The primers I designed back during my Bachelor's thesis days to target a notoriously difficult GC-rich UTR region of a housekeeping gene were better than whatever abomination this supposedly authoritative body spat out.
I used Primer3Plus to search for candidates, then screened their specificities using Primer-BLAST and verified their secondary structure profiles with IDT OligoAnalyzer. Each pair took three days and I had to design three pairs.
As for the CDC, I have no idea.
I worked there for a while a few years ago. We had to use Office 2003 with some of our stuff because other programs we used that were tied to it were so outdated that it wouldn't work with any newer versions. Our sample inventory and results were sloppily managed with an Excel spreadsheet up until the point where we had like tens of thousands of samples and results and it officially became a shitshow and caused a massive study to be delayed. We had a LIMS we were supposed to use, but it wasn't set up for our lab yet (they had one person doing it for the whole department, maybe even for other departments) and I never got to actually see it used after being there for years.
After the study delay they finally let us pay a contractor to develop a database thing to get us through until a more official solution was available. The thing worked, but it was so user-unfriendly that literally only one person in our group (the one that worked with the contractor to say what we needed) was able to fully understand it and she left around the same time I did so I have no idea how that turned out.
tl;dr: They don't seem the best about using the right program for things.
I used to work in a plant pathology lab. I was the fungus person, so I grew, isolated DNA from, and ran PCR on fungi over and over and over and over and... yeah. A year and a half of my life, full of daily PCR and gels.
Now I work in a zebrafish lab and OH MY GOD I LOVE DNA ISOLATION. It's so easy. And it's so fast. And it doesn't take 3 hours of consistent work. I don't even mind the PCR anymore.
That said I DID fuck up my PCR today so uh
He found warnings given by the free primer design software that everybody has access to.
The CDC has lost any ounce of respect I had left for it. I wouldn't have gotten away with this in an undergrad project. Even I used primer3 during two internships. Anyone can do it. It's hosted online!
The calculated dG on primer3 are a little more than I would be comfortable with but technically acceptable for a singleplex test.
Regardless of that however, they really should have done a more in depth bench test. The number of matches in those hairpins and dimers should be enough to give ANYONE familiar with PCR design some pause.
This isn't a very practical solution because the more primer/probe sets you test, the lower your throughput (fewer samples can be tested per plate) and the higher the cost. It's much better to take measures to eliminate contaminations to begin with.
Another thing that can happen is that the kit is manufactured correctly, but because the lab keeps running the same tests over and over (with positive controls), the amplicons end up in the air of the room. If anti-contamination measures aren't adequate (e.g. reactions are always mixed up in a different room from the RT-PCR machine), over time you start getting false positives no matter how many different regions you test for.
I know. It’s ridiculous. I learned that literally when I was a first quarter rotation student in grad school which was literally my first time in a biology lab. It was part of my PI teaching me “how to clone”.
Edit: it’s honestly such a dumb mistake I would never have thought to double check it.
I worked there for a few years. Before I started, I imagined it as this perfect place where the best and brightest worked (and that I had just somehow gotten in due to a fluke) but it was more or less as competent as any other lab I've worked in, which was slightly terrifying.
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