这项工作得到了阿卜杜拉国王科技大学（King Abdullah University of Science and Technology）通过核心资金和S.M.H.的国家学期大挑战（NTGC）的支持。

1. Taq聚合酶的表达和纯化
<ol><li>在酶的C末端构建具有可切割的六组氨酸标签的质粒。</li>
	<li>按照标准方案38将50ng表达载体转化为大肠杆菌BL21-（DE3）菌株。</li>
	<li>将转化的细胞接种在四个6L烧瓶中，每个烧瓶在37°C下装有2L2YT培养基肉汤，以170rpm振荡，直到OD 600为0.8或细胞数6.4×108 。</li>
	<li>用0.5mM异丙基-β-d-硫代吡喃半乳糖苷（IPTG）诱导Taq聚合酶表达，并在16°C下进一步摇动孵育18小时。</li>
	<li>通过在4°C下以7808× g 旋转10分钟来收获细胞。将沉淀重悬于200 mL冰冷的Taq聚合酶裂解缓冲液（表1）中，在5 mL / g细胞沉淀中。</li>
	<li>将细胞与溶菌酶（2mg / mL裂解缓冲液）和蛋白酶抑制剂孵育45分钟，然后将裂解物通过30kPsi的细胞破坏物，并在4°C下以22,040× g 离心30分钟以分离细胞碎片。</li>
	<li>在85°C下加热上清液15分钟。 在4°C下以95,834× g 旋转1小时。 收集上清液并使用0.45μm过滤器在冰上过滤。</li>
	<li>使用快速蛋白质液相色谱 （FPLC） 进行蛋白质纯化，首先使用 Taq 聚合酶缓冲液 A 将样品以 4 mL/min 的流速通过 Ni-NTA HP 5 mL 色谱柱（表 2;图1A）。</li>
	<li>用 10 列体积 （CV） 的 Taq 聚合酶缓冲液 A 和 4% 的 Taq 聚合酶缓冲液 B 洗涤（表 2）。在 100 mL 瓶中使用 Taq 聚合酶缓冲液 B 超过 20 CV 以线性梯度洗脱结合蛋白。</li>
	<li>使用 Taq 聚合酶缓冲液 C 将 100 mL 瓶中的洗脱液以 4 mL/min 的速度通过阳离子交换 5 mL 柱（表 2;图1A）。</li>
	<li>用20CV的100%Taq聚合酶缓冲液C和5%Taq聚合酶缓冲液D洗涤（表2）。</li>
	<li>使用Taq聚合酶缓冲液D（表2）从5%到100%的线性梯度洗脱。</li>
	<li>如前所述，通过进行SDS-PAGE凝胶电泳 39 ，然后进行凝胶染 色40 来检查洗脱的级分。简而言之，取每个馏分 10 μL 并加入等体积的 2x SDS 上样染料。样品在90°C下变性10分钟。将样品加载到10%SDS-PAGE凝胶上，并在200V下运行凝胶25分钟，使用考马斯亮蓝染色凝胶，然后去染色。</li>
	<li>收集含有纯化的Taq聚合酶的所有馏分，并在4°C下对taq聚合酶储存缓冲液（表3）进行透析。 简而言之，准备2L储存缓冲液，并将透析盒浸入缓冲液中2分钟以水合。使用针头将收集的馏分注入透析盒中，并在搅拌器上以200rpm的速度将其置于透析缓冲液中过夜。</li>
	<li>透析后，使用分光光度计测量蛋白质浓度，制备10μL等分试样，在液氮中快速冷冻并储存在-80°C。 注意：在将所有缓冲液加载到FPLC系统之前，使用0.45μm过滤器过滤所有缓冲液进行纯化。</li>
</ol>2. MMLV-RT在昆虫细胞表达系统中的表达及纯化
<ol><li>杆啶DNA的生成和分离<ol><li>如前所述，使用 C 末端可切割的 TEV-8xHis-Strep 标签克隆 MMLV-RT 序列41。</li>
		<li>将 100 ng 表达载体混合到 50 μL DH10Bac 细胞中，并通过敲击试管轻轻混合。</li>
		<li>将细胞在冰上孵育15分钟。在42°C下对细胞进行热休克1分钟。</li>
		<li>立即转移到冰上并加入 400 μL SOC 培养基。在37°C振荡孵育4小时。</li>
		<li>将 10 μL 和 15 μL 混合物放在含有三种抗生素的 LB 琼脂平板上;50 μg/mL 卡那霉素、10 μg/mL 四环素和 7 μg/mL 庆大霉素以及 40 μg/mL IPTG 和 100 μg/mL X-Gal 用于蓝白选择。</li>
		<li>将板在37°C孵育48小时。挑选几个白色菌落和一个蓝色菌落作为对照，并在含有上述抗生素的新鲜LB琼脂平板上重新划线。将板在37°C孵育过夜。</li>
		<li>确认白色表型后，挑选几个菌落，并将它们接种到含有 50 μg/mL 卡那霉素、10 μg/mL 四环素和 7 μg/mL 庆大霉素的 10 mL LB 培养基中，装在 50 mL 试管中。</li>
		<li>将培养物在37°C下以170rpm振荡孵育过夜。通过以 22,040 x g 旋转细胞 10 分钟来沉淀细胞。</li>
		<li>倒出上清液，并将沉淀重悬于来自小型制备试剂盒的 250 μL 重悬溶液中（该溶液需要按照制造商的说明处理），然后将溶液转移到 1.5 mL 微量离心管中。</li>
		<li>加入 250 μL 裂解液，轻轻混合并孵育 3 分钟。加入 350 μL 中和溶液，倒置 2x-3x，并以 22,040 x g 离心 10 分钟。</li>
		<li>将上清液转移到新鲜管中，加入等体积的冰冷异丙醇，并在-20°C下孵育30分钟。</li>
		<li>通过以 22,040 x g 旋转 10 分钟来沉淀沉淀的 DNA。弃去上清液，用700μL70%冰冷乙醇洗涤沉淀，2x。</li>
		<li>用移液管取出上清液，不要接触沉淀。让颗粒在层流罩中空气干燥 5-10 分钟或直到管中看不到液体。将沉淀溶解在 100 μL EB 缓冲液中。</li>
	</ol></li>
	<li>杆啶转染和病毒扩增<ol><li>P1病毒制备<ol><li>在 6 孔组织培养板中，将 ~ 9 x 105 个细胞/孔接种在 2 mL 昆虫细胞培养基中。</li>
			<li>在室温下孵育板~30分钟，以使细胞附着。</li>
			<li>按如下方式制备 Bacmid/Fugene 混合物：将 1 μg Bacmid DNA 和 300 μL 昆虫细胞培养基混合在一个试管中。在另一个试管中，混合 8 μL 转染试剂和 300 μL 昆虫细胞培养基。通过轻轻移液混合两种混合物，并在室温下静置~30分钟，以形成杆嘧胺/转染试剂复合物。</li>
			<li>向每个孔中滴加杆窜复合物混合物（~210μL），并用透明薄膜密封板。</li>
			<li>将板在27°C孵育3-4天。</li>
			<li>取含有病毒的培养基，加入FBS（终浓度2%），使用0.45μm过滤器过滤，并在4°C下避光储存，作为P1病毒原液。</li>
		</ol></li>
		<li>P2病毒制备<ol><li>在培养瓶中以 2 x 106 个细胞/mL 的速度转染 50 mL 昆虫细胞和 3 mL P1 病毒。</li>
			<li>在27°C下以100rpm振荡孵育4-6天，直到死细胞百分比达到25%-30%。</li>
			<li>以300× g 离心10分钟，收集含有病毒的上清液。</li>
			<li>加入FBS至终浓度为2%，通过0.45μm过滤器过滤，各等分试样1mL，并作为P2病毒原液储存在-80°C下。</li>
		</ol></li>
		<li>P3病毒制备<ol><li>在培养瓶中以 2 x 106 个细胞/mL 的速度将 100 mL 昆虫细胞与 2 mL P2 病毒一起转染。</li>
			<li>在27°C下以100rpm振荡孵育3-4天，直到死细胞的百分比达到15%-20%。</li>
			<li>将细胞以300× g 离心10分钟，并收集含有病毒的上清液。</li>
			<li>加入FBS至终浓度为2%。通过0.45μm过滤器过滤。可在4°C避光保存1个月。</li>
		</ol></li>
	</ol></li>
	<li>MMLV-RT在昆虫细胞中的表达和纯化<ol><li>将 5 mL P3 病毒以 2 x 106 个细胞/mL（700 mL/2L 烧瓶）的密度加入新鲜昆虫细胞中，共 6 个烧瓶。</li>
		<li>转染后55-60小时后，以7808×g旋转10分钟收获细胞。</li>
		<li>将细胞沉淀重悬于200mL MMLV-RT裂解缓冲液中（表4）。将裂解物通过15kPsi的细胞干扰物，并在4°C下以22,040× g 离心30分钟。</li>
		<li>将上清液转移到新管中，并在4°C下以95,834× g 再次旋转1小时。收集上清液并使用0.45μm过滤器在冰上过滤。</li>
		<li>首先使用MMLV-RT缓冲液A以4 mL/min的流速将样品通过Ni-NTA Excel 5 mL色谱柱（图1B），开始使用FPLC进行蛋白质纯化（表5）。</li>
		<li>用 10 CV 的 MMLV-RT 缓冲液 A 和 4% 的 MMLV-RT 缓冲液 B 洗涤色谱柱（表 5），并在 100 mL 瓶中以 20 CV 以上的 MMLV-RT 缓冲液 B 的线性梯度洗脱蛋白质。</li>
		<li>将洗脱的样品通过用MMLV-RT缓冲液C平衡的Strep 5 mL色谱柱（图1B）（表5）。</li>
		<li>用10CV的100%MMLV-RT缓冲液C洗涤色谱柱，并用20CV的缓冲液D以线性梯度洗脱蛋白质（表5）。</li>
		<li>按照步骤1.13.-1.14执行SDS-PAGE检查洗脱的馏分。并收集含有纯化的MMLV-RT的馏分，以便在4°C下根据MMLV-RT储存缓冲液（表6）进行透析。</li>
		<li>使用Nanodrop，等分试样，在液氮中快速冷冻并储存在-80°C下测量蛋白质浓度。 注意：在将所有缓冲液加载到FPLC系统之前，使用0.45μm过滤器过滤所有缓冲液进行纯化。</li>
	</ol></li>
</ol>3. 内部多重R3T一步法RT-qPCR试剂盒组分的制备
<ol><li>缓冲液混合物制备<ol><li>为RT-qPCR反应准备2x缓冲液，该缓冲液含有先前在37中显示的试剂。在无DNase / RNase的水中制备所有试剂，并将缓冲液混合物储存在-20°C冰箱中。</li>
	</ol></li>
	<li>引物和探针套件以及引物混合物制备 注意：使用的探针和引物列在 表7中。流感和 SARS-CoV-2 多重试剂盒包含 3 个探针和 4 个正向和反向引物组。探针在 5' 末端使用报告基因 6-羧基荧光素 （FAM） 标记 InfA，Texas Red-XN 标记 SARS-CoV-2（N 基因）和 Yakima Yellow 标记 InfB。MERS-CoV 和 SARS-CoV-2 多重试剂盒包含 3 个探针和 3 个正向和反向引物组。探针还使用报告基因Texas Red-XN标记MERS-CoV（ORF1a基因），VIC标记MERS-CoV（UpE基因）和6-羧基荧光素（FAM）SARS-CoV-2（N基因）。<ol><li>在 1.5 mL 试管中制备包含 表 7 中列出的所有引物和探针的引物混合物，每个正向和反向引物的终浓度为 6.7 μM，每个探针的终浓度为 1.25 μM。将引物混合物储存在-20°C冰箱中。使用洗脱缓冲液补足所需体积。</li>
	</ol></li>
	<li>酶混合物制备<ol><li>如 表3 所示，在Taq聚合酶储存缓冲液中制备Taq聚合酶（30U / μL）和MMLV-RT（60ng / μL）酶混合物，以弥补所需的体积。在冰上执行此步骤，并将酶混合物储存在-20°C。</li>
	</ol></li>
	<li>RNA模板<ol><li>将SARS-CoV-2、甲型H1N1流感、甲型H3N2流感、乙型流感和MERS-CoV的合成RNA分别重悬于100μL 1x Tris-EDTA缓冲液（10 mM Tri-Cl和1 mM EDTA（pH 8.0）中，制成1 x 106 RNA拷贝/μL的储备液。</li>
		<li>以以下配置混合用于 RT-qPCR 的合成 RNA：1） SARS-CoV-2、甲型流感和乙型流感，将每种病毒原液的 5 μL 加入 50 μL 体积，制成 1 x 105 个 RNA 拷贝/μL;2） SARS-CoV-2、MERS-CoV，将每种病毒原液的 5 μL 加入 50 μL 体积，制成 1 x 105 个 RNA 拷贝/μL。 对每种合成 RNA 混合物进行 10 倍连续稀释，范围从 1 x 105 个 RNA 拷贝/μL 到 10 个 RNA 拷贝/μL。 注意：确保使用冰冷的 DNase/RNase-Free 水来准备模板的连续稀释液。</li>
	</ol></li>
</ol>4. 内部多重 SARS-CoV-2、甲型流感、乙型流感和 SARS-CoV-2、MERS-CoV 一步法 RT-qPCR 检测
注意：对工作站表面进行消毒，并使用96孔板模板来规划PCR板布局。
<ol><li>96孔板的制备<ol><li>解冻 2x 缓冲液和引物混合物。将酶混合物放在冰上。</li>
		<li>向每个孔中加入 10 μL 2x 缓冲液混合物、1.5 μL 引物混合物、1 μL 酶混合物、6.5 μL DNase/RNase-free 水和 1 μL 需要测试的相应 RNA 混合物。每个孔的最终体积为 20 μL。请参阅准备好的布局以获取有关此步骤的帮助。 注意：建议根据包含除RNA样品以外的所有试剂的反应数量进行预混液，以避免移液偏差。此外，一式两份或一式三份地进行反应，以避免技术错误。</li>
		<li>用粘性PCR板密封板密封板，并短暂离心1分钟以收集板底部的所有液体。在将样品转移到qPCR机器之前，确保每个孔具有相同体积的液体并且没有气泡。 注意：所有PCR反应都需要在冰上设置。</li>
	</ol></li>
	<li>PCR程序<ol><li>打开real-time qPCR机器程序，选择以下实验特性： 模块类型：快速 96 孔 （0.1 mL） 实验设置：标准曲线 试剂：TaqMan 试剂 运行属性：标准。</li>
		<li>定义所有基因靶标及其报告染料，如 表7所示。此外，为要测试的每个反应定义样品名称，以便于分析扩增曲线。</li>
		<li>根据 96 孔板将靶标和样品分配给程序的板布局。</li>
		<li>在PCR仪器中设置以下循环程序，如下所示： 55°C10分钟 40 次循环：94 °C 持续 1 分钟 94°C10秒 68°C10秒 68°C20秒;这里获取荧光。</li>
		<li>将板转移到实时qPCR机器中，并将其放入支架中，确保板的方向正确，然后开始运行。</li>
		<li>选择文件位置以保存实验数据。</li>
	</ol></li>
	<li>数据分析<ol><li>首先检查qPCR程序产生的扩增图至关重要。分析检测限以评估可检测的最小RNA浓度。</li>
		<li>在x轴上绘制RNA拷贝数的对数，在y轴上绘制相应的平均Ct值，以评估测定的灵敏度。斜率和 R2 表示反应的可靠性和效率。</li>
	</ol></li>
</ol>

用于呼吸道病毒检测的多重 RT-qPCR 检测

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由于SARS-CoV-2、甲型/乙型流感和中东呼吸综合征冠状病毒变异株等常见呼吸道病毒的传播，感染率和死亡率高，给全球医疗系统带来了沉重的经济负担12,19,20。出于减轻这种负担的责任感，我们意识到需要一种快速、精确和可及的诊断检测方法，例如 RT-qPCR，以在一次测试中区分这些常见病毒。由于qRT-PCR的多重特性，诊断和区?...

正在进行的 2019 年冠状病毒病 （COVID-19） 的大流行是由称为严重急性呼吸系统综合症冠状病毒 2 （SARS-CoV-2）1 的新型冠状病毒引起的。由于 SAR-CoV-2 具有很强的传染性和快速传播能力，COVID-19 大流行出现在中国武汉市，并迅速蔓延到世界各地。这最终导致呼吸窘迫体征的开始，甚至死亡2,3,4。COVID-19 已在超过 213 个国家/地区被宣布为大流行，预计确诊病例数量将急剧增加，不同研究发表的论文证明了这一点 3,5.COVID-19主要通过感染者释放到环境中的小呼吸道飞沫传播，然后通过吸入或密切接触受污染的表面暴露给脆弱个体。当这些飞沫接触到眼睛、嘴巴或鼻子的粘膜时，一个人可能会被感染6.世界卫生组织（WHO）公布的统计数据显示，全球新冠肺炎确诊病例已超过7600万例，死亡人数达到惊人的700万例7。因此，联合国将 COVID-19 疾病引起的大流行归类为灾难，因为它对全球数十亿人的生活产生直接影响，并产生深远的经济、环境和社会影响。
包括彻底检测、早期发现、接触者追踪和病例隔离在内的公共卫生举措都已被证明对控制这一大流行至关重要8,9,10,11。冬季将增加其他呼吸道病毒的传播，如甲型和乙型流感，并伴有类似 COVID-19 的症状，因此难以及早识别、追踪和隔离 COVID-19 实例。每年，甲型和乙型流感的爆发始于深秋或一月初，季节性可预测12.SARS-CoV-2 和流感病毒具有许多流行病学特征。此外，在易感人群中也有相似之处，包括儿童、老年人、免疫功能低下以及患有慢性合并症（如哮喘、慢性阻塞性肺病、心肾衰竭或糖尿病）的个体12,13。这些病毒不仅具有易感人群，而且具有接触途径和呼吸道飞沫的传播途径14。预计随着流感季节接近 14，患者可能会感染不止一种呼吸道病毒。为此，需要在隔离有症状的患者之前对 SARS-CoV-2 和流感病毒进行筛查。由于全球缺乏核酸提取和诊断资源，无法对三种病毒（SARS-CoV-2、甲型流感和乙型流感）进行单独检测。为了在一次反应中筛选它们，需要开发一种方法或测试。
中东呼吸综合征（MERS）-CoV是人类冠状病毒（CoV）家族的成员。第一批中东呼吸综合征冠状病毒分离株来自沙特阿拉伯的一名住院患者，该患者于2012年9月因急性呼吸道疾病死亡15。有证据表明，中东呼吸综合征冠状病毒的主要宿主是单峰骆驼。已经证明，来自受感染的单峰骆驼的病毒是人畜共患的，因此可以感染人类16,17。感染这种病毒的人可以通过密切接触将其传播给他人18.截至2018年1月26日，全球已有2143例中东呼吸综合征冠状病毒感染实验室确诊病例，包括750例死亡19。中东呼吸综合征冠状病毒最典型的症状是咳嗽、发烧和呼吸急促。据报告，中东呼吸综合征冠状病毒感染还表现出肺炎、腹泻和胃肠道疾病症状20。目前尚无针对中东呼吸综合征冠状病毒的商业疫苗或特定治疗方法。因此，及时和准确的诊断对于预防中东呼吸综合征冠状病毒大范围暴发和区分中东呼吸综合征冠状病毒与SARS-CoV-2疾病至关重要。
迄今为止，已经提出了许多方法来检测这些病毒，例如多重 RT-PCR21、22、23、24、25、CRISPR/Cas1226、27、CRISPR/Cas928 和 CRISPR/Cas329、侧向层免疫测定30、纸质生物分子传感器31、一锅 SHERLOCK 测试32、DNA 适配体33、环介导的等温放大（LAMP）19,34等上述每种方法在灵敏度和特异性方面都有独特的优点和缺点。在这些方法中，基于核酸扩增的检测：多重qRT-PCR是最常见的，被认为是诊断SARS-CoV-2、甲型流感、乙型流感和MERS-CoV的金标准。
在这项研究中，我们设计并评估了各种引物组合和探针，以利用标准扭曲合成病毒 RNA 有效、准确和同时检测 SARS-CoV-2、甲型流感、乙型流感和 SARS-CoV-2、MERS-CoV。世界卫生组织 （WHO） 推荐针对 MERS-CoV 或 SARS-CoV-2 靶基因开发的多重检测方法。这些基因通常编码有助于形成复制/转录复合物 （RTC）35 的蛋白质和复合物，例如用于 MERS-CoV 检测的开放阅读框 1a （ORF1a） 内的区域。此外，结构蛋白由诊断测定中使用的基因编码，例如包膜基因 （upE） 和核衣壳基因 （N） 的上游区域，它们分别用于 MERS-CoV 和 SARS-Cov-2 测定35,36。我们使用内部的 R3T 一步法 RT-qPCR 试剂盒建立了用于检测病毒的 RT-qPCR37。使用标准扭曲合成RNA的10倍连续稀释液测试和评估我们的R3T一步法RT-qPCR试剂盒和引物组的病毒检测、灵敏度、特异性和动态范围。最低实际检测限为每次反应约10个转录本拷贝。因此，内部 R3T 一步法 RT-qPCR 试剂盒和引物/探针组可以成功用于 SARS-CoV-2、甲型流感、乙型流感和 SARS-CoV-2、MERS-CoV 的常规同时诊断。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m filter cups</td>
			<td>Thermo Scientific</td>
			<td>291-4545</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Glycine SDS running buffer</td>
			<td>Novex</td>
			<td>LC2675</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culturing plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>A701-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Corning</td>
			<td>61-238-RH</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange (HiTrap SP HP) 5 mL</td>
			<td>Cytiva</td>
			<td>17-1152-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Biotin, 98+%</td>
			<td>Thermo Scientific</td>
			<td>A14207.60</td>
			<td></td>
		</tr>
		<tr>
			<td>DH10Bac competent cells</td>
			<td>Fisher Scientific</td>
			<td>10361012</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (Snakeskin 10,000 MWC)</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase/Rnase Free Distilled Water</td>
			<td>Ambion</td>
			<td>AM9930</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Thermo Scientific</td>
			<td>R0192</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BL21(DE3) competent cells</td>
			<td>Invitrogen</td>
			<td>C600003</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>ESF 921 insect cell culture medium (Insect cells media)</td>
			<td>Expression Systems</td>
			<td>96-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Solution</td>
			<td>Gibco</td>
			<td>A38400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene (transfection reagent)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Fisher Scientific</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma Aldrich</td>
			<td>I8896-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma Aldrich</td>
			<td>56750-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A (H1N1) synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103001</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A (H3N2)&#160; synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103002</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza B synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gold Biotechnology</td>
			<td>I3481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Gibco</td>
			<td>11815-032</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth media</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma Aldrich</td>
			<td>L6876-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>13152-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>MERS-CoV synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>103015</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-well Reaction plates with Barcode (0.1 mL)</td>
			<td>Applied Biosystems</td>
			<td>10310855</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini- PROTEAN TGX Precast Gel</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Excel (HisTrap Excel) 5 mL</td>
			<td>Cytiva</td>
			<td>17-3712-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA HP (HisTrap HP) 5 mL</td>
			<td>Cytiva</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Adhesice Covers (PCR Compatible,DNA/Rnase/PCR Inhibitors Free</td>
			<td>Applied Biosystems</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Mini tablets EDTA-Free</td>
			<td>Thermo Scientific</td>
			<td>A32955</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein marker</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>RT-qPCR machine (QuantStudio 7 Flex)</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C medium</td>
			<td>Fisher Scientific</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-+A2:C442 synthetic RNA</td>
			<td>Twist Bioscience</td>
			<td>102024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 insect cells</td>
			<td>Gibco</td>
			<td>A35243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>StrepTrap XT 5 mL</td>
			<td>Cytiva</td>
			<td>29401323</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>IBI Scientific</td>
			<td>IB02200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base Molecular Biology Grade</td>
			<td>Promega</td>
			<td>H5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Affymetrix</td>
			<td>22676</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Invitrogen</td>
			<td>B1690</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of multiplex real-time rt-qpcr assays for the detection of sars-cov-2, influenza a/b, and mers-cov

导致 2019 冠状病毒病 （COVID-19） 的严重急性呼吸系统综合症冠状病毒 2 （SARS-CoV-2） 对公众健康构成严重威胁。在流感季节，SARS-CoV-2和其他呼吸道病毒的传播可能导致全人群难以控制的呼吸道疾病负担。为此，在即将到来的秋冬季节，需要仔细监测呼吸道病毒SARS-CoV-2、甲型流感、乙型流感和中东呼吸综合征（MERS-CoV），特别是在SARS-CoV-2、甲型流感和乙型流感的情况下，它们具有相似的流行病学因素，如易感人群、传播方式和临床综合征。如果没有靶标特异性检测，由于这些病毒的相似性，区分这些病毒的病例可能具有挑战性。因此，一种灵敏且有针对性的多重检测方法可以很容易地区分这些病毒靶标，这对医疗保健从业者很有用。在这项研究中，我们利用内部开发的 R3T 一步法 RT-qPCR 试剂盒开发了一种基于实时逆转录酶 PCR 的检测方法，用于同时检测 SARS-CoV-2、甲型流感、乙型流感和 SARS-CoV-2、MERS-CoV。只需 10 个拷贝的合成 RNA，我们就可以以 100% 的特异性同时成功识别 SARS-CoV-2、甲型流感、乙型流感和 MERS-CoV 靶标。该测定被发现是准确、可靠、简单、灵敏和特异性的。所开发的方法可用作医院、医疗中心和诊断实验室中优化的 SARS-CoV-2、甲型流感、乙型流感和 SARS-CoV-2、MERS-CoV 诊断检测以及研究目的。

近年来，使用PCR方法检测常见呼吸道病毒的诊断方法取得了重大进展21,22,23,24,25。然而，尽管取得了这些进步，但允许在一次测试中检测多种病毒的多重方法尚未得到广泛实施，特别是在RT-qPCR平台中。该方法已使用合成RNA病毒和反应组分的内部优化成功实现37</s...

Watch this Scientific Journal Video about Advancements in Multiplex Detection of Respiratory Viruses at JoVE.com

作者聚焦：呼吸道病毒多重检测的进展 

我们推出了两种基于探针的内部一步法RT-qPCR试剂盒，用于常见呼吸道病毒。第一种检测是针对 SARS-CoV-2 （N）、甲型流感（H1N1 和 H3N2）和乙型流感。第二种是SARS-Cov-2（N）和MERS（UpE和ORF1a）。这些检测可以在任何专业实验室中成功实施。

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

development-multiplex-real-time-rt-qpcr-assays-for-detection-sars-cov

Research

JoVE Journal

Biology

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

842 次观看.  King Abdullah University of Science and Technology (KAUST). 我们推出了两种基于探针的内部一步法RT-qPCR试剂盒，用于常见呼吸道病毒。第一种检测是针对 SARS-CoV-2 （N）、甲型流感（H1N1 和 H3N2）和乙型流感。第二种是SARS-Cov-2（N）和MERS（UpE和ORF1a）。这些检测可以在任何专业实验室中成功实施。 

视频: 作者聚焦：呼吸道病毒多重检测的进展 

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

科研

生物学

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

城市环境 -环境水库SARS-CoV-2采样和检测管道

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

量子点偶联SARS-CoV-2穗状三聚体的高通量共聚焦成像，以追踪HEK293T细胞中的结合和内吞作用

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

用化学发光法实时检测水稻免疫反应中活性氧的产生

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

使用比色法和 RT-LAMP 分析检测 SARS-CoV-2 

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

作者聚焦：使用标准化 LAMP 推进病原体诊断

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

通过逆转录环介导的等温扩增检测 SARS-CoV-2 病毒

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...