I noticed that the RNAse P design for Covid-19 kit is same as H1N1, Influenza A (other RNA virus inputs from the CDC’s past docs).
I also noted that the assay only targets exon 1 of this human gene, so it would not allow one to know that whether amplification was from RNA or gDNA if the isolation of nucleic acids was not entirely RNA. So why is there not clear directions in the covid-19 IFU (or prior IFU’s for H1N1 etc.) to ensure that gDNA is not part of the viral/human RNA isolation kit/protocol?
In fact, some of the kits the CDC recommend are total nucleic acid offerings (i.e. Roche) so how does one ensure that you would avoid a false negative that the RNA from the sample was not efficiently isolated and the RNAse P signal is coming from gDNA? I know the CDC suggest to spike in RNA for extraction and that this positive control needs to pass, but many clients of mine are confused by the total nucleic acid isolation approach versus ensuring one only has the viral/human RNA isolated. Why isn’t this clearly communicated in the IFU from the CDC? This would certainly pose issue if one wanted to try to quantitate covid-19 results using RNAse P as a Cq reference gene no?
Mr. Mustafa
I appreciate your post.
Per my nucleic acid extraction concerns, the kit your doc states to use is one of the two Qiagen kits recommended by CDC. What I am getting at is why doesn't the CDC's IFU clearly state to have either an up front DNA removal step or emphasize DNAse treatment or something of the sort? I know they state up and down its for mRNA of RNAse P, but my concern is for clients that use kits that are total nucleic acid or do not specifically remove human genomic DNA, that RNAse P signal will be present from DNA in addition to the RNA. Make sense?
Niko
Dr Niko Tsatsos your concern is very true. In fac most of the kits are providing cloned DNA as positive controls, which can only verify if the PCR worked. However, there are very less kits which include an RNA control to verify the RT step. I also think that an internal control preferably a human transcript target should be included to work as internal control, RT & PCR control as well as for normalizing the virus quantification.
SD
I think you are concerned that primers for RNAseP will amplify both from cDNA and from DNA. I agree with you. And I don’t know why don’t use primers only form human cDNA that not amplifie DNA. Will be a better control. But I think if there is no RNA in the extraccion the Ct will be much later.
I had the same issue in another FDA EUA kit where there was a problem in amplification of positive cases while the internal control is still giving me a good Ct value. Fortunately I was validating with another kit and I know that they were positive and when I reviewed the kit I find this defect (RNAse P as int control). If the reverse transcriptase for any reason deteriorated false negative results may be not detected.
The problem that the positive control of the kit is carried on plasmid , so still keeping positive also with a less than 20 ct
Yes, the RNAse P CDC internal control is horribly flawed, see this for details and a corrected version of the RNAse P internal control that ignores gDNA contamination:
https://www.biorxiv.org/content/10.1101/2020.05.13.094839v2.full.pdf
Best thing to do is include an inactivated viral control as a positive extract control since RT failure would be detectable with this, but not DNA-based positive extraction controls.