RT-PCR amplification for the discrimination between wild type and mutation alleles in SARS-CoV-2 genomes from wastewater v1

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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.

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