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RT-LAMP assays using in-house produced enzymes show promise for accurate and affordable SARS-CoV-2 testing

In the efforts to limit the spread of SARS-CoV-2, extensive testing has played a crucial role. However, traditional reverse transcription PCR (RT-PCR) testing requires specialized equipment and reagents that are difficult to apply on a large scale. A more accessible form of PCR known as reverse transcription loop-mediated isothermal amplification (RT-LAMP), has been developed, but distribution of molecular biology reagents remains limited.

In a recent publication in Nature Scientific Reports, A. Alekseenko, et al. developed a set of protocols for production of DNA polymerases that are simple to make and ideal for use in RT-LAMP-based detection of SARS-CoV-2. The authors performed RT-LAMP assays to compare the performance of the in-house produced enzymes with commercially available alternatives. They used EvaGreen® Dye to track DNA amplified from synthetic SARS-CoV-2 RNA templates. Results show EvaGreen® Dye provided a higher dynamic range of fluorescence than SYBR® Green I during the optimization of the RT-LAMP assay. Overall, they found that their optimized mix of primers and in-house enzymes offered comparable performance to the commercially available master mixes.

The authors then evaluated the accuracy of their RT-LAMP assay by directly testing unextracted nasopharyngeal patient samples. They obtained positive and negative COVID-19 samples and found that their optimized RT-LAMP mix was successful at detecting samples with a high viral load of SARS-CoV-2. However, the samples with low to medium viral loads of SARS-CoV-2 led to false negative results, suggesting the need for further sample purification and RNA concentration to achieve RT-qPCR sensitivity.

The authors concluded that for SARS-CoV-2 detection, their in-house produced enzyme mixes are an inexpensive and practical alternative to commercially available enzymes, demonstrating comparable performance.

Benchmarking of in-house produced enzymes against commercial alternatives. Synthetic RNA templates (iLACO and As1e) amplified with the corresponding primers and either WarmStart, Bst3.0 with SSIV, Saphir Bst2.0 Turbo with SSIV, v5.9 with MashUp-RT, or v7.16 with MashUp-RT (same conditions for v5.9 and v7.16 as in Fig. 2B). All experiments were run for 1 h at 65 °C in a thermocycler, tracked by Eva Green fluorescence. Credit: A. Alekseenko, et al. reproduced under the Creative Commons license.

Learn more about Biotium’s versatile nucleic acid dyes, EvaGreen® Dye and EvaGreen® Plus Dye, as well as the Forget-Me-Not™ EvaGreen® qPCR Master Mixes.

Full Citation:

Alekseenko, A., Barrett, D., Pareja-Sanchez, Y. et al. Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples. Sci Rep 11, 1820 (2021).