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by yread 1650 days ago
Couldn't you run RT-qPCR twice with two different sets of primers? Does RT-qPCR give you the spike gene target failure rate?
1 comments

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-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.