Functionalized Co₂FeAl Nanoparticles for Detection of SARS CoV-2 Based on Reverse Transcriptase Loop-Mediated Isothermal Amplification

Abstract

Loop-mediated isothermal amplification (LAMP) is a sensitive, efficient, and rapid nucleic acid amplification technique resulting in a large number of amplicons; however, it suffers from a high incidence of false positives due to carry-over and aerosol. Herein, we report a 10 min nano-capture system that is used in conjunction with a modified reverse transcriptase-LAMP (RT-LAMP) assay for the accurate detection of SARS CoV-2 virus. The nano-capture system employs in-house-designed probe-functionalized magnetic nanoparticles Co2FeAl (cobalt-based Heusler alloy) for efficient capture of contaminating amplicons from the reaction mixture preceding RT-LAMP. The nano-cleaned RT-LAMP assay along with engineered primers successfully detected the presence of 10 copies of SARS CoV-2 virus while completely eliminating the incidence of false positives. The presented contaminant-capture method has been compared with other approaches for elimination of contaminants and was found to be more effective. The insight brought in this work is the design of a rapid nano-capture system that hybridizes with contaminating amplicons (carry-over) with high specificity to enable easy removal from the assay for elimination of false positives. The method has been proven to be successful for RT-LAMP assays in the rapid and highly specific detection of SARS CoV-2, which is currently a major challenge for global health. To the best of our knowledge, this is the first work involving a nano-based cleaning strategy for reliable and rapid diagnosis using isothermal amplification approaches.