作者感谢FDA微生物方法验证小组委员会（MMVS）和细菌分析手册（BAM）理事会的成员对 沙门氏菌 LAM方法验证研究的批判性审查。

注:LAMP反应混合物包含DNA聚合酶、缓冲器、MgSO 4、dNTP、引物、DNA模板和水。前四个试剂包含在同热主混合物（材料表）中。引物在内部预混合，成为引物组合 （10 倍）。DNA模板可以从动物食品样品的浓缩汤中制备，用于筛选目的，也可以从假定的沙门氏菌分离物培养物中制备，以达到确认目的。此外，每个LAM运行中都包括阳性控制（从任何沙门氏菌参考菌株中提取的DNA，例如，沙门氏菌肠血清素ATCC 19585 [LT2]）和无模板控制（NTC;无菌分子级水）。
1. 制备DNA模板
<ol><li>要准备动物食物浓缩的DNA模板，请遵循以下步骤。<ol>
<li>将25克动物食品样品（如干猫粮、干狗食品、牛饲料、马饲料、家禽饲料和猪饲料）重达25克（材料表）放入无菌过滤袋（材料表）或等价物中。将袋子放入一个大容器或货架中，以在孵化过程中获得支持。</li>
		<li>加入225 mL的无菌缓冲肽水（BPW）。通过旋转和简短的手部按摩混合好。让在室温下站立60±5分钟。</li>
		<li>通过旋转很好地混合，确定pH与试卷。如有必要，将 pH 调整为 6.8 ± 0.2，无菌 1 N NAOH 或 1 N HCL. 孵化在 35 ± 2 °C，24 ± 2 小时。</li>
		<li>将装有动物食物浓缩汤的袋子旋转好混合。将 1 mL 从袋的过滤侧转移到微中心管。漩涡短暂。</li>
		<li>使用样品制剂（材料表）提取DNA如下。<ol>
<li>离心机在900 x g 1分钟，以去除大颗粒和转移超纳坦到一个新的微中心管。</li>
			<li>离心机在16，000 x g 2分钟，并丢弃超纳坦。</li>
			<li>将颗粒在样品制备试剂的 100 μL 中悬中，在 100 ± 1 °C 下在干热块中加热 10 分钟。</li>
			<li>冷却至室温，并将DNA提取物样本储存在-20°C。</li>
		</ol>
</li>
	</ol>
</li>
	<li>要从假定的 沙门氏菌 培养物中编写 DNA 模板，请遵循以下步骤。<ol>
<li>在FDA的BAM第5章沙门氏菌D节:沙门氏菌27的隔离之后，获得所有食物中假定的沙门氏菌隔离。</li>
		<li>接种假定的 沙门氏菌 隔离在非选择性琼脂板（如血液琼脂，营养琼脂，和尝试大豆琼脂），并在35±2°C孵育24±2小时。</li>
		<li>将几个单一菌落转移到 5 mL 的尝试性大豆汤 （TSB） 或脑心脏输液 （BHI） 汤，并在 35 ± 2 °C 下孵育 16 ± 2 小时。 注意:如果假定的 沙门氏菌 培养纯洁，此步骤可以是可选的。在这种情况下，DNA 模板可以通过在 5 mL 的 TSB 中暂停几个单一菌落和在干燥的热块中以 100 ± 1 °C 加热 500 μL 的悬架来制备。继续下面的步骤 5。</li>
		<li>将 500μL 的隔夜培养转移到微中心电管中，在 100 ± 1 °C 下在干热块中加热 10 分钟。</li>
		<li>冷却至室温，并将脱氧核糖核酸提取物储存在-20°C。</li>
	</ol>
</li>
	<li>要准备正对照DNA，请遵循上述类似步骤，用额外的稀释步骤从假定的 沙门氏菌 培养物中准备DNA模板。<ol>
<li>接种 S. Typhimurium ATCC 19585 （LT2） 或任何 沙门氏菌 参考菌株在非选择性琼脂板 （如血琼脂， 营养琼脂， 和尝试酱油） 和孵育在 35 ± 2 °C 24 ± 2 小时.</li>
		<li>将几个单一菌落转移到 5 mL 的 TSB 或 BHI 汤中，并在 35 ± 2 °C 下孵育 16 ± 2 小时，达到 +109 CFU/mL。</li>
		<li>连续稀释0.1%的肽水的隔夜文化，以获得+107 CFU/mL。</li>
		<li>将 500 μL 的稀释转移到微中心电管中，在 100 ± 1 °C 下加热 10 分钟。</li>
		<li>冷却至室温，并将正对照DNA存储在-20°C。</li>
	</ol>
</li>
</ol>2. 准备入门混合 （10 倍）
<ol><li>获得商业合成的LAM底漆（Sal4-F3，Sal4-B3，萨尔4-FIP，萨尔4-BIP，萨尔4-LF和萨尔4-LB）与标准脱盐净化（表1）。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>引号</td>
			<td>描述</td>
			<td>序列 （5'-3'）</td>
			<td>长度（bp）</td>
		</tr>
<tr>
<td>萨尔4-F3</td>
			<td>向前外部底漆</td>
			<td>加茨格特格格加格特</td>
			<td>18</td>
		</tr>
<tr>
<td>萨尔4-B3</td>
			<td>向后外部底漆</td>
			<td>克加塔格特</td>
			<td>18</td>
		</tr>
<tr>
<td>萨尔4-菲普</td>
			<td>前进内引入</td>
			<td>格茨卡塔-塔特加特</td>
			<td>38</td>
		</tr>
<tr>
<td>萨尔4-比普</td>
			<td>向后内引入</td>
			<td>格加克加格格茨格- 茨加加格查加克</td>
			<td>38</td>
		</tr>
<tr>
<td>萨尔4-LF</td>
			<td>循环向前入门</td>
			<td>特卡特卡 - 特克特格</td>
			<td>25</td>
		</tr>
<tr>
<td>萨尔4-LB</td>
			<td>循环向后引入</td>
			<td>阿加格塔格</td>
			<td>21</td>
		</tr>
</tbody></table>表1:用于在动物食品中筛查沙门氏菌和确认沙门氏菌免受文化隔离的LAM入门。引材根据沙门氏菌 invA序列（GenBank 入世编号 M90846）设计。
<ol start="2"><li>通过用适量的无菌分子级水补充引物，准备每个引物（100 μM）的库存溶液。混合好10s的漩涡和存储在-20°C（-80°C的长期存储）。</li>
	<li>根据工作表 （表 2）准备底漆混合 （10 倍）。将适量的入门库存溶液和无菌分子级水加入微中心水管中。混合所有的试剂以及通过漩涡为10s。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>库存凝结 （μM）</td>
			<td>引体混合凝结 （μM）</td>
			<td>体积 （μL）</td>
		</tr>
<tr>
<td>萨尔4-F3引水器</td>
			<td>100</td>
			<td>1</td>
			<td>10</td>
		</tr>
<tr>
<td>萨尔4-B3引水器</td>
			<td>100</td>
			<td>1</td>
			<td>10</td>
		</tr>
<tr>
<td>萨尔4-FIP入门</td>
			<td>100</td>
			<td>18</td>
			<td>180</td>
		</tr>
<tr>
<td>萨尔 4 - 比普入门</td>
			<td>100</td>
			<td>18</td>
			<td>180</td>
		</tr>
<tr>
<td>萨尔4-LF引漆</td>
			<td>100</td>
			<td>10</td>
			<td>100</td>
		</tr>
<tr>
<td>萨尔 4 - lb 入门</td>
			<td>100</td>
			<td>10</td>
			<td>100</td>
		</tr>
<tr>
<td>分子级水</td>
			<td>不适用</td>
			<td>不适用</td>
			<td>420</td>
		</tr>
<tr>
<td>总</td>
			<td>不适用</td>
			<td>不适用</td>
			<td>1000</td>
		</tr>
</tbody></table>表2:用于准备LAM底漆混合（10倍）的工作表。 引入已列在 表 1中。
<ol start="4"><li>Aliquot 的 10 倍入门混合到每微中心管 500μL，并存储在 -20 °C。</li>
</ol>3. 灯反应的组装
注:为防止交叉污染，强烈建议将用于制备LAM主混合和添加DNA模板的区域物理分离。图 1是一个灯图。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61239/61239fig1v2.jpg"> 图1:沙门氏菌LAM工作流程的示意图。<a href="https://www.jove.com/files/ftp_upload/61239/61239fig1v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<ol><li>准备和运行设置<ol>
<li>清洁的长凳与异丙醇和DNA和DNA和DNase降解溶液（材料表）。清洁移液器和管带支架（材料表）与DNA和DNase降解溶液。</li>
		<li>解冻同热主混合，引漆混合（10倍），分子级水，正对照DNA和DNA模板在室温下。</li>
		<li>打开 LAMP 仪器（材料表），点击打开屏幕即可访问主屏幕。按照以下步骤创建运行。 注:LAMP仪器的一个模型有2个方块（A和B），每个块有8个样本，另一个模型有一个可容纳8个样品的方块（材料表）。<ol>
<li>点击 LAMP+安妮， 并选择 编辑 输入样本信息。 注:默认的LAM运行配置文件包括30分钟在65°C的放大和从98°C到80°C的安妮相，每秒0.05°C的减小。</li>
			<li>点击每个示例行以激活光标并输入相关示例信息，使用 AB 块 图标在两个 LAMP 仪表块之间切换。</li>
			<li>输入所有样本信息后，点击 "检查 "图标。 注:可选地，可保存运行设置（称为"配置文件"，其中包含示例信息以及默认的 LAMP 运行配置文件），供以后使用。点击 "保存 "图标，给配置文件一个独特的名称。下次测试同一组样品时，可以使用保存的配置文件启动新的运行。点击主屏幕左下角 的文件夹 图标，并选择 "配置文件夹" 以加载已保存的配置文件。</li>
		</ol>
</li>
	</ol>
</li>
	<li>灯反应组件 注:使用两个LAM仪表块（A和B，共16个样本）时，为18个样品准备LAM主组合。如果只使用一个LAM仪表块（共8个样本），则准备10个样品的LAM主组合。对于其他样本编号，相应地调整体积以适应管道损失。每次 LAMP 运行中始终包含正控和 NTC。建议在独立的LAM运行中对每个样品进行重复测试。<ol>
<li>根据工作表（表3）准备LAM主混合。将适量的同热主混合、引水混合和分子级水加入微中心水管和涡流中，轻轻搅拌3s. 离心机。</li>
		<li>将管带放置在带状支架中，并将 23μL 的 LAMP 主混合分配给每个井。</li>
		<li>漩涡所有DNA模板和离心机短暂。将 2 μL 的 DNA 模板添加到适当的井中，并紧紧盖住盖子。</li>
		<li>从支架上取下管带，轻拂手腕，以确保所有试剂都汇集在管子底部。</li>
		<li>将管带加载到 LAMP 仪表块中，确保盖子在关闭盖子之前是安全的。</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>工作委员会。</td>
			<td>最终反应凝结。</td>
			<td>每个样本的体积 （μL）</td>
			<td>18 个样本的体积 （μL）</td>
			<td>10 个样本的体积 （μL）</td>
		</tr>
<tr>
<td>ISO-001异热主混合</td>
			<td>1.67 倍</td>
			<td>1x</td>
			<td>15</td>
			<td>270</td>
			<td>150</td>
		</tr>
<tr>
<td>入门混合</td>
			<td>10倍</td>
			<td>1x</td>
			<td>2.5</td>
			<td>45</td>
			<td>25</td>
		</tr>
<tr>
<td>分子级水</td>
			<td>不适用</td>
			<td>不适用</td>
			<td>5.5</td>
			<td>99</td>
			<td>55</td>
		</tr>
<tr>
<td>主混合子图</td>
			<td>不适用</td>
			<td>不适用</td>
			<td>23</td>
			<td>414</td>
			<td>230</td>
		</tr>
<tr>
<td>DNA 模板</td>
			<td>不适用</td>
			<td>不适用</td>
			<td>2</td>
			<td>不适用</td>
			<td>不适用</td>
		</tr>
</tbody></table>表3:用于准备LAM反应组合的工作表。 底漆组合 （10 倍）是根据 表 2 编写的，使用表 1中列出的入门的库存解决方案。
4. 灯跑
注意:在LAM运行期间，使用FAM通道获取荧光读数。当荧光比达到放大速率曲线的最大值时，仪器会自动确定时间到峰值值（Tmax: min） 。Tm （°C） 是最终放大产品的熔化/退化温度。
<ol><li>单击屏幕右上角的 "运行" 图标，然后选择包含管带的块（s）以启动 LAMP 运行。</li>
	<li>可选地，当反应正在进行中时，点击 温度、 放大和 Anneal 选项卡，查看 LAMP 运行过程中各种参数的动态变化。</li>
	<li>运行完成后，点击 放大 和 安妮尔 选项卡，查看完整的放大和报解曲线，然后点击 结果 选项卡查看结果。</li>
	<li>可选地，用于记录，使用"仪器串行number_run号"的格式（例如"GEN2-2209_0030"）记录位于屏幕左上角的运行号码。</li>
</ol>5. 对LAM结果的解释
注:LAMP 结果可直接在 LAMP 仪表板上查看和/或使用 LAMP 软件（材料表）。
<ol><li>要解释仪表板上的 LAMP 结果，请遵循以下步骤。<ol>
<li>点击主屏幕左下角 的文件夹 图标，然后选择 "日志" 以导航到文件位置以加载感兴趣的 LAMP 运行。 注:LAMP 运行按日期组织，从年初开始。</li>
		<li>观察与每次运行相关的五个选项卡:配置文件、温度、放大、安妮和结果。 注:随着LAMF反应的进行， 配置文件 和 温度 选项卡分别显示样品井中的编程温度和实际温度。 放大 和 安妮尔 选项卡分别显示放大和退化阶段的荧光读数和荧光变化。 结果 选项卡显示 LAMP 结果的表格视图。</li>
		<li>点击 结果 选项卡，观察每口井的 LAMP 结果。 注:有三个列（嗯，放大和安妮尔）。"放大"列显示每个样品（"嗯"）的时间到峰值值（T最大值:分钟:秒），"安妮"列显示该井中任何放大产品的熔化/退化温度（Tm:°C）。</li>
		<li>解释 LAMP 结果并报告最终的 LAMP 结果如下。<ol>
<li>首先检查控制井。NTC井应有空白T最大值，而Tm可以为空白（两个LAMG仪表型号）或<83°C（仅用于带两个方块的LAMG仪表模型）。正控制井应最大T在5至10分钟之间，T米约90°C。</li>
			<li>检查样本井。所有具有正确T m（约90°C）和T最大值（5~30分钟）的样品均被视为沙门氏菌阳性。</li>
			<li>根据重复运行的结果报告最终的 LAMP 结果。如果重复运行的结果一致，则可以报告最终的 LAMP 结果。如果重复运行不一致，则单独重复两次运行。如果结果仍然不一致，样本应被视为 沙门氏菌 的假定阳性，并且需要经过培养确认。</li>
		</ol>
</li>
	</ol></li>
	<li>要使用该软件解释 LAMP 结果，请按照以下步骤操作。<ol>
<li>单击左面板上的 计算机 图标，导航到文件位置以加载感兴趣的 LAMP 运行。 注:安装软件的计算机不需要连接到LAMB仪器来分析LAMB结果，即远程访问可用。LAMP 运行按日期组织。</li>
		<li>观察与每次运行相关的七个选项卡:配置文件、温度、放大、放大速率、安妮、安妮衍生物和结果。 注:与仪表板视图类似，简介和温度选项卡分别显示样本井中的编程温度和实际温度，以及 LAMP 反应的进行。放大/放大率和安妮/安妮/安妮衍生物选项卡分别显示放大和安妮阶段的荧光读数或荧光变化。结果选项卡显示与仪表板视图略有不同的 LAMP 结果的表格视图。</li>
		<li>点击 放大速率 选项卡，按时间查看荧光比的图形显示。单击屏幕右上角的 "设置" 图标，将"峰值检测阈值比"从 0.020 调整到 0.010。 注:调整是必要的，以确保确定所有有效的峰值，以及使用软件获得的结果与仪表板上显示的结果匹配。</li>
		<li>点击 "结果 "选项卡，观察每口井的 LAMP 结果。 注:有四个列（图形名称、井号、井号和峰值值）。"峰值值"列的上半部分显示每个样品的"放大时间"（T最大值:最小:秒），而底部部分显示"安妮衍生产品"（Tm: °C）用于该井中的任何放大产品。</li>
		<li>解释 LAMP 结果并报告最终 LAMP 结果，按照类似步骤报告最终 LAMP 结果，就像使用仪表板时一样，但有一个例外，即 NTC 井和其他负样本应具有空白Tm，因为 LAMP 软件设置消除了这些Tm < 83 °C 结果。同样，所有具有正确Tm（约90°C）和T最大值（5~30分钟）的样品均被视为沙门氏菌阳性。</li>
	</ol></li>
</ol>

<ol>
	<li>Notomi, T., 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=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Research. 28 (12), 63 (2000).</li><li>Kaneko, H., Kawana, T., Fukushima, E., Suzutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+of+loop-mediated+isothermal+amplification+to+a+culture+medium+and+biological+substances.">Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances.</a> Journal of Biochemical and Biophysical Methods. 70 (3), 499-501 (2007).</li><li>Francois, P., 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=Robustness+of+a+loop-mediated+isothermal+amplification+reaction+for+diagnostic+applications.">Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications.</a> FEMS Immunology and Medical Microbiology. 62 (1), 41-48 (2011).</li><li>Yang, Q., Wang, F., Prinyawiwatkul, W., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustness+of+Salmonella+loop-mediated+isothermal+amplification+assays+for+food+applications.">Robustness of Salmonella loop-mediated isothermal amplification assays for food applications.</a> Journal of Applied Microbiology. 116 (1), 81-88 (2014).</li><li>Nagamine, K., Watanabe, K., Ohtsuka, K., Hase, T., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+reaction+using+a+nondenatured+template.">Loop-mediated isothermal amplification reaction using a nondenatured template.</a> Clinical Chemistry. 47 (9), 1742-1743 (2001).</li><li>Zhang, X., Lowe, S. B., Gooding, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+review+of+monitoring+methods+for+loop-mediated+isothermal+amplification+(LAMP).">Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP).</a> Biosensors &amp; Bioelectronics. 61, 491-499 (2014).</li><li>Nagamine, K., Hase, T., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+reaction+by+loop-mediated+isothermal+amplification+using+loop+primers.">Accelerated reaction by loop-mediated isothermal amplification using loop primers.</a> Molecular and Cellular Probes. 16 (3), 223-229 (2002).</li><li>Yang, Q., Domesle, K. J., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+Salmonella+detection+in+food+and+feed:+Current+applications+and+future+directions.">Loop-mediated isothermal amplification for Salmonella detection in food and feed: Current applications and future directions.</a> Foodborne Pathogens and Disease. 15 (6), 309-331 (2018).</li><li>Mori, Y., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+Expansion+of+its+practical+application+as+a+tool+to+achieve+universal+health+coverage.">Loop-mediated isothermal amplification (LAMP): Expansion of its practical application as a tool to achieve universal health coverage.</a> Journal of Infection and Chemotherapy. 26 (1), 13-17 (2020).</li><li>Mansour, S. M., Ali, H., Chase, C. C., Cepica, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+diagnosis+of+18+World+Organization+for+Animal+Health+(OIE)+notifiable+viral+diseases+of+ruminants,+swine+and+poultry.">Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.</a> Animal Health Research Reviews. 16 (2), 89-106 (2015).</li><li>Kumar, Y., Bansal, S., Jaiswal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+A+rapid+and+sensitive+tool+for+quality+assessment+of+meat+products.">Loop-mediated isothermal amplification (LAMP): A rapid and sensitive tool for quality assessment of meat products.</a> Comprehensive Reviews in Food Science and Food Safety. 16 (6), 1359-1378 (2017).</li><li>Kundapur, R. R., Nema, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification:+Beyond+microbial+identification.">Loop-mediated isothermal amplification: Beyond microbial identification.</a> Cogent Biology. 2, 1137110 (2016).</li><li>. Compliance Policy Guide Sec. 690.800 Salmonella in Food for Animals Available from: <a target="_blank" href="https://www.fda.gov/downloads/iceci/compliancemanuals/compliancepolicyguidancemanual/ucm361105.pdf">https://www.fda.gov/downloads/iceci/compliancemanuals/compliancepolicyguidancemanual/ucm361105.pdf</a> (2013)</li><li>Bird, P., 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=Evaluation+of+the+3M+molecular+detection+assay+(MDA)+2+-+Salmonella+for+the+detection+of+Salmonella+spp.+in+select+foods+and+environmental+surfaces:+collaborative+study,+first+action+2016.01.">Evaluation of the 3M molecular detection assay (MDA) 2 - Salmonella for the detection of Salmonella spp. in select foods and environmental surfaces: collaborative study, first action 2016.01.</a> Journal of AOAC International. 99 (4), 980-997 (2016).</li><li>D'Agostino, M., 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=Validation+of+a+loop-mediated+amplification/ISO+6579-based+method+for+analysing+soya+meal+for+the+presence+of+Salmonella+enterica.">Validation of a loop-mediated amplification/ISO 6579-based method for analysing soya meal for the presence of Salmonella enterica.</a> Food Analytical Methods. 9 (11), 2979-2985 (2016).</li><li>Ge, B., 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=Multi-laboratory+validation+of+a+loop-mediated+isothermal+amplification+method+for+screening+Salmonella+in+animal+food.">Multi-laboratory validation of a loop-mediated isothermal amplification method for screening Salmonella in animal food.</a> Frontiers in Microbiology. 10, 562 (2019).</li><li>D'Agostino, M., Diez-Valcarce, M., Robles, S., Losilla-Garcia, B., Cook, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loop-mediated+isothermal+amplification-based+method+for+analysing+animal+feed+for+the+presence+of+Salmonella.">A loop-mediated isothermal amplification-based method for analysing animal feed for the presence of Salmonella.</a> Food Analytical Methods. 8 (10), 2409-2416 (2015).</li><li>Galan, J. E., Ginocchio, C., Costeas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+characterization+of+the+Salmonella+invasion+gene+invA:+homology+of+InvA+to+members+of+a+new+protein+family.">Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family.</a> Journal of Bacteriology. 174 (13), 4338-4349 (1992).</li><li>Chen, S., Wang, F., Beaulieu, J. C., Stein, R. E., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+viable+salmonellae+in+produce+by+coupling+propidium+monoazide+with+loop-mediated+isothermal+amplification.">Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification.</a> Applied and Environmental Microbiology. 77 (12), 4008-4016 (2011).</li><li>Yang, Q., Chen, S., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Salmonella+serovars+in+shell+eggs+by+loop-mediated+isothermal+amplification.">Detecting Salmonella serovars in shell eggs by loop-mediated isothermal amplification.</a> Journal of Food Protection. 76 (10), 1790-1796 (2013).</li><li>Yang, Q., 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=Evaluation+of+loop-mediated+isothermal+amplification+for+the+rapid,+reliable,+and+robust+detection+of+Salmonella+in+produce.">Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.</a> Food Microbiology. 46, 485-493 (2015).</li><li>Yang, Q., Domesle, K. J., Wang, F., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Salmonella+in+food+and+feed+by+coupling+loop-mediated+isothermal+amplification+with+bioluminescent+assay+in+real-time.">Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time.</a> BMC Microbiology. 16 (1), 112 (2016).</li><li>Domesle, K. J., Yang, Q., Hammack, T. S., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+Salmonella+loop-mediated+isothermal+amplification+assay+in+animal+food.">Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.</a> International Journal of Food Microbiology. 264, 63-76 (2018).</li><li>Bustin, S. A., 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=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clinical Chemistry. 55 (4), 611-622 (2009).</li></ol>

作者宣称他们没有相互竞争的经济利益。本手稿中表达的观点是作者的观点，不一定反映卫生与公众服务部、美国食品和药物管理局或美国政府的官方政策。提及任何商业材料、设备或工艺绝不构成食品和药物管理局的批准、认可或建议。

我们在这里分别介绍了一种简单、快速、具体和敏感的LAM方法，用于在动物食品和纯文化中筛查和确认沙门氏菌。由于含有四个关键试剂的同热主混合物和现成的内部预制引物混合物的便利性，组装 LAMP 反应只需要几个管道步骤（图 1）。包括放大和退影相在内的总运行时间小于 38 分钟（图2A、B和图 3A、B）。阳性结果通过实时荧光（图2C和图3C，D）进行监测，最早可在5分26秒检测到。退火阶段作为 LAMP 特异性的额外确认，因为只有具有正确Tm（约 90 °C） 的样本报告为阳性（图 2D、E和图 3E+G）。先前26日曾报道过1个纯培养物中的沙门氏菌细胞和1CFU/25克动物食品的敏感性。
由于LAML非常有效，并产生大量的DNA1，因此使用最佳实验室实践防止交叉污染至关重要，这可能包括物理分离准备LAMG主混合物和添加DNA模板的区域，避免产生气溶胶，使用滤波器提示，经常更换手套，以及避免在放大后打开LAM反应管。

在整个LAM方法开发、评价、预科研究和多实验室验证的生命周期中，我们观察到LAM对各种动物食品或食品矩阵和文化介质4、19、22、23、24中的抑制剂的高耐受性，突出了该方法的稳健性，并在全球范围开展了许多其他研究。这是优于PCR或实时PCR，这通常需要内部放大控制，以确保负结果不会由于矩阵抑制28。此外，在绝大多数研究中，LAMP 与 PCR 或实时 PCR 相比，表现出类似的（或优越的）特异性和灵敏度。LAMP试剂的成本约为每反应1美元。本协议中使用的LAM仪器体积小，维护低，便携。它们可以处理任何采用荧光测量（包括LAM）进行目标检测的同热放大方法。使用LAMB软件，可以以多种格式生成综合报告（pdf、文本和图像）。
方法验证是采用新方法进行日常使用的关键步骤。值得注意的是，这里报告的LAM协议已经成功地完成了多实验室验证19。随着最近将这种LAM协议纳入美国FDA的BAM第5章 沙门氏菌27，预计该方法将得到更广泛的应用，既作为动物食品的快速筛选方法，也作为一个可靠的确认方法，推定 沙门氏菌 从所有食品类别分离。

循环介质等热放大（LAMP）是日本科学家1于2000年发明的一种新型的等热核酸放大试验（iNAAT）。通过在初始步骤中形成目标特异性干细胞环DNA结构，LAMP使用链状脱氧核糖核酸聚合酶，以准指数高效放大这种启动材料，在不到1小时1小时的时间内产生109个目标副本。与广泛使用的NAAT聚合酶链反应（PCR）相比，LAMP具有几个优点。首先，LAMP 反应是在同热条件下进行的。这就不需要精密的热循环仪器。其次，LAMP对培养素和生物物质具有高度的耐受性2，临床和食品应用都表现出稳健性。这简化了样品准备，最大限度地减少了虚假的阴性结果5。第三，LAMP 可适应多种检测平台，如浊度、色度、生物发光、荧光和微流体6。第四，LAMP 非常具体，因为它使用四到六个专门设计的引漆来瞄准6到 8 个特定区域1、7。第五，LAMP是超敏感的，许多研究已经报告了它对PCR或实时PCR8的优越灵敏度。最后，LAMP 更快，许多检测现在采用 30 分钟的标准运行时间，而 PCR 型检测通常需要 1+2 h8。
这些有吸引力的功能推动了LAM在广泛的病原体检测领域的应用，包括体外诊断9，动物疾病诊断10，食品和环境测试11。值得注意的是，世界卫生组织已建议使用结核病杆菌（LAMP）作为痰涂片显微镜的有效替代测试，用于12号外围环境下的肺结核诊断。LAMP的应用也扩展到微生物鉴定之外，还包括过敏原、动物物种、耐药性、转基因生物和杀虫剂的检测。
非足部沙门氏菌是一种人为病原体，具有重大的食品安全和公共卫生问题。它也已被确定为动物食物中重要的微生物危害（即动物食品）15，16。为了防止沙门氏菌疾病/疫情受到污染的人类食物和动物食品，必须拥有快速、可靠和可靠的方法在各种矩阵中检测沙门氏菌。在过去十年中，国际上在各种食品基质中开发和应用沙门氏菌LAM检测技术方面作出了相当大的努力，最近在一份广泛的审查报告8中总结了这一点。一些沙门氏菌LAM检测，包括这里提出的一个，已经成功地完成了多实验室验证后，既定的国际准则17，18，19，20。
我们的沙门氏菌LAM检测特别针对沙门氏菌入侵基因ivA（GenBank入世号M90846）21，是快速，可靠，健壮的多个食品矩阵4，22，23，24，25，26。该方法已在6个动物食品矩阵中验证在合作研究26和干狗食品在多实验室合作研究19。因此，这里介绍的沙门氏菌LAM方法最近被纳入美国食品和药物管理局（FDA）的细菌分析手册（BAM）第5章沙门氏菌27，以服务于两个目的，一个是作为动物食品中沙门氏菌存在的快速筛查方法，另一个是作为从所有食物中分离出的沙门氏菌的可靠确认方法。

<table>
	<tbody>
		<tr>
			<td>Brain heart infusion (BHI) broth</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>299070</td>
			<td>Liquid growth medium used in the cultivation of Salmonella.</td>
		</tr>
		<tr>
			<td>Buffered peptone water (BPW)</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>218105</td>
			<td>Preenrichment medium for the recovery of Salmonella from animal food samples.</td>
		</tr>
		<tr>
			<td>DNA AWAY</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>7010</td>
			<td>Eliminates unwanted DNA and DNase from laboratory bench, glassware, and plasticware without affecting subsequent DNA samples.</td>
		</tr>
		<tr>
			<td>Genie Explorer software</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>Version 2.0.6.3</td>
			<td>Supports remote operation of Genie instruments including LAMP runs and data analysis.</td>
		</tr>
		<tr>
			<td>Genie II or Genie III (LAMP instrument)</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>GEN2-02 or GEN3-02</td>
			<td>A small instrument capable of temperature control up to 100 &#176;C with &#177; 0.1 &#176;C accuracy and simultaneous fluorescence detection via the FAM channel. Genie II has 2 blocks (A and B) with 8 samples in each block. Genie III has a single block that accommodates 8 samples.</td>
		</tr>
		<tr>
			<td>Genie strip</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>OP-0008</td>
			<td>8-well microtube strips with integral locking caps and a working volume of 10 to 150 &#181;l.</td>
		</tr>
		<tr>
			<td>Genie strip holder</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>GBLOCK</td>
			<td>Used to hold Genie strips when setting up a LAMP reaction, the aluminum holder can also be used as a cool block.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl) solution, 1 N</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>SA48-500</td>
			<td>Adjusts pH of animal food samples after adding BPW and prior to overnight enrichment.</td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>88-860-022</td>
			<td>Heats samples at 100 &#177; 1 oC for DNA extraction.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>3960</td>
			<td>Standard laboratory incubator.</td>
		</tr>
		<tr>
			<td>ISO-001 isothermal master mix</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>ISO-001</td>
			<td>An optimized master mix to simplify the assembly of a LAMP reaction, containing a strand-displacing GspSSD DNA polymerase large fragment from Geobacillus spp., thermostable inorganic pyrophosphatase, reaction buffer, MgSO4, dNTPs, and a double-stranded DNA binding dye (FAM detection channel).</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>A416</td>
			<td>Disinfects work surfaces.</td>
		</tr>
		<tr>
			<td>LAMP primers</td>
			<td>Integrated DNA Technologies Inc., Coralville, IA</td>
			<td>Custom</td>
			<td>LAMP primers with detailed information in Table 1.</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf North America, Hauppauge, NY</td>
			<td>22620207</td>
			<td>MiniSpin plus personal microcentrifuge.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>05-408-129</td>
			<td>Standard microcentrifuge tubes.</td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>AM9938</td>
			<td>Used in making primer stocks, primer mix, and LAMP reaction mix.</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH) solution, 1 N</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>SS266-1</td>
			<td>Adjusts pH of animal food samples after adding BPW and prior to overnight enrichment.</td>
		</tr>
		<tr>
			<td>Nonselective agar (e.g., blood agar, nutrient agar, and trypticase soy agar)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>R01202</td>
			<td>Solid growth medium used in the cultivation of Salmonella.</td>
		</tr>
		<tr>
			<td>Peptone water</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>218071</td>
			<td>Dilutes overnight Salmonella cultures to make positive control DNA.</td>
		</tr>
		<tr>
			<td>Pipettes and tips</td>
			<td>Mettler-Toledo Rainin LLC, Oakland CA</td>
			<td>Pipet Lite LTS series</td>
			<td>Standard laboratory pipettes and tips.</td>
		</tr>
		<tr>
			<td>PrepMan Ultra sample preparation reagent</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>4318930</td>
			<td>A simple kit used for the rapid preparation of DNA templates for use in a LAMP reaction.</td>
		</tr>
		<tr>
			<td>Salmonella reference strain LT2</td>
			<td>ATCC, Manassas, VA</td>
			<td>700720</td>
			<td>Salmonella reference strain used as positive control.</td>
		</tr>
		<tr>
			<td>Trypticase soy broth (TSB)</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>211768</td>
			<td>Liquid growth medium used in the cultivation of Salmonella.</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries, Inc., Bohemia, NY</td>
			<td>SI-0236</td>
			<td>Standard laboratory vortex mixer.</td>
		</tr>
		<tr>
			<td>Whirl-pak filter bag</td>
			<td>Nasco Sampling Brand, Fort Atkinson, WI</td>
			<td>B01318</td>
			<td>Filter bags to hold animal food samples for preenrichment.</td>
		</tr>
	</tbody>
</table>

loop-mediated isothermal amplification for screening salmonella in animal food and confirming salmonella from culture isolation

循环介质异热放大 （LAMP） 已成为快速检测多种细菌、真菌、寄生虫和病毒制剂的强大核酸放大测试。 沙门氏菌 是全球食品安全关注的细菌病原体，包括动物食品。这里介绍的是一个多实验室验证的 沙门氏菌 LAM协议，可用于快速筛查动物食品是否存在 沙门氏菌 污染，也可用于确认从所有食品类别中回收的 假定沙门氏菌 分离物。LAMP检测特别针对 沙门氏菌 入侵基因（invA），快速、灵敏、特异性。模板DNAs是由动物食品的浓缩肉汤或纯文化的假定 沙门氏菌 分离。LAMP试剂混合物是通过结合同热主混合物、入门、DNA模板和水制备的。LAMP 检测在 65 °C 的恒定温度下运行 30 分钟。通过实时荧光监测阳性结果，最早可在 5 分钟内检测到。LAMP检测对动物食品或培养基中的抑制剂具有很高的耐受性，是一种快速、可靠、健壮、经济、方便用户的方法，用于筛查和确认 沙门氏菌。LAMP方法最近被纳入美国食品和药物管理局的 细菌分析手册 （BAM）第5章。

图 2 和 图 3 显示两个平台上显示的具有代表性的 LAMP 图形/表。在此 LAMP 运行中，样本 S1 到 S6 是 S. 肠 血清婴儿 ATCC 51741 的 10 倍序列稀释，每个反应从 1.1 x 106 CFU 到 11 CFU 不等。正对照是 S. 肠 血清钛 ATCC 19585 （LT2） 在 1.7 x 104 CFU 每反应和 NTC 是分子级水。

在同一组样品的重复运行之后，将报告这些样品的最终 LAMP 结果。具有代表性的LAM运行表明，LAMP成功地检测出样品中浓度广泛的 沙门氏菌 。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg"> 图2:在LAM仪表板上显示的代表性LAM结果。（A） "个人资料" 选项卡显示编程的温度轮廓。（B） 温度 标签显示样品井中的实际温度，随着LAMB反应的进行。（C） 放大 选项卡显示 LAMP 放大过程中的荧光读数。（D） 安妮标签 显示在退星阶段荧光（衍生物）的变化。（E） 结果 选项卡显示 LAMP 结果的表格视图。 <a href="https://www.jove.com/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg"> 图3:在LAM普软件中查看的代表性LAM结果。（A） "个人资料" 选项卡显示编程的温度轮廓。（B） 温度 标签显示样品井中的实际温度，随着LAMB反应的进行。（C） 放大 选项卡显示 LAMP 放大过程中的荧光读数。（D） 放大率 选项卡显示 LAMP 放大过程中荧光（荧光比）的变化。（E） 安妮标签 显示在安妮阶段的荧光读数。（F） 安妮衍生产品 标签显示在退星阶段荧光（衍生物）的变化。（G） 结果 选项卡显示 LAMP 结果的表格视图。 <a href="https://www.jove.com/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation at JoVE.com

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

循环介质异热放大 （LAMP） 是一种同热核酸放大测试 （iNAAT），已引起病原体检测领域的广泛兴趣。在这里，我们提出了一个多实验室验证的 沙门氏菌 LAM协议，作为一种快速、可靠和可靠的方法，用于在动物食品中筛查 沙门氏菌 ，并确认 假定的沙门氏菌 来自培养隔离。

循环介制的对热放大，用于在动物食品中筛查沙门氏菌，并确认来自文化隔离的沙门氏菌

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

loop-mediated-isothermal-amplification-for-screening-salmonella

Research

JoVE Journal

Biology

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

7.9K 次观看.  U.S. Food and Drug Administration. 循环介质异热放大 （LAMP） 是一种同热核酸放大测试 （iNAAT），已引起病原体检测领域的广泛兴趣。在这里，我们提出了一个多实验室验证的 沙门氏菌 LAM协议，作为一种快速、可靠和可靠的方法，用于在动物食品中筛查 沙门氏菌 ，并确认 假定的沙门氏菌 来自培养隔离。

视频: Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

慢性沙门氏菌感染小鼠模型

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

科研

生物学

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

在小鼠和大鼠中确认青春期发作和执行生育力测定生殖能力

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

食品和环境样品中沙门氏菌的准总体基因体分析

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

从成年小鼠肾脏中分离细胞核以进行单核RNA测序

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

检测植物-真菌相互作用中果胶的双重染色方法

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.