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.

Keywords: Delta variant; R203M mutation; RT-LAMP; SARS-CoV-2; rapid diagnosis.

Figure 1

. RT-LAMP strategy for SARS-CoV-2 Delta and selection of target and primers. (A) The principle of detecting SNPs using RT-LAMP. SNP genotyping could be performed by tracking the time gap between these two reactions, which could be visually reported using the quantitative fluorescent Cq-difference. (B) The pipeline of the Cq ratio-based RT-LAMP method in detecting Delta. The extracted RNA from clinical samples was amplified with two sets of primers (R203M and conserved primer). (C) Schematic view of two sets of RT-LAMP primer target sites. The R203M mutation (G to T transition) in the mutant primer is indicated in the black box, G refers to the wild allele and T is the mutant allele in the Delta (sequence from GenBank: MN908947.3). (D) Heat map of the observed mutations within the N protein as of February 2022. Each row corresponds to the variant of SARS-CoV-2 listed on the left; each column indicates mutations listed on the top. (E) The specific differences in the R203M, R203K/G204R and T205I mutations in the N gene sequence.

Figure 2.

R203M primer screening, and the analytical specificity and detection limit of the two sets of RT-LAMP primers. (A) Primer sequence comparisons for six viruses (SARS-CoV, MERS-CoV, Bat SARA CoV HKU3, H1N1, Influenza B, HPIV-1 and HRV-A66) The red “T” in the LF of the R203M primer set is the mutation site of R203M. (B) Selection of loop primers in the mutant primer set. The selected LF4 shows the best distinguishing effect for R203M. (C) The analytical specificity analyses of RT-LAMP. Numbers 1–4: the amplified curve of the R203M, R203K/G204R, T205I mutant-containing template and wild template using the conserved primer set. Numbers 5–8: the amplified curve of the R203M, R203K/G204R, T205I mutant-containing template and wild template using the R203M primer set. Numbers 9–20 indicate RT-LAMP reactions using the conserved primer set for detecting 12 pathogenic plasmids consecutively (SARS-CoV, MERS-CoV, H1N1/H3N2 of influenza A viruses, influenza B viruses, MP, EV-U/71, HPIV-1/2/3, AdV-B/E, CA16, Cpn, RSV and HCMV). Numbers 21–32 refer to RT-LAMP reactions using the R203M primer set for these 12 pathogens. Numbers 33 and 34 were DEPC H₂O (negative control) with the conserved primer set and the R203M primer set, respectively. The RT-LAMP assay did not cross-react with other human-pathogenic coronaviruses and common viral pathogens. (D) The 95% limits of detection (LOD95) of the RT-LAMP. The nucleic acid concentration was determined from 2000 copies/reaction to 7.83 copies/reaction. The LOD95 of the conserved region primer set (left) and the LOD95 of the R203M primer set (right). A probit regression model was used to estimate the LOD95. A cycle threshold value of less than 55 was defined as positive.

Figure 3.

The Cq ratio-based RT-LAMP method. (A) The real-time quantitative fluorescent RT-LAMP result of the conserved and R203M primers over a range of templates from 10³ −10⁸ copies/mL. The amplification curves: wild-type plasmid using the conserved primers (solid lines), wild-type plasmid using the mutant primers (dotted lines), and R203M mutant plasmid using mutant primers (dashed lines). (B) The Cq ratio of the mutant plasmid and wild-type plasmid (left). The Cq ratio of the Delta variant and non-Delta variant (right). Box plots indicate the minimum and maximum value (whiskers), median (middle line), and 25th and 75th percentiles (box). The cutoff of the Cq ratio is set at 1.80. The statistical analysis was performed using the Wilcoxon/Kruskal-Wallis test, ***P < 0.001.

Figure 4.

Analysis of RT-LAMP results and Delta genotyping methods from clinical samples. (A) Comparison of the Cq values for R203M and conserved primers on the amplification of clinical samples. Box plots indicate the minimum and maximum value (whiskers), median (middle dotted line), and 25th and 75th percentiles (box). *Low-concentration samples: The four low-concentration non-Delta samples circled by the red dotted line have no corresponding amplification Cq value using R203M primers. (B) Comparison of the Cq ratio between Delta and non-Delta variants. The cutoff of the Cq ratio is set at 1.80 (dotted line). The Cq ratio values are significantly different in Delta and non-Delta variants. Statistical analysis was performed using the Wilcoxon/Kruskal-Wallis test, ***P < 0.001. (C) ROC (receiver operating characteristics with area under curve) curves for Delta variant identification as measured by the developed RT-LAMP method.