Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community
Abstract
1. Introduction
2. Materials and Methods
2.1. IPS COVID Lab Accreditation and Admission Proceedings
2.2. Sample Collection and Storage
2.3. Sample Pooling and Procedure Validation
2.4. Multiplex RT-qPCR and Validation
3. Results and Discussion
3.1. Sample Pooling
3.2. Multiplex RT-qPCR
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fluorophore | Excitation Max (nm) | Emission Max (nm) | Extinction Coefficient * | Color ** | Quencher |
---|---|---|---|---|---|
FAM | 495 | 520 | 75,850 | Yellow-Green | BHQ-1 |
HEX | 535 | 556 | 98,000 | Yellow | TAMRA |
ROX | 575 | 602 | 82,000 | Orange-Red | BHQ-2 |
Gene Target | Fluorophore 5′ | Sequence (5′ → 3′) | Quencher 3′ |
---|---|---|---|
RP | HEX | TTC TGA CCT GAA GGC TCT GCG CG | TAMRA |
N1 | FAM | ACC CCG CAT TAC GTT TGG TGG ACC | BHQ-1 |
N2 | ROX | ACA ATT TGC CCC CAG CGC TTC AG | BHQ-2 |
RP 1 | N1 | N2 | ||||
---|---|---|---|---|---|---|
Probe | Standard | Multiplex | Standard | Simplex | Standard | Simplex |
Efficiency | 109 ± 2% | 126 ± 14% | 117 ± 3% | 95 ± 4% | 114 ± 3% | 84 ± 2% |
Detection limit (Cp < 35) | DF 2 625 | DF 2 625 | ≈6.4 cp/µL 3 DF 2 15,625 | ≈6.4 cp/µL 3 DF 2 15,625 | ≈6.4 cp/µL 3 DF 2 15,625 | ≈6.4 cp/µL 3 DF 2 15,625 |
[RNA] (cp/µL) | Dilutions Performed in RNA Matrix * | Dilutions Performed in Water | ||||||
---|---|---|---|---|---|---|---|---|
Triplex (RP + N1 + N2) | Duplex (N1 + N2) | Duplex (RP + N1) | Duplex (RP + N2) | Triplex (RP + N1 + N2) | Duplex (N1 + N2) | Duplex (RP + N1) | Duplex (RP + N2) | |
20,000 | ||||||||
4000 | ||||||||
800 | ||||||||
160 | ||||||||
32 | ||||||||
6.4 |
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Miguel, F.; Baleizão, A.R.; Gomes, A.G.; Caria, H.; Serralha, F.N.; Justino, M.C. Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community. BioTech 2024, 13, 26. https://doi.org/10.3390/biotech13030026
Miguel F, Baleizão AR, Gomes AG, Caria H, Serralha FN, Justino MC. Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community. BioTech. 2024; 13(3):26. https://doi.org/10.3390/biotech13030026
Chicago/Turabian StyleMiguel, Fernanda, A. Raquel Baleizão, A. Gabriela Gomes, Helena Caria, Fátima N. Serralha, and Marta C. Justino. 2024. "Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community" BioTech 13, no. 3: 26. https://doi.org/10.3390/biotech13030026
APA StyleMiguel, F., Baleizão, A. R., Gomes, A. G., Caria, H., Serralha, F. N., & Justino, M. C. (2024). Strategies for Increasing the Throughput of Genetic Screening: Lessons Learned from the COVID-19 Pandemic within a University Community. BioTech, 13(3), 26. https://doi.org/10.3390/biotech13030026