Development of multiplex allele-specific RT-qPCR assays for differentiation of SARS-CoV-2 Omicron subvariants.

Quick differentiation of current circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmissions. However, the widely applied gene sequencing method is time-consuming and costly especially when facing recombinant variants, because a large part or whole genome sequencing is required. Allele-specific reverse transcriptase real time RT-PCR (RT-qPCR) represents a quick and cost-effective method for SNP (single nucleotide polymorphism) genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 5 multiplex allele-specific RT-qPCR assays targeting 20 key mutations for quick differentiation of the Omicron subvariants (BA.1 to BA.5 and their descendants) and the recombinant variants (XBB.1 and XBB.1.5). Two parallel multiplex RT-qPCR reactions were designed to separately target the prototype allele and the mutated allele of each mutation in the allele-specific RT-qPCR assay. Optimal annealing temperatures, primer and probe dosage, and time for annealing/extension for each reaction were determined by multi-factor and multi-level orthogonal test. The variation of Cp (crossing point) values (ΔCp) between the two multiplex RT-qPCR reactions was applied to determine if a mutation occurs or not. SARS-CoV-2 subvariants and related recombinant variants were differentiated by their unique mutation patterns. The developed multiplex allele-specific RT-qPCR assays exhibited excellent analytical sensitivities (with limits of detection (LoDs) of 1.47-18.52 copies per reaction), wide linear detection ranges (109-100 copies per reaction), good amplification efficiencies (88.25 to 110.68%), excellent reproducibility (coefficient of variations (CVs) < 5% in both intra-assay and inter-assay tests), and good clinical performances (99.5-100% consistencies with Sanger sequencing). The developed multiplex allele-specific RT-qPCR assays in the present study provide an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants. KEY POINTS: • A panel of five multiplex allele-specific RT-qPCR assays for quick differentiation of 11 SARS-CoV-2 Omicron subvariants (BA.1, BA.2, BA.4, BA.5, and their descendants) and 2 recombinant variants (XBB.1 and XBB.1.5). • The developed assays exhibited good analytical sensitivities and reproducibility, wide linear detection ranges, and good clinical performances, providing an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants.

Development of a panel of three multiplex allele-specific qRT-PCR assays for quick differentiation of recombinant variants and Omicron subvariants of SARS-CoV-2.

Quick differentiation of the circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmission. However, the widely used gene sequencing method is time-consuming and costly when facing the viral recombinant variants, because partial or whole genome sequencing is required. Allele-specific real time RT-PCR (qRT-PCR) represents a quick and cost-effective method in SNP genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 3 multiplex allele-specific qRT-PCR assays targeting 12 key differential mutations for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2). Two parallel multiplex qRT-PCR reactions were designed to separately target the protype allele and the mutated allele of the four mutations in each allele-specific qRT-PCR assay. The variation of Cp values (ΔCp) between the two multiplex qRT-PCR reactions was applied for mutation determination. The developed multiplex allele-specific qRT-PCR assays exhibited outstanding analytical sensitivities (with limits of detection [LoDs] of 2.97-27.43 copies per reaction), wide linear detection ranges (107-100 copies per reaction), good amplification efficiencies (82% to 95%), good reproducibility (Coefficient of Variations (CVs) < 5% in both intra-assay and inter-assay tests) and clinical performances (99.5%-100% consistency with Sanger sequencing). The developed multiplex allele-specific qRT-PCR assays in this study provide an alternative tool for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2).

Asymmetric Michael addition of 1-acetylindolin-3-ones to ß-nitrostyrenes catalyzed by bifunctional thioureas: a simple access to 2-functionalized indoles.

The first asymmetric Michael addition of 1-acetylindolin-3-ones to ß-nitrostyrenes has been developed. 2-Substituted indolin-3-one derivatives were obtained with excellent yields (up to 99%) and good stereoselectivities (up to 28:1 dr and 92% ee), which could be transformed into 2-functionalized indoles easily without racemization. This achievement might further contribute to the chemistry and pharmacology of indole-related compounds.