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.
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). https://doi.org/10.1038/s41598-020-80352-8