Development of Loop-mediated Isothermal Amplification (LAMP) Assays Using Five Primers Reduces the False-positive Rate in COVID-19 Diagnosis

Abstract

The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a cheaper and faster testing alternative for detecting SARS-CoV-2. However, high false-positive rate due to misamplification is one of the major limitations. To overcome misamplifications, we developed colorimetric and fluorometric RT-LAMP assays. The assay performances was verified by the gold-standard RT-qPCR technique on 150 clinical samples. Compared to other primer sets with six primers (N, S, andRdRp), E-ID1 primer set, including five primers, performed superbly on both colorimetric and fluorometric assays, yielding sensitivities of 89.5% and 100%, respectively, with a limit of detection of 20 copies/µL. The colorimetric RT-LAMP had a specificity of 97.2% and an accuracy of 94.5%, while the fluorometric RT-LAMP obtained 96.9% and 98%, respectively. No misamplification was evident even after 120 minutes, which is crucial for the success of this technique. These findings are important to support the use of RT-LAMP in the healthcare systems in fighting COVID-19.

Detection of feline immunodeficiency virus by neutral red-based loop-mediated isothermal amplification assay

“…Compared to typical PCR assays with a higher detection limit, false-positive results may occur when NR-LAMP is used in veterinary practice. This is likely due to the minimal quantity of proviral DNA, sample quality, time of sample collection from disease onset, and the use of six novel LAMP primers specific to the FIV gag gene [57,58]. To confirm FIV-infected cats, nucleotide sequencing is required.…”