Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART)

Abstract

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Fig 1

A. SARS-RT-LAMP-BART assay data derived via real-time monitoring the lights on the tube. Serial 10-fold-diluted synthetic SARS-CoV-2 RNA including the target region of RdRp and N genes (5×10⁴, 5×10³, 5×10², 10², 5×10, and 5 RNA copies) were assayed. The real-time monitoring was performed using Real-time LAMP-BART apparatus (PCRuN, Biogal Galed Lab, Israel). The assay was identified the light output peak in accordance with the manufacturer’s protocol. B. The Real-time RT-PCR (TaqMan probe method) data performed using a commercially available kit, One Step Prime Script RT-PCR kit (Takara Bio, Shiga, Japan), and LightCycler480 (Roche Diagnostics, Basel, Switzerland). Its reaction time was 130 min. (A) Serial 10-fold-diluted synthetic SARS-CoV-2 RNA including the target region of RdRp and N genes (5×10⁴, 5×10³, 5×10², 5×10, and 5 RNA copies) plus 2 copies were assayed. (B) The relation between the Crossing point (Cp) of each sample and the log of the amount of initial template RNA.

Fig 2

A. L452R-RT-LAMP-BART assay data with PNA and without PNA derived via real-time monitoring the lights on the tube using 3M™ Molecular Detection Instrument (MDS100, 3M, USA). Assayed samples were synthetic S gene RNA of SARS-CoV-2 with L452R (T1355G) (positive control, 5×10⁴ RNA copies), SARS-CoV-2 RNA (wild-type, JPN/AI/1-004, 5×10⁶ RNA copies), and DW. B. L452R-RT-LAMP-BART assay data derived via real-time monitoring the lights on the tube. Serial 10-fold-diluted samples (synthetic S gene RNA of SARS-CoV-2 with L452R (T1355G); 5×10⁴, 5×10³, 5×10², 10², 5×10, and 5 RNA copies) were assayed. The real-time monitoring was performed using Real-time LAMP-BART apparatus (MDS100, 3M, USA). The assay was identified the light output peak in accordance with the manufacturer’s protocol.