Jan 30, 2023
Whole genome amplification of West Nile virus lineage 2
- 1National Reference Laboratory for Viral Zoonoses, National Public Health Center
Protocol Citation: Anna Nagy 2023. Whole genome amplification of West Nile virus lineage 2. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjopdlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2023
Last Modified: January 30, 2023
Protocol Integer ID: 76038
Keywords: West Nile virus, lineage 2, Whole genome amplification, one-step RT-PCR
Abstract
West Nile virus is one of the most important endemic arbovirus in Hungary. For next-generation whole genome sequencing of West Nile virus lineage 2, a one-step reverse transcription PCR assay was developed. PCR amplicons can be used for further amplicon based sequencing protocols on Illumina MiSeq platform. The genome of the West Nile virus is a single-stranded, positive-sense, capped RNA of approximately 10–11 kb in length. The whole genome amplification was carried out by twelve overlapping PCR amplicons. For ampification of the whole genome primer sets were designed by Geneious Prime (version 2021.2.2) primer design tool.
Nucleic acid extraction
Nucleic acid extraction
Use Qiagen QIAamp Viral RNA Mini Kit (cat. no. 52904 or 52906) for nucleic acid extraction. Nucleic acid extraction should be done by following the manufacturer's instructions. The total volume of the extracted viral RNA is 60 µl.
One-Step Reverse transcription (RT) PCR Setup
One-Step Reverse transcription (RT) PCR Setup
Reagent name: Invitrogen™ SuperScript™ III One-Step RT-PCR System with Platinum™TaqHigh Fidelity DNA Polymerase (cat. no. 12574030 or 12574035)
Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them to the preheated thermal cycler and immediately start the RT–PCR program. West Nile virus (WNV) whole genome can be amplified by 12 overlapping fragments for each sample. Detailed description of primer sets for the whole genome amplification of WNV lineage 2 can be found in the Word file (Primers_WNV_whole_genome_amplification) attached below.
Primers_WNV_whole_genome_amplification.docx PCR setup:
Add the following to a 0.2–mL, nuclease-free, thin-walled PCR tube on ice:
| A | B | |
| Components | Volume (µl) for 1 x rxn | |
| SuperScript™ III RT/ Platinum™ Taq High Fidelity Enzyme Mix | 0.5 | |
| 2X Reaction Mix (a buffer containing 0.4 mM of each dNTP, 2.4 mM MgSO4 ) | 12.5 | |
| Autoclaved distilled water | 5.0 | |
| Sense primer (10 μM) | 1.0 | |
| Anti-sense primer (10 μM) | 1.0 | |
| Total volume | 20.0 | |
| Template RNA (1 pg to 1 μg) | 5.0 | |
| Final volume | 25.0 |
PCR master mix components for 25.0 µl reactions.
For multiple reactions, you can prepare a master mix to minimize reagent loss and enable accurate pipetting.
Program the thermal cycler so that cDNA synthesis is followed immediately with PCR amplification automatically:
| A | B | C | D | |
| Reverse transcription | 55°C | 30 mins | 1x | |
| Activation/initial denaturation | 94°C | 2 mins | 1x | |
| Amplification | 94°C | 15 sec | 40x | |
| 59°C | 30 sec | |||
| 68°C | 4 mins 30 sec | |||
| Final extension | 68°C | 5 mins | 1x | |
| HOLD | 12°C | infinite | infinite |
Cycling conditions for whole genome amplification of West Nile virus lineage 2
Gently mix and make sure that all the components are at the bottom of the amplification tube. Centrifuge briefly if needed. Place the reaction tubes in the preheated thermal cycler programmed as described above.
Agarose gel electrophoresis
Agarose gel electrophoresis
For visualization and interpretation of the results 1% agarose gel should be used.