A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA

Abstract

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL−1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.

Advancements in Testing Strategies for COVID-19

“…Here, in contrast to TMSD assays, the toehold of M1 present at the c domain of the loop region facilitates the proper quenching. NISDA enabled SARS-CoV-2 with a limit of detection of 10 copies/μL [ 93 ].…”

“…Quenched 6-FAM fluorophore (bhq-1) restores fluorescence upon detecting target (viral RNA/DNA) after 30 min at 42 °C. Letters labels represent domains, while prime labeled domains donated complementary sequences Readapted with permission [ 93 ]. Copyright © 2021, The Authors.…”

A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction

“…Some studies have pointed out that toehold-mediated recognition is restricted by the folding format of the gene region. The selection of target is important for the clinical application of toehold based methods [39,40]. Therefore, we tested three speci c target site using t-PER for HPV detection, including capsid protein coding gene (CPCG), L1 gene, and L2 gene (Fig.…”

Target-triggered cascade signal amplification for sensitive electrochemical detection of SARS-CoV-2 with clinical application

“…Among them, proper diagnosis using sensitive, conveniently-operated, and accessible technologies is the premise for isolation and therapy [ 4 , 5 ]. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) has been recognized as the current gold standard for SARS-CoV-2 detection for its robust analytical performance [ [6] , [7] , [8] ]. However, it meets some technical drawbacks, such as long assay time, high-cost instruments, and involvement of trained personnel.…”