SARS-CoV-2 variant identification by RT-qPCR assays focusing on mutations

In a latest examine posted to the medRxiv* preprint server, researchers investigated the efficacy of diagnostic checks for figuring out extreme acute respiratory syndrome coronavirus (SARS-CoV-2) variants of concern (VOCs).

The coronavirus illness 2019 (COVID-19) pandemic has induced an unprecedented well being emergency for the previous two years leading to greater than 447 million infections and over 6 million deaths so far. SARS-CoV-2, the etiologic agent of COVID-19, has mutated all through the pandemic, with new and extra infectious variants continuously rising. In consequence, the World Well being Group (WHO) has categorized these variants into variants of concern (VOC) or curiosity (VOI), or below monitoring (VUM).

Study: Identifying SARS-CoV-2 Variants of Concern through saliva-based RT-qPCR by targeting recurrent mutation sites. ​​​​​​​Image Credit: NIAIDExamine: Figuring out SARS-CoV-2 Variants of Concern by means of saliva-based RT-qPCR by focusing on recurrent mutation websites. ​​​​​​​Picture Credit score: NIAID

The mutation fee of SARS-CoV-2 is estimated to be 1 x 10-3 substitutions per base per 12 months below impartial genetic drift situations. Though most substitutions are insignificant, some confer selective benefits for (enhanced) transmission and immune evasion. Complete-genome sequencing (WGS) can differentiate SARS-CoV-2 variants with distinctive decision. Though WGS is taken into account the gold customary for variant differentiation, it isn’t possible for real-time software of large-scale surveillance as it’s costly and time-consuming. These limitations necessitate an economical methodology for the surveillance of SARS-CoV-2 variants at a inhabitants stage.

Reverse transcription-quantitative polymerase chain response (RT-qPCR) has been carried out for public surveillance of SARS-CoV-2 variants such because the Alpha, Beta, Gamma, and Delta VOCs. Because the Alpha VOC was detected based mostly on S-gene goal failure (SGTF), researchers exploited goal gene failure for detecting single nucleotide polymorphisms (SNPs) or deletions amongst VOCs. Curiously, utilizing aggressive probes for mutation and reference sequences in RT-qPCR assay will increase its specificity.

The examine

The current examine evaluated TaqPath (spike SNP) assays for saliva samples for the K417T, E484Q, E484K, and L452R mutations. RT-qPCR assays for ORF1aΔ3675-3677 and SΔ69-70 have been developed in-house. As the most recent Omicron variant carries the SΔ69-70 and L452R mutations, these assays have been used to establish Omicron’s emergence in December 2021. The cross-validation of the assays was carried out utilizing WGS outcomes.

Artificial ribonucleic acids (RNAs) of six SARS-CoV-2 strains (B.1, B.1.1.7, B.1.351, P.1, B.1.617.1, and B.1.617.2) have been serially diluted 10-fold for estimating analytical sensitivity of the RT-qPCR assays. Equally, analytical specificity was assessed by finishing up SΔ69-70 and ORF1aΔ3675-3677 deletion assays and spike SNP assays for K417T, E484K, E484Q, and L452R on the artificial RNAs from six strains.

Application and interpretation of differential VOC assays. 3A. VOC strain typing by mutation site. Each strain will produce a different combination of results from the six assays. Strains with an alternate allele at the mutation site will produce inconclusive results. 3B. Example strain typing workflow using minimal steps. Saliva samples that are determined positive by routine diagnostic testing are analyzed by various assays that produce differential results for each VOC.

Utility and interpretation of differential VOC assays. 3A. VOC pressure typing by mutation website. Every pressure will produce a distinct mixture of outcomes from the six assays. Strains with an alternate allele on the mutation website will produce inconclusive outcomes. 3B. Instance pressure typing workflow utilizing minimal steps. Saliva samples which are decided constructive by routine diagnostic testing are analyzed by numerous assays that produce differential outcomes for every VOC.


The authors estimated the effectivity of mutation probes to be within the vary of 89.5 – 112.04%. The detection restrict (LoD) for the SΔ69-70 deletion assay was 40 genome copies per assay, whereas it was 4 genome copies per assay for ORF1aΔ3675-3677, E484K, K417T, L452R, and E484Q, together with the management gene (nucleocapsid [N] gene).

No amplification failure or cross-reactivity was detected for any deletion assay. The fluorescent output diverse with a low output for the ORF1aΔ3675-3677 assay. Though SNP assays confirmed no cross-reactivity, amplification failure, as anticipated, for strains with out each mutation and reference sequences. These outcomes indicated the excessive specificity of the SNP assays to artificial RNAs.

The deletion and spike SNP assay outcomes have been in contrast with WGS outcomes to establish their scientific sensitivity and specificity. Whole accuracy, particular person probe accuracy, and constructive (PPV) and unfavorable predictive values (NPV) have been additionally estimated for the deletion and spike SNP assays.

SΔ69-70 assay yielded a complete accuracy of 93.6%, whereas it was 68% for the ORF1aΔ3675-3677 assay. Scientific sensitivities have been 94.82% (SΔ69-70 assay) and 95.65% (ORF1aΔ3675-3677 assay), and the scientific specificity and PPV have been 100% for each the deletion assays. Whole accuracy was larger than 96.4% for all spike SNP assays; scientific sensitivity and NPV have been 100% for all assays besides L452R. Additionally, scientific specificity ranged between 95.05 – 99.44% for SNP assays.

Moreover, 162 SARS-CoV-2-positive samples have been collected between December 7 – 16, 2021. Of that, 13 samples have been recognized with the L452R assay for reference sequence at this locus. Subsequent, the SΔ69-70 assay carried out on these 13 specimens recognized deletions in 11 of them. WGS confirmed that these 13 sequences have been of the Omicron variant. Equally, 183 further samples have been screened between December 17 – 22, 2021, and the authors discovered 107 potential Omicron-positive samples.


The present examine evaluated the efficacy of RT-qPCR assays for six mutation websites of SARS-CoV-2 variants on saliva samples. The authors designed two deletion assays for SΔ69- 70 and ORF1aΔ3675-3677 websites and validated 4 TaqPath Spike SNP assays.

The SNP assays produced correct outcomes even in samples with low viral load suggesting that saliva samples don’t confound the assays’ constancy. Nevertheless, the present technique is inadequate for complete pressure typing, and rising the variety of mutation websites may maximize pressure identification. To this finish, the authors are engaged on validating SNP assays for the N501Y, G339D, and K417N mutations.

*Essential discover

medRxiv publishes preliminary scientific experiences that aren’t peer-reviewed and, subsequently, shouldn’t be considered conclusive, information scientific apply/health-related conduct, or handled as established data.

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