Li Bao, Lin Baobao, Zeng Wu, Gu Yin, Zhao Yulan, Liu Peng
School of Biomedical Engineering, Tsinghua University, Haidian District, Beijing 100084, China.
Changping Laboratory, Beijing, China.
Lab Chip. 2025 Jan 28;25(3):454-464. doi: 10.1039/d4lc00770k.
Rapid and accurate molecular diagnostics are crucial for preventing the global spread of emerging infectious diseases. However, the current gold standard for nucleic acid detection, reverse transcription polymerase chain reaction (RT-PCR), relies heavily on traditional magnetic beads or silica membranes for nucleic acid extraction, resulting in several limitations, including time-consuming processes, the need for trained personnel, and complex equipment. Therefore, there is an urgent need for fully integrated nucleic acid detection technologies that are simple to operate, rapid, and highly sensitive to meet unmet clinical needs. In this study, we developed a novel, integrated microfluidic cartridge featuring a unique needle-plug/piston microvalve, which enables stable long-term reagent storage and flexible liquid handling for on-site nucleic acid analysis. Coupled with tetra-primer recombinase polymerase amplification (tp-RPA), we achieved highly sensitive nucleic acid detection with a remarkable limit of detection of 20 copies per mL (0.02 copies per μL) and a short turnaround time of less than 30 minutes. To validate this assay, we tested 48 oropharyngeal swab samples. The positive detection rate reached 64.58% (31/48), significantly exceeding the approximately 50% positive detection rate of the traditional RT-PCR method. Furthermore, our assay demonstrated a 100% concordance rate with RT-PCR in detecting positive samples. Thus, we believe our microfluidic nucleic acid analysis system represents a promising approach for enabling rapid and ultrasensitive nucleic acid detection of pathogenic microorganisms in resource-limited settings and low-income areas.
快速准确的分子诊断对于预防新发传染病的全球传播至关重要。然而，目前核酸检测的金标准——逆转录聚合酶链反应（RT-PCR），在核酸提取方面严重依赖传统磁珠或硅胶膜，导致了一些局限性，包括耗时的流程、对专业人员的需求以及复杂的设备。因此，迫切需要一种操作简单、快速且高度灵敏的全集成核酸检测技术，以满足未被满足的临床需求。在本研究中，我们开发了一种新型的集成微流控芯片，其具有独特的针塞/活塞微阀，能够实现试剂的稳定长期储存以及灵活的液体处理，用于现场核酸分析。结合四引物重组酶聚合酶扩增（tp-RPA），我们实现了高灵敏度的核酸检测，检测限低至每毫升20拷贝（每微升0.02拷贝），周转时间短至不到30分钟。为了验证该检测方法，我们检测了48份口咽拭子样本。阳性检出率达到64.58%（31/48），显著超过传统RT-PCR方法约50%的阳性检出率。此外，我们的检测方法在检测阳性样本时与RT-PCR的一致性率达到100%。因此，我们相信我们的微流控核酸分析系统是一种很有前景的方法，能够在资源有限的环境和低收入地区实现对病原微生物的快速、超灵敏核酸检测。
