DIY Covid-19 testing at work...
March 13, 2020 1:37 AM Subscribe
... would be difficult for most, but I am working in a life science lab. But as I have no experience in
this kind of diagnostics I have some questions:
1. It seems that kits to isolate viral RNA are getting scarce, and anyways, I would not want to "take away" Kits that are needed by hospitals etc. We isolate RNA from our tissue samples with Trizol and chloroform, is this sensitive enough to get adequate quantities of RNA for qPCR or PCR? Here is the kit we use.
2. Which protocol to use? I had a look at the WHO website , and there are several protocols. The one from CDC seems to be the most straightforward, but we have no positive control. Will we still be able to interpret the ct value?
3. The one from Japan is the only one that doesn't use qPCR, it uses a nested PCR. Is this considered a good test? At the end of the PCR they send the product for sequencing, was this done to validate the test, or are the bands enough to say the test is positive?
BTW, this is intended as a semi-kind-of-fun experiment amongst the 4 collegues in the lab and not intended for serious diagnostics purposes. We would also experiment with swabs from door handles, bus doors etc.
2. Which protocol to use? I had a look at the WHO website , and there are several protocols. The one from CDC seems to be the most straightforward, but we have no positive control. Will we still be able to interpret the ct value?
3. The one from Japan is the only one that doesn't use qPCR, it uses a nested PCR. Is this considered a good test? At the end of the PCR they send the product for sequencing, was this done to validate the test, or are the bands enough to say the test is positive?
BTW, this is intended as a semi-kind-of-fun experiment amongst the 4 collegues in the lab and not intended for serious diagnostics purposes. We would also experiment with swabs from door handles, bus doors etc.
Response by poster: Let's assume we can get one.
posted by SweetLiesOfBokonon at 3:39 AM on March 13, 2020
posted by SweetLiesOfBokonon at 3:39 AM on March 13, 2020
Best answer: Yeah, it would probably work. Trizol is fine (kits are easier and more scalable, and don't involve handling Trizol), although I don't know how you would extract from a swab with your particular kit. I think nested PCR is there for situations where you don't have a qPCR machine/reagents - you will get a band from the nested PCR and you can sequence to check it is the expected product, qPCR cycles are easier to interpret and set a cutoff than looking at a low-positive band to decide if it is real. Whether the nested PCR is 'good enough' is going to be relative - what are you going to conclude from a positive result? You can't make any proper diagnostic conclusions, because you are not running then in a qualified lab, but it will be good enough for research interest if you can show it works with the proper controls. But unless you can get a positive control, you can't interpret anything at all. And if you are handling viral swabs which may have SARS-CoV-2 to extract the RNA you should be doing this using the appropriate biosafety protocols and risk assessment.
posted by penguinliz at 4:23 AM on March 13, 2020 [4 favorites]
posted by penguinliz at 4:23 AM on March 13, 2020 [4 favorites]
Hopefully you've seen some of the other messages going around, but the RNA Society now has a page up to coordinate efforts and reagents. Colorado labs are dropping various supplies specifically requested by state public health officials tomorrow morning. The list of wanted items included RNA extraction kits, filter tips, nuclease free tubes, gloves, and 200 proof ethanol.
posted by deludingmyself at 11:07 PM on March 13, 2020 [2 favorites]
posted by deludingmyself at 11:07 PM on March 13, 2020 [2 favorites]
This thread is closed to new comments.
posted by emd3737 at 3:28 AM on March 13, 2020 [3 favorites]