Ultra-sensitive colorimetric detection of SARS-CoV-2 by novel gold nanoparticle (AuNP)-assisted loop-mediated isothermal amplification (LAMP) and freezing methods.

Loop-mediated isothermal amplification (LAMP) is a molecular diagnosis technology with the advantages of isothermal reaction conditions and high sensitivity. However, the LAMP reactions are prone to producing false-positive results and thus are usually less reliable. This study demonstrates a gold nanoparticle (AuNP)-assisted colorimetric LAMP technique for diagnosing SARS-CoV-2, which aims to overcome the false-positive results. The AuNPs were functionalized with E gene probes, specifically tailored to bind to the amplified E-gene LAMP product, using the freezing method. Varied salt concentration and AuNP/probe combinations were tested for the highest visual performance. The experiments were conducted on synthetic SARS-CoV-2 RNA (Omicron variant), as well as on clinical samples. The assay showed an exceptional sensitivity of 8.05 fg of LAMP amplicon mixture (0.537 fg/µL). The average reaction time was ~ 30 min. In conclusion, AuNP-assisted LAMP detection will not identify any potential unspecific amplification, which helps to improve the efficiency and reliability of LAMP assays in point-of-care applications. The freezing method to functionalize the AuNPs with probes simplifies the assay, which can be utilized in further diagnostic studies.