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A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification
Takahiro Iijima1, Jun Sakai2, Dai Kanamori1
1Division of Pediatric Dentistry, Department of Human Development and Fostering, Meikai University School of Dentistry, Sakado 350-0283, Japan.
International Journal of Molecular Sciences
|July 14, 2023
Summary
A new rapid assay, RT-LAMP-BART, accurately detects SARS-CoV-2 variants like Omicron by identifying key mutations. This simple and sensitive method is faster and more effective than traditional PCR for variant surveillance.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, including Alpha, Delta, and Omicron, possess distinct mutations in the spike (S) gene.
- Accurate and rapid detection of these SARS-CoV-2 variants is crucial for effective COVID-19 control.
- Previous development of RT-LAMP-BART successfully detected the L452R mutation.
Purpose of the Study:
- To establish and evaluate novel LAMP primers and PNA probes for detecting N501Y and Q493R/Q498R mutations in the SARS-CoV-2 S gene.
- To assess the specificity and sensitivity of the developed RT-LAMP-BART assays.
- To compare the performance of the RT-LAMP-BART assay with conventional real-time RT-PCR for SARS-CoV-2 variant detection.
Main Methods:
- Design of LAMP primer sets and peptide nucleic acid (PNA) probes targeting specific mutations (N501Y, Q493R/Q498R) on the SARS-CoV-2 S gene.
- Evaluation of assay specificity using five viral and four bacterial reference strains.
- Assessment of assay sensitivity using synthetic RNAs and spiked clinical samples, with results compared against real-time RT-PCR.
Main Results:
- The RT-LAMP-BART assays accurately identified the N501Y and Q493R/Q498R mutations.
- The novel assay detected 100-200 copies of target genes within 30 minutes, significantly faster than real-time RT-PCR (130 minutes, 500-3000 copies).
- Real-time RT-PCR failed to detect Omicron variants (BA.1, BA.2) despite the presence of the N501Y mutation, highlighting a limitation.
Conclusions:
- The developed RT-LAMP-BART assay demonstrates high specificity and superior sensitivity compared to conventional real-time RT-PCR.
- This simple, inexpensive, and rapid assay is a valuable tool for identifying SARS-CoV-2 variants of concern.
- The assay's efficiency supports enhanced global surveillance and control of emerging viral threats.
