# Development of a Novel Nested–RT–LAMP Assay for the Rapid and Accurate Coronavirus Disease-2019 Diagnosis

## TL;DR

This paper introduces a new, fast, and cost-effective test for diagnosing COVID-19 that is more sensitive than existing methods.

## Contribution

A novel nested–RT–LAMP assay is developed with higher sensitivity and lower cost than RT–PCR and standard RT–LAMP.

## Key findings

- The nested–RT–LAMP assay detected as few as 5 copies/μL of the virus, outperforming RT–PCR and standard RT–LAMP.
- The assay achieved 100% sensitivity and 98% specificity in confirmed samples.
- The one-step version of the assay had 94% sensitivity and 97% specificity without RNA extraction.

## Abstract

Background and Aims: Coronavirus disease 2019 (COVID-19), an emerging life-threatening viral disease, has rapidly spread worldwide, exerting a detrimental impact on public health. We aimed to devise an innovative platform based on the loop-mediated isothermal amplification (LAMP) method, having priorities over real-time PCR (RT–PCR) in terms of sensitivity, specificity, and low running costs.

Methods: To develop a novel assay, a new primer set plus four primer sets were designed targeting the N gene of the COVID-19 agent, resulting in the sensitivity reinforcement. The limit of detection (LOD) of the developed approach was determined and compared to those of the standard RT–LAMP and RT–PCR. Two hundred confirmed positive and negative samples initially tested by RT–PCR were recruited to assess the nested–RT–LAMP assay. Furthermore, for the one-step nested–RT–LAMP assay, positive samples were tested directly without the need for RNA extraction.

Results: The LOD of nested–RT–LAMP, LAMP, and RT–PCR were 5, 15, and 15 copies/μL, respectively. The findings of the investigation illustrated 100% sensitivity and 98% specificity for both LAMP assays. Moreover, respectively, 94% and 97% sensitivity and specificity were determined regarding the one-step nested–RT–LAMP assay.

Conclusion: We offered a novel approach with more sensitivity compared to RT–PCR and common RT–LAMP, not only being a simple, accurate, cost-effective alternative diagnostic tool for RT–PCR but also being able to detect asymptomatic or mildly symptomatic patients more accurately in 2 h by naked eyes.