稠，趋于在移液以产生气泡。它是难以除去你气泡，并且它们导致在微孔板阅读器值的变化。因此，要首先放置在样品中的板和仅确保所有的气泡?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
The authors thank Dr. Yoav Barak from the Chemistry Faculty, Weizmann Institute, for support and advice. This study was supported by the Israel Science Foundation (grant # 913/12), and by the Minerva Foundation with funding from the Federal German Ministry for Education and Research.
Materials
| Filter sampling | |||
| HiVol 3000 – High Air Volume Sampler | Ecotech | ||
| Quartz Microfiber Filters | Whatman | 1851-865 | 203mm X 254mm |
| ELF – Laboratory Chamber Furnaces | Carbolite | ELF 11series | |
| Aluminum foil | Opal | ||
| Name | Company | Catalog Number | Comments |
| Endotoxin | |||
| Ethanol | Sigma Aldrich | 16368 | Laboratory Reagent, 96% |
| Airstream Class II Biological Safety Cabinet AC-4E1 | ESCO | 10011712 | |
| Pyrotell -T | Associates of Cape Cod, Inc. | T0051 | |
| Control Standard Endotoxin | Associates of Cape Cod, Inc. | E0005-1 | Escherichia coli O113:H10, 0.5 µg/vial 1 Pack |
| LAL Reagent Water | Associates of Cape Cod, Inc. | W0051 | |
| 10 mL sterile syringe with Luer-Lok Tip | Becton-Dickinson & Co. | 309605 | |
| BD Precisionglide syringe needle | Becton-Dickinson & Co. | 305129 | Sterile |
| Parafilm-M sealing tape | Parafilm | P7543 | Sigma catalog number |
| Microtubes | Axigen | MCT-200-C | 2ml, pyrogen free |
| 1.12 cm diameter Cork Borer | Boekel Scientific | 1601 BD Series – Steel | Part of a cork borer set containing borers with various diameters. |
| 50 mm Petri Dish | Miniplast Ein-Shemer | 72050-01 | Aseptic |
| Vortex Genie 2 | Scientific Industries, inc. | SI-0297 | |
| Microcentrifuge 5415 D | Eppendorf | 22621408 | |
| TC MicroWell 96 F SI w/lid | Nunc | 167008 | Flat bottom wells (with lid (individually wrapped)), sterile, pyrogen free |
| Synergy HT Multi-Detection Microplate Reader | Biotek | 7091000 | |
| Name | Company | Catalog Number | Comments |
| DNA | |||
| DNA away | Sigma Aldrich | 7010 | |
| Standard DNA of the microbial species of interest | ATCC or other culture collection | Either the appropriate microbial strain for DNA extraction or the extracted DNA | |
| Neubauer-improved | Marienfeld | 640030 | hemocytometer |
| TE buffer, Low EDTA | Life Technologies | 12090-015 | 10 mM Tris-HCl (pH 8.0) 0.1 mM EDTA |
| Nuclease-free PCR-grade water | Sigma Aldrich | 3315959001 | |
| PCR primers | Sigma Aldrich | Targets the microbial species of interest | |
| Dual-Labeled Probes | Sigma Aldrich | Targets the microbial species of interest | |
| Screw cap tubes | Axigen | ST-200-SS | 2 mL |
| PowerSoil DNA extraction kit | Mo Bio Laboratories | 12888-100 | |
| Glass beads, acid-washed 425-600 Microns | Sigma Aldrich | G8772-100G | |
| Glass beads, acid-washed <106 microns | Sigma Aldrich | G4649-100G | |
| PowerSoil Solution C1 | Mo Bio Laboratories | 12888-100-1 | Cell lysis buffer , Power soil Kit |
| Magic Touch ice bucket | Bel-Art | 18848-4001 | |
| Mini-Beadbeater-16 | BioSpec | 607EUR | |
| StepOnePlus Real-Time PCR System | Applied Biosystems | 4376600 | |
| Fast SYBR Green Master Mix | Applied Biosystems | 4385612 | |
| TaqMan Gene Expression Master Mix | Applied Biosystems | 4370048 | |
| MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL | Applied Biosystems | 4346906 | |
| MicroAmp Splash-Free 96-Well Base | Applied Biosystems | 4312063 | |
| MicroAmp Optical Adhesive Film | Applied Biosystems | 4311971 | |
| Centrifuge 5810 R | Eppendorf | 5811 000.010 | Rotor A-4-62 with MTP buckets |
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Tags
Air SamplingFilter AnalysisEndotoxinsDNA ContentBiological Aerosol ResearchHigh Volume SamplingQuartz Fiber FiltersOrganic And Biological Compound SamplingBaked FiltersHigh Volume SamplerBiosafety CabinetEndotoxin Spiked FiltersPyrogen Free TubesMicrocentrifugeEndotoxin TestMicroplate ReaderKinetic Reaction
Citar este artículo
Lang-Yona, N., Mazar, Y., Pardo, M., Rudich, Y. Air-sampled Filter Analysis for Endotoxins and DNA Content. J. Vis. Exp. (109), e53444, doi:10.3791/53444 (2016).