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You need a quantitative real-time PCR machine, not just a PCR machine. Not cheap. EDIT: You can't just put this on a gel. Something that comes up at 35 cycle
by gravelc 7y ago
You need a quantitative real-time PCR machine, not just a PCR machine. Not cheap.
EDIT: You can't just put this on a gel. Something that comes up at 35 cycles (likely negative - needs re-running) looks exactly the same as something that comes up at 15 cycles (likely positive). The quantitative part is important.
2nd EDIT: it uses dual-labelled probe - end point PCR is absolutely not good enough.
- copperx 7y agoWhy is that? Is it really impossible to do the test on a simple PCR machine, or is it just too slow?
- gravelc 7y agopPCR is quantitative - measures fluorescence. End point PCR just measures whether you have the sequence there or not. It's slower, more work, and prone to false positives (see edit above).
- mjfl 7y agoOr you could just run the PCR product on a gel for much cheaper... Edit: A gel gives you the ability to select for size as well, which a qpcr machine does not. Not sure why I have to reply in the form of edits.
- jevinskie 7y agoI saw a whole kit including laptop, software, and license for $3,500 buy-it-now on eBay last week.
- hcknwscommenter 7y agoTotally wrong. Take a sample out in 15 cycles. Take a sample in 30. Run a gel. Compare. Done. Most of the issues you think exist are because an RT pcr machine cannot distinguish between primer-dimer and pcr product. A gel can distinguish.
- gravelc 7y agoThis argument is pointless as the assay relies on a dual-labelled probe. Look up how that works [1]. Anyway, an end-point PCR is not quantitative. You could do semi-quantitative on a gel, but it's a rubbish technique that was rightfully forgotten years ago, as it requires a lot of optimisation and has very little reproducibility. [1] https://www.sigmaaldrich.com/technical-documents/articles/biology/dual-labeled-probes.html https://www.sigmaaldrich.com/technical-documents/articles/bi...
- test7777 7y agoyou really think anyone cares how much of the corona you got in the particular blob of snot you gave for testing?
- hcknwscommenter 7y agoYou deserve a 1000 upvotes for your succinct and accurate comment.
- hcknwscommenter 7y agoYou are totally wrong. Your comment is dangerous misinformation. NO coronavirus assay requires quantitation.
- hcknwscommenter 7y agoYou seem to miss the point entirely. The point is that the assay doesn't NEED or REQUIRE dual labelled probes at all. The only need for quantification at all is to avoid mistaking signal for spurious noise (e.g., primer dimer). The gel provides both semi-quantification and provides amplicon size, which massively increases specificity of an end-point version of the assay. It is perfectly adequate. The larger point is that there are many ways the test could have been designed around any possible combination of supply roadblocks (no DNA extraction reagent, choose phenol/chloroform; no cotton swabs, use paper; I could go on and on), so there are no excuses as to the massive and unwarranted and fatal delay in rolling out comprehensive testing in the U.S. Sure some work arounds are less desirable, but the alternative we have chosen is to fail a number (~>5,000) of people who didn't have to die by letting this thing spread without adequate testing.
- test7777 7y agoon a gel you see the size, something coming up with exactly the right size even at 35 cycles indicates that there was at least some of what you designed the primers on in the sample. if you want higher certainty, with three primers you can get two different sizes ruling out any doubt. with the rtpcr machine readout which only gives you the signal corresponding to how much was amplified over time you need tricks like the dual labelling.