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)

• Published in: PloS one 2022-03, Vol.17 (3), p.e0265748-e0265748
• Main Authors: Iijima, Takahiro, Ando, Shinnosuke, Kanamori, Dai, Kuroda, Kazumichi, Nomura, Tsutomu, Tisi, Laurence, Kilgore, Paul E, Percy, Neil, Kohase, Hikaru, Hayakawa, Satoshi, Seki, Mitsuko, Hoshino, Tomonori
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Analysis; Assaying; Avidity; Binding Sites; Biology and life sciences; Bioluminescence; California; Coronaviruses; COVID-19; COVID-19 - diagnosis; Early Diagnosis; Gene amplification; Gene sequencing; Genetic transcription; Humans; India; Leucine; Limit of Detection; Luminescent Measurements; Medicine and health sciences; Middle East respiratory syndrome; Missense mutation; Molecular Diagnostic Techniques; Mutation; Mutation, Missense; Nucleic Acid Amplification Techniques; Nucleic acids; Nucleotides; Peptide nucleic acids; Peptide Nucleic Acids - genetics; Physical Sciences; Polymerase chain reaction; Real time; Real-Time Polymerase Chain Reaction; Receptors; Research and Analysis Methods; Reverse Transcription; Ribonucleic acid; RNA; Saliva; SARS-CoV-2 - classification; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Sensitivity and Specificity; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus - chemistry; Spike Glycoprotein, Coronavirus - genetics; Spike protein; Strains (organisms); Viral diseases
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0265748

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.