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RT-qPCR is indeed the common detection method right now. RT (reverse transcriptase) converts RNA->DNA, PCR (polymerase chain reaction) doubles DNA by replicatin
by entee 7y ago
RT-qPCR is indeed the common detection method right now. RT (reverse transcriptase) converts RNA->DNA, PCR (polymerase chain reaction) doubles DNA by replicating it over and over in heat cool cycles. To a first approximation, you get 2^n copies of the RNA (it was converted to DNA) where n is the number of heat cool cycles. By adding a fluorescent tracer, you get the q (Quantitative), so you can see how much DNA exists at each cycle. You can then fit a curve and see how much RNA (via its DNA complement) existed in the original sample. It's not actually that slow, you can do this in a couple hours if you have the right setup. The bottleneck is you do need time for the heating and cooling cycles. You also need to extract RNA from the sample first, which could be another bottleneck. I'm not totally clear on the relative cost, a good ELISA would eventually be cheaper, but qPCR itself isn't very expensive. The benefit is that qPCR is extremely sensitive, you WILL see if there is virus in the bloodstream. You can even use swabs and avoid blood altogether. Also, the minute you have a viral genome, you can make a qPCR test, you just need the sequence. That said, if you cleared the infection you will be negative under RT-qPCR.
An ELISA works differently. In this case you would present a part of the viral protein, the patient's antibodies will bind to it, and you then use another antibody (this one is conjugated to some sort of readout method, fluorescent or biochemical) which binds to generic human antibody. Now you have a stack: viral protein <> patient antibody <> readout antibody. You read out the signal provided by your readout, usually some sort of colorimetric thing (see home pregnancy tests, also an ELISA, slightly different configuration though). This works fast, can be manufactured in bulk, BUT requires do you have a protein(s) (in this case the viral protein) that is/are universally recognized by patient antibodies. It's a little trickier to develop. You also need to produce that protein at scale which can take a little time as well. Major benefit: with a good ELISA you WILL see not only whether someone IS infected but whether someone WAS infected for some period after illness regardless of symptoms. This is essentially the only way you'll get a really good number for baseline infection rate. That said, you likely actually need a blood draw.
In short, we need both, we probably could do a lot better than we are doing with qPCR in testing volumes and we could also really use an ELISA.
Source: PhD biochemist, have personally run these assays in various forms.
EDIT: Acronym expansion, source
- maxerickson 7y agoWhat's the approximate setup and validation time once you have a sequence? (that is, what's the scale of the fuck up that the machine testing is still being brought online)
- entee 7y agoI used to set up and run a large plate of qPCR (we didn't use the RT step in our particular use case) in an hour or so. Mind you this was for something like 200 samples. It took 2-3h to run the actual machine. If I was setting up such an assay, I'd order a bunch of different primers (you need DNA to make DNA) and run a bunch of plates with as many samples as I could to see what primers worked best. You'd include a bunch of negative controls, ideally some controls from patients/cultures infected with similar but not identical virus (for example a different coronavirus). To develop an RT-qPCR assay would take a week or two (rough guess), I think it took about a week to develop the WHO assay. From what I can tell (mostly anecdotal and other data from friends I trust) the CDC assay used bad primers, they were noisy, showing a bunch of false positives. They also used a potentially dodgy fluorescence readout. I used to use the same readout, but our scenario was full of internal controls and the input sample was far more consistent. For human sample data a different fluorescent method that is more reliable should have been used. Important ways this can be screwed up: it's helpful to use the same machine, the same protocol every time. This is because qPCR is an exponential assay, which means small errors can have big repercussions. TLDR: Scale of fuckup here is massive. This really isn't that hard, and we could just have used what the WHO/others were using. I have no idea why they decided to go their own way. more info: https://www.propublica.org/article/cdc-coronavirus-covid-19-test https://www.propublica.org/article/cdc-coronavirus-covid-19-...
- gewa 7y agoMaybe one should note, that there are two possible Elisa based strategies. Yes, You can present some viral protein to detect antibodies produced by the body after serum conversion, as you said. This serum conversion takes at least a week to happen and you need a blood sample. On the other hand you can also immobilize antibodies specific to the viral proteins and directly detect the virus. This is possible immediately after infection and fluids like sputum or saliva can be used. Both strategies are under development for quick tests right now.