Comparison of RT-LAMP and real-time RT-PCR in SARS-CoV-2 diagnostic testing

Abstract

Since the novel coronavirus disease (COVID-19) emerged in late 2019 and subsequently evolved into the pandemic, accurate and rapid diagnostic tests of the disease have been developed. Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) amplifies target sequences within cDNA using a strand-displacement-type DNA polymerase under a constant reaction temperature and can detect SARS-CoV-2 within 1 hour. In this study, we compared RT-LAMP and the conventional real-time reverse transcription polymerase chain reaction (RT-PCR) in SARS-CoV-2 diagnostic testing. We prepared a 10-fold dilution series of SARS-CoV-2 N-gene synthetic RNA and found that RT-LAMP and real-time RT-PCR detected the gene in up to 10²- and 10³-fold diluted materials, respectively. Next, we randomly selected 12 RT-LAMP-positive nasopharyngeal swabs and prepared a 10-fold dilution series from purified nucleic acids of these specimens. The concordance rate of RT-LAMP and real-time RT-PCR for positive results was 97%. Comparing the threshold time (Tt) value of RT-LAMP and the cycle threshold (Ct) value of real-time RT-PCR, both values showed a positive correlation (correlation coefficient, 0.89, P < 0.001) and samples with a Ct value of 37 cycles or less were detectable with RT-LAMP. Between Ct values of 29-40 cycles and Tt values of 11-20 minutes, the approximation of Ct value = 1.1 × Tt value + 17 could be applied. On the other hand, as the Tt value corresponding to the Ct value of 40 cycles was calculated as 20 minutes and 55 seconds, when the Tt value in RT-LAMP exceeds 20 minutes, we should be cautious of assessing the material as positive.