8 years after asking this group for advice, I've finally done my first viral diagnostics qPCR run at home. I used the Biomeme Franklin platform to test my family for Sars-CoV-2 and I'm convinced it's the first taste of what many people will be doing before long. Here's some details written for a general audience. Please forgive my over simplifications, I know many of you here are experts in all of this.
On Saturday, April 4, 2020 at 4:04:56 PM UTC-4, Reginald Smith wrote:
-- I also participated in a COVID Q&A with Max Perleman, co-founder of Biomeme. First I was pretty shocked there wasn't anyone else chomping at the bit to ask him about how technology like his can help massively scale access to COVID testing. But more importantly I was excited by the new device he gave a sneak peak of (at time 1:03). It uses a 9-well cartridge that does integrated sample prep. So you put in a raw sample and hit run and you get up to 27 assays in a single automated run. I'll definitely be buying this with a respiratory virus panel when it becomes available. Does it still count as "DIY" if there's essentially nothing to do but swab and press run? :-)
My hope is that if there's any upside to the pandemic, it will be that it creates a consumer awareness and market for simple viral diagnostics. And if many people have a qPCR machine in their home and test kits are cheap, think of all the other science that could be done just as easily, as well as being so much better prepared for the next pandemic!
I would love to find out if the Biomeme kits can be effectively used with sample pooling techniques to effectively reduce the cost per sample by 10x or more (when infection prevalence is below 1%). In addition, recent research suggests that saliva may be even more sensitive and reliable as a sample source than nasopharyngeal swabs. I wonder if essential businesses could be doing regular practical mass screening of their employees with point-of-care devices like this by using saliva samples pooled 10x or more per test.
But I know all of this is best explored by professional researchers and public health experts. But it seems to me like there may still be a role for biohackers and small companies to contribute and safely explore a broader array of ideas which which aren't getting the attention they deserve. If saliva really is more sensitive than NP swabs, why are we learning this >3 months into the pandemic? I like the suggestion in this thread about environmental sampling, has anyone actually tried it yet? At the least I could probably be sampling my mail and groceries to see if I can find any evidence of viral RNA at all (being careful not to imply that that is infectious of course).
We're all taking risks just trying to live day-to-day, might as well get some research out of it too! Thoughts?
Thanks,
Rick
On Saturday, April 4, 2020 at 4:04:56 PM UTC-4, Reginald Smith wrote:
Thanks, I am not super experienced in the subtleties of qPCR evaluation since I just have an old school PCR and gel. I thought it would be relevant to those here talking about testing surfaces, etc. for viral RNA which surely has concentrations much lower than the threshold the paper describes. I had read the Korean paper you refer to a couple of weeks back. I remember they were most favorable towards N2 and the Japanese primer for the N gene (both of which basically overlap the same area on the N gene within a couple dozen bp I think). Also the Orf1ab primer from the Chinese CDC.Could I ask which primer sets you are using on your qPCR? One thing I find interesting is the CDC seems to be the only test just using one gene, the N gene. Not sure if there is a reason for this such as number of relatively conserved regions, etc.Reggie
On Saturday, April 4, 2020 at 6:30:14 AM UTC-4, Michael Crone wrote:I would not read too much into the paper. There was a much better Korean paper released a while ago comparing different primer sets (https://www.biorxiv.org/content/10.1101/2020.02.25. ). The "background amplification" that would cause this in N2 and N3 seems like it could just be contamination and they don't have a proper explanation as to why this would happen (it could potentially occur with primer-probe interaction too, but Occam's razor). With a primer-probe set you really shouldn't see any fluorescent signal unless you amplify your specific target, for SYBR green yes, but probes no. Additionally, spiking raw RNA onto nasopharyngeal swabs and then drawing conclusions based on that is just not very good science. We've got down to 2.5 ul copies per reaction in our qPCR. Please don't believe anything in that paper... peer review is there for a reason.964775v1.full.pdf MichaelOn Fri, 3 Apr 2020 at 13:00, Reginald Smith <rsm...@supremevinegar.com> wrote:--FYI, for those interested in this topic, a team at Yale on Wednesday released an evaluation of the SARS-CoV-2 primer sets out of the US, China, Hong Kong University, and Germany. The results are interesting though this is a MedRxiv paper that hasn't gone through peer review yet. I am sure it will review quickly due to the urgency of this issue.For those interested in the "false negative" reports in the news recently I have not read into all the details on that but the paper reports a lower detection limit of 100 SARS-CoV-2 genome equivalents per uL. Not way off from other PCR amplification requirements but if swabbing/RNA extraction is not optimal I can see where the issues start.In short, all the primers could detect the viral RNA but some of the primers were prone to being unable to properly distinguish between low levels of viral RNA (less than 100 genome equivalents per uL) and controls with no viral RNA. So these could theoretically give a false positive since results from no virus and low virus concentration are both below the detection cutoff for "positive" results (qPCR CT<40). This was an issue for the China primers and two of the CDC primers (N2 and N3, the latter which has already been taken out of the kit by the CDC a month ago due to the problems it was causing). The Hong Kong University primers performed the best and did not give false positive issues.Reggie
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1 comments:
Nice post, keep up the good work, thanks
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