Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

Abstract

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID₅₀ ml^-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

Keywords: RT-LAMP; SARS-CoV-2; nasopharyngeal swabs; universal transport media.

Fig. 1.

Limit of detection of N1-STOP-LAMP and establishing optimal sample template volume and inactivation conditions. (a) Plot showing performance of the N1 LAMP assay across 5-log₁₀ dilution of purified viral RNA. Y-axis is time-to-positive (Tp) and the x-axis is an estimate of viral genomes/reaction based on starting RNA concentration. The number of replicates per dilution (n) and number of positive replicates per dilution is indicated. (b) Example N1-STOP-LAMP amplification plots for SARS-CoV-2 positive clinical sample No. 52, showing inhibitory impact of 5 µl of a neat sample matrix on LAMP and the effect of diluting the UTM in water. (c) N1-STOP-LAMP amplification plots for three SARS-CoV-2 positive clinical samples, showing no loss in detection sensitivity after specimen heat-treatment of 60 °C for 30 min to inactivate virus.

Fig. 2.

Comparison of E-gene RT-qPCR with N1-STOP-LAMP and limit of detection. Titred virus was serially diluted 10-fold in UTM in triplicate and RNA was extracted from each replicate dilution. A 1 µl aliquot of each UTM dilution was also tested directly by N1-STOP-LAMP. (a) Calibration curve for the E-gene RT-qPCR. A curve was interpolated using linear regression. (b) Comparative performance of N1-STOP-LAMP with 5 µl purified RNA versus 1 µl or 5 µl of UTM directly added to the reaction.

Fig. 3.

Clinical evaluation of N1-STOP-LAMP. (a) Establishing limit of detection according to FDA Emergency Use Authorisations (EUA) guidelines. Twenty, 1 µl replicates of SARS-CoV-2 virus in UTM at a concentration of 54 TCID₅₀ ml⁻¹ were tested by direct N1-STOP-LAMP. All data points plotted, with average and 95% CI indicated. (b) Assessment of assay against FDA EUA clinical performance criteria. Thirty healthy volunteer dry nasopharyngeal swabs eluted in 1.5 ml of PBS were screened by N1-STOP-LAMP. Shown are time-to-positives (Tp). Mean and 95% CI are indicated. (c). Plot showing correspondence between 107 E-gene RT-qPCR positive and N1-STOP-LAMP, where 1 µl of clinical specimen in UTM was added directly to the RT-LAMP reaction. The y-axis is LAMP Tp and the x-axis is RT-qPCR cycle threshold (Ct). The dotted line indicates Tp 30 min. Results plotted above this line (encircled) were RT-qPCR positive but N1-STOP-LAMP negative. (d) Results of four-way interlaboratory comparison of N1-STOP-LAMP. Ten clinical samples previously tested positive by E-gene RT-qPCR were used to create a test panel for comparing assay performance between laboratories. The sample with Ct of 15 was the positive control, purified SARS-CoV-2.