A Clinical Assessment of the RT-LAMP-Based Colorimetric Diagnostic Method for COVID-19 with Improved Primers Sets

A cost-effective and robust diagnosis of an infectious agent is essential during a pandemic outbreak. While RT-qPCR-based methods are widely used, alternative methods that can be used outside the laboratory setup would enable better disease control and widen the surveillance efforts. The loop-mediated isothermal amplification (LAMP) method is an alternate method that has not yet been commercially deployed widely. The colorimetric LAMP can be adapted for various pathogens and would be helpful for high-throughput screening in a limited resource setting. Moreover, it is quicker and less labor-intensive. In our study, improved RT-LAMP assay primer sets were designed to detect SARS-CoV-2: N gene and ORF1a gene fragments. RT-LAMP assay primers were also designed to detect human RNase P to ensure RNA integrity. RT-LAMP reaction tubes/plates were incubated at 65°C for 30 min. Cell phone cameras were used to record results. RT-LAMP results were assessed by comparing the results obtained with RT-qPCR. RT-LAMP detected ~100 RNA copies, and its sensitivity was on par with RT-qPCR. Out of 60 patient samples with extracted RNA, a positive agreement of 98.2% was observed between RT-LAMP and RT-qPCR. On a cautionary note, severe discrepancies were observed if the same oligos were procured from different vendors, drastically affecting the test results. Therefore, mandatory quality control of primers is indispensable.