我们要感谢 Helty Adisetiyo, 博士和 Shangxin 杨博士的发展的协议。对国际妇幼青少年艾滋病临床试验组 (IMPAACT) 的总体支助是由国家卫生研究院 (NIH) 国家过敏和传染病研究所 (NIAID) 根据奖励数字提供的。UM1AI068632 (IMPAACT), UM1AI068616 (IMPAACT SDMC) 和 UM1AI106716 (IMPAACT LC), 与来自尤尼斯·肯尼迪-国家儿童健康和人类发展研究所 (发育研究院) 和国家心理健康研究所 (镍氢) 的共同资助。内容完全是作者的责任, 不一定代表 NIH 的官方观点。

对于所有的协议步骤, 必须佩戴适当的个人防护设备 (PPE), 并需要采取严格的防污染措施。观察从预放大工作区到放大后工作区域的工作流程, 以尽量减少样品的污染。所有使用的用品是无菌的, 没有 RNase, DNase, DNA 和热原。所有吸管提示都被过滤。提供了协议步骤的流程图 (图 1)。
1. 样品裂解
注: 样品裂解和核酸提取是在洁净室环境中使用 DNA/RNA 萃取试剂盒进行的, 在这两个工程和程序控制都到位, 以尽量减少向样品中引入环境细菌。
<ol><li>
工作区准备
	<ol>
<li>清洁生物安全柜 (BSC) 工作区与适当的表面清洁剂, 以消除任何核酸污染。</li>
		<li>打开温度控制的 vortexer, 并将其设置为37摄氏度。</li>
	</ol>
</li>
	<li>
胍硫氰酸盐裂解缓冲液的制备 (见材料表)
	<ol>
<li>检查沉淀的溶解缓冲。在37摄氏度的温度下重新溶解沉淀。</li>
		<li>为每个样品准备600µL 的裂解缓冲液和6µL β巯基乙醇 (β i)。考虑每个样本额外的20% 卷。</li>
	</ol>
</li>
	<li>
样品准备
	<ol>
<li>如果整个牛奶都结冰了, 就在冰上解冻。整除5毫升的全脂牛奶成15毫升或5毫升不育管在平衡计分卡, 并保持在冰上。</li>
		<li>旋转5毫升牛奶整除10分钟在 5000 x g 在4°c 到颗粒细胞。</li>
		<li>去除脂肪层, 现在的顶层在管, 用塑料铲或大口径吸管尖。</li>
		<li>不干扰颗粒, 除去所有上清, 除100µL。</li>
		<li>用 resuspending 在1毫升无菌磷酸盐缓冲盐水 (PBS) 中清洗颗粒。</li>
		<li>通过将1毫升不育 PBS 添加到5毫升管中, 准备1负控制。</li>
		<li>将悬浮液转移至清洁的1.5 毫升离心管, 并在离心中旋转1分钟 (室温 (RT) 5000 x g)。</li>
		<li>使用1000µL 无菌过滤吸管提示丢弃整个上清/脂肪层。</li>
		<li>如果不提取同一天, 通过将细胞颗粒放入乙醇/干冰泥浆中, 并立即将其转移到-80 摄氏度冷藏库中, 将其冻结。</li>
	</ol>
</li>
	<li>
样品中断和均匀化 (珠敲打)
	<ol>
<li>加入600µL 的裂解缓冲液, 其中含有β i 的颗粒, 并将悬浮液转移到珠管上。</li>
		<li>每个抽取批次12包含10个样本, 1 个负控制 (在步骤3.7 中编写), 1 个正控制 (在下一步中准备)。<ol>
<li>用裂解缓冲剂和20µL 的细菌模拟样品制备1阳性对照 (模拟社区使用的浓度, 一旦提取, 大约 0.2 ng/µL 的 DNA)。</li>
		</ol>
</li>
		<li>漩涡所有珠管大力十五年代。</li>
		<li>在温度控制的 vortexer 的热样品在37°c 为10分钟, 与震动在 700-800 转每分钟。</li>
		<li>将管道加载到自动的示例干扰器适配器集中。</li>
		<li>珠打1分钟在30赫兹。</li>
		<li>拆卸适配器集, 并手动将适配器集与从左机械手到仪器的右侧机械臂的样本板一起切换。</li>
		<li>珠打1分钟, 在30赫兹。</li>
		<li>离心管在 17200 x g, 3 分钟在25摄氏度。</li>
		<li>用200µL 吸管取出所有上清液, 小心不要在样品的顶端或试样底部的珠子残留物上得到任何脂肪层, 并应用于均质体柱。</li>
		<li>离心机均质柱最大速度为 17200 x 克, 3 分钟25摄氏度。</li>
		<li>将350µL 的洗脱液转移到2毫升制造商的离心管中, 用于纯化 DNA 和 RNA 的自动仪器 (不要使用任何其他管)。注意不要转移任何残留的脂肪层。</li>
		<li>如果流经体积小于350µL, 添加裂解缓冲与β-我到最后的体积350µL。</li>
		<li>在使用自动 DNA 纯化仪进行核酸隔离之前, 将样品留在 RT 上长达2小时, 或在-80 摄氏度处冻结。</li>
	</ol>
</li>
	<li>
清洁工作区
	<ol>
<li>清洁所有表面使用非酶净化溶液, 离开10分钟, 然后喷雾与70% 乙醇和擦拭表面。</li>
	</ol>
</li>
</ol>2. 分离 DNA/RNA
<ol><li>
工作区准备
	<ol>
<li>打开散热块, 将温度设置为70摄氏度。</li>
		<li>打开温度控制的 vortexer, 将温度设置为37摄氏度。</li>
		<li>预热洗脱缓冲 (EB) 包含10毫米三氯, pH 8.5, 在50毫升管到70°c。</li>
		<li>加热350µL 冷冻样品裂解物2毫升管在37°c, 直到完全解冻没有任何沉淀 (约10分钟)。</li>
	</ol>
</li>
	<li>
DNA 纯化
	<ol>
<li>漩涡和离心机2毫升管与样品简要地 (3000 x g 十年代)。</li>
		<li>根据制造商的说明, 在自动 DNA/RNA 净化仪器的加载图后, 将2毫升管插入到振动筛中。</li>
		<li>根据样品数量, 获取转子适配器并将其设置在托盘上。</li>
		<li>根据样品的标识 (ID) 对每个转子适配器进行标签。</li>
		<li>切下盖子, 平滑各个旋转柱的边缘, 用于 DNA 和 RNA。</li>
		<li>插入没有收集管的 DNA 旋转柱进入转子适配器。丢弃收集管。</li>
		<li>标签1.5 毫升收集管, 并插入到转子适配器。</li>
		<li>根据自动 DNA/RNA 纯化仪的加载图, 将转子适配器设置为离心机。</li>
		<li>插入制造商的1000µL 过滤器-提示到尖端机架。</li>
		<li>根据样品数量 (根据特定机器协议指令), 在2毫升制造商的离心管中添加 RNase 水。</li>
		<li>将管插入管插槽 "A" 的离心管插槽。</li>
		<li>丢弃试剂瓶中剩下的试剂, 并填充最小体积10毫升。</li>
		<li>将试剂瓶插入试剂瓶架中 (EB 瓶除外)。</li>
		<li>从50毫升管中加入热 EB 到试剂瓶位置6。</li>
		<li>检查1.5 毫升管放在转子适配器紧密。</li>
		<li>关闭仪器的盖子并选择: "RNA" → "提取套件" → "动物, 组织和细胞" → "套件的名称350µL 部分自定义 DNA" →编辑洗脱量为100µL (默认) 或50µL 低生物量样品→ "开始"。</li>
		<li>完成后, 从离心机上取出转子接头, 放在托盘上。</li>
		<li>从转子适配器位置3丢弃 DNA 自旋柱。</li>
		<li>不要丢弃转子适配器, RNA 在位置2。</li>
		<li>去除1.5 毫升收集管含有洗脱 DNA 在位置 3, 并存储在−20°c。</li>
		<li>收集从振动筛和存储在−20°c 样品, 如果任何样品留在, 否则丢弃。</li>
		<li>继续与协议 "试剂盒的名称350µL 部分 B rna" 进一步纯化 RNA。</li>
		<li>RNA 纯化将由大约350µL 流经, 在转子适配器的中间位置。</li>
	</ol>
</li>
	<li>
RNA 纯化
	<ol>
<li>插入 RNA 旋转柱没有它的收集管和盖子到转子适配器。</li>
		<li>标签新的1.5 毫升收集管和插入到转子适配器, 如手册中所示。</li>
		<li>在自动 DNA/RNA 纯化仪的加载图后, 将转子适配器设置为离心机。</li>
		<li>关闭仪器的盖子, 并选择: "RNA" → "制造商的套件" → "动物, 组织和细胞" → "标准部分 B RNA" → "开始"。</li>
		<li>完成后, 从离心机上取出转子接头, 放在托盘上。</li>
		<li>从位置3丢弃 RNA 自旋列。</li>
		<li>去除1.5 毫升收集管包含30µL 洗脱 RNA 在位置 3, 并且存放在−80°c。</li>
		<li>取出试剂瓶。</li>
		<li>通过适当的危险废物通道处理转子适配器的内容。</li>
	</ol>
</li>
	<li>
清洁工作站
	<ol>
<li>喷雾所有的自动 DNA/RNA 净化仪器的配件, 如试剂架, 托盘, 和任何其他表面使用非酶净化解决方案, 离开10分钟, 并冲洗与去离子 (DI) 水, 然后让他们干。</li>
		<li>喷雾自动仪器只有制造商批准的非酶净化解决方案, 擦拭内部的离心机连同所有的表面使用, 离开10分钟, 然后擦拭70% 乙醇。不要使用其他类型的净化解决方案, 因为他们可以损坏仪器。</li>
	</ol>
</li>
</ol>3. 靶向 16S PCR 设置
注: 16S PCR 的设置是在清洁室内指定的预放大工作区进行的。试剂和样品的制备, 然后加载到一个液体处理程序, 以执行 PCR 为每个样本三 (30 样品, 其中包括真实样品和提取阳性和阴性对照, 加上 2 PCR 水控制, 总共96组合的样本和控件)。一旦 PCR 反应组装和密封, 样品板被转移到一个热循环仪位于后放大区域的自行车。
<ol><li>
工作区准备
	<ol>
<li>清理 PCR 工作站。喷雾所有表面使用 RNase, DNase, DNA decontaminant, 其次是 DI 水两次, 最后70% 乙醇。</li>
		<li>使用精确的样本列表准备 16S pcr 工作表 (参见目标 16S pcr 工作表), 并为每个示例9分配不同的条形码引物。打印出工作表和板块图 (见板 1)。</li>
	</ol>
</li>
	<li>
PCR 主混合制剂 注: 16S 底漆选择是基于感兴趣区域的特定研究, 并可通过研究或样本类型改变。因此, 在订购底漆之前, 重要的是要确认 16S rRNA 的哪个区域最好能放大特定研究中感兴趣的微生物。本协议的编写是为了放大 16S V4 区域。如果选择不同的引物/区域, 则热循环中的退火温度可能需要调整。<ol>
<li>在 PCR 工作站工作, 准备一切。</li>
		<li>从-20 摄氏度取出 50-100 µL 的 DNA 样本, 以及所有需要的试剂, 然后在冰上解冻。漩涡和简要地旋转下来。</li>
		<li>引物预稀释至5µM 的工作浓度, 最小体积为20µL。</li>
		<li>根据工作表上的计算, 在特定的5毫升管中准备 PCR 主混合, 只用正向底漆。</li>
	</ol>
</li>
	<li>
为自动 PCR 装置准备机器人液体处理程序
	<ol>
<li>对于样品, 拿出一个32井仪器的样品适配器, 并根据制造商的指示, 按照96井板图载入 50-100 µL 的 DNA 样本。<ol>
<li>对于每一个 pcr 板, 设置2阴性控制通过放置30µL PCR 水在一个干净的样品管。</li>
			<li>将所有样品放置在32井仪器的样品适配器上, 并将瓶盖锁定在打开位置。</li>
		</ol>
</li>
		<li>对于反向引物9<ol>
<li>删除每个反向底漆的上限与特定的条码 # 的一个在一次 (更换手套之间, 以避免交叉污染)。</li>
			<li>在仪器的试剂适配器上放置最多32个底漆。</li>
		</ol>
</li>
		<li>拿出一个96良好的 PCR 板, 并标签它的研究名称, 样本数, 日期和技术员的缩写。</li>
		<li>装载机器人液体处理程序<ol>
<li>将试剂适配器放置在 B1 位置。为了避免边缘效应, 小心地将适配器的一端放在手柄一侧, 然后慢慢地将另一边缘向下。一定要把适配器的所有角落都推上。</li>
			<li>将样品适配器放置在 C1 位置。一定要把适配器的所有角落都推上。</li>
			<li>涡流5毫升主混合, 打开帽, 并将其放置在仪器的主混合和试剂块位置 A。</li>
			<li>将 pcr 盘放在96井仪器的适配器上, 用于控制半掠过的 pcr 板。</li>
			<li>启动运行并另存为新文件。</li>
			<li>按照提示和复选标记每个提示: 一个, 提示可用, 两个, 废物箱可用, 三, 开始。</li>
		</ol>
</li>
		<li>运行完成后, 通过检查计算机中是否有错误消息来验证没有错误。</li>
		<li>用12或8条带帽密封板, 漩涡大力, 并向下旋转1分钟使用96井板微调, 并把它放在冰或冰箱。</li>
		<li>除去含有反向底漆和样品的管子。</li>
		<li>将96井板带到后放大室, 并根据热循环条件表将该板加载到热循环仪并循环 (请参见表1)。</li>
	</ol>
</li>
</ol>4. 采用基于胶带的凝胶电泳平台进行靶向16S 后 PCR 质量控制
注: PCR 后质量控制 (QC) 和所有后续步骤都在实验室指定的后放大区域进行。dna 被分析在一个自动化的 dna/RNA 片段分析仪。
<ol><li>
工作区准备
	<ol>
<li>通过喷洒 RNase、DNase、DNA decontaminant 的所有表面, 然后用二次水, 最后70% 乙醇, 清洁工作站。</li>
		<li>收集所有需要的用品和设备。</li>
	</ol>
</li>
	<li>
制备试剂
	<ol>
<li>将样品缓冲器和梯子放在温度控制的 vortexer 中, 在摄氏25摄氏度, 最小为30分钟。</li>
	</ol>
</li>
	<li>当试剂正在升温时, 通过将每个样品的 3 pcr 复制汇集到一根管子中, 制备 pcr 样品。</li>
	<li>在仪器上装载样品。</li>
	<li>简要地涡旋和自旋向下管与汇集 PCR 产品。</li>
	<li>将仪器的96井板放在 RT 上的机架上并贴上标签。</li>
	<li>简要涡旋和向下旋转样本缓冲区和梯子。</li>
	<li>将3µL 样本缓冲区添加到96井板的每个井 (至少需要53µL/丝网磁带)。</li>
	<li>运行偶数样本;如果有奇数的样本, 将4µL 的样本缓冲区添加到空井中。</li>
	<li>在梯子上加1µL 梯子。</li>
	<li>按照地图, 将1µL 的聚合 PCR 产品添加到其指定的井中。</li>
	<li>铝箔密封盖板</li>
	<li>用仪器的 vortexer 和适配器在2000转1分钟内涡流板。</li>
	<li>如果有气泡再次旋转, 向下旋转以将样品放置在管子的底部。</li>
	<li>启动软件。</li>
	<li>加载屏幕磁带和加载提示到仪器。</li>
	<li>将样品装入带96井适配器的碎片分析仪中。</li>
	<li>设置与控制器软件的运行。</li>
	<li>一定要选择连编号井。</li>
	<li>编写示例 id。</li>
	<li>检查屏幕磁带的所有油井是否以灰色突出显示。</li>
	<li>请确保分析器识别所有的吸管提示。</li>
	<li>选择 "开始" 并指定一个文件名以保存结果 (研究名称、示例编号和日期)。</li>
	<li>有关详细信息, 请参阅制造商的说明。</li>
	<li>运行完成后, 丢弃笔尖、屏幕胶带和管子。</li>
	<li>分析数据为16S 峰值 nM (315-450 bp 为 V4)。此峰值将根据所选引物的不同而有所不同。</li>
</ol>5. 图书馆的计算、汇集、清理和 QC
<ol><li>在确定了所有示例的扩增子大小和浓度之后, 请将库池存储起来, 以实现池库的最终所需卷和 nM (请参见示例计算)。</li>
	<li>根据制造商的协议 (请参阅材料表), 清理并集中使用基于硅膜的 PCR 产品纯化试剂盒的池库。<ol>
<li>洗脱的 DNA 的最终体积为50µL。</li>
	</ol>
</li>
	<li>根据制造商的协议, 使用双链 DNA 高灵敏度套件对荧光计, 立即测量清理后的库的浓度。<ol>
<li>稀释2µL 的汇集和被清洗的图书馆与198µL 稀释缓冲器加上染料 (1:100 稀释)。</li>
		<li>从荧光器件中记录测量值, 并根据稀释因子进行转换。</li>
	</ol>
</li>
	<li>使用集中阅读 (ng/µL) 的汇集库, 计算图书馆浓度在 nM。使用1µL 未稀释的图书馆测量 microvolume 分光光度计上的 OD260/280。 注: 值 1.8-2.0 表示库的纯度足够。</li>
	<li>将最终库存储在-20 摄氏度, 直到为下一代排序做好准备。</li>
</ol>

<ol>
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Mock microbial communities Available from: <a target="_blank" href="https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1">https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1</a> (2017)</li></ol>

作者没有什么可透露的。

16S rRNA 的目标下一代测序是一种广泛使用的、快速的微生物特征18技术。但是, 许多因素, 包括批处理效果、环境污染、样本交叉污染、灵敏度和重现性都会对实验结果产生负面影响, 并混淆其解释7,19,20. 为了最好地促进强健的16S 分析, 微生物组工作流必须包括良好的实验设计、使用适当的控制、工作流步骤的?...

殖民人类的微生物群落被认为对人类健康和疾病影响新陈代谢、免疫发展、对疾病的易感性以及对疫苗接种和药物治疗的反应至关重要1, 2. 为了解微生物群对人类健康的影响而作出的努力目前强调识别与定义的解剖隔间 (即、皮肤、肠道、口腔、等) 相关的微生物, 以及内部的本地化站点。这些舱室3,4。这些调查工作的基础是迅速出现并增加了下一代测序技术的可获得性, 为样品的微生物遗传量 (微生物) 分析提供了一个大规模的并行平台。对于许多生理样本, 相关的菌群既复杂又丰富 (即, 大便), 但对于一些样本, 微生物群是由低生物量 (即, 人乳, 下呼吸道) 所代表的, 其中敏感度、实验性文物和可能的污染成为主要问题。微生物研究和适当实验设计的共同挑战是多个评论文章的主题5,6,7,8。
本文介绍了一种基于 rRNA 16S V4 区域9的目标测序的鲁棒实验管道, 以表征人乳的微生物群。微生物菌群分析的人奶不仅是复杂的先天低生物量的10, 但另外由高级别的人类 DNA 背景11,12,13,14和PCR 抑制剂的潜在结转15,16在提取的核酸。该协议依赖于商业上可用的提取工具包和半自动平台, 可帮助最大限度地减少样本准备批次之间的可变性。它包含了一个定义良好的细菌模拟社区, 它与样品一起处理, 作为质量控制来验证协议中的每一步, 并提供一个独立的管道健壮性度量。虽然所描述的协议是特定于人乳样本, 它很容易适应其他样本类型, 包括大便, 直肠, 阴道, 皮肤, 晕和口服拭子10,17, 并可以作为一个起点希望进行微生物分析的研究人员。

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">AllPrep RNA/DNA Mini Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">80204</td>
			<td style="width:395px;">DNA/RNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Eliminase</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:153px;">435532</td>
			<td>RNase, DNase, DNA decontaminant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Thermo Mixer</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:153px;"></td>
			<td>temperature-controlled vortexer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Buffer RLT plus</td>
			<td style="width:115px;">Qiagen</td>
			<td>1053393</td>
			<td>guanidinium thiocyanate lysis buffer/ Part of Allprep kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#223;-Mercaptoethanol&#160;</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td>63689-25ML-F</td>
			<td>&#223;-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">LME Beads</td>
			<td style="width:115px;">MP Biomedicals</td>
			<td>116914050</td>
			<td>bead tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAgen TissueLyzer</td>
			<td style="width:115px;">Qiagen</td>
			<td>85300</td>
			<td>automated sample disruptor adapter set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAshredder column</td>
			<td style="width:115px;">Qiagen</td>
			<td>&#160;79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAgen RB tube</td>
			<td style="width:115px;"></td>
			<td></td>
			<td>manufacturer&#39;s microcentrifuge tube in kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAcube and related plasticware</td>
			<td style="width:115px;">Qiagen</td>
			<td>9001292</td>
			<td>automated DNA/RNA purification instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DNA exitus plus</td>
			<td style="width:115px;">Applichem</td>
			<td>A7089</td>
			<td>non-enzymatic decontamination solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EB Buffer</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">19086</td>
			<td>elution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAgility and related plasticware</td>
			<td style="width:115px;">Qiagen</td>
			<td>9001532</td>
			<td>robotic liquid handler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR water</td>
			<td style="width:115px;">MO BIO</td>
			<td>17000-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5PRIME HotMasterMix</td>
			<td style="width:115px;">Quantabio</td>
			<td>2200400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Barcoded reverse primers</td>
			<td style="width:115px;">Eurofin</td>
			<td style="width:153px;">No Catalog #&#39;s</td>
			<td>designed and ordered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">&#160;96 well PCR plate</td>
			<td style="width:115px;">USA scientific</td>
			<td style="width:153px;">1402-9708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tapestation 2200 and related plasticware</td>
			<td style="width:115px;">Agilent</td>
			<td>G2964AA</td>
			<td>automated DNA/RNA fragment analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D1000 reagents for Tapestation&#160;</td>
			<td style="width:115px;">Agilent</td>
			<td>5067-5585</td>
			<td>Sample buffer and ladder are part of this kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneStep PCR Inhibitor Removal Kit&#160;</td>
			<td style="width:115px;">Zymo Research</td>
			<td style="width:153px;">50444470</td>
			<td>PCR inhibitor removal is done per the manufacturer&#39;s instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">QIAquick PCR Purification Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:153px;">28104</td>
			<td>DNA clean up kit: silica-membrane-based purification of PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:153px;">Q32854</td>
			<td>dimethylsulfoxide-based dilution buffer and dye are part of this kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Qubit Fluorometer</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NanoDrop</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:153px;"></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MiSeq 300 V2 kit</td>
			<td style="width:115px;">Illumina</td>
			<td>15033624/15033626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MiSeq&#160;&#160;&#160;</td>
			<td style="width:115px;">Illumina</td>
			<td style="width:153px;">No Catalog #&#39;s</td>
			<td>next generation sequencer</td>
		</tr>
	</tbody>
</table>

a method for targeted 16s sequencing of human milk samples

随着相对便宜、快速和高吞吐量测序的发展, 微生物群落的研究变得越来越普遍。然而, 与所有这些技术一样, 可重现的结果取决于实验室工作流, 其中包含适当的预防措施和控制。这是特别重要的低生物量样本, 污染细菌 DNA 可以产生误导的结果。本文详细介绍了一种半自动的工作流程, 即使用16S 核糖体 RNA (rRNA) V4 区域的目标测序, 在低到中吞吐量范围内, 识别出人类母乳样本中的微生物。该协议描述了从全牛乳样品的制备方法, 包括: 样品裂解、核酸提取、16S rRNA 基因 V4 区的扩增和质量控制措施的库准备。重要的是, 该议定书和讨论考虑了对低生物量样品的制备和分析具有显著意义的问题, 包括适当的阳性和阴性对照、PCR 抑制剂去除、环境样品污染、试剂或实验源和实验性最佳实践, 旨在确保重现性。虽然所描述的议定书是特定于人乳样本, 但它适用于许多低和高生物量样本类型, 包括收集在拭子上的样本, 冷冻整齐, 或稳定在保存缓冲区。

此处介绍的协议包括重要的质量控制 (QC) 步骤, 以确保所生成的数据符合协议灵敏度、特异性和污染控制的基准。该协议的第一个 QC 步骤是 16S V4 区域的 PCR 放大 (图 2)。通过电泳分析了每种样品的 PCR 产物的一个µL, 以确认其在 315-450 bp (图 2、红色箭头) 的预期尺寸范围内。一些人奶样本产生了较低数量的特定产品 (<strong class="xf...

Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

A Method for Targeted 16S Sequencing of Human Milk Samples

提出了一种半自动化工作流, 用于从人乳和其他低生物量样品类型中进行 16S rRNA 的目标排序。

人乳样品靶向16S 测序方法的研究

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

a-method-for-targeted-16s-sequencing-of-human-milk-samples

Research

JoVE Journal

Immunology and Infection

9.7K 次观看.  University of California at Los Angeles. 提出了一种半自动化工作流, 用于从人乳和其他低生物量样品类型中进行 16S rRNA 的目标排序。

视频: A Method for Targeted 16S Sequencing of Human Milk Samples

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...