RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant

Abstract

The highly infectious Delta variant strain of SARS-CoV-2 remains globally dominant and undermines COVID-19 vaccines. Rapid detection of the Delta variant is crucial for the identification and quarantine of infected individuals. In this study, our aim was to design and validate a genotyping RT-LAMP method to detect Delta variants specifically. R203M in the N gene of SARS-CoV-2 was chosen as the Delta variant-specific mutation for genotyping. To target the R203M-harboring region and the conserved sequence of the N gene, two sets of primers were designed, and a Cq (quantification cycle) ratio-based RT-LAMP for SARS-CoV-2 and R203M detection was developed by analyzing the significant discrepancy in amplification efficiency of the two sets of primers. We validated the RT-LAMP method on 498 clinical specimens in parallel with RT-qPCR, and 84 Delta variants from 198 positive samples were determined by sequencing. Compared with traditional RT-qPCR analyses, RT-LAMP appears to be 100% accurate in detecting SARS-CoV-2 clinical samples. RT-LAMP has a good ability to distinguish between Delta and non-Delta variants under a Cq ratio threshold of 1.80. Furthermore, the AUC (area under the curve) of this method was 1.00; the sensitivity, specificity and accuracy were all 100%. In summary, we have proposed a rapid, accurate and cost-effective RT-LAMP method to detect SARS-CoV-2 and Delta variants, which may facilitate the surveillance of COVID-19.

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers

“…The authors used molecular beacons and additional oligonucleotides to block nucleic acids. Another study reported an RT-LAMP-based genotyping method focusing on R203M in the SARS-CoV-2 N gene to detect Delta variants specifically 33 . Although the test did not use additional oligonucleotides, the excellent ability to distinguish between Delta and non-Delta variants occurred by calculating a ratio between the Cts of two different reactions per sample.…”

Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA-PfAgo Method

“…According to the fluorescence signals, with as low as 10 0 copies of the SARS-CoV-2 mutant (MT) RNA, the L452R mutation could be identified (Figure ). The single-copy sensitivity is the highest sensitivity that can be achieved theoretically, and is also the highest sensitivity as compared to the available SARS-CoV-2 genotyping assays. − According to the reports, the nucleic acid amplification and probe-based assays had sensitivities in the range of tens to hundreds of copies per reaction. − The CRISPR-based assays had sensitivities of around 10 copies per reaction. , By fully utilizing Pf Ago’s specific recognition and cleavage property, the RPA- Pf Ago method exhibited a solid advantage in terms of sensitivity with which a single copy could be sufficient for the mutation identification (Figure ). The single-copy sensitivity is the highest sensitivity that can be achieved theoretically and is also the highest sensitivity as compared to the available SARS-CoV-2.…”

Fast, low-cost and highly specific colorimetric RT-LAMP assays for inference of SARS-CoV-2 Omicron BA.1 and BA.2 lineages

“…Sherrill-Mix et al (2021) 32 used a pair of molecular beacons with RT-LAMP to specifically detect B.1.1.7 and other variants that contain a characteristic deletion in the gene encoding the viral spike protein. Yang et al (2022) 33 designed and validated a genotyping RT-LAMP method to detect Delta variants specifically by detecting the R203M in the N gene of the SARS-CoV-2 Delta variant. Both studies reported good specificity and a 95% confidence limit of detection (LOD 95) at approximately 4.0 × 10 2 copies per reaction.…”

“…Although it presents significant advantages in terms of simplicity, time, cost, and instrumental need, the proposal to use LAMP to infer variants of SARS-CoV-2 is still discrete. 33 designed and validated a genotyping RT-LAMP method to detect Delta variants specifically by detecting the R203M in the N gene of the SARS-CoV-2 Delta variant. Both studies reported good specificity and a 95% confidence limit of detection (LOD 95) at approximately 4.0 × 10 2 copies per reaction.…”

“…Recently, RT-LAMP methodologies have emerged with the opposite strategy, using primers designed to identify specific lineage-defining mutations. [32][33][34] In this way, RT-LAMP has been proposed as a viable alternative to detect mutations to help in the surveillance of VOCs. In this study, we designed two sets of primers targeting specific mutations in either the Omicron lineage BA.1 or lineage BA.2.…”