Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection

Abstract

Background: Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low / middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals.

Methods: Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP reactions were performed using Warm Start 2X Master Mix and real-time PCR machine at 65 °C for 60 cycles with 15 s for each cycle. Results were read by visualizing turbidity under ultraviolet light and real time fluorescence detection through FAM channel of the real time PCR machine. We tested a total of 320 including 240 SARS CoV-2 positive (Ct values <40) and 80 SARS CoV-2 negative samples as tested by a real time RT-PCR using the newly developed LAMP assay.

Results: A total of 206 out of 240 SARS CoV-2 positive samples were tested positive by the newly developed LAMP assay with a sensitivity of 86%. All 80 SARS CoV-2 negative samples were tested negative by the newly developed LAMP assay with a specificity of 100%.

Conclusion: The newly developed real time LAMP assay has a sensitivity of 86% and specificity of 100% compared to the real time RT-PCR for the detection of SARS CoV-2. The new assay will be useful to screen large number of samples if adopted to minimize the time and cost.

Keywords: Loop mediated amplification; SARS CoV-2, Severe acute respiratory syndrome coronavirus-2; SARS-CoV-2; Sensitivity; Specificity; rtRT-PCR; rtRT-PCR, Real time reverse transcription polymerase chian reaction.

Fig. 1

Amplification of viral RNA detected through real time relative fluorescence units (RFU) per cycle. A - Amplification of the positive samples at 65 °C for 60 cycles with 15 s per cycle (30 min). B - Amplification of the negative samples under similar conditions.

Fig. 2

Images of SARS CoV-2 positive and negative samples with multi-colour filter (UV light) in a gel documentation system (Applied Bio system). Positive sample showed a yellow colour (Tubes A, B & C) whereas the negative samples did not show yellow colour (Tubes D, E & F).

Fig. 3

SARS-CoV-2 RNA detected by direct visualization for the presence and absence of turbidity. A - Positive samples showing turbidity. B -Negative samples not showing turbidity.

Fig. 4

Receiver operating characteristic curve (ROC curve) to study the performance of the newly developed LAMP assay for the detection of SARS CoV-2.

Fig. 5

Comparison of turnaround time for real time RT-LAMP and real time RT-PCR for the detection of SARS CoV-2.