3 ms·

Would be interesting if they could also tell apart different variants--I'd be interested to see if any of the recent increase was Omicron-related.

reaperducer 5y ago
They can. I've seen on the news that at least two southwest cities that are currently using wastewater specifically for omicron surveillance.
gentleman11 5y ago
Omicron is not a significant spreader yet, this is still Delta. Omicron hasn’t had enough time, it’s only just arrived Eg, in the uk there’s less than 300 cases of it
- aardvark92 5y ago
  We found Omicron genomes in my county's wastewater. Give it like, two days and it'll hit the news. SOURCE: I work in a leading U.S. wastewater testing lab.
aardvark92 5y ago
We're working on it! Demultiplexing the signal is tricky though. Currently, labs have two main tools available - RT-qPCR and tiled amplicon sequencing. RT-qPCR provides cycle threshold (Ct) values which quantify the amount of virus RNA in a sample. RT-qPCR is not practical for finding and quantifying de novo variants as it relies on standard primer sets. Sequencing based approaches are less quantitative but provide more insight into the diversity of RNAs and mutations present in a sample - and thus which variants are present. https://www.gisaid.org/hcov19-variants/ https://www.gisaid.org/hcov19-variants/ has a variant dashboard based on sequencing data, including Omicron. It's not as quantitative qPCR, but still may be of interest.
- yread 5y 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?
  - 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.

reaperducer 5y ago

They can. I've seen on the news that at least two southwest cities that are currently using wastewater specifically for omicron surveillance.

gentleman11 5y ago

Omicron is not a significant spreader yet, this is still Delta. Omicron hasn’t had enough time, it’s only just arrived Eg, in the uk there’s less than 300 cases of it

aardvark92 5y ago
We found Omicron genomes in my county's wastewater. Give it like, two days and it'll hit the news. SOURCE: I work in a leading U.S. wastewater testing lab.

aardvark92 5y ago

We're working on it! Demultiplexing the signal is tricky though. Currently, labs have two main tools available - RT-qPCR and tiled amplicon sequencing. RT-qPCR provides cycle threshold (Ct) values which quantify the amount of virus RNA in a sample. RT-qPCR is not practical for finding and quantifying de novo variants as it relies on standard primer sets. Sequencing based approaches are less quantitative but provide more insight into the diversity of RNAs and mutations present in a sample - and thus which variants are present. https://www.gisaid.org/hcov19-variants/ https://www.gisaid.org/hcov19-variants/ has a variant dashboard based on sequencing data, including Omicron. It's not as quantitative qPCR, but still may be of interest.

yread 5y 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?
- 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.