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

Figure 1

E gene multiple sequence alignment (MUSCLE) by MEGA11 version 0.1. The region of primers and probe was 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: 1. NC_045512.2: E gene (nucleotide sequence: 26245–26472, 228 bp), SARS-CoV-2 isolate Wuhan-Hu-1, SARS-CoV-2 Ref. Genome; 2. OM287553.1: E gene (nucleotide sequence: 26170–26397, 228 bp), SARS-CoV-2/human/NLD/EMC-Omicron-1/2021; 3. OK091006.1: E gene (nucleotide sequence: 26218–26445, 228 bp) SARS-CoV-2/human/JPN/SARS-CoV-2, B.1.617.2 lineage, Delta variant/2021; 4. MZ427312.1: E gene (nucleotide sequence: 26214–26441, 228 bp), SARS-CoV-2/human/DEU/SARS-CoV-2_P.1 (Gamma)_VeroE6_210419_P3/2021; 5. MZ888515.1: E gene (nucleotide sequence: 26197–26424, 228 bp) SARS-CoV-2/human/THA/AFRIMS-COV0087/Alpha variant/2021; 6. MG772933.1: E gene (nucleotide sequence: 26150–26377, 228 bp), bat SARS-like coronavirus isolate bat-SL-CoVZC45; 7. NC_004718.3: E gene (nucleotide sequence: 26117–26347, 231 bp), SARS coronavirus Tor2; and 8. NC_014470.1: E gene (nucleotide sequence: 26018–26248, 231 bp), bat coronavirus BM48-31/BGR/2008.