Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV‑2 Detection Using the HFman Probe

• Published in: ACS sensors Vol. 7; no. 3; pp. 730 - 739
• Main Authors: Dong, Yajuan, Zhao, Yongjuan, Li, Shenwei, Wan, Zhenzhou, Lu, Renfei, Yang, Xianguang, Yu, Guoying, Reboud, Julien, Cooper, Jonathan M., Tian, Zhengan, Zhang, Chiyu
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25.03.2022
• Subjects: COVID-19 - diagnosis; COVID-19 Testing; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques - methods; Point-of-Care Systems; RNA, Viral - analysis; RNA, Viral - genetics; SARS-CoV-2 - genetics; Sensitivity and Specificity; HFman probe; high-fidelity DNA polymerase; multiplex LAMP; COVID-19/SARS-CoV-2; non-specific amplification; point-of-care testing (POCT)
• ISSN: 2379-3694; 2379-3694
• DOI: 10.1021/acssensors.1c02079

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single “pot”. Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay’s use in decentralized testing.