这项工作部分由 FDOH-7ZK15 和 NIAID 1R21AI128188-01 支助。该出版物中报告的研究部分由国家过敏和传染性疾病研究所支持, 部分由佛罗里达卫生部的生物医学研究计划提供。内容完全是作者的责任, 不一定代表 NIH 或佛罗里达卫生部的官方意见。动态组合化学有限责任公司对该项目的支持和贡献表示认可。
1登革热病毒 (应变 BOL-KW010) 是由佛罗里达州卫生局实验室提供的。疾病控制和预防中心慷慨地提供了 Zika 病毒和亚洲基孔肯亚病毒血统。基孔肯亚病毒的印度洋血统是由罗伯特. Tesh (世界新兴病毒和虫媒病毒中心) 提供的, 通过得克萨斯州加尔维斯顿的德克萨斯大学医学分支机构向超滤-FMEL。我们感谢贝拉米, b. 伊斯特蒙, 贝莱斯, k. 威金斯, R. ZimLer 和 k. Zirbel 协助感染研究。我们还感谢金硕士提供了 Q 纸。

注意: 蚊子是这项研究中唯一直接使用的动物。管理鸡的程序, 其血液被用来喂养受感染的蚊子, 被批准为 IACUC 协议 #201507682 由佛罗里达大学机构动物护理和使用委员会. 病毒传播和蚊虫感染研究是在佛罗里达州的维罗海滩医学昆虫实验室的 BSL-3 设施进行的 RT 灯实验在 FfAME 和国产生物分子科学有限责任公司在阿拉丘瓦, 佛罗里达州共享的 BSL-2 实验室进行。
1. 引物和绞线置换探头的设计
<ol><li>从广研所数据库23提取1登革热病毒序列;NIAID 病毒病原体数据库和分析资源的 Zika 和基孔肯亚序列24。<ol>
<li>使用软件肌肉 v3.8.3125为感兴趣的序列创建多个序列对齐 ()。使用生成的合作关系在目标的保守区域内搜索光源引物集, 并避免非相关或无意的目标, 如目标子类型之间的区别。</li>
	</ol>
</li>
	<li>按照在线 (http://loopamp.eiken.co.jp/e/lamp/) 中描述的灯引物的设计规则, 并允许使用底漆中的混合基来覆盖病毒发散26。为了避免引物集的交叉反应性, 请使用 NCBI27将生成的光源引到 NCBI RNA 病毒数据库。比较每个设置, 以消除引物, 将 dimerize 在复用试验中使用 NCBI 爆炸以及。</li>
	<li>将绞线置换探针的双股链部分与任何病毒基因组序列和蚊子基因组序列进行筛选。分别对 Zika、基孔肯亚和登革热1的荧光 amidite (carboxytetramethylrhodamine)、六角染料和 5-(TAMRA) 染料进行目标结合探针 5 ' 端的修改。<ol>
<li>包括一个积极的控制底漆集为尿样。设计用于靶向人类线粒体 DNA 的光源引物。标签线置换探头与春节在 5 ' 年底。标签淬火探针, 它们部分地补充了在 3 ' 端 (表 1) 上标有淬火 (例如, 爱荷华州黑色) 的每个荧光标记探针。</li>
	</ol>
</li>
</ol>2. 病毒分离和感染的蚊子样本
<ol><li>使用 Zika 病毒 (波多黎各菌株)、基孔肯亚病毒 (La 留尼汪岛或英属维尔京群岛菌株) 和登革热 serotype-1 病毒 (主要西株) 的分离。使用斑块测定法测定病毒滴度28或定量 rt-pcr29。利用商业上可用的 RNA 提取试剂盒提取病毒 rna。 注意:表 2表示本研究中使用的每个隔离病毒的病毒滴度。</li>
	<li>按照芽儿et . 发布的文章, 了解有关Ae. 蚊雌性病毒感染的详细协议 (Zika 和基孔肯亚)20。有关感染 Zika 病毒的蚊子样本的病毒滴度, 请参见表 2 。</li>
</ol>3. RT 灯加上 Thermolabile 尿 DNA Glycosylase
<ol><li>
10X. 底漆混合制剂。
	<ol>
<li>准备100µM 的每一个底漆和探针的库存解决方案 (见步骤 1) 添加核酸酶自由水。漩涡井和存储在-20 °c, 直到使用。</li>
		<li>10X 底漆混合为每 Zika, 登革热 1, 或线粒体 DNA 靶, 混合16µL 的 FIP, 16 µL, µL F3, 2 µL B3, 5 µL 的 LF, 2 µL 的 lb, 3 µL 荧光标记探针, 4 µL 的淬火探针, 和50µL 的核酸酶水, 共提供100µL 容积。</li>
		<li>对于10X 底漆混合基孔肯亚, 混合16µL 的 FIP, 16 µL, 2 µL 的 F3, 2 µL B3, 5 µL 的 LB, 2 µL 的 lf, 3 µL 的 lf 荧光标记探针, 4 µL 的淬火探头, 50 µL 的核酸酶水, 给总共100µL 量。 注: 上述探针浓度使用300纳米的最终荧光探针和400纳米的淬火探针。或者, 使用 80 nm 的荧光标记探针和 200 nm 的淬火探针进行实时分析。</li>
	</ol>
</li>
	<li>添加5µL 10X 底漆混合 (3 丛, 添加5µL 的每10X 底漆混合), 5 µL 10X 等温放大缓冲器 (1X 缓冲成分:20 毫米三 HCl 缓冲 pH 8.8, 50 毫米氯化钾, 10 毫米 (NH4)2所以4, 8 毫米 MgSO4, 0.1% Tween-20, 1 毫米), 7 µL 的 dNTP 混合物 (10 毫米的 dATP, dCTP 和 dGTP; 5 毫米 dTTP 和 dUTP), 16 单位的 DNA 聚合酶, 15 个单位逆转录, 80 单位的重组核糖核酸抑制剂, 2 单位 thermolabile UDG, 1-2 µL 的不同数量的 viral rna 和水使总容量达到50µL 在0.25 毫升 PCR 管 (参见材料目录)。</li>
	<li>在这个阶段包括阴性控制化验通过替换病毒 RNA 容量与水。在65-68 摄氏度之间孵化样品45分钟到1小时, 然后通过运行5µL 在2.5% 琼脂糖凝胶上, 溴溴化物 (0.4 µg/毫升) 的1X 缓冲液。在凝胶上使用 25 bp 或 50 bp DNA 梯子作为标记。 注意: 添加非目标特定模板是可选的。</li>
	<li>为了检测致病 RNA 的存在, 通过改变温度和/或镁浓度来测试每个底漆集及其无模板控制, 因为每个 RT 灯底漆集设计为在不同温度下工作 (65 °c 到68°c) 或镁浓度 (6 至10毫米决赛)。在室温下准备实验。</li>
</ol>4. 使用病毒 RNA 尖尿的 RT 灯
<ol><li>测试 RT 灯底漆在不同的最终浓度尿 (0%, 10%, 20% 和 50%) 在50µL 的总反应量。对于10% 最终尿浓度, 添加1µL 病毒 RNA 到5µL 尿。对于20% 最终尿浓度, 添加1µL 病毒 RNA 到10µL 尿。对于50% 最终尿浓度, 添加1µL 病毒 RNA 到25µL 尿。</li>
	<li>对于10% 尿液中的 RT 灯进行实时监测, 请使用具有不同荧光过滤器的实时 PCR 仪器 (参见材料表)。<ol>
<li>要读取 Zika 标记的 amplicons 的荧光信号, 请使用从483到 533 nm 不等的过滤器;对于基孔肯亚的十六进制标记 amplicons, 请使用从523到 568 nm 不等的过滤器;对于登革1的 TAMRA 标记 amplicons, 请使用从558到 610 nm 不等的过滤器;对于 amplicons 标记的线粒体 DNA, 使用从523到 568 nm 的过滤器。 注意: 家族的励磁和发射最大值分别为 495 nm 和 520 nm。六角的激发和发射最大值分别为 538 nm 和 555 nm。TAMRA 的激发和发射最大值分别为 559 nm 和 583 nm。其激发和发射最大值分别为 522 nm 和 539 nm。</li>
	</ol>
</li>
	<li>使用96井板, 用透明的塑料片封住盘子, 然后在65-68 摄氏度上孵化出60-90 分钟的样品, 每三十年代使用光循环仪记录荧光。最初使用80毫微米荧光标记探针, 然后测试300毫微米荧光标记探针。<ol>
<li>通过在最终尿浓度中加入序列稀释病毒 rna, 确定每个靶的检测限值10%。 注: 使用实时 RT 灯确定最佳温度、镁浓度、荧光标记探针浓度和反应时间。</li>
	</ol>
</li>
	<li>为了可视化由不可替代探针产生的荧光, 使用蓝色 LED 光源从光循环仪与励磁在470毫微米 (任何凝胶电泳盒与内置蓝光光源将工作, 见材料表), 并记录图像用手机相机在室温下在黑暗中。 注: 使用 300 nM 的荧光标记探针, 当所有三颜色的可视化使用蓝色 LED 的眼睛是需要的。</li>
</ol>5. 用 Q 纸技术检测 Zika 感染蚊虫的 RT 灯
<ol><li>根据先前发布的方法, 对30进行轻微修改, 准备季铵盐改性滤纸 (Q 纸)。<ol>
<li>浸入1克纤维素滤纸圈 (参见材料表), 直径为3.5 厘米, 50 毫升1.8% 的水氢氧化钠溶液, 用于激活15分钟。</li>
		<li>通过真空过滤收集活化的纸圈, 然后在室温下将其浸入 (23-epoxypropyl) 铵氯 (0.28 克) 的40毫升水溶液中。 注: 保持 (23-epoxypropyl) 铵氯的质量比, 过滤纸在0.28 到1。</li>
		<li>通过真空过滤收集产生的阳离子纸, 中和50毫升1% 的乙酸。</li>
		<li>用乙醇清洗纸张三次, 并在罩下风干, 保持气流。</li>
	</ol>
</li>
	<li>使用纸冲床或剪刀将 Q 纸板切成小矩形 (3 毫米 x 4 毫米)。粉碎Ae. 蚊在每张纸上都有可能感染 Zika 的雌性蚊子, 微杵。</li>
	<li>添加到每张纸20µL 1 米水氨溶液 (pH ≈ 12), 并等待5分钟。然后用20µL 50% 乙醇, 一次用20µL 的核酸酶水洗一次纸。在 BSL-2 生物安全柜中风干纸张约1小时。 注: 在这一点上, 样品可以存储在-20 °c 过夜, 直到使用。</li>
	<li>使用镊子, 将每张纸放在100µL 的 RT 反应混合物中, 在65-68 摄氏度处孵化45分钟. 请务必将纸张完全浸入溶液中;它不应该浮动。使用蓝色 LED 光源和橙色或黄色滤镜, 读取并记录 Zika 病毒产生的荧光。</li>
</ol>6. 100 µL 反应体积的 RT 灯试剂冻干
<ol><li>根据3.1 节的规定, 准备10X 灯底漆混合物, 并根据3.2 节的规定制备含 dUTP 的 dNTP 混合物。贮存底漆混合物和 dNTPs 在-20 °c, 直到使用。</li>
	<li>准备10毫升的酶透析缓冲液去除甘油, 通过混合100µL 1 米三盐酸 pH 值 7.5, 500 µL 的1米氯化钾, 2 µL 的0.5 米 EDTA, 100 µL 10% 海卫四 X-100, 100 µL 0.1 m 和9.198 毫升无核酸酶水。将透析缓冲液贮存在摄氏4摄氏度。 注: 一定要过滤 (0.2 微米过滤器) 所有的缓冲试剂解决方案之前使用和存储在4摄氏度。<ol>
<li>使用具有 10 kDa 截止限制的超滤膜 (请参阅材料表)。放置350µL 透析缓冲器, 4 µL 32 单位的 DNA 聚合酶, 2 µL 30 单位的反向逆转录酶, 2 µL 80 单位 RNase 抑制剂成超滤膜和离心机在 1.4万 x g 5 摄氏度。</li>
		<li>在超滤膜和离心机 (1.4万 x g) 中添加另外350µL 的透析缓冲液, 再加5分钟. 清空收集管并重复此步骤, 然后离心机 (1.4万 x g) 为3分钟。</li>
		<li>对于洗脱, 倒置膜和地方在一个新的收集管。旋转 1000 x g 2 分钟测量洗脱量;如果少于8µL, 则通过添加透析缓冲器, 使体积达到8µL。在4摄氏度处存储洗脱, 然后立即进行下一步。 注: 本协议适用于单管冻干, 但每次使用相同的超滤膜制备至少10反应管, 使用相同量的透析缓冲液。</li>
	</ol>
</li>
	<li>混合10µL 10X 灯底漆如果 singleplex (为3丛, 增加10µL 每10X 底漆混合), 14 µL dNTP 混合物, 8 µL 透析酵素混合, 2 µL 2 个单位 thermolabile UDG 和66µL 核酸酶自由水 (为3丛, 使用46µL) 在0.5 毫升离心管和保持盖子打开。相反, 添加另一个盖子刺穿了针, 以允许蒸汽逃脱。</li>
	<li>立即冻结管在-80 °c 为2小时, 并 lyophilize 管 4 h. 用铝箔盖住冻干室, 以保护光线。当试剂干燥, 取出穿刺盖子, 关闭原来的盖子, 并储存在4摄氏度的冻干试剂在黑暗中的管。 注: 如有必要, 试剂可以在一夜之间冻干。</li>
</ol>7. 测试含 Zika 的尿液和蚊子样本的冻干 RT 试剂
<ol><li>使用工具包中的以下项目 (请参见材料表) Zika: 一个3维打印的带有橙色过滤器的观察盒 (长通滤波器, 切割 ~ 540 nm), 2 个用于观察盒的 AAA 电池, 冻干反应管标记为 ZV Zika 检测, 以及1.1X. 1.5 毫升螺旋顶管的补液缓冲液。</li>
	<li>用 DNA 聚合酶 (参见材料表)、3.3 毫升100毫米 MgSO4、110µL 的 0.5 M 和41.09 毫升的核酸酶水来混合5.5 毫升的10X 等温放大缓冲器, 准备50毫升的1.1X 补液缓冲器。混合好, 整除1毫升的这个解决方案, 1.5 毫升螺丝顶管, 并存储在4°c, 直到使用。</li>
	<li>在每个反应管 (0.5 毫升) 中添加90µL 的1.1X 补液缓冲液。确保液体填满含有冻干试剂的管子的底部 (通过震动将其溶解, 然后简单地向下旋转 (1000 x g))。添加10µL 的尿样与病毒 RNA 的反应管 (见4.1 节详细)。<ol>
<li>如果测试蚊子, 添加一个长方形的 Q 纸持有蚊子尸体 (如5.2 和5.3 节所述步骤所准备), 连同10µL 纯净水, 而不是尿样。 注: 或者, 添加10µL 唾液或血清样本, 而不是尿液。如果样品被提取脱氧核糖核酸或 RNA, 增加2-5 µL 样品入水化混合物并且完成以核酸酶无水到10µL 最后样品容量。</li>
	</ol>
</li>
	<li>关闭反应管的盖子。放置在热块/浴缸 (或孵化器) 预先设置在65-68 摄氏度。孵育30-45 分钟, 但不超过1小时。孵化后, 从热中取出管子。为了防止未来化验的污染, 确保这些管子保持闭合。</li>
	<li>在观察箱中放置反应管;温度会下降到环境温度 (最好是25摄氏度)。打开观察框背面的开关。通过橙色过滤器观察样本, 并通过手机相机捕获图像, 该摄像头在图像中填充80% 的视野。 注意: 在低光环境中实现更好的可视化。</li>
	<li>可视化完成后, 关闭开关。在黑暗中储存密封的管子, 最好是用冷冻, 如果以后需要可视化。</li>
</ol>

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一些作者和他们的机构拥有与此化验相关的知识产权。

包括 Zika、基孔肯亚和登革热在内的蚊虫传播病毒威胁到公共卫生, 最近的 Zika 暴发突出表明需要低成本的护理点检测替代品来进行患者诊断, 以及进行蚊虫监测。等温放大方法是作为可负担的替代 PCR 系统开发的。特别是, 基于 RT 灯的平台已被用于检测范围广泛的病原体。然而, 等温平台的使用主要限于单目标检测。这里报告的方法使用一个改进的 RT 灯允许同时检测三个不同的目标在一个单一的?...

蚊虫传播的病毒感染, 包括登革热、基孔肯亚和 Zika 病毒正在上升, 需要立即管理战略。登革热和基孔肯亚病毒已经在许多热带地区流行, 在那里 Zika 现在在西半球传播1。Zika 病毒, 如登革热, 是Flaviviridae家族的成员, 原产于非洲, 有一个亚洲和两个非洲遗传血统2。尽管 Zika 病毒的鉴定可以追溯到 1947, 但在太平洋和美洲出现前半个世纪, 人类的 Zika 感染仍然是零星的。第一次报告说, 2007年在密克罗尼西亚联邦发生的 Zika 热爆发, 随后是在2013年和2014法属波利尼西亚。美洲第一次大规模暴发发生在2015年巴西。
Zika、基孔肯亚和登革热病毒主要由伊蚊蚊和白纹伊蚊传播。然而, Zika 有额外的下游人到人传输的可能性, 可能通过性接触传播, 母婴互动, 并通过母乳喂养3,4,5。Zika 发烧最初被认为只导致轻度疾病。然而, 它后来与成人的格林-巴利综合征, 新生儿小头和慢性肌肉骨骼疾病, 可能持续数月至数年。诊断 Zika 疾病可能是挑战, 因为 Zika 感染的症状类似于其他蚊子传播病毒6。这些病毒的常见联合感染使鉴别诊断更加具有挑战性7,8。因此, 需要对 Zika 和其他病毒的核酸进行快速、可靠的检测, 以便实时了解流行病学, 启动控制和预防措施, 并管理病人护理9。
目前对这些病毒的诊断测试包括血清学测试、病毒分离、病毒测序和反向转录 pcr (rt-pcr)。标准血清学方法往往受到不充分的敏感性, 结果可能是复杂的交叉反应的病人谁曾感染其他 flaviviruses。
因此, 核酸检测仍然是检测和鉴别这些病毒最可靠的方法。Zika 和其他蚊虫传播病毒的检测通常是使用 rt-pcr 或实时 rt-pcr 在各种生物液中进行, 如血清、尿液、唾液、精子、母乳和脑液10,11。尿液和唾液样本通常比血液更可取, 因为它们的 PCR 抑制较少, 病毒负载更高, 病毒存在时间较长, 而且收集和处理的易用性提高了12,13。然而, 基于 RT PCR 的诊断测试包括大量的样品准备步骤和昂贵的热循环设备, 使其对护理点的优化不那么理想。
反向转录环介导的等温放大 (rt 灯) 已成为一个强大的 rt-pcr 替代方案, 由于其高灵敏度和特异性14, 它对生物样品中的抑制物质的容忍15, 和单一温度下的操作, 大大降低了检测的复杂性和相关成本, 使之适合低资源环境。RT 灯, 因为它是经典的实施, 包括六引物, 绑定到八个不同的区域内的目标 RNA。它运行在恒定的温度在60°c 和70°c 之间, 并且使用一条反向逆转录酶和一根有强的链置换活动的脱氧核糖核酸 pcr。
在 RT 灯的初始阶段, 向前和向后内部引物 (FIP 和保险,图 1A) 连同外向前和向后引物 (F3 和 B3) 形成哑铃结构, 种子结构的指数灯放大。放大是进一步加速的循环向前和向后引物 (LF 和磅), 这是设计来绑定的单一搁浅地区的哑铃, 并导致形成 concatemers 与多个重复循环16。基于浊度或锂染料读数的经典光源检测方法并不完全适合 Zika 的点检, 在那里需要某种程度的多路复用17,18,19。在这些系统中, 多路复用不容易获得, 因为由于目标放大, 它们容易产生误报。
为了管理这些问题, 文献以 "线置换探针" 的形式向经典的 RT 灯体系结构20、21、22添加了一个附加组件。每个探针都有一个序列特定的双链区域和一个单链启动区域。该探针与单绞区被标记为 5 '-荧光, 互补探针是修改与 3 '-端淬火。在没有目标的情况下, 由于互补探针链的杂交, 没有发现荧光, 从而使荧光和淬火接近。在目标存在的情况下, 荧光探针的单链部分与靶上的补体相结合, 然后通过链置换聚合酶进行扩展。进一步的聚合酶引伸反向引物导致分离的淬火链从其互补荧光标记链, 允许发射荧光(图 1B)。通过这种设计, 在哑铃形成后产生信号, 减少了假阳性信号的几率。
链置换探针的双绞线可以是任意序列, 当应用多路复用时, 相同的序列可以与不同的荧光-淬火对一起使用。通过这种体系结构, 直接将病毒污染的尿液、血清或蚊子样本压扁在纸上, 而不进行样品制备。在 30-45 分钟内产生肉眼可见的三色荧光读出, 并使用蓝色 LED 和橙色过滤器在3维打印的观察框中可视化信号。冷冻干燥的 RT 灯试剂启用此工具包的部署, 以降低资源设置, 不需要制冷。

<table><tbody><tr><td>SafeBlue Illuminator/ Electrophoresis System, MBE-150-PLUS</td><td>Major Science</td><td>MBE-150</td><td>Gel electrophoresis</td></tr><tr><td>G:BOX F3</td><td>Syngene</td><td>G:BOX F3</td><td>Gel imaging</td></tr><tr><td>LightCycler 480 Instrument II, 96-well</td><td>Roche Applied Science</td><td>05 015 278 001</td><td>Real-time PCR</td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>Millipore Sigma</td><td>UFC501096</td><td>ultrafiltraton membrane for dialysis</td></tr><tr><td>Eppendorf 5417C Centrifuge</td><td>Marshall Scientific</td><td>EP-5417C</td><td>centrifuge</td></tr><tr><td>Myblock Mini Drybath</td><td>Benchmark Scientific</td><td>BSH200</td><td>drybath</td></tr><tr><td>FreeZone Plus 6 Liter Cascade Console Freeze Dry System</td><td>Labconco</td><td>7934020</td><td>lyophilizer</td></tr><tr><td>all priers and probes</td><td>IDT</td><td>custom</td><td>RT-LAMP primers and probes</td></tr><tr><td>dNTP set</td><td>Bioline</td><td>BIO-39049</td><td></td></tr><tr><td>Deoxyuridine Triphosphate (dUTP)</td><td>Promega</td><td>U1191</td><td></td></tr><tr><td>Bst&amp;nbsp;2.0 WarmStart&amp;nbsp;DNA Polymerase</td><td>New England Biolabs</td><td>M0538L</td><td>enzyme</td></tr><tr><td>WarmStart RTx Reverse Transcriptase</td><td>New England Biolabs</td><td>M0380L</td><td>enzyme</td></tr><tr><td>RNase Inhibitor, Murine</td><td>New England Biolabs</td><td>M0314L</td><td>enzyme</td></tr><tr><td>Antarctic Thermolabile UDG</td><td>New England Biolabs</td><td>M0372L</td><td>enzyme</td></tr><tr><td>50bp DNA Step Ladder</td><td>Promega</td><td>G4521</td><td>marker</td></tr><tr><td>LightCycler 480 Multiwell Plate 96, white</td><td>Roche Applied Science</td><td>4729692001</td><td>real-time RT-LAMP analysis</td></tr></tbody></table>

multiplexed isothermal amplification based diagnostic platform to detect zika, chikungunya, and dengue 1

Zika、登革热和基孔肯亚病毒是由蚊子传播的, 引起类似患者症状的疾病。然而, 他们有不同的下游病人对病人的传输潜能, 并需要非常不同的病人治疗。因此, 最近的 Zika 暴发使人们迫切需要开发快速鉴别这些病毒的患者和被困蚊子的工具, 选择正确的病人治疗, 并实时了解和管理他们的流行病学。
不幸的是, 目前的诊断测试, 包括那些收到2016紧急使用授权和快速轨道状态, 检测病毒 RNA 的反向转录聚合酶链反应 (RT PCR), 这需要检测, 训练有素的用户, 并大量的样品准备。因此, 他们必须被送往 "批准" 的参考实验室, 需要时间。事实上, 在 2016年8月, 疾病控制中心 (CDC) 正在询问那些被蚊子叮咬的孕妇, 并在得知他们是否感染之前, 在2周的时间里, Zika 的皮疹来等待一个不可接受的4星期。我们非常需要测试, 可以在现场进行, 很少的资源, 并由训练有素但不一定是授权的人员。
本视频演示了一种符合这些规范的检测方法, 即使用尿液或血清 (为病人) 或被粉碎的蚊子尸体 (用于环境监测), 所有这些都没有大量的样品准备。在运载季铵盐组 (Q 纸) 的纸上捕获蚊子的尸体, 然后用氨水处理来管理生物危害。这些都是直接的, 没有 RNA 分离, 放入化验管中含有冷冻干燥试剂, 无需冷藏链。改进形式的反向转录回路介导的等温放大与目标特定的荧光标记不可替代探针产生读数, 在30分钟, 作为一个三色荧光信号。这是可视化的手持式电池供电设备与橙色过滤器。前污染是防止与密封管, 并使用 thermolabile 尿 DNA glycosylase (UDG) 在存在 dUTP 在放大混合物。

首先, 用凝胶电泳法对各 RT 灯底漆(表 1)及其相应的病毒 RNA 基底和阴性对照进行了性能评估。用于 Zika 和登革1的 NS5 区域 (rna 依赖性 rna 聚合酶) 和 nsP2 区域 (非结构化蛋白 P2) 用于基孔肯亚的 RT 灯引物。模板是从非洲绿猴肾脏 (维罗) 细胞培养的病毒种群中提取的总 RNA。在一种情况下, 从基孔肯亚感染的雌性Ae 蚊 (表 2)中提取总核酸 (DNA 和 RN...

Watch this Scientific Journal Video about Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1 at JoVE.com

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

目前用于检测 Zika、基孔肯亚和登革热病毒的复用诊断程序需要复杂的样本准备和昂贵的仪器, 而且在低资源环境下很难使用。我们显示一个诊断, 使用等温放大与目标特定的链不可替代探针, 以检测和区分这些病毒具有高灵敏度和特异性。

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

multiplexed-isothermal-amplification-based-diagnostic-platform-to

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

14.2K 次观看.  Foundation for Applied Molecular Evolution (FfAME). 目前用于检测 Zika、基孔肯亚和登革热病毒的复用诊断程序需要复杂的样本准备和昂贵的仪器, 而且在低资源环境下很难使用。我们显示一个诊断, 使用等温放大与目标特定的链不可替代探针, 以检测和区分这些病毒具有高灵敏度和特异性。

视频: Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

科研

遗传学

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

生物工程

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

免疫与感染

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

纸基脚趾开关诊断开发和患者验证的设计到实施研究

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

高通量抗病毒检测筛查寨卡病毒复制抑制剂

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

生物学

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...