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Study finds minimal impact of SARS-CoV-2 genome variability on current diagnostic molecular systems

*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

The authors of a new study under review at the journal Scientific Reports and currently posted to the Research Square preprint* server proposed a novel analysis pipeline to help identify mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-polymerase chain reaction (RT-PCR) primer regions.

To this end, they comprehensively evaluated the genetic variability in 53 publically available SARS-CoV-2 diagnostic assays using 16 different types of primer sets in their probe-binding regions.

Study: Identification of mutations in SARS-CoV-2 PCR primer regions. Image Credit: Small365 / Shutterstock


As the number of mutations in the SARS-CoV-2 genome continues to rise due to its continuous evolution in the past two years of the coronavirus disease 2019 (COVID-19) pandemic, the diagnostic accuracy of the RT-PCR test, considered a gold standard diagnostic test for SARS-CoV-2, has declined considerably. Nevertheless, it remains the primary diagnostic tool for identifying SARS-CoV-2-infected people using naso- or oro-pharyngeal swab samples.

As different variants emerge with multiple genetic mutations, it might reduce the effectiveness of a specific RT-PCR primer set targeted to detect them in the SARS-CoV-2 viral populations spread across different geographical regions furthermore. Also, it might result in false-negative results during RT-PCR detection. Thus, it is crucial to continuously monitor the genetic variations (or mutations) in RT-PCR primer/probe regions using genomic sequences from SARS-CoV-2 isolates collected worldwide.

Developers typically use an assortment of primer systems in a single RT-PCR test, called a primer set, to reliably detect the genome of a virus (here SARS-CoV-2) in a test sample. This set is a collection of the forward and reverse primers (and the probe) designed to amplify and detect a single target region (TR) in the viral genomic, though it might also have two or three TRs in the virus genome.

The total number of mutations overlapping the TR, their genomic location within the TR, and their type (point mutation, insertion/deletion, transversion, or transition) are the main determinants of the efficacy of RT-PCR tests.

About the study

In the present study, researchers analyzed 16 SARS-CoV-2 RT-PCR primer sets to detect genomic variations compared to the original SARS-CoV-2 reference genomic sequence of Wuhan-Hu1 and find overlaps in the TRs of all 16 RT-PCR primer sets.

They retrieved 987,138 samples from the CoVEO database collected between 1 January and 31 December 2021. This database compiles data of good-quality genomic samples of highly-reliable variants, which helped the researchers distinguish between harmless/neutral and possibly damaging mutations. In addition, the CoVEO database provided a fast and direct mutation retrieval compared to databases containing only the consensus sequences of the samples.

The raw sequencing data of the samples also allowed for the direct filtering of genomic positions based on alternate allele frequency and sequencing depth. The researchers applied the same post-processing workflow on the Global Initiative on Sharing All Influenza Data (GISAID) database to check their results on another dataset. In this way, the researchers compiled a comprehensive, raw list of mutations overlapping PCR primer TRs in the investigated samples, which could be further filtered when exploring the effects of yet-to-emerge mutations on RT-PCR performance or designing new PCR primer sets.

Researchers find viral genomic samples prone to misclassification at a daily rate of nearly 2%; however, the daily ratio of these samples with susceptibility to misclassification within a given primer set could reach as high as 90%. So, the team calculated the ratio of samples with one, two, three, or more genomic variations in the TRs of a given primer system.

It helped them differentiate between mutations likely to damage the RT-PCR efficiency and ones anticipated to be harmless (or neutral) for a given RT-PCR primer set based on the effect of mutations. Likewise, the researchers investigated whether there were samples with damaged TRs in multiple primer systems of specific primer sets.

To monitor whether samples with high-risk mutations in the TRs of the different primer sets were becoming more frequent, they plotted the proportion of samples having a slight chance of misclassification and being susceptible to misclassification. In addition, examining the type and location of the detected variants in the TRs of different primer systems helped the researchers categorize them as high or moderate-risk mutations. Finally, the researchers analyzed the possibility of predicting specific primer sets becoming redundant due to emerging mutations in SARS-CoV-2.


The final good-quality SARS-CoV-2 genomic sample set comprised 665,325 samples, most of which belonged to Alpha or Delta variants of concern (VOCs). In the investigated SARS-CoV-2 samples, the researchers found 1,826 genetic variations in 2,188 genomic positions, overlapping 141 primer binding sites (or TRs).

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Number of mutations and their possible effect on PCR amplification. a. The ratio and number of samples with one (green bars), two (yellow bars) and three or more (red bars) variants in the TRs of different primer systems. b. The ratio and number of samples with variants in the TRs of different primer systems. Samples that contain a variant in at least one „high risk” position in the TRs of the given primer system are marked with red, other samples having only „moderate risk” mutations in the given TRs are presented in blue. For further details on mutation classification, see Methods. Primer system names are based on the nomenclature: [first author last name]-[target gene name]-[id, when multiple primer systems target the same gene]. Samples with no variants in the given TRs are not shown.

On average, test samples had 1.16 mutations in the TRs of the investigated primer systems. However, most samples had only a single variant position over the TRs. Fewer samples in every primer system had two or more variations in the TRs, but they accounted for less than 0.5% of all samples. Among point mutations, 1,677 transition mutations and 1,510 transversions affected TRs, while a much lower number of deletions and insertions (79 vs. 23) affected TRs.  Intriguingly, different primer sets targeted the same genomic sections in many cases. 

Strikingly, the frequency of samples prone to misclassification changed during the study period with the advent of SARS-CoV-2 VoCs with highly diverse mutational profiles. Thus, there is a need for continuously reevaluating RT-PCR efficacy and updating the primer sets used in clinical and commercial settings, as required.

Remarkably, most of the samples with any number of mutations in the TRs of any given primer system mostly contained variants with no drastic effect on RT-PCR efficiency based on their location. Also, in most cases, very few TRs of a primer set were damaged simultaneously in all test samples.

Further, the study results showed that most samples with any variation in the TRs of a probe binding region generally had a single point mutation, which, in most cases, was unlikely to influence the sensitivity of the RT-PCR process. However, the researchers found the most frequent single nucleotide polymorphisms (SNPs) overlapping any TRs in over 50% of the samples, mainly from the Delta VOC. Also, this SNP was found to be high risk in two forward primers.

During the predominance of the Alpha VOC, deletion mutation His69_Val70del overlapped the Young-S forward primer TR, detected in a relatively high number of samples. The Omicron strains, BA.1 and BA.3, also had this deletion mutation, which might have reduced the RT-PCR efficiency of two primer sets belonging to the TaqPath kit and Young-set. Although genomic variations in the TRs of the 16 investigated primer sets were primarily low, and the ratio of affected samples remained under 1%, an easy-to-deploy pipeline for surveying mutation frequency in the TRs is much-needed.

Given that primer sets perform differently across variant groups, it is important to continuously surveil the ratio of samples prone to misclassification to determine whether the given primer set is suitable for the detection of SARS-CoV-2 samples of the presently spreading lineage.


The current study highlighted the need for continuous surveillance of mutations in the SARS-CoV-2 genome that could reduce or hamper the efficacy of primer sets used in the current RT-PCR diagnostic assays. Although the researchers observed that TRs of many of the investigated primers were prone to mutations in the analyzed samples, further investigations are warranted to determine if these variations had the potential to reduce PCR sensitivity in a clinical setting.

A single RT-PCR assay uses multiple primer systems to simultaneously target more than one genomic region in a viral genome. However, within a primer set, more than one primer system could have a damaged TR in the sequenced genome of all investigated samples. Thus, a single RT-PCR assay having a damaged TR in more than 50% of the employed primer systems could yield a positive test result.

To summarize, based on previous evidence and the bioinformatics data of this study, the known genetic variations in the SARS-CoV-2 population had minimal or no impact on the sensitivity of RT-PCR diagnostic assays. Also, since most of the observed variants were neutral, they could not potentially disrupt the RT-PCR process. However, the researchers noted three exceptions: one mutation spanning nucleotides G28881A, G28882A, and G28883C, one SNP in a primer TR near G15451A’s 3’ end, and one deletion in the ATACATG21764A sequence, all of which occurred at a high rate in the test samples.

Nevertheless, the study results confirmed that the RT-PCR test based on any of the 16 primer sets investigated in this study could most reliably detect the SARS-CoV-2 Alpha and Delta VOCs.

*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Preliminary scientific report. Anikó Mentes, Krisztián Papp, Dávid Visontai et al. Identification of mutations in SARS-CoV-2 PCR primer regions, 22 July 2022, PREPRINT (version 1) available at Research Square, DOI:,

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Tags: Allele, Assay, Bioinformatics, Coronavirus, Coronavirus Disease COVID-19, covid-19, Diagnostic, Efficacy, Evolution, Frequency, Gene, Genetic, Genome, Genomic, Influenza, Mutation, Nucleotide, Nucleotides, Omicron, Pandemic, Point mutation, Polymerase, Polymerase Chain Reaction, Research, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Single Nucleotide Polymorphisms, Syndrome, Transcription, Virus

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Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.

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