False positive RT-PCR in Viral Detection due to Human genome?
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7 weeks ago
Naja • 0

Hello, I am Critical Care Physician.

I have done a background research (https://www.eurosurveillance.org/con...0.25.3.2000045) on how first RT-PCR test for Covid19 virus was developed and how genetic sequences of various coronaviruses were compared to target a specific gene unique to Covid19 virus in developing the RT-PCR assay that would be specific for Covid19 virus.

My question is- suppose there is a hypothetical virus (DNA/RNA). While developing an RT-PCR test for this Virus, Is the genome of the Virus compared with Human Genome? Especially RT-PCR assays target a 'specific nucleotide sequence' which is unique to the Virus (and not to the related Viruses from the same family)

However what if this 'specific nucleotide sequence' matches with the Human DNA sequence? ( If it is a RNA virus, RT-PCR assay used to detect this virus could technically detect Human RNA- synthesized by the corresponding human DNA) & vice versa for DNA Virus.

I know that theoretically speaking, the genes targeted in the RT -PCR assay should not match with the Human Genome, but practically speaking, there could be a possibility that the nucleotide sequences that RT-PCR assays target could match with the Human genome.

What are the chances of RT-PCR assay showing false positive due to Human DNA/RNA and are specific analysis done to compare Whole human Genome while developing a RT-PCR assay?

Any help in answering the question will be much appreciated!

False-Positive RT-PCR • 399 views
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7 weeks ago
Michael 54k

I know that theoretically speaking, the genes targeted in the RT -PCR assay should not match with the Human Genome, but practically speaking, there could be a possibility that the nucleotide sequences that RT-PCR assays target could match with the Human genome.

That's a theoretical question indeed but it is common practice to check for the presence of alternative matches of the primer sequences used. It is very easy to do and you could easily do it via a BlastN search.

Here is a review about that: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221722/

For you, this page by the CDC might also be useful: https://www.cdc.gov/coronavirus/2019-ncov/hcp/testing-overview.html

Also, it is not one gene but multiple including several controls that are targeted:

According to Centers for Disease Control and Prevention (CDC) and WHO guidelines, the RNA samples are reverse-transcribed into cDNA using different primers specific for the open reading frame 1ab (ORF1ab), ORF8, RNA-dependent RNA polymerase (RdRp), hemagglutinin-esterase (HE), and the nucleocapsid genes N1, N2, envelope genes (E), spike genes (S), and transmembrane gene (M), while human RNase P is used as control (Table S1). Some other controls in use for each reaction are no template control, 2019-nCoV positive control, and human specimen control (CDC 2020) [119,120,121]. Additionally, ORF1ab and RdRp are included in RT-qPCR reactions to rule out any potential cross-reactivity, which may occur with other coronaviruses, and to avoid chances of genetic drift in the SARS-CoV-2 genome [122].

Further, it is not sufficient to find a similar sequence in the genome, but to be detected and lead to false positives, it also needs to be in a transcribed exon of a gene.

You can also be sure that these tests have been carefully validated and a 100% false-positive rate (which your assumption implies) would have been detected very early.

So in theory, yes it is worth checking for crossreactivity (mostly to related virus) when developing a new test indeed following the process described in the review as a model. In practice the scenario (crossreactivity with human) you describe isn't possible in the COVID19 case with the way the current test protocol is implemented.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10267656/#:~:text=The%20RNase%20P%20gene%20is,is%20easily%20detectable%20%5B4%5D.

So human RNA p gene is used as internal control that distinguishes human genome from viral genome.

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In my understanding, this transcript is targeted as a positive control to see if the sampling, reverse transcription and amplification worked at all. So, it's not really for distinguishing viral from human RNA. It is needed as a ubiquitous positive control similar to the positive control dye of the antigen tests. In case there is no signal for it, the test needs to be repeated.

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Thanks for the info

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