6 ms·

So, how does the PCR-based testing work? Is it just the usual gene amplification steps? * Get DNA out of cells and unwrapped from their histones. * Cleave the

So, how does the PCR-based testing work? Is it just the usual gene amplification steps? * Get DNA out of cells and unwrapped from their histones. * Cleave the DNA sample with restriction enzymes at boundary sites which should mark the start/end of a stretch of viral DNA. * Amplify the sample with PCR, using primers which match stretches of viral DNA. * Run a gel electrophoresis and look for a mass of DNA that matches the expected size of the stretch of viral DNA. Is that how things work, or are these kinds of diagnostics done differently?

mbreese 7y ago
It's an RNA virus, so you start by isolating RNA and then using a reverse transcription reaction to make cDNA from the RNA. Then you use viral-specific primers in a traditional PCR. Most of the tests that I've seen presented have then be real-time qPCR tests as opposed to gels, but for the most part, you're not far off. Also, restriction digests aren't required. Note: I haven't seen any of the protocols, just what I've been able to learn from press reports. Edit: Here's a link to the CDC's panel protocol: https://www.fda.gov/media/134922/download https://www.fda.gov/media/134922/download And to add Wikipedia's page on real-time PCR (not to be confused with RT-PCR, which is also used here): https://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction https://en.wikipedia.org/wiki/Real-time_polymerase_chain_rea... . However, the crux of it is that the reaction contains a chemical that fluoresces in the presence of double-stranded DNA. As the PCR reaction progresses, if there is a valid template in the original mixture (i.e. if there is virus present), then there will be a detectable amount of DNA present after a number of PCR cycles. The lower the number of PCR cycles required to detect this fluorescence, the more viral RNA was present in the initial sample.
- leggomylibro 7y ago
  Neat, thanks - it's amazing how quickly sequencing technologies have evolved.
  - mbreese 7y ago
    This isn't sequencing... you'd use sequencing to identify novel mutations or strains of the virus. This is a set of known PCR primers that amplify the virus's RNA. You could (in theory) also use a similar approach to identify specific strains of the virus, but this shouldn't be the priority at the moment. While you could have an assay that used sequencing, it wouldn't be nearly as fast or cost efficient.
    - VectorLock 7y ago
      This CDC document was an interesting read. I found this part particularly fascinating. >RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed to cDNA and subsequently amplified in the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS version 1.4 software. In the process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by Applied Biosystems 7500 Fast Dx Real-Time PCR System with SDS version 1.4 software.
    - leggomylibro 7y ago
      Right, sorry...I sort of think of PCR as 'grep for genomes', even though it doesn't really 'read' the DNA.
  - kylebenzle 7y ago
    You mean in the last 20 years? Yeh, it was super fast.
    - pbhjpbhj 7y ago
      As someone outside the industry it _appears_ [emphasis there] to have gone from "we can take a few weeks and test you for a specific disease susceptibility" to "place your finger here and then grab a coffee ... sorry for the wait, OK we have details on 100 different diseases now" in a matter of 2 or 3 years.
projektfu 7y ago
Sort of. There are now reagents that can fluoresce as the strand is elongated allowing instantaneous reading. Also, for an RNA virus, it starts with reverse transcriptase. https://en.m.wikipedia.org/wiki/Real-time_polymerase_chain_reaction https://en.m.wikipedia.org/wiki/Real-time_polymerase_chain_r...
vikramkr 7y ago
qPCR doesn't use a gel, and you get specificity by using primers to the viral dna (which are what tell you the part you start and end amplification at), not restriction enzymes, but yes conceptually similar sort of idea. You set up reaction conditions so that at the end of the run, if there was any viral dna at the start, there's now a lot more for you to detect, while if there wasn't, you just see the background since nothing else should have been replicated. I believe the reagent issue with the original run of tests from the CDC had to do with the controls you set up to show that nothing amplifies if you don't have any virus (since you can't just run one tube and see if there's a signal, you've got to run a few for reundancy and run samples where you should and should not see signals to make sure you are seeing signals where you should, and the reaction worked, so your signal from the patient samples are accurate)

mbreese 7y ago

It's an RNA virus, so you start by isolating RNA and then using a reverse transcription reaction to make cDNA from the RNA. Then you use viral-specific primers in a traditional PCR. Most of the tests that I've seen presented have then be real-time qPCR tests as opposed to gels, but for the most part, you're not far off. Also, restriction digests aren't required. Note: I haven't seen any of the protocols, just what I've been able to learn from press reports. Edit: Here's a link to the CDC's panel protocol: https://www.fda.gov/media/134922/download https://www.fda.gov/media/134922/download And to add Wikipedia's page on real-time PCR (not to be confused with RT-PCR, which is also used here): https://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction https://en.wikipedia.org/wiki/Real-time_polymerase_chain_rea... . However, the crux of it is that the reaction contains a chemical that fluoresces in the presence of double-stranded DNA. As the PCR reaction progresses, if there is a valid template in the original mixture (i.e. if there is virus present), then there will be a detectable amount of DNA present after a number of PCR cycles. The lower the number of PCR cycles required to detect this fluorescence, the more viral RNA was present in the initial sample.

leggomylibro 7y ago
Neat, thanks - it's amazing how quickly sequencing technologies have evolved.
- mbreese 7y ago
  This isn't sequencing... you'd use sequencing to identify novel mutations or strains of the virus. This is a set of known PCR primers that amplify the virus's RNA. You could (in theory) also use a similar approach to identify specific strains of the virus, but this shouldn't be the priority at the moment. While you could have an assay that used sequencing, it wouldn't be nearly as fast or cost efficient.
  - VectorLock 7y ago
    This CDC document was an interesting read. I found this part particularly fascinating. >RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed to cDNA and subsequently amplified in the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS version 1.4 software. In the process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by Applied Biosystems 7500 Fast Dx Real-Time PCR System with SDS version 1.4 software.
  - leggomylibro 7y ago
    Right, sorry...I sort of think of PCR as 'grep for genomes', even though it doesn't really 'read' the DNA.
- kylebenzle 7y ago
  You mean in the last 20 years? Yeh, it was super fast.
  - pbhjpbhj 7y ago
    As someone outside the industry it _appears_ [emphasis there] to have gone from "we can take a few weeks and test you for a specific disease susceptibility" to "place your finger here and then grab a coffee ... sorry for the wait, OK we have details on 100 different diseases now" in a matter of 2 or 3 years.

projektfu 7y ago

Sort of. There are now reagents that can fluoresce as the strand is elongated allowing instantaneous reading. Also, for an RNA virus, it starts with reverse transcriptase. https://en.m.wikipedia.org/wiki/Real-time_polymerase_chain_reaction https://en.m.wikipedia.org/wiki/Real-time_polymerase_chain_r...

vikramkr 7y ago

qPCR doesn't use a gel, and you get specificity by using primers to the viral dna (which are what tell you the part you start and end amplification at), not restriction enzymes, but yes conceptually similar sort of idea. You set up reaction conditions so that at the end of the run, if there was any viral dna at the start, there's now a lot more for you to detect, while if there wasn't, you just see the background since nothing else should have been replicated. I believe the reagent issue with the original run of tests from the CDC had to do with the controls you set up to show that nothing amplifies if you don't have any virus (since you can't just run one tube and see if there's a signal, you've got to run a few for reundancy and run samples where you should and should not see signals to make sure you are seeing signals where you should, and the reaction worked, so your signal from the patient samples are accurate)