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&quot; 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 &amp;micro;m, 37 mm</td><td>EMD Millipore</td><td>FSLW03700</td><td>Contains: 37 mm, 3.0 &amp;micro;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 &amp;micro;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

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

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遗传学

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 ...

环境科学

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

免疫与感染

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

不同环境条件下生物气溶胶的组成及分布分析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

生物学

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

实验性曲霉病肺真菌负担的组织学定量测定

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

从土壤中分离可培养酵母和霉菌以研究真菌种群结构

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

分离、表征和总 DNA 提取以鉴定真菌异养植物中的内生真菌

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and microbial dispersal. Mycelial networks created by filamentous fungi or other filamentous microorganisms (e.g., Oomycetes) act as 'fungal highways' that can be utilized by bacteria for transport throughout heterogeneous environments, greatly facilitating their mobility and granting them access to regions that may be challenging or impossible to reach on their own (e.g., due to air pockets within the soil). Several devices and experimental protocols have been created to study these fungal highways, including fungal highway columns. The fungal highway column designed by our group can be used for a variety of in situ or in vitro applications, as well as with diverse environmental and host-associated sample types. Herein, we describe the methods for performing experiments with these columns, including designing, printing, sterilizing, and preparing the devices. The options for analyzing data obtained from the use of these devices are also discussed here, and troubleshooting advice regarding potential pitfalls associated with experiments using fungal highway columns is offered. These devices can be used to gain a more comprehensive understanding of the diversity, mechanisms, and dynamics of fungal highway BFIs to provide valuable insights into the structural and functional dynamics within complex environments (e.g., soils) and across diverse habitats in which bacteria and fungi co-exist.

This protocol provides detailed instructions on how to construct, sterilize, assemble, utilize, and reuse the fungal highway columns to enrich bacterial-fungal pairs interacting through fungal highways from diverse environmental substrates.

使用真菌 Highway 色谱柱在不同环境和基材中研究细菌-真菌相互作用

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and ...