I have no experience in conducting PCR or qPCR. I am trying to understand some basic information about qPCR testing for SARS-CoV-2, and hence was reading through a typical protocol https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf.
In the protocol it is mentioned that specificity of the primers are tested against other coronaviruses. Shouldn't the specificity also be tested against human mRNA?
From reading some material online (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-research-areas/virus-detection-using-real-time-pcr.html, https://www.qiagen.com/us/applications/human-identity-and-forensics/investigator-solutions/sample-preparation/qiaamp-viral-rna-mini-kit/), it seems that viral RNA can be specifically isolated, however I do not find a description of how. I notice that DNA can be removed from the reaction using DNase I treatment, but I am unable to find a similar explanation for mRNA.
Any help is greatly appreciated. Thanks!
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The primers were tested of course, in primer design, after having some candidate primers, you need to validate the specificity, in this case, you can blast the primers to the human genome and transcriptome. Viral RNA is commonly captured by hybridization methods, not sure on the protocols you posted.
Thanks for the response.
My question arises because the protocol doesn't indicate any tests against the human transcriptome, while it presents tests against coronaviruses. They also compare the primer targets to other coronavirues through alignment and the results are shown in the protocol paper. I couldn't find a similar comparison against the human transcriptome in the report. So if they did BLAST against the human transcriptome, they don't mention it. However, I wonder whether these are implicit assumptions when developing these tests.
They did mention, "RNA was extracted from clinical samples by using the MagNA Pure 96 system (Roche) and from cell culture supernatants by the viral RNA mini kit (Qiagen)." I am looking up what these are for. May be they perform the hybridization step you mentioned (?)
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