RT-PCR amplification for the discrimination between wild type and mutation alleles in SARS-CoV-2 genomes from wastewater
Ana C Reis, Daniela Pinto, Mónica V. Cunha
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.
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.
Steps
RT-PCR amplification
Prepare the amplification mixture as follows:
| A | B |
|---|---|
| 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
| A | B | C | D |
|---|---|---|---|
| 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.
