Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations

Spieß, Katja; Gunalan, Vithiagaran; Marving, Ellinor; Nielsen, Sofie Holdflod; Jørgensen, Michelle Grace Pinto; Fomsgaard, Anders; Nielsen, Line; Alfaro-Núñez, Alonzo; Karst, Søren Michael; Mortensen, Shila; Rasmussen, Morten; Lassaunière, Ria; Rosenstierne, Maiken Worsøe; Polacek, Charlotta; Fonager, Jannik; Cohen, Arieh; Nielsen, Claus Henrik

doi:10.1128/spectrum.03591-22

Cited by 8 publications

(8 citation statements)

References 32 publications

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“…In our study, we made a head-to-head comparison of the RT-PCR variants assays with WGS. Previous studies [ 15 ; 19 ] have also explored the use of variant detection by RT-PCR from nasopharyngeal and from waste-water, with high levels of concordance. However, our study provides new information and insights of special importance for patient management: this is the first study to use variant assays for both SARS-CoV-2 diagnosis and variant identification, vital for treatment decisions, especially in severe cases where certain Omicron variants may resist monoclonal antibodies [ 12 ; 20 ]; we also, showed excellent assay specificity and positive predictive values, pivotal for clinical decisions; finally, our data support these assays to bolster regional and national variant surveillance, particularly beneficial for low- and middle-income countries with limited access to whole genome sequencing.…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of a combined detection of SARS-CoV-2 and its variants using real-time allele-specific PCR strategy: an advantage for clinical practice

Chaves-Blanco,

de Salazar,

Fuentes

et al. 2023

Epidemiol. Infect.

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This study aimed to assess the ability of a real-time reverse transcription polymerase chain reaction (RT-PCR) with multiple targets to detect SARS-CoV-2 and its variants in a single test. Nasopharyngeal specimens were collected from patients in Granada, Spain, between January 2021 and December 2022. Five allele-specific RT-PCR kits were used sequentially, with each kit designed to detect a predominant variant at the time. When the Alpha variant was dominant, the kit included the HV69/70 deletion, E and N genes. When Delta replaced Alpha, the kit incorporated the L452R mutation in addition to E and N genes. When Omicron became dominant, L452R was replaced with the N679K mutation. Before incorporating each variant kit, a comparative analysis was carried out with SARS-CoV-2 whole genome sequencing (WGS). The results demonstrated that RT-PCR with multiple targets can provide rapid and effective detection of SARS-CoV-2 and its variants in a single test. A very high degree of agreement (96.2%) was obtained between the comparison of RT-PCR and WGS. Allele-specific RT-PCR assays make it easier to implement epidemiological surveillance systems for effective public health decision making.

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Section: Discussionmentioning

confidence: 99%

“…RT-PCR allows to obtain and report the results on the same day, and may be easily implemented in the routine of a clinical microbiology laboratory [ 14 ; 15 ]. In this paper, we aimed to demonstrate the ability of RT-PCR to detect SARS-CoV-2 and its variants in a single test, increasing the number of samples that can be analysed.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a combined detection of SARS-CoV-2 and its variants using real-time allele-specific PCR strategy: an advantage for clinical practice

Chaves-Blanco,

de Salazar,

Fuentes

et al. 2023

Epidemiol. Infect.

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“…Clinical samples were sequenced with targeted NGS and screened with these RT-PCR assays and showed a ~99.5% concordance. Another study designed two flexible, multiplexed RT-qPCR platforms for small- and large-scale screening, using MGB probes, to detect the L452R, E484K, N501Y and ΔH69/V70 deletion in clinical samples from Denmark ( Spiess et al., 2023 ). Relative to our study, a similar approach was taken where the multiplexed assays comprised two probes – one to detect the wild-type nucleotide sequence and one to detect the mutation.…”

Section: Discussionmentioning

confidence: 99%

“…Despite this limitation, we showed that all probes were specific for their cognate sequences and to independently confirm the SNP calling, regions harbouring respective mutation in the S -gene were amplified and Sanger sequenced. Other studies have used either Sanger sequencing or WGS for confirmation ( Erster et al., 2021 ; Aralis et al., 2022 ; Moisan et al., 2023 ; Spiess et al., 2023 ; Zare Ashrafi et al., 2023 ). A large proportion of samples, particularly from Clinical Site 2, could not be confirmed as PCR failed to generate amplicons for sequencing.…”

Section: Discussionmentioning

confidence: 99%

Development of primer-probe sets to rapidly distinguish single nucleotide polymorphisms in SARS-CoV-2 lineages

Ealand,

Gordhan,

Machowski

et al. 2023

Front. Cell. Infect. Microbiol.

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Ongoing SARS-CoV-2 infections are driven by the emergence of various variants, with differential propensities to escape immune containment. Single nucleotide polymorphisms (SNPs) in the RNA genome result in altered protein structures and when these changes occur in the S-gene, encoding the spike protein, the ability of the virus to penetrate host cells to initiate an infection can be significantly altered. As a result, vaccine efficacy and prior immunity may be diminished, potentially leading to new waves of infection. Early detection of SARS-CoV-2 variants using a rapid and scalable approach will be paramount for continued monitoring of new infections. In this study, we developed minor groove-binding (MGB) probe-based qPCR assays targeted to specific SNPs in the S-gene, which are present in variants of concern (VOC), namely the E484K, N501Y, G446S and D405N mutations. A total of 95 archived SARS-CoV-2 positive clinical specimens collected in Johannesburg, South Africa between February 2021 and March 2022 were assessed using these qPCR assays. To independently confirm SNP detection, Sanger sequencing of the relevant region in the S-gene were performed. Where a PCR product could be generated and sequenced, qPCR assays were 100% concordant highlighting the robustness of the approach. These assays, and the approach described, offer the opportunity for easy detection and scaling of targeted detection of variant-defining SNPs in the clinical setting.

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“…Although it is not quite clear whether the Omicron sub-variants differ from each other in the severity of the clinical outcome, local diagnostic laboratories still need to constantly monitor their epidemiology. Due to their continuous evolution and extensive simultaneous spread worldwide, there are very limited molecular detection tests available that are also capable of distinguishing specific Omicron sub-variants [ 27 , 28 , 29 , 30 ]. In some cases, the escape from molecular detection, by S dropout (the so-called “S gene target failure” or SGTF) has been related to the detection of BA.4 and BA.5 sub-lineages.…”

Section: Discussionmentioning

confidence: 99%

An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants

Bozidis,

Petridi,

Gartzonika

2023

Pathogens

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As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, BA.2, BA.3, BA.4, and BA.5. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) that will be able to distinguish the basic sub-variants of Omicron in a rapid and specific way. Full genome sequences of Omicron strains with high frequency and wide geographical distribution were retrieved by the NCBI Virus and ENA databases. These sequences were compared to each other in order to locate single nucleotide polymorphisms common to all strains of the same sub-variant. These polymorphisms should also be capable of distinguishing Omicron sub-variants not only from each other but from previously circulating variants of SARS-CoV-2 as well. Thus, specific primers targeting characteristic polymorphisms of the four Omicron main branches BA.1, BA.2, BA.4, and BA.5 were designed according to the principles of the amplification refractory mutation system (ARMS) and with the ability to react under multiplex PCR conditions. According to our results, the ARMS-multiplex PCR could successfully distinguish all Omicron sub-variants that carry the corresponding mutations.

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Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations

Abstract: Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark.

Cited by 8 publications

References 32 publications

Evaluation of a combined detection of SARS-CoV-2 and its variants using real-time allele-specific PCR strategy: an advantage for clinical practice

Evaluation of a combined detection of SARS-CoV-2 and its variants using real-time allele-specific PCR strategy: an advantage for clinical practice

Development of primer-probe sets to rapidly distinguish single nucleotide polymorphisms in SARS-CoV-2 lineages

An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants

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