Re: [DIYbio] Re: DIY COVID-19 PCR test

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/10.1101/2020.02.25.964775v1.full.pdf). 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.

Michael

On 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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