A Comparison of Seegene Technologies Novaplex SARS-CoV-2 Variants I, II, and IV Assays with Spike Gene Sequencing for Detection of Known Severe Acute Respiratory Syndrome Coronavirus 2 Variants

Nielsen, Marisa C.; Machado, Rafael Rahal Guaragna; Mitchell, Brooke; McConnell, Allan; Saada, Nehad; Weaver, Scott C.; Ren, Ping

doi:10.1016/j.jmoldx.2022.02.001

Cited by 9 publications

(7 citation statements)

References 13 publications

(9 reference statements)

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“…highly consistent with WGS Limpopo data reported by the South African Network for Genomic Surveillance (NGS-SA) as well other reports covering the time period we analysed [15,20]. Furthermore, our results are consistent with similar studies that have recently evaluated Seegene Variant assays [21,22]. This observation reinforces the predictive value of commercial multiplex PCR genotyping assays in epidemiological surveillance and highlights the diagnostic utility this approach can play in supporting WGS in resource-scarce and under-represented settings, as has been shown in other studies [23][24][25][26][27][28][29].…”

Section: Plos Onesupporting

confidence: 93%

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Evaluation of a commercial SARS-CoV-2 multiplex PCR genotyping assay for variant identification in resource-scarce settings

Umunnakwe¹,

Makatini

Maphanga³

et al. 2022

PLoS ONE

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The rapid emergence and spread of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants across the globe underscores the crucial need for continuous SARS-CoV-2 surveillance to ensure that potentially more pathogenic variants are detected early and contained. Whole genome sequencing (WGS) is currently the gold standard for COVID-19 surveillance; however, it remains cost-prohibitive and requires specialized technical skills. To increase surveillance capacity, especially in resource-scarce settings, supplementary methods that are cost- and time-effective are needed. Real-time multiplex PCR genotyping assays offer an economical and fast solution for screening circulating and emerging variants while simultaneously complementing existing WGS approaches. In this study we evaluated the AllplexTM SARS-CoV-2 Variants II multiplex real-time PCR genotyping assay, Seegene (South Korea), and implemented it in retrospectively characterizing circulating SARS-CoV-2 variants in a rural South African setting between April and October 2021, prior to the emergence of the Omicron variant in South Africa. The AllplexTM SARS-CoV-2 Variants II real-time PCR assay demonstrated perfect concordance with whole-genome sequencing in detecting Beta and Delta variants and exhibited high specificity, sensitivity and reproducibility. Implementation of the assay in characterization of SARS-CoV-2 variants between April and October 2021 in a rural South African setting revealed a rapid shift from the Beta to the Delta variant between April and June. All specimens successfully genotyped in April were Beta variants and the Delta variant was not detected until May. By June, 78% of samples genotyped were Delta variants and in July >95% of all genotyped samples were Delta variants. The Delta variant continued to predominate through to the end of our analysis in October 2021. Taken together, a commercial SARS-CoV-2 variant genotyping assay detected the rapid rate at which the Delta variant displaced the Beta variant in Limpopo, an under-monitored province in South Africa. Such assays provide a quick and cost-effective method of monitoring circulating variants and should be used to complement genomic sequencing for COVID-19 surveillance especially in resource-scarce settings.

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Section: Plos Onesupporting

confidence: 93%

“…Commercial multiplex PCR genotyping kits can be combined to maximize variant detection capacity and identify novel variants. There are a number of variant PCR assays which, when combined, allow for detection of multiple SAR-CoV-2 variants [21,22,28,29,33,34].…”

Section: Plos Onementioning

confidence: 99%

Evaluation of a commercial SARS-CoV-2 multiplex PCR genotyping assay for variant identification in resource-scarce settings

Umunnakwe¹,

Makatini

Maphanga³

et al. 2022

PLoS ONE

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“…Whole-genome or complete spike gene sequencing is the gold standard for identifying the strain of SARS-CoV-2. Rapid detection methods with simultaneous detection of variants using real-time multiplex PCR are commercially available and widely used to screen SARS-CoV-2 variants before sequencing con rmation by NGS [12][13][14]. In a recent study, a similar technology (Multiplex PCR-Mass spectrometry Minisequencing) was developed for the differentiation of three SARS-CoV-2 VOCs, including Alpha, Beta, and Delta, using 9 mutation sites of the spike gene [10].…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology

Wacharapluesadee

Hirunpatrawong

Petcharat

et al. 2023

Preprint

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The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/ µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

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Section: Introductionmentioning

confidence: 99%

“…To address the greater need and demand for population‐level SARS‐CoV‐2 variant surveillance, nucleic acid amplification tests (NAATs) such as reverse transcription polymerase chain reaction (RT‐PCR) have been explored. 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 The PCR‐based strategies differentiate among variants through the detection of unique, characteristic single‐nucleotide polymorphisms (SNPs). Several approaches demonstrate the SNP‐recognition capabilities inherent to PCR when primers or hydrolysis probes have single base mismatches that alter the quantification cycle (Cq) value at which amplification occurs for a target.…”

Section: Introductionmentioning

confidence: 99%

Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

Nelson

Shilts

Pakala

et al. 2022

Influenza Resp Viruses

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Background COVID‐19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. Methods A ligation‐based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS‐CoV‐2) by identifying the presence of characteristic single‐nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real‐time polymerase chain reaction (PCR) instrumentation for assay performance. Results The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation‐based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. Conclusions The assay demonstrates the potential for robust variant typing with performance comparable with next‐generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

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A Comparison of Seegene Technologies Novaplex SARS-CoV-2 Variants I, II, and IV Assays with Spike Gene Sequencing for Detection of Known Severe Acute Respiratory Syndrome Coronavirus 2 Variants

Cited by 9 publications

References 13 publications

Evaluation of a commercial SARS-CoV-2 multiplex PCR genotyping assay for variant identification in resource-scarce settings

Evaluation of a commercial SARS-CoV-2 multiplex PCR genotyping assay for variant identification in resource-scarce settings

Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology

Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

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