Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages
Abstract
:1. Introduction
2. Materials and Methods
2.1. Biological Material Samples
2.2. Whole-Genome and Sanger Sequencing Conditions
2.3. Data Analysis
2.4. Real-Time PCR Target Selection
2.5. Development of Multiplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for Genotyping of Omicron SARS-CoV-2 Sublineages
2.6. Primer Synthesis for PCR Techniques
2.7. Statistical Analysis
3. Results
3.1. Epidemiological Analysis of the Prevalence of Omicron Sublineages in Russia
3.2. Results Obtained by Using One-Step Multiplex PCR
4. Discussion
5. Patents
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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SARS-CoV-2 S-Gene Mutations | 5′-Sequence-3′ |
---|---|
L452R | (F) GGC TGC GTT ATA GCT TGG AAT TCT |
(R) CCG GCC TGA TAG ATT TCA GTT GAA (P) * (R6G)AAT TAC CGG TAT AGA T (BHQ1) | |
N501Y | (F) CTG AAA TCT ATC AGG CCG GTA |
(R) GCT GGT GCA TGT AGA AGT TCA AAA G (P) * (FAM)CCC ACT TAT GGT G (BHQ1) | |
T95I | (F) GGT TTG ATA ACC CTG TCC TAC CA |
(R) GGG ACT GGG TCT TCG AAT CTA A (P) * (ROX)GCT TCC ATT GAG AA (BHQ-2) | |
delHV69-70 | (F) GGA CTT GTT CTT ACC TTT CTT TTC CAA TG |
(R) TGG AAG CAA AAT AAA CAC CAT CAT TAA AT (P) * (R6G)TCC ATG CTA TCT CTG GGA C (BHQ1) | |
delLPP24-26 | (F) CAC TAG TCT CTA GTC AGT GTG T |
(R) TGT CAG GGT AAT AAA CAC CAC G (P) * (FAM)GAA CTC AAT CAT ACA CT (BHQ-1) | |
Ins214EPE | (F) GGA CCT TGA AGG AAA ACA GGG TAA (R) CCA ATG GTT CTA AAG CCG AAA AAC C (P) * (ROX)TAG TGC GTG AGC CAG AA (BHQ2) |
P681R | (F) GTA GGC AAT GAT GGA TTG ACT AGC TAC |
(R) TGC AGG TAT ATG CGC TAG TTA TCA GA (P) * (R6G)GCC GAC GAG AA (BHQ1) | |
E484A | (F) TTC AAC TGA AAT CTA TCA GGC CG |
(R) AGT TGC TGG TGC ATG TAG AAG TTC A (P) * (FAM)GTG TTG CAG GTG (BHQ-1) |
BA.1 | BA.2 | BA.3 | BA.4/BA.5 | Delta | |
---|---|---|---|---|---|
L452R | - | - | - | + | + |
P681R | - | - | - | - | + |
N501Y | + | + | + | + | - |
delHV69-70 | + | - | + | + | - |
Ins214EPE | + | - | - | - | - |
E484A | + | + | + | + | - |
delLPP24-26 | - | + | - | + | - |
T95I | + | - | + | - | - * |
N501Y | L452R | T95I | Del LPP24-26 | 69-70 | Ins214 | E484A | P681R | |
---|---|---|---|---|---|---|---|---|
NGS/Sanger negative | 10 | 26 | 245 | 22 | 71 | 120 | 16 | 110 |
True Negative (TN) | 8 | 26 | 241 | 20 | 63 | 120 | 14 | 110 |
False Positive (FP) | 2 | 0 | 4 | 2 | 8 | 0 | 2 | 0 |
Specificity/(CI) | 80.0% (49.0–94.4%) * | 100% (87.1–100%) * | 98.4% (95.9–99.6%) | 90.9% (72.2–97.5%) * | 88.7% (79.0–95.0%) | 100% (97.0–100%) | 87.5% (64.0–96.5%) * | 100% (97.4–100%) |
NGS/Sanger positive | 213 | 192 | 46 | 235 | 276 | 3 | 255 | 0 ** |
True Positive (TP) | 206 | 183 | 43 | 235 | 270 | 3 | 251 | - |
False Negative (FN) | 7 | 9 | 3 | 0 | 6 | 0 | 4 | - |
Sensitivity/(CI) | 96.7% (93.4–98.7%) | 95.3% (91.3–97.8%) | 93.5% (82.1–98.6%) | 100% (98.4–100%) | 97.8% (95.3–99.2%) | 100% (43.9–100%) * | 98.4% (96.0–99.6%) | - |
Total | 223 | 218 | 291 | 257 | 347 | 123 | 271 | 110 |
Accuracy | 96.0% (92.5–98.1%) | 95.9% (92.3–98.1%) | 97.6% (95.16–99.06%) | 99.26% (97.26–99.96%) | 96% (93.36–97.8%) | 100% (97.1–100%) | 97.8% (95.2–99.2%) | 100% (96.7–100%) |
BA.1 | BA.2 | BA.3 | BA.4/5 | Delta | Not Detected | Total | Confidence Level/Margin of Error * | |
---|---|---|---|---|---|---|---|---|
July | 2 (4.3%) | 17 (37.0%) | 0 (0.0%) | 27 (58.7%) | 0 (0.0%) | 0 (0.0%) | 46 (100.0%) | 90%/10% |
August | 0 (0.0%) | 2 (3.8%) | 0 (0.0%) | 51 (96.2%) | 0 (0.0%) | 0 (0.0%) | 53 (100.0%) | 90%/10% |
September | 0 (0.0%) | 2 (1.4%) | 0 (0.0%) | 139 (98.6%) | 0 (0.0%) | 0 (0.0%) | 141 (100.0%) | 95%/2% |
October | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 181 (95.3%) | 2 (1.1%) | 7 (3.7%) | 190 (100.0%) | 99%/1% |
November | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 52 (100.0%) | 0 (0.0%) | 0 (0.0%) | 52 (100.0%) | 90%/10% |
December | 0 (0.0%) | 3 (6.0%) | 0 (0.0%) | 47 (94.0%) | 0 (0.0%) | 0 (0.0%) | 50 (100.0%) | 90%/10% |
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Esman, A.; Dubodelov, D.; Khafizov, K.; Kotov, I.; Roev, G.; Golubeva, A.; Gasanov, G.; Korabelnikova, M.; Turashev, A.; Cherkashin, E.; et al. Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages. Genes 2023, 14, 1218. https://doi.org/10.3390/genes14061218
Esman A, Dubodelov D, Khafizov K, Kotov I, Roev G, Golubeva A, Gasanov G, Korabelnikova M, Turashev A, Cherkashin E, et al. Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages. Genes. 2023; 14(6):1218. https://doi.org/10.3390/genes14061218
Chicago/Turabian StyleEsman, Anna, Dmitry Dubodelov, Kamil Khafizov, Ivan Kotov, German Roev, Anna Golubeva, Gasan Gasanov, Marina Korabelnikova, Askar Turashev, Evgeniy Cherkashin, and et al. 2023. "Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages" Genes 14, no. 6: 1218. https://doi.org/10.3390/genes14061218
APA StyleEsman, A., Dubodelov, D., Khafizov, K., Kotov, I., Roev, G., Golubeva, A., Gasanov, G., Korabelnikova, M., Turashev, A., Cherkashin, E., Mironov, K., Cherkashina, A., & Akimkin, V. (2023). Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages. Genes, 14(6), 1218. https://doi.org/10.3390/genes14061218