Keywords
PCR primers - SARS-CoV-2 detection - false positives
Introduction
Defining the relationship(s) between infectious agents and the human host is a crucial
topic in immunology, microbiology, and infectious medicine. Although it has been proposed
that genetic factors might play a role,[1]
[2] the exact mechanisms of chronic infections and occasional (re)activation of pathogens
in the human host are largely misunderstood and poorly studied. The issue became even
more relevant in light of the recent Ebola virus, Dengue virus, and SARS outbreaks
associated with high morbidity and mortality.[3]
[4]
[5] In this context, there is a need not only for knowing the molecular basis of infections
to define effective and safe preventive and therapeutic interventions but also for
sensitive and specific diagnostic tools. Indeed, accurate screening of asymptomatic,
presymptomatic, and symptomatic subjects might be key to effective epidemiological
measures during pandemics. However, especially in analyzing SARS-CoV-2 as a paradigmatic
example, contrasting data have been reported on the analytical performance of SARS-CoV-2
detection methods and claims about the rates of false negatives and false positives
have been published.[6]
[7]
[8]
[9]
[10]
[11]
On the basis of all these, this study focused on the possible genetic basis of potential
false polymerase chain reaction (PCR) results by comparing the nucleotide sequence
of proposed/used SARS-CoV-2 primers versus the human genome. The scientific rationale
is that—given the high level of amino acid sequence sharing between SARS-CoV-2 proteins
and the human proteome[12]
[13]
[14]
[15]—parallel sequence matching at the nucleotide level might exist between the SARS-CoV-2
primer sequences and the human genome, in this way possibly explaining the generation
of false-positive SARS-CoV-2 detection results. Data are reported here that confirm
the likelihood of the research hypothesis.
De facto, using the nucleotide Basic Local Alignment Search Tool (BLASTn) program from NCBI
(http://blast.ncbi.nlm.nih.gov,[16]
[17] a sample of 12 primers retrieved from literature,[18]
[19] proposed/used even by government health institutions[19] to detect SARS-CoV-2, and described here in [Table 1], was analyzed for nucleotide sequence sharing with the human genome. BLASTn analyses
documented a relevant viral versus human oligonucleotide overlap, with shared primer
sequences repeatedly present in the human genome, disseminated among different chromosomes,
and located in plus strands, minus strands, mRNAs, pseudogenes, etc. Due to space
constraints, an in extenso description of the complete nucleotide sequence sharing is practically not possible,
and only a synthetic snapshot is shown in [Table 2].
Table 1
Nucleotide sequence of primers used/proposed for PCR detection of SARS-CoV-2[a]
|
Primer no.
|
Target gene[b]
|
Primer direction
|
Primer nucleotide sequence
|
|
1
|
S 2
|
F
|
CCACTAGTCTCTAGTCAGTGTGTTAAT
|
|
2
|
S 2
|
R
|
AAACTGAGGATCTGAAAACTTTGTC
|
|
3
|
8
|
F
|
GGAGCTAGAAAATCAGCACCTTTAA
|
|
4
|
8
|
R
|
TCGATGTACTGAATGGGTGATTTAG
|
|
5
|
E
|
F
|
ACAGGTACGTTAATAGTTAATAGCGT
|
|
6
|
E
|
R
|
ATATTGCAGCAGTACGCACAGA
|
|
7
|
N
|
F
|
GACCCCAAAATCAGCGAAAT
|
|
8
|
N
|
R
|
TCTGGTTACTGCCAGTTGAATCTG
|
|
9
|
N
|
F
|
GGGGAACTTCTCCTGCTAGAAT
|
|
10
|
N
|
R
|
CAGACATTTTGCTCTCAAGCTG
|
|
11
|
N
|
R
|
TAATCAGACAAGGAACTGATTA
|
|
12
|
N
|
F
|
TGGCAGCTGTGTAGGTCAAC
|
Abbreviations: F, forward; PCR, polymerase chain reaction; R, reverse.
a Primers retrieved from Gadkar et al[18] and Qasem et al,[19] and further details and references therein.
b Gene names given according to Uniprot.[20]
Table 2
Oligonucleotide sharing between the human genome and PCR primers proposed/used to
detect SARS-CoV-2: a few examples[a]
|
|
a Twelve primers described in [Table 1] and derived from [[18]
[19]] were analyzed for sharing of nucleotide sequences with the human genome. BLASTn
[[16]
[17]] was used to find and localize regions of identity in the human nucleotide collection
covering genomic and transcript sequences; further details are available at http://blast.ncbi.nlm.nih.gov. The 12 primers are listed with shared nucleotide sequences given underlined.
In conclusion, this communication highlights the likelihood that viral versus human
nucleotide sequence overlap can interfere with nucleic acid amplification testing
and generate PCR false-positive results in SARS-CoV-2 detection, in this way affecting
medical diagnoses.
