Yuan Yuan, Ellis Perry, Tao Ye, Bikos Dimitri A, Loveday Emma K, Thomas Mallory M, Wilking James N, Chang Connie B, Ye Fangfu, Weitz David A
Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health) Wenzhou Institute University of Chinese Academy of Sciences Wenzhou Zhejiang China.
John A. Paulson School of Engineering and Applied Sciences Harvard University Cambridge Massachusetts USA.
Smart Med. 2024 Jun 5;3(2):e20240008. doi: 10.1002/SMMD.20240008. eCollection 2024 Jun.
Nucleic acid amplification testing (NAAT) remains one of the most reliable methods for pathogen identification. However, conventional bulk NAATs may not be sufficiently fast or sensitive enough for the detection of clinically-relevant pathogens in point-of-care testing. Here, we have developed a digital droplet RT-LAMP (ddRT-LAMP) assay that rapidly and quantitatively detects the SARS-CoV-2 viral E gene in microfluidic drops. Droplet partitioning using ddRT-LAMP significantly accelerates detection times across a wide range of template concentrations compared to bulk RT-LAMP assays. We discover that a reduction in droplet diameter decreases assay times up to a certain size, upon which surface adsorption of the RT-LAMP polymerase reduces reaction efficiency. Optimization of drop size and polymerase concentration enables rapid, sensitive, and quantitative detection of the SARS-CoV-2 E gene in only 8 min. These results highlight the potential of ddRT-LAMP assays as an excellent platform for quantitative point-of-care testing.
核酸扩增检测（NAAT）仍然是病原体鉴定最可靠的方法之一。然而，传统的批量NAAT在即时检测中对临床相关病原体的检测可能不够快速或灵敏。在此，我们开发了一种数字液滴RT-LAMP（ddRT-LAMP）检测方法，可在微流控液滴中快速、定量地检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒E基因。与批量RT-LAMP检测相比，使用ddRT-LAMP进行液滴分区可在广泛的模板浓度范围内显著加快检测时间。我们发现，液滴直径减小会使检测时间缩短，直至达到一定大小，超过该大小后，RT-LAMP聚合酶的表面吸附会降低反应效率。优化液滴大小和聚合酶浓度能够仅在8分钟内实现对SARS-CoV-2 E基因的快速、灵敏和定量检测。这些结果凸显了ddRT-LAMP检测作为定量即时检测的优秀平台的潜力。
