Question: (Closed) No concern of human mRNA contamination in viral qPCR tests?
0
gravatar for oddjobs
6 months ago by
oddjobs10
oddjobs10 wrote:

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!

ADD COMMENTlink written 6 months ago by oddjobs10
1

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.

ADD REPLYlink written 6 months ago by JC12k

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 (?)

ADD REPLYlink modified 6 months ago • written 6 months ago by oddjobs10

Hello anandatanjali!

We believe that this post does not fit the main topic of this site.

Sorry, but this is not a bioinformatics issue. Pure wetlab.

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.

Cheers!

ADD REPLYlink written 6 months ago by ATpoint41k
Please log in to add an answer.
The thread is closed. No new answers may be added.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 2010 users visited in the last hour