即用型qPCR，用于检测 克氏锥虫 或其他致病微生物的DNA
Özet
本工作描述了生产用于 克氏锥虫 DNA检测的即用型qPCR的步骤，该qPCR可以预先加载到反应容器中并在冰箱中储存数月。
Abstract
实时荧光定量PCR（qPCR）是一种非常灵敏和精确的技术，可以从大量样品中扩增微量的核酸靶标。它已广泛应用于许多研究领域，并在转基因作物的人体诊断和性状选择等领域实现工业应用。然而，qPCR不是一种防错技术。将所有试剂混合到单个预混液中，随后分配到常规qPCR板的96个孔中可能会导致操作人员错误，例如试剂混合不正确或分配到孔中不准确。这里介绍了一种称为凝胶化的技术，其中预混液中存在的大部分水被试剂取代，这些试剂在提交真空时形成溶胶-凝胶混合物。结果，qPCR试剂在室温下有效保存数周或在2-8 °C下保存数月.此处显示了制备每种溶液的详细信息以及旨在检测克氏锥虫 卫星DNA（satDNA）的凝胶化反应的预期方面。类似的程序可以应用于检测其他生物。开始凝胶化qPCR运行就像从冰箱中取出板，将样品添加到各自的孔中并开始运行一样简单，从而将全板反应的设置时间减少到加载样品所需的时间。此外，可以批量生产和控制凝胶化PCR反应的质量，从而节省时间并避免在运行常规PCR反应时出现常见的操作错误。
Introduction
恰加斯病是在20世纪初在 巴西农村地区发现的，那里的贫困普遍存在1，2。即使在今天，这种疾病仍然与美洲健康的社会和经济决定因素有关。恰加斯病是双相的，包括急性期和慢性期。它是由 克氏锥 虫寄生虫感染、通过昆虫媒介传播、通过先天途径输血或口服摄入受污染的食物引起的3，4。
恰加斯病的诊断可以通过观察临床症状（尤其是罗马尼亚征）、血涂片显微镜检查、血清学和分子检测（如实时荧光定量 PCR （qPCR） 或等温扩增4，5，6，7，8，9）进行。临床症状和血涂片显微镜检查用于疑似急性感染病例，而寻找抗体则用作无症状患者的筛查工具。由于其敏感性和特异性，qPCR 已被建议用作慢性患者的监测工具、正在接受治疗的急性患者测量血液中寄生虫负荷以及作为治疗失败的替代标志物6，8，10，11，12.尽管qPCR比目前可用的测试更具敏感性和特异性，但由于运输和储存需要冷冻温度，qPCR在全球贫困地区被有效地阻止了诊断工具13，14，15。
为了绕过这一障碍，已经探索了冻干和凝胶化等保护技术16，17。虽然冻干可提供多年的保存，但它需要不含甘油的特殊试剂，甘油通常用于酶稳定/保存18。虽然凝胶化已被证明可以提供数月的保存，但它允许使用常规试剂19。凝胶化溶液包括四种组分，每种组分在过程中都有特定的作用：糖海藻糖和黑糖糖在干燥过程中通过还原溶液中的游离水分子来保护生物分子，糖原产生更广泛的保护基质，氨基酸赖氨酸用作自由基清除剂以抑制生物分子羧基之间的氧化反应， 氨基和磷酸基团。这些组分定义了溶胶-凝胶混合物，可防止在干燥过程中损失三级或四级结构，从而有助于维持生物分子在补水时的活性19。一旦在反应管内稳定，反应可以在2-8°C下储存几个月或在21-23°C下储存数周，而不是常规的-20°C。 这种方法已被纳入旨在帮助诊断恰加斯病、疟疾、利什曼病、结核病和环孢子虫病等疾病的测试中13，14，15，20。
本工作描述了为凝胶化程序准备所需解决方案的所有步骤、过程中的陷阱以及八管条带内即用型凝胶化qPCR的预期最终方面。相同的方案可以适用于单管或96孔板。最后， 克氏锥虫 DNA的检测将显示为对照运行。
Protocol
Representative Results
Discussion
近年来，人们强调需要找到更敏感和更具体的技术来帮助诊断热带和被忽视的疾病。虽然寄生虫学（光学显微镜）和血清学检测对流行病学控制很重要，但局限性，特别是在敏感性和床旁适用性方面。DNA扩增技术，如PCR，等温扩增和相应的变异，长期以来一直在实验室环境中使用，但技术障碍使其无法在现场环境中使用。主要障碍之一是试剂的运输和储存需要-20°C的温度。为了补救这种情况，已?…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
作者感谢Aline Burda Farias为真空烘箱提供的技术援助，并感谢巴拉那分子生物学研究所（IBMP，巴西库里提巴）的行政部门允许使用上述设备。这项工作的部分资金来自 CNPq 445954/2020-5 赠款。
Materials
| Bentonite clay bags (activated) | Embamat Global Packaging Solutions (Barcelona, Spain) | 026157/STD | Not to be confused with silica gel packs |
| Glycogen | Amersham Bioscience | Cat# US16445 | |
| Lysine | Acros Organic | Cat# 365650250 | |
| Melezitoze | Sigma-Aldrich | Cat# 63620 | |
| Nuclease-free water | preferred vendor | ||
| Oligonucleotides | preferred vendor | ||
| PCR mastermix | preferred vendor or Instituto de Biologia Molecular do Paraná (IBMP, Curitiba, Brazil) | Chagas NAT kit | |
| PCR thermocycler | preferred vendor | ||
| software for vacuum oven | Memmert Gmbh | Celsius v10.0 | |
| Trehalose | Sigma-Aldrich | Cat# T9531 | |
| Trypanosoma cruzi DNA | from in-house cultivated parasites, or purchased from accredited vendors such as ATCC | ||
| Vacuum oven | Memmert Gmbh | VO-400 |
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