An alternative real-time fluorescence reverse transcription loop-mediated isothermal amplification assay for the...

COVID-19, caused by SARS-CoV-2 virus infection, remains a public health concern in many countries. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and cost-effective alternative test for COVID-19 diagnosis. In this study, we developed and evaluated a real-time RT-LAMP (rRT-LAMP) assay coupled with a melting curve analysis to detect SARS-CoV-2. The reaction was carried out in a real-time thermal cycler at 63 °C for 45 min to amplify the region of SARS-CoV-2 orf8; real-time monitoring of amplification was performed by fluorescence detection. The performance was assessed by comparing it to a real-time reverse transcription-polymerase reaction (rRT-PCR) as a reference. The rRT-LAMP could detect as few as 15 copies of SARS-CoV-2 RNA per reaction. Positive results appeared within 30 min, while the melting-temperature analysis could verify the amplification specificity. No positive results from non-SARS-CoV-2 templates and no mis-amplification were observed. The comparative analysis using 262 RNA extracted from nasopharyngeal swab samples revealed the overall accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) of the rRT-LAMP at 88.55% (95% CI: 77.52-100%), 84.13% (95% CI: 71.56-98.27%), 100% (95% CI: 78.38-100%), 100% (95% CI: 85.06-100%), and 70.87% (95% CI: 55.55-89.11%), respectively. The greatest sensitivity was as high as 98-100% for specimens with threshold rRT-PCR cycle (Ct) values of less than 30 cycles. Overall, this rRT-LAMP showed good performance for the rapid detection of SARS-CoV-2. It is proposed as a potential method for real-time amplification detection, offering increased laboratory capacity for SARS-CoV-2 testing in a cost-effective and timely manner.