1. Introduction
1. Introduction
Single RT-LAMP is performed to confirm whether N gene of SARS CoV-2 is appropriate as a target gene.
2. Materials and equipment
2. Materials and equipment
Equipment
QuantStudio5(ABI device)
Sequence of Primer and Probe
| Target | Name | Sequence (5' → 3') | Length |
|---|
| SARS CoV-2 (N gene) | N F3 | TGGACCCCAAAATCAGCG | 18 |
| N B3 | GCCTTGTCCTCGAGGGAAT | 19 |
| N FIP | CCACTGCGTTCTCCATTCTGGTAAATGCACCCCGCATTACG | 41 |
| N BIP | CGCGATCAAAACAACGTCGGCCCTTGCCATGTTGAGTGAGA | 41 |
| N BLP | GGTTTACCCAATAATACTGCGTCTT | 25 |
| N FLP | TGAATCTGAGGGTCCACCAAA | 21 |
| N BLP Probe 2 | [Cy5]- GTCAGTGCAGGCTCCCGTGTTAGGACGAGGGTAGGGGTTTACCCAATAATACTGCGTCTT | 60 |
| Quencher probe 2 | CCTACCCTCGTCCTAACACGGGAGCCTGCACTGAC-[BHQ2] | 35 |
3. Procedure
3. Procedure
① manufacture of the N gene Primer Mix 100µℓ
| Reagent added | Volume |
|---|
| F3 | 4 uL |
| B3 | 4 uL |
| FIP | 3.3 uL |
| BIP | 3.3 uL |
| LF | 10 uL |
| LB | 4 uL |
| LB probe | 6 uL |
| DW | 65.4 uL |
| Total Volume | 100 uL |
② N gene RT-LAMP reagent composition
| Reagent | Volume |
|---|
| 2X Reaction buffer | 12.5 uL |
| Enzyme mix | 2.0 uL |
| Primer mix | 1.2 uL |
| Quencher2 solution | 0.06 uL |
| RNA Template | 2.5 uL |
| DW | 6.74 uL |
| Total Volume | 25 uL |
③ Dilute the template stock solution of 10 6 [copy/uL] up to 10 0 [copy/uL] by ten times each.
④ For three positive controls and one negative control (4 in total), pre-mixing is prepared as a reagent except for RNA template and then mix and spin down is performed after including one extra reagent (22.5uL x 5=112.5uL).
⑤ Put 22.5uL of pre-mixture into 4 tubes.
⑥ After adding 2.5uL COVID-19 RNA template to only three tubes, add 2.5uL of DW to one tube to complete the reagent.
⑦ Put reagent in Quant Studio (ABI device), select Cy5 dye as a reporter with a condition of 60°C and 40 minutes for RT-LAMP, and start amplification and quantitative analysis (CT value).
4. Results
4. Results
| Template concetration [copy/uL] | CT value |
|---|
| 10^6 | 20.14 |
| 10^6 | 20.46 |
10^6
| 20.52 |
| 0 (Negative Control) | Undetermined |
5. Precautions
5. Precautions
1) Pay attention to pipetting so that RNA or PCR reagents are not contaminated during the experiment.
2) Be careful not to make a mistake in the temperature or the reagent amount s during the experiment is performed.
6. References
6. References
Jang, Woong Sik et al. “Development of a multiplex Loop-Mediated Isothermal amplification (LAMP) assay for on-site diagnosis of SARS CoV-2.” PloS one vol. 16,3 e0248042. 3 Mar. 2021, doi:10.1371/journal.pone.0248042
Contents
1.Introduction
2.Materials and equipment
3. Procedure
4. Results
5. Precautions
6. References
1. Introduction
1. Introduction
Single RT-LAMP is performed to confirm whether N gene of SARS CoV-2 is appropriate as a target gene.
2. Materials and equipment
2. Materials and equipment
Equipment
QuantStudio5(ABI device)
Sequence of Primer and Probe
3. Procedure
3. Procedure
① manufacture of the N gene Primer Mix 100µℓ
② N gene RT-LAMP reagent composition
③ Dilute the template stock solution of 10 6 [copy/uL] up to 10 0 [copy/uL] by ten times each.
④ For three positive controls and one negative control (4 in total), pre-mixing is prepared as a reagent except for RNA template and then mix and spin down is performed after including one extra reagent (22.5uL x 5=112.5uL).
⑤ Put 22.5uL of pre-mixture into 4 tubes.
⑥ After adding 2.5uL COVID-19 RNA template to only three tubes, add 2.5uL of DW to one tube to complete the reagent.
⑦ Put reagent in Quant Studio (ABI device), select Cy5 dye as a reporter with a condition of 60°C and 40 minutes for RT-LAMP, and start amplification and quantitative analysis (CT value).
4. Results
4. Results
5. Precautions
5. Precautions
1) Pay attention to pipetting so that RNA or PCR reagents are not contaminated during the experiment.
2) Be careful not to make a mistake in the temperature or the reagent amount s during the experiment is performed.
6. References
6. References
Jang, Woong Sik et al. “Development of a multiplex Loop-Mediated Isothermal amplification (LAMP) assay for on-site diagnosis of SARS CoV-2.” PloS one vol. 16,3 e0248042. 3 Mar. 2021, doi:10.1371/journal.pone.0248042