Molecular Diagnostics of Dengue by Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) in Disposable Polyester-Toner Microdevices

Dengue is one of the most prevalent infectious tropical diseases in the world, with high incidence in over 100 countries. Rapid and reliable diagnosis of dengue is of great importance for public health. To simplify molecular diagnostics, isothermal amplification techniques have recently emerged as an alternative to conventional methods of deoxyribonucleic acid (DNA) amplification. Here, we developed a one-step method for dengue virus detection from real sample based on RT-LAMP (reverse transcription-loop mediated isothermal amplification) in a disposable microdevice. The reaction was thermally controlled with a thermoblock for 15 min at 72 ºC. At the end of the incubation time, we either removed the solution for detection of fragments by gel electrophoresis or added DNA intercalator for visual detection on-chip. Our results demonstrated that it is possible to detect dengue virus through RT-LAMP directly from a serum sample, without previous ribonucleic acid (RNA) extraction. The success of RT-LAMP was confirmed in reactions initiated with 0.8 fg µL-1 of RNA, which represents 200 copies of RNA per µL. RT-LAMP in a polyester-toner (PeT) microdevice is a simple and inexpensive method that allows for rapid detection of dengue virus with high reliability and great potential for point-of-care applications.登革热是全球范围内流行最为广泛的热带传染病之一，在逾100个国家呈现高发态势。快速且可靠的登革热诊断对于公共卫生事业具有关键意义。为简化分子诊断流程，等温扩增技术近来已成为传统脱氧核糖核酸（deoxyribonucleic acid，DNA）扩增方法的替代方案。本研究基于逆转录环介导等温扩增（reverse transcription-loop mediated isothermal amplification，以下简称RT-LAMP），在一次性微装置中开发了一种可直接从真实样本中检测登革病毒的一步法检测方案。该反应通过热块在72℃下控温15分钟完成。孵育结束后，既可取出反应液通过凝胶电泳检测扩增片段，也可加入DNA嵌合剂实现在片可视化检测。研究结果表明，无需预先提取核糖核酸（ribonucleic acid，RNA），即可直接从血清样本中通过RT-LAMP检测登革病毒。当以0.8 fg·μL⁻¹的RNA（即每微升200份RNA拷贝）启动反应时，可成功实现RT-LAMP扩增，验证了该方法的有效性。基于聚酯-墨粉（polyester-toner，PeT）微装置的RT-LAMP方法简便廉价，能够快速、高可靠地检测登革病毒，在即时检测（point-of-care）应用场景中具有巨大潜力。