Multifunctional self-priming hairpin probe-based isothermal nucleic acid amplification and its applications for COVID-19 diagnosis

• Published in: Biosensors & bioelectronics 2024-06, Vol.253, p.116147-116147, Article 116147
• Main Authors: Kim, Hansol, Lee, Seoyoung, Ju, Yong, Kim, Hyoyong, Jang, Hyowon, Park, Yeonkyung, Lee, Sang Mo, Yong, Dongeun, Kang, Taejoon, Park, Hyun Gyu
• Format: Article
• Language: English
• Subjects: COVID-19; Isothermal amplification; Molecular diagnostics; SARS-CoV-2; Self-priming hairpin probe
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2024.116147

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings.