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

Tao Zheng,Mohsen Mohammadniaei,Ulf Bech Christensen,Finn Erland Nielsen,Jon Ashley,Lennart Jan Friis-Hansen,Jens Henning Rasmussen,Jan Gorm Lisby,Ellen Bøtker Pedersen,Maryam Naseri,Anne Christine Rye Olinger,Thomas Lars Benfield,Ming Zhang,Wentao Wang,Rasmus Gregersen,Yi Sun,Lærke Tørring Kolding,Jan Gorodkin

doi:10.1038/s41467-021-25387-9

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.

Highlights

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
For RNA-dependent RNA polymerase (RdRP) gene, the whole binding site was in a structured region, whereas for the N gene, the toehold part of the binding site was in a high probable unpaired region (Supplementary Fig. 6b, c)
We employed IntaRNA31 at 42 °C on the RdRP and N gene pairs, respectively. We found that both the hybridization and the energy of the duplex was lower for the N gene than the RdRP (Supplementary Fig. 8)

Summary

Introduction

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. 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. To keep up with the high transmission rate of the COVID-19, SARS-CoV-2 tests have been developed at a record-breaking pace, and the in vitro diagnostic (IVD) market experienced an unprecedented dynamic[5]. Chest computed tomography (CT) and serology tests are being used for COVID-19 diagnosis[7,8], the gold standard for SARS-CoV-2 RNA detection is still quantitative reverse transcription-polymerase chain reaction (qRT-PCR)[9].

Methods

Results

Conclusion