Jul 10, 2023
RT-PCR amplification for the discrimination between wild type and mutation alleles in SARS-CoV-2 genomes from wastewater
- 1Centre for Ecology, Evolution and Environmental Changes (cE3c) & CHANGE - Global Change and Sustainability Institute, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal
Protocol Citation: Ana Reis, Daniela Pinto, Mónica Cunha 2023. RT-PCR amplification for the discrimination between wild type and mutation alleles in SARS-CoV-2 genomes from wastewater. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3ww1vzp/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: July 10, 2023
Last Modified: July 10, 2023
Protocol Integer ID: 84761
Keywords: mutation alleles in sar, reverse transcription of the sar, applied biosystems taqman snp genotyping assay, target sequence region in each mutation assay, isolated rna from sar, mutation assay, reverse transcription, pcr for the allelic discrimination, mutation sequence, pcr reaction, pcr amplification for the discrimination, wastewater this protocol, mutation allele, positive wastewater sample, mutation alleles in specific position, rna, wastewater sample, pcr amplification, genome, isolated rna, pcr, wastewater, known sar, reverse primer, sar, target sequence region
Funders Acknowledgements:
“Systematic Surveillance of SARS-CoV-2 and its variants in wastewaters, Portugal (Ref. 060701/2021/864489/SUB/ENV.C2)”, financed by The European Commission through the Emergency Support Instrument
Grant ID: 060701/2021/864489/SUB/ENV.C2
Abstract
This protocol describes the procedure of RT-PCR for the allelic discrimination between wild type and mutation alleles in specific positions of the SARS-CoV-2 genome. Starting with isolated RNA from SARS-CoV-2 positive wastewater samples, this workflow combines Applied Biosystems TaqMan SNP Genotyping Assays with a one-step RT-PCR reaction to detect whether there are known SARS-CoV-2 mutations present in the wastewater samples.
Sequence-specific forward and reverse primers amplify the target sequence region in each mutation assay. The reverse primer also primes reverse transcription of the SARS-CoV-2 genomic RNA sequences. Each mutation assay includes two TaqMan minor groove binder probes with nonfluorescent quenchers: one VIC dye labelled probe to detect the reference sequence and one FAM dye labelled probe to detect the mutation sequence.
Materials
- TaqPath 1-Step RT-qPCR Master Mix CG, ThermoFisher Scientific, Catalog A15299
- AcroMterix SARS-CoV-2 Control, ThermoFisher Scientific, Catalog 954519
- Mutation Assay: S.G339D.GGT.GAT, ThermoFisher Scientific, Catalog CV47VRX
- Mutation Assay: S.Q493R.CAA.CGA, ThermoFisher Scientific, Catalog CVH49P2
- Mutation Assay: S.L452R.CTG.CGG, ThermoFisher Scientific, Catalog CVAAAAD
- Mutation Assay: S.T547K.ACA.AAA, ThermoFisher Scientific, Catalog CVYMJGA
- Mutation Assay: M.D3N.GAT.AAT, ThermoFisher Scientific, Catalog CVAAAAK
- Mutation Assay: ORF7b.L11F.TTG.TTT, ThermoFisher Scientifc, Catalog CVCE3VH
Before start
Wipe bench surfaces with RNAse Away and keep working environment clean. Maintain the RNA samples on ice, and prepare the RT-PCR mixture on ice.
RT-PCR amplification
Prepare the amplification mixture as follows:
| Reagent | Concentration in the reaction | |
| TaqPath 1-Step RT-qPCR Master Mix CG (4X) | 1X | |
| Mutation assay (40X) | 2X | |
| DNAse/RNAse free water | up to 15 µL |
To the 15 μl of reaction mix, add 5 μl of wastewater RNA sample, or no-template control (DNAse/RNAse free water), or a wild-type AcroMetrix Coronavirus 2019 (COVID-19) RNA control, to complete 20 μl of reaction volume.
Thermocycling conditions
| Step | Temperature (ºC) | Time | No. Cycles | |
| Reverse transcription | 45 | 15' | ||
| Taq polymerase activation | 95 | 2' | ||
| Denaturation | 95 | 15'' | 45 | |
| Annealing | 58 | 45'' | ||
| Post-read | 60 | 30' |
Set the detection module for FAM and VIC labelled probes.
Data analysis
The allelic discrimination results can be graphed on a scatter plot contrasting reporter dye florescence (i.e., allele X versus allele Y), and analyzed with the QuantStudio Design and Analysis Software, using the genotyping analysis module with real-time data.