Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly contagious disease. To tame the continuously raging outbreak of COVID-19, developing a cheap, rapid and sensitive testing assay is absolutely imperative. Herein, we developed a one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA. The assay integrates Si-OH magnetic beads (MBs)-based fast RNA extraction and rapid isothermal amplification in a single tube, thus bypassing the RNA elution step and directly amplifying on-beads RNA molecules with the visualized results. This one-tube assay has a limit of detection (LOD) as low as 200 copies/mL for sample input volumes of up to 600 μL, and can be performed in less than 1 hour from sample collection to result readout. This assay demonstrated an 100% concordance with the gold standard test RT-qPCR test by using 29 clinical specimens and showed high specificity. This one-tube colorimetric RT-LAMP assay can serve as an alternative platform for a rapid and sensitive diagnostic test for COVID-19 and is particularly suitable for use at community clinics or township hospitals.

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Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/μL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 μL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 μL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 in samples with low viral loads can be improved by increasing the sample input volume.

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