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。

1. 制备 NIOSH 气溶胶取样器
注: NIOSH 气溶胶取样器是一个两级旋风气溶胶取样机, 收集 bioaerosols 使用两个取样管和聚四氟乙烯 (PTFE) 过滤。
<ol><li>装配前, 仔细检查取样器。检查取样器, 确保它没有损坏, 所有螺钉都到位, 舒适, 并在两个部分形成的接缝周围的密封胶带完好无损。检查取样器 O 形环, 以确保它没有任何刻痕, 裂缝或眼泪, 它有一个非常轻涂硅脂。 注: 该取样器包括37毫米3µm 聚四氟乙烯过滤器在聚苯乙烯或聚丙烯三片滤盒。<ol>
<li>通过在基片的网格化表面放置纤维素或塑料过滤器支撑垫 (包括在过滤器中) 来组装过滤盒 (请参见图 1)。使用过滤钳将过滤器放在过滤器支撑垫的顶部, 并朝上收集端。</li>
		<li>使用手动或气压印刷机密封过滤盒。手动关闭将允许空气在过滤器周围泄漏, 并降低收集效率。插入环形件 (延伸罩), 并使用按下紧密和均匀地向下推。按下扩展罩到过滤器足够紧密, 以确保空气不泄漏周围的过滤器。 注: 取样时不使用盒顶部, 但在取样完成后应保存在过滤器上。</li>
		<li>将组装的卡带装到取样器的顶部, 尽量将其推下。将19毫米 (3/4 英寸) 胶带绕在过滤盒和取样器的外部, 将过滤盒装在原处, 并作为备用密封来防止泄漏。</li>
		<li>拧紧每管, 并充分进入取样器, 直到它的底部和包装密封胶带周围的每管作为一个次要的密封, 以防止泄漏。 注: 第一管, 15 毫升聚丙烯管, 收集非吸入颗粒的空气动力学直径 > 4 µm。第二管, 1.5 毫升聚丙烯离心管, 收集在1和4µm 之间的可吸入颗粒。剩余的较小粒子 < 1 µm 在 PTFE 过滤器上收集 (请参见图 2)。</li>
	</ol>
</li>
	<li>在每次使用前用校准罐校准气流通过 NIOSH 取样器, 以区别滤波器和取样装置会导致气流变化。<ol>
<li>要使用校准罐, 请将 jar 的鲁尔管接头插入过滤盒的顶部, 将取样器放在罐子中, 然后密封罐子。</li>
		<li>将校准罐连接到校准流量计和取样泵。打开流量计, 让它预热几分钟。请注意, 流量计必须始终有一个过滤器连接到其入口, 以避免污染流量计和取样。</li>
		<li>打开取样泵, 并允许它运行几分钟, 以热身和稳定。</li>
		<li>调整泵以在3.5 升/分钟内设置正确的流量。 注: NIOSH 气溶胶取样器的流速通常设置为3.5 升/分。在此流速下, 取样器符合可吸入粒子取样的 ACGIH/ISO 标准16。如果需要不同的截止尺寸或降低噪音水平, 则可以使用其他流量, 但要知道, 如果使用不同的流速, 取样器将不再符合可吸入的取样标准。</li>
		<li>校准完成后, 关闭泵和流量计。</li>
	</ol>
</li>
</ol>2. 静态和个人气溶胶取样
<ol><li>为个人或静态区域取样设置 NIOSH 气溶胶取样器。 注: 静态取样是指当取样器和泵安装在正在研究的人员上时, 使用气溶胶取样机, 而将其连接到三脚架或其他持有装置 (请参见图 3), 与个人抽样相比 (请参阅图 4)。<ol>
<li>对于静态区域取样, 请将取样器尽可能靠近所研究的特定区域。保持取样器远离空气入口, 房间入口和可能在气流路径的地方。 注: 气溶胶浓度即使在短距离内也会有很大的变化, 特别是当气溶胶源在房间17中时。</li>
		<li>对于个人抽样, 在人的呼吸区域内设置取样器。将取样器连接到翻领、肩部或胸部, 并将取样泵放在腰部或背包中。 注: 呼吸区通常被认为是一个30厘米半球围绕一个人的鼻子, 其中大部分空气是在吸入17。</li>
	</ol>
</li>
	<li>确保取样器管的取样口很清楚, 没有任何东西阻碍进水口或干扰气流进入取样。取样管不能夹紧或纠结。任何堵塞都会导致泵停止。</li>
	<li>打开水泵。一两分钟后, 检查泵是否仍在运行。 注意: 根据环境10、18、19, 对时间段进行采样, 范围从10分钟到完全8小时的工作班次不等。图 6中显示的结果在室内环境中具有60分钟采样周期的代表性。</li>
	<li>空气取样完成后, 采集取样管和过滤器。取下密封胶带, 拧开管子, 盖上盖子。将过滤盒的第三片放在过滤器上。将样品存放在4摄氏度, 直到准备好进行加工。</li>
</ol>3. 从空气样品中提取基因组 DNA
<ol><li>从 NIOSH BC251 取样器的每个阶段提取基因组 DNA (gDNA)。分别处理空气取样收集管和过滤器, 以便测定样品中可吸入和不可吸入的微生物气溶胶。 注: 或者, 如果样品接种太小, 可以组合和提取三个阶段。<ol>
<li>在第二类生物安全柜或层流清洁工作台, 灌装移除过滤器。用70% 乙醇擦拭取样盒, 用盒式打开工具撬开取样盒。使用过滤器升降机向上推过滤器和支撑垫。用过滤钳抓住过滤器, 把它放在无菌培养皿中。将过滤器切成6块, 放在2毫升加筋管中, 含300毫克玻璃珠 (0.2-0.5 毫米)。</li>
		<li>三十年代将含有过滤器的管子放置在液氮中, 并立即将其放置在4.5 米/秒的珠磨机上。</li>
		<li>重复步骤3.1.2 一次或两次, 直到过滤器被粉碎。小块完整的过滤器可能仍然存在。添加0.5 毫升的裂解缓冲液 (4 米尿素, 200 毫米三, 20 毫米氯化钠, 200 毫米乙二胺乙酸 (EDTA), pH 7.4)。</li>
		<li>在15毫升和1.5 毫升空气取样器管中加入0.3 毫升的裂解缓冲液。涡流管为 10-十五年代, 而直立, 然后另 10-十五年代倒置。将每管的内容转移到含有300毫克玻璃珠的2毫升增强管中。 注: 在每个取样管收集的大部分材料将积聚在管顶部附近。</li>
		<li>处理所有管在珠轧机均质机在4.5 米/秒三十年代和离心他们在 2万 x g 1 分钟在22°c。转移上清液到无菌的1.5 毫升离心管和离心管在 2万 x g 再次1分钟在22°c。重复此步骤。</li>
	</ol>
</li>
	<li>添加30µL 裂解试剂 (见材料表) 到每管和孵化的管在37°c 15 分钟。</li>
	<li>添加0.2 毫升的捆绑缓冲 (10 米尿素, 6 米胍盐酸, 10 毫米三盐酸, 20% 海卫 X-100, pH 4.4) 和蛋白酶 K (100 µg/毫升) 到每管和孵化管在70°c 为10分钟. 将异丙醇的100µL 添加到每管。</li>
	<li>将萃取液转移到玻璃纤维过滤管 (700 µL 容量) 放置在2毫升收集管和离心机收集管在 2万 x g 三十年代在22°c。丢弃收集管并将过滤管放入新的2毫升收集管中。</li>
	<li>添加0.5 毫升抑制剂去除缓冲 (5 米胍盐酸, 20 毫米三 hcl, pH 值 6.6, 38% 乙醇) 到每管和离心机收集管在 2万 x g 三十年代在22°c。丢弃收集管并将过滤管放入新的2毫升收集管中。</li>
	<li>增加0.5 毫升洗涤缓冲 (20 毫米氯化钠, 2 毫米三 HCl, pH 值 7.5, 80% 乙醇) 对每管和离心机收集管在 2万 x g 三十年代在22°c。丢弃收集管并将过滤管放入新的2毫升收集管中。重复此过程。</li>
	<li>离心机收集管在 2万 x g 为一个额外的1分钟22°c, 以消除任何残留洗涤缓冲。丢弃收集管并将过滤管放入新的收集管中。</li>
	<li>添加100µL 温 (≥70°c) 洗脱缓冲 (10 毫米三 HCl, pH 8.5) 到每管和孵化他们在室温下为 1-2 分钟. 离心机收集管在 2万 x g 三十年代在22°c。将空滤管转移到无菌的1.5 毫升离心管, 并从步骤3.8 中重新应用 eluates。在室温下孵育 1-2 分钟离心机在 2万 x g 三十年代在22摄氏度。 注: Eluates 可以立即用于步骤4或存储在-20 °c, 直到准备使用。基因组 DNA 可以储存长达一年的摄氏-20 摄氏度。如果需要长期贮存, 则建议将 DNA 储存在-80 摄氏度。</li>
</ol>4. 真菌核糖体 DNA 的扩增
<ol><li>使用普遍的真菌底漆放大其区域1和2从提取的 gDNA。 注意: 对于本协议, 底漆对 Fun18Sf (5 '-TTGCTCTTCAACGAGGAAT-3 ')/ITS4R (5 '-TCCTCCGCTTATTGATATGC-3 ') 用于提供其区域的最大覆盖率4,5。其他底漆集, 例如那些放大 ITS1 或 ITS2 区域单独, 可以使用。<ol>
<li>建立聚合酶链反应 (PCR) 为每一个样品在无菌0.5 毫升 pcr 管或96井 pcr 板50µL 反应: 5 µL 提取的模板 DNA (从步骤 3), 33.3 µL pcr 级水, 5 µL 10x pcr 缓冲器, 1.5 µL 50 毫米氯化镁2, 1 µL 10 毫米 2 '-deoxynucleoside 5 '-triphosphates, 0.5 µL 20 µM Fun18Sf 向前底漆, 0.5 µL 20 µM ITS4R 反向底漆, 0.2 µL 的Taq DNA 聚合酶。</li>
		<li>在热循环仪中进行反应:95 °c 变性3分钟;6周期变性 (96 °c, 三十年代) 退火 (50 °c, 四十五年代) 和底漆引伸 (72 °c, 3 分钟);20周期变性 (96 °c, 三十年代), 退火 (50 °c, 四十五年代) 和底漆引伸 (72 °c, 1 分钟);和底漆延伸在72°c 10 分钟保持反应混合物在4°c, 直到下一步。</li>
	</ol>
</li>
	<li>用硅胶膜纯化试剂盒将三项 PCR 反应提纯。 注: 本程序允许去除 PCR 成分 (底漆, dNTPs, 酶, 盐,等) 和其他污染物的扩增 DNA 制剂。<ol>
<li>将每个样品的三反应 (150 µL 总) 结合在无菌1.5 毫升离心管中。在5x 体积 (750 µL) 上添加捆绑缓冲 (5 米胍-HCl, 30% 异丙醇), 并用吹打上下十次混合溶液。</li>
		<li>添加450µL 的混合物, 旋转柱放置在收集管和离心管在 17900 x g 三十年代. 丢弃滤液。将余下的450µL 添加到列和离心机在 17900 x g 三十年代在22摄氏度。 注: 自旋柱含有硅膜, 允许将 DNA 吸附到柱上。</li>
		<li>丢弃滤液并将750µL 洗涤缓冲器 (10 毫米三盐酸, pH 7.5, 80% 乙醇) 添加到自旋柱上。离心机列在 17900 x g 三十年代在22°c。 注: 此过程去除上述 PCR 反应中的污染物。</li>
		<li>丢弃滤液并离心 17900 x g 的空柱, 在22摄氏度处1分钟。 注意: 此步骤是删除任何可能干扰下游应用程序的残余洗涤缓冲区。</li>
		<li>将旋转柱转移到无菌1.5 毫升离心管。添加45µL 洗脱缓冲器 (10 毫米三氯, pH 8.5), 并在室温下孵化管5分钟. 离心机的旋转柱在 17900 x g 1 摄氏度。</li>
		<li>立即使用洗脱 DNA 的步骤4和5或存储在-20 °c, 直到准备使用。 注: amplicons 可以存储长达一年的-20 摄氏度。如果需要长期贮存, 则建议将 DNA 储存在-80 摄氏度。</li>
	</ol>
</li>
</ol>5. 琼脂糖凝胶电泳法验证真菌的扩增
<ol><li>铸造1% 琼脂糖凝胶含有1µg/毫升溴溴化物。在沸腾的1x 三乙酸乙酯-EDTA (泰) 缓冲液中溶解琼脂糖。一旦溶液冷却到约50摄氏度, 加入溴溴化, 倒入凝胶浇铸。</li>
	<li>琼脂糖凝胶固化后, 将凝胶浸泡在1x 泰中, 并准备在凝胶上装 amplicons。对8µL 的扩增 DNA, 添加2µL 5x 加载缓冲器。 注意: 这些卷可以根据所需的加载缓冲区的浓度进行调整。</li>
	<li>载入样品, 所有10µL, 到凝胶和 DNA 梯子为大小参考。在 75 v (6 伏/厘米) 处运行凝胶, 大约90分钟, 可视化带, 通常见于750和1000基对之间, 使用紫外线 (请参见图 5)。</li>
</ol>6. 真菌的区域排序和分析
<ol><li>序列提取的 gDNA 或扩增真菌的区域和分析序列使用当前和适当的方法。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+DNA+extraction+methodologies+used+for+assessing+fungal+diversity+via+ITS+sequencing.">Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing.</a> J Environ Monit. 14 (3), 766-774 (2012).</li><li>ACGIH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Documentation of the threshold limit values and biological exposure indices. Appendix C: Particle size-selective sampling criteria for airborne particulate matter. , (2001).</li><li>Rodes, C. E., Thornburg, J. W., Ruzer, L. S., Harley, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosols handbook: measurement, dosimetry, and health effects. , (2005).</li><li>Broadwater, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+a+persistent+odor+at+an+aircraft+seat+manufacturer.">Investigating a persistent odor at an aircraft seat manufacturer.</a> J Occup Environ Hyg. 13 (10), D159-D165 (2016).</li><li>Couch, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Health Hazard Evaluation Program: Evaluation of Potential Hazards during Harvesting and Processing Cannabis at an Outdoor Organic Farm. Report No. 2015-0111-3271. , (2017).</li><li>Feinstein, L. M., Sul, W. J., Blackwood, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bias+associated+with+incomplete+extraction+of+microbial+DNA+from+soil.">Assessment of bias associated with incomplete extraction of microbial DNA from soil.</a> Appl Environ Microbiol. 75 (16), 5428-5433 (2009).</li><li>Keswani, J., Kashon, M. L., Chen, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+interference+to+conventional+and+real-time+PCR+for+detection+and+quantification+of+fungi+in+dust.">Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust.</a> J Environ Monit. 7 (4), 311-318 (2005).</li><li>Bellemain, E., Carlsen, T., Brochmann, C., Coissac, E., Taberlet, P., Kauserud, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITS+as+an+environmental+DNA+barcode+for+fungi:+an+in+silico+approach+reveals+potential+PCR+biases.">ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases.</a> BMC Microbiol. 10, 189 (2010).</li><li>Farrelly, V., Rainey, F. A., Stackebrandt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+and+rrn+gene+copy+number+on+PCR+amplification+of+16S+rRNA+genes+from+a+mixture+of+bacterial+species.">Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species.</a> Appl Environ Microbiol. 61 (7), 2798-2801 (1995).</li><li>Vesper, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Environmental+Relative+Moldiness+index+for+US+homes.">Development of an Environmental Relative Moldiness index for US homes.</a> J Occup Environ Med. 49 (8), 829-833 (2007).</li></ol>

本报告的结论和结论是作者的意见, 不一定代表国家职业安全与健康研究所、疾病控制和预防中心的官方立场。

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...

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。

<table>
	<tbody>
		<tr>
			<td>NIOSH BC251 bioaerosol cyclone sampler</td>
			<td>NIOSH</td>
			<td>BC251</td>
			<td>The NIOSH sampler is not yet commercially available. Please contact William Lindsley, PhD (wlindsley@cdc.gov) for information on obtaining the NIOSH sampler</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>02-681-373</td>
			<td>1.5 mL polypropylene microcentrifuge tubes for air sampling; screw top threading must match the threading of the NIOSH sampler</td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352096</td>
			<td>15 mL polypropylene tubes for air sampling</td>
		</tr>
		<tr>
			<td>Clean Room Vinyl Tape, Easy-Remove, 1/4&#34; Width</td>
			<td>McMaster-Carr</td>
			<td>76505A1</td>
			<td>sealing tape</td>
		</tr>
		<tr>
			<td>Filter Cassette, Clear Styrene, 37 mm</td>
			<td>SKC Inc.</td>
			<td>225-3LF</td>
			<td>3-piece sampling cassette (no filter). Contains: cassette base, extension cowl, cassette cap and inlet/outlet plugs</td>
		</tr>
		<tr>
			<td>PTFE hydrophobic fluoropore membrane filters, 3.0 &#181;m, 37 mm</td>
			<td>EMD Millipore</td>
			<td>FSLW03700</td>
			<td>Contains: 37 mm, 3.0 &#181;m PTFE filters and support pads</td>
		</tr>
		<tr>
			<td>Fisherbrand filter forceps</td>
			<td>Fisher Scientific</td>
			<td>09-753-50</td>
			<td>filter forceps</td>
		</tr>
		<tr>
			<td>Model 502 Precision PanaPress</td>
			<td>PanaVise</td>
			<td>502</td>
			<td>pneumatic cassette press is constructed from this precision arbor press</td>
		</tr>
		<tr>
			<td>Scotch Super 33+ vinyl electrical tape</td>
			<td>McMaster-Carr</td>
			<td>76455A21</td>
			<td>19 mm tape</td>
		</tr>
		<tr>
			<td>Multi-purpose Calibration Jar, Large</td>
			<td>SKC Inc.</td>
			<td>225-112</td>
			<td>calibration jar</td>
		</tr>
		<tr>
			<td>Universal PCXR4 Sample Pump</td>
			<td>SKC Inc.</td>
			<td>224-PCXR4</td>
			<td>sampling pump</td>
		</tr>
		<tr>
			<td>Mass Flowmeter 4140</td>
			<td>TSI Inc.</td>
			<td>4140</td>
			<td>flow meter</td>
		</tr>
		<tr>
			<td>Roche High Pure PCR Template Kit</td>
			<td>Roche Diagnostics</td>
			<td>11796828001</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Fisherbrand 2 mL Reinforced Polypropylene Screw Cap Tubes with Caps</td>
			<td>Fisher Scientific</td>
			<td>15340162</td>
			<td>2 mL reinforced tubes for bead homogenization</td>
		</tr>
		<tr>
			<td>Glass beads, acid washed, 212-300 &#181;m</td>
			<td>Sigma-Aldrich</td>
			<td>G1277</td>
			<td>glass beads</td>
		</tr>
		<tr>
			<td>Fisher Scientific Bead Mill 24 Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>15-340-163</td>
			<td>bead homogenizer</td>
		</tr>
		<tr>
			<td>CelLytic B Cell Lysis Reagent, 10X</td>
			<td>Sigma-Aldrich</td>
			<td>C8740</td>
			<td>lysis reagent</td>
		</tr>
		<tr>
			<td>Platinum Taq polymerase</td>
			<td>Invitrogen</td>
			<td>10966-018</td>
			<td>Contains: Platinum Taq polymerase, 10X PCR buffer (no MgCl2), 50 mM MgCl2, KB Extender</td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Invitrogen</td>
			<td>18427-088</td>
			<td>10 mM dNTP mix</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Owl EasyCast Mini Gel Electrophoresis System</td>
			<td>Thermo Fisher</td>
			<td>B1 or B2</td>
			<td></td>
		</tr>
		<tr>
			<td>TrackIt 1 KB Plus DNA Ladder</td>
			<td>Thermo Fisher</td>
			<td>10488-085</td>
			<td>DNA ladder</td>
		</tr>
	</tbody>
</table>

collection and extraction of occupational air samples for analysis of fungal dna

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。

通过确定空气样品中确定的每个 OTU 的克隆数, 可以利用相对丰度评估环境中的物种分布。图 6是一个克朗图, 代表了在60分钟的空气取样后, 室内环境中分类放置的物种。可以观察到, 在两种主要的真菌门、Ascomycota 和 Basidiomycota 中, 以及属于 Zygomycota (根霉 microsporus) 的物种中, 环境中含有多种物种。传统的评估方法偏向于 Ascomycota 物种, 因为许多...

Watch this Scientific Journal Video about Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA at JoVE.com

采集和提取职业空气样本用于真菌 DNA 分析 |环境 |视频

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

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。

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

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12.3K 次观看.  Centers for Disease Control and Prevention. 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。

视频: Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

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A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

内毒素和DNA含量的空气采样滤波器分析

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

提取及土壤微生物磷脂脂肪酸分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

自动化，高清晰度移动采集系统对NO的氮同位素分析<sub系列&gt; X</sub

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...