LAMP
A significant limitation of PCR is the need not only to control but rapidly change the temperature of the reaction. Normally one uses a PCR machine to do this. These are expensive (especially with optics) and you can't just carry one around in your backpack. People are looking for POC (point of care) tests that could be done on check-in at a local clinic, or in the home.
Twenty years ago a PCR method that works at constant temperature was invented (and patented). It's called LAMP: loop-mediated isothermal amplification of DNA.
The precise details are a bit too complicated to explain. There are diagrams in this wikipedia article.
The fundamental idea is to make copies of the target DNA that contain single-stranded loops on the ends. This allows a DNA primer for the next round of amplification to find and bind to its complementary sequence (within the loop) without the need to denature the double-stranded part of the molecule.
Another thing that's needed for this to work is a helicase activity that can open up the double-stranded DNA ahead of the polymerase in order for the new synthesis to occur, like the activity symbolized by the blue arrow in this diagram:
How that's done is proprietary, AFAIK. People have described, for example, fusion proteins that contain both activities.
These systems can make a lot of DNA. So much that the product can be detected by physical properties.
When a nucleotides is assembled into DNA, two phosphates joined together (called pyrophosphate) are released. These accumulate and can be detected as a precipitate with Mg2+. And here.
I believe this is the principle behind the Abbott POC (point of care) test for SARS-CoV-2. The problem with Abbott's test is it requires an Abbott machine, uses expensive cartridges (vendor lock-in), and only runs a single test at a time.
We are currently running in the neighborhood of a few hundred thousand tests a day. We need to be running millions to have a hope of controlling the pandemic since we have failed with the shutdown.
STOPCovid
"STOPCovid is an effort to develop point-of-care and at-home tests for COVID-19."
website
What they've done is to add an additional layer to the first stage isothermal LAMP process to detect the product. The second step uses CRISPR, technology that has been in the news a lot.
To simplify a great deal, a short DNA probe is constructed for the CRISPR reaction which has an antigen A on one end and a biotin molecule B on the other. If the probe binds a complementary sequence from step 1, then CRISPR cleaves it, separating A and B.
In the development phase, an antibody for A that is itself attached to colloidal gold is added.
With me so far?
CoV-positive: Gold-antibody-A- plus free B
CoV-negative: Gold-antibody-A-DNA-B
The solution flows up a slide by capillary action.
For a negative sample, the entire thing is captured on a strip of streptavidin low down on the slide, forming a visible line from the gold on the antibody. (Streptavidin binds B, biotin).
For a positive sample, the Gold-antibody-A- doesn't stop at the streptavidin but flows further up the slide, to be captured by ____
It's cheap, does not require a PCR reaction, and could be scaled. They are working to validate with clinical samples, but preliminary tests show 97% sensitivity and 100% specificity. They never declare a ball to be a strike (never say someone is infected when he's not), but they do sometimes call a strike a ball.
It's about as easy to use as a pregnancy test.
[Their server is currently down. I will update when it comes back online.]
They may have to change the name. It's being applied to an app for tracing, used in France.
