A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.
A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.
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DOI：
10.1371/journal.pone.0131701
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Wong S
中科院分区：
文献类型：
--
作者：
Wong G;Wong I;Chan K;Hsieh Y;Wong S
许多靶向核酸的现代分子诊断测定通常限于发达国家或发展中国家的国家参考实验室。如果能够以便携、低成本的方式广泛提供传染病快速诊断技术，将标志着全球卫生领域向前迈出革命性的一步。许多分子检测也是基于聚合酶链反应（PCR）开发的，这需要相对较重（>20磅）的热循环仪并且需要持续的电力。最经济的热循环仪的升温速度相对较慢（2至3°C/s），因此聚合酶链反应可能需要1至2小时。最重要的是，这些热循环仪对于低资源设置用途来说仍然太贵（2k到4k美元）。在这篇文章中，我们展示了一种低成本和快速水浴的热循环仪，不需要主动温度控制或连续电源在PCR过程中的发展。这个单位的成本130美元，建立使用商业现成的项目。使用两个或三个真空绝缘的不锈钢保温瓶食品罐含有热水（用于变性和退火/延伸步骤）和一层油在水面上允许显着稳定的温度进行PCR。使用基于Arduino的微控制器，我们自动化了在水浴之间手动转移PCR管的“古老”方法。我们证明，这种创新的装置可以提供高速PCR（每个PCR循环17秒），有可能超过本研究中测试的1,522 bp长的扩增子，并且可以从模板扩增到每个反应至少20个拷贝。该装置也接受常规PCR管和玻璃毛细管。我们的热循环仪的PCR效率与其他商业热循环仪没有区别。当与快速核酸检测方法相结合时，热循环仪（TTC）可以在低资源环境中实现现场分子诊断。
Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3°C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the “archaic” method of hand-transferring PCR tubes between water baths. We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.
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