A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test

Abstract

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

Figure 1

Amplification curves of SARS-CoV-2 RT-LAMP runs using an intercalating fluorescent dye for detection. During the 30 min isothermal incubation (65 °C), the fluorescence signal is read minutely on the FAM channel. A threshold is applied to identify positive samples and calculate the threshold time (Tt) value.

Figure 2

Comparison of the SARS-CoV-2 RT-LAMP Tt values and the RT-PCR Ct values. The dashed lines represent the negative cut-offs. Artificial Ct and Tt values above these cut-offs were assigned to negative samples for data visualization purpose. Spearman correlation results are shown. Only true positive samples were included into the correlation analysis. (A) SARS-CoV-2 RT-LAMP with RNA isolation compared to the in-house RT-PCR. (B) SARS-CoV-2 RT-LAMP with RNA isolation compared to the LabsystemsDx RT-PCR (E gene target). (C) SARS-CoV-2 RT-LAMP with RNA isolation compared to the LabsystemsDx RT-PCR (N gene target). (D) SARS-CoV-2 RT-LAMP with RNA isolation compared to the LabsystemsDx RT-PCR (ORF1ab gene target). (E) SARS-CoV-2 RT-LAMP without RNA isolation compared to the in-house RT-PCR.