Colorimetric and fluorometric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for diagnosis of SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since it infected humans almost 3 years ago. Improvements of current assays and the development of new rapid tests or to diagnose SARS-CoV-2 are urgent. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and propitious assay, allowing to detect both colorimetric and/or fluorometric nucleic acid amplifications. This study describes the analytical and clinical evaluation of RT-LAMP assay for detection of SARS-CoV-2, by designing LAMP primers targeting N (nucleocapsid phosphoprotein), RdRp (polyprotein), S (surface glycoprotein), and E (envelope protein) genes. The assay’s performance was compared with the gold standard RT-PCR, yielding 94.6% sensitivity and 92.9% specificity. Among the tested primer sets, the ones for S and N genes had the highest analytical sensitivity, showing results in about 20 min. The colorimetric and fluorometric comparisons revealed that the latter is faster than the former. The limit of detection (LoD) of RT-LAMP reaction in both assays is 50 copies/µl of the reaction mixture. However, the simple eye-observation advantage of the colorimetric assay (with a color change from yellow to red) serves a promising on-site point-of-care testing method anywhere, including, for instance, laboratory and in-house applications. Supplementary Information The online version contains supplementary material available at 10.1007/s10142-022-00900-5.

Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis

“…In this work, two RT-LAMP assays (colorimetric and fluorometric) targeting the E gene of SARS-CoV-2 were developed. The colorimetric RT-LAMP has the advantage of simple and cost-effective reaction conditions in addition to visual interpretation of the results 16 . Its diagnostic accuracy, however, must be comparable to the gold standard RT-PCR to consider this technique reliable to avoid false results and their undesired consequences.…”

“…It provides simple detection techniques, being also cheaper and faster than the gold standard 7,9,10 . The efficiency of RT-LAMP in COVID-19 diagnosis was demonstrated by many researchers [11][12][13][14][15][16] , and many validated the performance of FDA-approved colorimetric RT-LAMP kits for emergency use authorization 17,18 . However, Optimization of the colorimetric and fluorometric RT-LAMP assays.…”

“…It provides simple detection techniques, being also cheaper and faster than the gold standard 7 , 9 , 10 . The efficiency of RT-LAMP in COVID-19 diagnosis was demonstrated by many researchers 11 – 16 , and many validated the performance of FDA-approved colorimetric RT-LAMP kits for emergency use authorization 17 , 18 . However, because of the number of primers, the most common limitation of the RT-LAMP technique is the misamplifications that arise from unwanted secondary structures.…”

Current strategies for SARS-CoV-2 molecular detection

“…From then on, the Q-PCR assay quickly became the main detection assay for SARS-CoV-2 until now. The TaqMan probe-based Q-PCR is one of the most popular diagnostic methods applied for the detection of SARS-CoV-2 at present, 29 and the detection steps are elaborated in [Fig. 3].…”

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

“…The amplification reaction in this technique takes place isothermally in a single tube without requiring bulky instruments and provides simple detection techniques, being also cheaper and faster than the gold standard (Notomi, 2000, Notomi et al, 2015; (Alhamid and Tombuloglu, 2022). The efficiency of RT-LAMP in COVID-19 diagnosis was demonstrated by many researchers (Aoki et al, 2021, Bokelmann et al, 2021, Dao Thi, 2020, de Oliveira Coelho et al, 2021, Lu et al, 2020; Alhamid et al, 2022), and many validated the performance of FDA-approved colorimetric RT-LAMP kits for emergency use authorization (Baba et al, 2021, Promlek et al, 2022, Color SARS-CoV-2 RT-LAMP Diagnostic Assay -EUA Summary , 2021). However, because of the number of primers, the most common limitation of the RT-LAMP technique is the misamplifications that arise from unwanted secondary structures.…”