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Yes! Looking for gene target failure rates is a valid, albeit indirect way of quantifying variants. You will only be able to detect variants this way if the var
by aardvark92 5y ago
Yes! Looking for gene target failure rates is a valid, albeit indirect way of quantifying variants. You will only be able to detect variants this way if the variant contains a mutation within the primer binding region and if that mutation happens to affect primer binding causing signal drop-off (a lot of 'ifs'!).
Some labs quantify amount of variant by running multiple RT-qPCRs using primers specific to mutations unique to each variant (see https://www.promega.com/products/pcr/qpcr-and-rt-qpcr/sars-cov-2-rt-qpcr-kit-for-wastewater/?catNum=CS3174B02 https://www.promega.com/products/pcr/qpcr-and-rt-qpcr/sars-c...).
Both of these strategies leave much to be desired. For one, it's terribly costly to run multiple RT-qPCRs in parallel. It also fails to account for any novel variants whose mutations lie outside your primer binding region(s).
From what I've gathered, RT-qPCR is useful for quantifying what is already known while sequencing helps you discover what is unknown.
I guess if you had unlimited time and money you could order new primer/probe sets from IDT every time a new variant comes into play...kind of like a home-brew microarray? Honestly, I'm kind of surprised there aren't SARS-CoV-2 variant microarrays on the market yet...we're all just spitballing here.