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Thanks for the question! The goal of skipping RNA extraction is to decrease the amount of labor necessary for processing samples and also to eliminate a depende
by dvdt 6y ago
Thanks for the question! The goal of skipping RNA extraction is to decrease the amount of labor necessary for processing samples and also to eliminate a dependency on RNA extraction reagents that have recently become difficult to find. The FDA is very strict about the specific brand and model of kit you use, so showing that you can swap out one RNA kit for another is actually very useful because you will have alleviated some of the supply chain strain (although I agree at high enough load both supply chains will then become limiting).
The way currently available COVID-19 testing works is by detection of viral RNA. Since the amount of viral RNA in a patient sample is too low to detect directly, we first need to amplify it by PCR. However, this viral RNA is packaged within all sorts of proteins and lipids that could make it inaccessible to amplification unless they are first purified away. Furthermore, the sample is shipped in "viral transport medium", which is essentially a cocktail of chemicals designed to preserve the virus. Unfortunately, these preservatives often have the side effect of interfering with PCR amplification, so these too need to be purified from the sample.
However, since RNA extraction is usually the most laborious part of the assay, there has been a lot of interest in optimizing the amplification so that it is resilient to all of these impurities. The preprint referenced in our manuscript (https://www.biorxiv.org/content/10.1101/2020.03.20.001008v1 https://www.biorxiv.org/content/10.1101/2020.03.20.001008v1) gave us the initial idea that this could be possible, and much of it comes down to the choice of amplification method (e.g. choice of enzymes and buffers) that you choose.
However, even when you choose a "good" enzyme and buffer, you will still suffer an amplification penalty, and this will cause you to return a false-negative on some affected samples because there was so little virus in the sample to begin with. The innovation we have is to spike-in a correspondingly low level of DNA to the reaction mixture. That way, if you see the low level of DNA without seeing any viral signal, you can be assured that the amplification still worked and that there truly is no virus in the sample.
- abhisuri97 6y agoThank you so much for that explanation!
- treis 6y ago>the amount of viral RNA in a patient sample is too low to detect directly, How many tests have you run on patient samples?
- mattmanser 6y agoIn the UK they're saying there's a shortage of swabs and pipettes even, do you not need these too? Also, in the UK our independent and uni labs have been saying for almost a week they could extract the RNA differently but the NHS have a fixed approved way that they won't change. - Are the chemicals you're using more common or would there just be a new shortage of different chemicals? - Is there a risk you'd be creating a test that didn't work very well, and the US would end up with a bunch of useless tests (e.g. Italy had to abandon a bunch of Chinese sourced tests, UK's anti-body tests are ineffective)?
- dvdt 6y agoOur technique would still be affected by shortages in specimen collection (like swabs). Purely speculative, but I think if swabs remain an issue for too long, alternatives could start coming online, such as even using qtips + saline (no idea if it works, it's just an example). The current swab + Universal / Viral Transport Medium combo is optimized for flexibility; it is designed to work across a very broad range of viruses and bacteria that have different viral loads and shedding characteristics. The current pandemic is pretty much COVID-19 only, so I think it's a priori feasible that a specimen collection procedure can be found that uses common materials. We did try early on to see if saline or other buffers affected the performance of the assay, and it worked fine in those conditions. We use fairly standard chemicals. I haven't heard from our suppliers about shortages for the chemicals we use. Chemicals and enzymes tend to be relatively fast to scale up for bulk manufacturing. There's always manufacturing risk that a product will not work as expected. In fact, the first COVID-19 test developed by the CDC did not work as expected, and this delayed testing by several weeks. We de-risk this as much as possible by performing experiments as early as possible, akin to the fail fast mentality of checking for the highest risk failure modes first. Since we don't have a national healthcare system in the US, the manufacturer takes on the vast majority of the risk of a defective product.
- abfan1127 6y agoif the virus is known to live on cardboard or plastic for 48-72 hours, is the viral transport medium even necessary, assuming rapid shipping and processing?
- amirhirsch 6y agoThe spike-in DNA injection is clever. How did you develop that technology? It seems like the kind of thing that just hits you one day.
- Blahah 6y agoIt's standard practise in lots of kinds of sequencing experiments to use a spike-in. Makes perfect sense to use it here - in fact all the other sequencing based SARS-CoV-2 tests I've seen also use spike-ins.
- t_serpico 6y agohttps://en.wikipedia.org/wiki/DNA_spiking https://en.wikipedia.org/wiki/DNA_spiking -- this seems like an old idea, no?
- deleted 6y ago[deleted]