Rational Primer and Probe Construction in PCR-Based Assays for the Efficient Diagnosis of Drifting Variants of SARS-CoV-2

Figure 2

N gene designated fragment—MSA (MUSCLE) by MEGA11 version 0.1. The region of primers and probe is designed by [1]. Star () signs denote perfect nucleotide position match, and red dots (.) denote mismatch. The black rectangle shape denotes the amplicon region for forward primer design, the blue rectangle shape denotes the amplicon region for probe design, and the pink rectangle shape denotes amplicon position for reverse primer position. Analyzed NCBI GenBank accession IDs for MSA for the entire E gene represent as follows: analyzed NCBI GenBank accession IDs for multiple sequence alignment for the N gene fragment represent as follows: 1. NC_045512.2: N gene designated fragment (nucleotide sequence: 28671–28850, 180 bp), SARS-CoV-2 isolate Wuhan-Hu-1, SARS-CoV-2 ref. Genome; 2. OM287553.1: N gene designated fragment (nucleotide sequence: 28587–28766, 180 bp), SARS-CoV-2/human/NLD/EMC-Omicron-1/2021; 3. OK091006.1: N gene designated fragment (nucleotide sequence: 28644–28823, 180 bp), SARS-CoV-2/human/JPN/SARS-CoV-2, B.1.617.2 lineage, Delta variant/2021; 4. MZ427312.1: N gene designated fragment (nucleotide sequence: 28644–28823, 180 bp), SARS-CoV-2/human/DEU/SARS-CoV-2_P.1 (Gamma)_VeroE6_210419_P3/2021; 5. MZ888515.1: N gene designated fragment (nucleotide sequence: 28622–28801, 180 bp), SARS-CoV-2/human/THA/AFRIMS-COV0087/Alpha variant/2021; 6. MG772933.1: N gene designated fragment (nucleotide sequence: 28576–28755, 180 bp), bat SARS-like coronavirus isolate bat-SL-CoVZC45; 7. NC_004718.3: N gene designated fragment (nucleotide sequence: 28520–28695, 180 bp), SARS coronavirus Tor2; and 8. NC_014470.1: N gene designated fragment (nucleotide sequence: 28059–28238, 180 bp), bat coronavirus BM48-31/BGR/2008.