A Smart Single-Loop-Mediated Isothermal Amplification Facilitates Flexible SNP Probe Design for On-Site Rapid Differentiation of SARS-CoV-2 Omicron Variants

Abstract

Rapid on-site typing methods for SARS-CoV-2 variants of concern are crucial for its effective surveillance and control. Herein, a smart single-loop-mediated isothermal amplification (ssLAMP) method with the absence of an inner primer but the addition of a swarm primer for differentiation of SARS-CoV-2 Omicron variants is developed. This unique primer design strategy offers greater flexibility in introducing single nucleotide polymorphism (SNP) identification probes and enables multiple detection assays for SARS-CoV-2 Omicron variants including BA.1, BA.2, BA.3, BA.4, and BA.5. A 3D-printed portable dual fluorescence visualization device and smartphone app are developed to enable point-of-care testing. This assay is rapid (within 90 min), highly sensitive (100 copies/reaction), and specific (identification of SNP) for SARA-CoV-2 Omicron variants. The ssLAMP method identifies five BA.5-positive samples among 97 nasopharyngeal swab samples from the clinic, with a 100% concordance rate with Sanger sequencing. The ssLAMP assay system is expected to be utilized for on-site, highly specific, and rapid visualization detection of SARS-CoV-2 and its variants, with great application potential in pathogen genotyping, early cancer screening, and other areas of SNP mutation detection.

Keywords: SARS‐CoV‐2 variants of concern; SNP detection; nucleic acid amplification; ssLAMP.

Figure 1

The principle and workflow of ssLAMP method. A) The composition of the ssLAMP primer set. B) The comparison between ssLAMP and LAMP. The ssLAMP has a larger SNP probe configuration area than LAMP C. In the initial period, FIP, aided by F3 and LF, invades the DNA double strand, initiates the ssLAMP reaction, and generates the essential single‐loop product that initiates the cyclic reaction. In the cycling phase, some of the products generated by the ssLAMP reaction will be employed as substrates for the subsequent round of reaction, thereby facilitating exponential amplification of the ssLAMP reaction. In the ssLAMP reaction, the identification of single‐nucleotide polymorphisms (SNPs) can be achieved by introducing an SNP detection probe. When the ribonucleotide base in the SNP probe aligns with the template base, it will be cleaved by Rnase H2, resulting in the generation of a fluorescent signal.

Figure 2

The validation of the principle of ssLAMP method. A) Evaluation of the effect of amplification product length in the ssLAMP system. B) Evaluation of the effect of F3B3 primers in the ssLAMP system. C) Comparison of ssLAMP amplification with LF primer and without LF primer. D) Optimization of the concentration of LF primer. E) Agarose gel electrophoresis analysis of ssLAMP method driven by different primer sets. The asterisk (*) marks the specific band that was excised from the gel and subjected to sequencing analysis. F) Sequence analysis of expected bands for the ssLAMP method. Bands were sequenced using the FIP and LIR2 primer.

Figure 3

Effects of accelerating primers and optimization of reaction conditons on ssLAMP. A) Comparison with ssLAMP, LAMP, and SAMP. B) Sensitivity test of ssLAMP and LAMP with high GC contant (gE gene) plasmid template and linear regression between the indicated dilutions of plasmid and TT value. C) Sensitivity test of ssLAMP and LAMP with low GC contant (invA gene) plasmid template and linear regression between the indicated dilutions of plasmid and TT value.

Figure 4

Working principle of ssLAMP‐based rapid testing of the SARS‐CoV‐2 Omicron variant. A) Schematic of SARS‐CoV‐2 S gene with annotation of the mutations or region targeting different VOC (highlighted in red colors). The specific mutations or region recognized by the different probes are also shown for each detection system. B) Genotyping strategy for SARS‐CoV‐2 Omicron variants with 3 individual reaction systems.

Figure 5

Sensitivity and specificity tests of ssLAMP‐based rapid testing of the SARS‐CoV‐2 Omicron variants. A) Sensitivity test of the systemII multiplex ssLAMP assay with BA.1 plasmid. B) Sensitivity test of the systemII multiplex ssLAMP assay with BA.3 plasmid. C) Sensitivity test of the systemIII multiplex ssLAMP assay with BA.4 plasmid. D) Sensitivity test of the systemIII multiplex ssLAMP assay with BA.5 plasmid. E) Specificity test of the systemII multiplex ssLAMP assay with different plasmid. F) Specificity test of the systemIII multiplex ssLAMP assay with different plasmid.

Figure 6

Point‐of‐care testing of SARS‐CoV‐2 variants using ssLAMP method with a portable device and a smart phone. A) Workflow of SARS‐CoV‐2 Omicron variant detection in clinical swabs when using the ssLAMP assay combine with the portable device and smart phone. B) Plasmid sample tested by ssLAMP and the protable device. C) Interface of the ssLAMP smart analyzing APP. D) 3D rendering of the protable detection device. Parts of the image were designed with BioRender.com, with permission.

Figure 7

Comparison between qPCR method and ssLAMP‐based rapid testing of clinical nasopharyngeal swab sample.