Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices.
Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices.
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在多孔基质中用于用于护理点诊断设备的多孔基质中的等温核酸扩增的基于酶的试剂的长期干燥储存。
DOI：
10.1039/d0an01098g
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发表时间：
2020-10-26
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影响因子：
--
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中科院分区：
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基于核酸扩增测试(NAAT)的护理点(POC)设备正在迅速增长，以用于低资源环境。然而，关键的挑战是在设备中以干燥形式存储酶基试剂的能力以及这些试剂在高温下的长期稳定性，特别是在环境温度可能高达45°C的情况下。在这里，我们描述了一套辅料，包括海藻糖、聚乙二醇和葡聚糖的组合，以及一种允许在多孔基质中长期干储用于等温链置换扩增(ISDA)反应的酶基试剂的方法。测试了包括硝基纤维素、纤维素和玻璃纤维在内的各种多孔材料。ISDA的共同干燥试剂通常包括那些在金黄色葡萄球菌中扩增ldh1基因的试剂(一个聚合酶和一个缺口酶，4个引物，dNTPs和一个缓冲液)。试剂还包括捕获探针和扩增后检测侧向流动(LF)所需的链霉亲和素-Au标记，或用于实时检测的荧光探针。在含有10%海藻糖和2.5%葡聚糖的玻璃纤维基质中，试剂的稳定性最好。侧向流动检测和凝胶电泳法测定试剂在~22℃下稳定一年以上。该试剂在45°C恒温360h后仍表现出良好的稳定性；侧向流动检测法检测到10个拷贝的ldh1基因靶标，实时荧光检测法检测到50个拷贝的ldh1基因靶标。这些结果表明，将干形式的扩增试剂加入到护理点式设备中，可在广泛的环境中使用。
Nucleic acid amplification test (NAAT)-based point-of-care (POC) devices are rapidly growing for use in low-resource settings. However, key challenges are the ability to store the enzyme-based reagents in dry form in the device and the long-term stability of those reagents at elevated temperatures, especially where ambient temperatures could be as high as 45 °C. Here, we describe a set of excipients including a combination of trehalose, polyethylene glycol and dextran, and a method for using them that allows long-term dry storage of enzyme-based reagents for an isothermal strand displacement amplification (iSDA) reaction in a porous matrix. Various porous materials, including nitrocellulose, cellulose, and glass fiber, were tested. Co-dried reagents for iSDA always included those that amplified the ldh1 gene in Staphylococcus aureus (a polymerase and a nicking enzyme, 4 primers, dNTPs and a buffer). Reagents also either included a capture probe and a streptavidin-Au label required for lateral flow (LF) detection after amplification, or a fluorescent probe used for real-time detection. The reagents showed the best stability in a glass fiber matrix when stored in the presence of 10% trehalose and 2.5% dextran. The reagents were stable for over a year at ~22 °C as determined by lateral flow detection and gel electrophoresis. The reagents also exhibited excellent stability after 360 h at 45 °C; the assay still detected as few as 10 copies of ldh1 gene target by lateral flow detection, and 50 copies with real-time fluorescence detection. These results demonstrate the potential for incorporation of amplification reagents in dry form in point-of-care devices for use in a wide range of settings.
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影响因子：
2.8
作者：
Mahalanabis M;Do J;ALMuayad H;Zhang JY;Klapperich CM
通讯作者：
Klapperich CM
影响因子：
2.9
作者：
Christiansen, Alexander;Wang, Qian;Wittung-Stafshede, Pernilla
通讯作者：
Wittung-Stafshede, Pernilla
影响因子：
2.9
作者：
Mazzobre, MF;Buera, MD;Chirife, J
通讯作者：
Chirife, J
影响因子：
4.6
作者：
Magro L;Jacquelin B;Escadafal C;Garneret P;Kwasiborski A;Manuguerra JC;Monti F;Sakuntabhai A;Vanhomwegen J;Lafaye P;Tabeling P
通讯作者：
Tabeling P
影响因子：
5.2
作者：
Chander Y;Koelbl J;Puckett J;Moser MJ;Klingele AJ;Liles MR;Carrias A;Mead DA;Schoenfeld TW
通讯作者：
Schoenfeld TW