Multiple thermocycles followed by LAMP with only two primers for ultrasensitive colorimetric viral RNA testing and tracking at single-base resolution

Abstract

Rapid, accurate and high throughput measurement of infectious viruses is an urgent need to prevent viral transmission. Loop-mediated isothermal amplification (LAMP) is an attractive isothermal amplification method for nucleic acid detection, especially for point-of-care (POC) testing, but it needs at least four primers and its sensitivity is also limited when integrating with visual detection methods. Herein, by designing only two primers to precisely recognize the four regions of the target, we developed a multiple thermocycles-based LAMP method (MTC-LAMP) for sensitive and specific testing and tracking of viral RNA. We also introduced a novel SYBR Green I (SG)-assisted stable colorimetric assay induced by the amplification products through the charge neutralization effect of positively charged SG toward gold nanoparticles (AuNPs). The ultralow nonspecific background of the double exponential amplification improved the detection sensitivity to near single-molecule level (1 aM, 3 copies in 5 μL solution), which was higher than RT-PCR and RT-LAMP. After adding AuNPs, a significant color difference between target and blank was immediately observed by naked eye. By introducing a peptide nucleic acid (PNA) clamp into our colorimetric MTC-LAMP assay, the specific distinguish of virus variants at single-base resolution was observed without the requirement of any equipment. This assay shows great potential for large-scale screening and tracking of the threatening viruses with ultrahigh sensitivity and pronounced colorimetric output, which is of great importance for pandemic control.