Name: loop-mediated isothermal amplification for screening salmonella in animal food and confirming salmonella from culture isolation
Uploaded: 2020-05-20T14:30:00.000+00:00
Duration: 8 min 24 s
Description: 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

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>
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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 &amp;deg;C with &amp;plusmn; 0.1 &amp;deg;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 &amp;micro;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 &amp;plusmn; 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

Universidad San Sebastian

Research

JoVE Journal

Biology

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

8.0K 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

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

从个人污秽苍蝇检测食源性细菌病原体

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

科研

环境科学

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫与感染

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

一个用于标识Allelotyping PCR扩增沙门氏菌肠炎血清型，哈达尔，海德堡和鼠伤寒沙门氏菌

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical &amp; Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively.

智能高通量抗菌药敏检测/噬菌体筛选系统及Lar耐药性指数的应用

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the ...

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 ...

生物学

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

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

摘要

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此视频中的章节

从个人污秽苍蝇检测食源性细菌病原体

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

一个用于标识Allelotyping PCR扩增沙门氏菌肠炎血清型，哈达尔，海德堡和鼠伤寒沙门氏菌

智能高通量抗菌药敏检测/噬菌体筛选系统及Lar耐药性指数的应用

慢性沙门氏菌感染小鼠模型

沙门氏菌筛选的高通量平台.

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体