Comparative Analysis of Five Multiplex RT-PCR Assays in the Screening of SARS-CoV-2 Variants

Pace, Vanessa De; Bruzzone, Bianca; Orsi, Andrea; Ricucci, Valentina; Domnich, Alexander; Guarona, Giulia; Randazzo, Nadia; Stefanelli, Federica; Battolla, Enrico; Dusi, Pier Andrea; Lillo, Flavia; Icardi, Giancarlo

doi:10.3390/microorganisms10020306

Cited by 22 publications

(28 citation statements)

References 25 publications

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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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Section: Nucleic Acid‐based Sars‐cov ‐2 Detectionmentioning

confidence: 99%

“…The overall mean Ct value of the five kits was 23.6 ± 3.8, with accuracy ranging from 96.9% to 100%, among which the SARS‐CoV‐2 Variants II Assay Allplex (for L452R, W152C, K417T, and K417N) kits had 100% sensitivity and specificity. 44 Novel whole‐genome sequencing technologies based on the EasySeqTM RC‐PCR SARS‐CoV‐2 WGS kit and RT‐PCR have proven to be useful for high‐throughput detection of mutant strains of SARS‐CoV‐2. 46 Vega‐Magaña et al designed three specific primers and probes for qRT‐PCR detection based on the N501Y, 69‐70del, K417N, and E484K S mutations, which played an important role in detecting the E484K mutation and P.2 mutant strains.…”

Section: Nucleic Acid‐based Sars‐cov ‐2 Detectionmentioning

confidence: 99%

An updated review of SARS‐CoV‐2 detection methods in the context of a novel coronavirus pandemic

Zhang

Huang

Zhu

et al. 2022

Bioengineering & Transla Med

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The World Health Organization has reported approximately 430 million confirmed cases of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), worldwide, including nearly 6 million deaths, since its initial appearance in China in 2019. While the number of diagnosed cases continues to increase, the need for technologies that can accurately and rapidly detect SARS‐CoV‐2 virus infection at early phases continues to grow, and the Federal Drug Administration (FDA) has licensed emergency use authorizations (EUAs) for virtually hundreds of diagnostic tests based on nucleic acid molecules and antigen–antibody serology assays. Among them, the quantitative real‐time reverse transcription PCR (qRT‐PCR) assay is considered the gold standard for early phase virus detection. Unfortunately, qRT‐PCR still suffers from disadvantages such as the complex test process and the occurrence of false negatives; therefore, new nucleic acid detection devices and serological testing technologies are being developed. However, because of the emergence of strongly infectious mutants of the new coronavirus, such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), the need for the specific detection of mutant strains is also increasing. Therefore, this article reviews nucleic acid‐ and antigen–antibody‐based serological assays, and compares the performance of some of the most recent FDA‐approved and literature‐reported assays and associated kits for the specific testing of new coronavirus variants.

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“…Moreover, as new variants have emerged, diagnostic panels are based on targets designed for detection of nucleotide changes that yield specific amino acid substitutions and call variants based on distinct target result combinations (18)(19)(20). However, most of these assays distinguish viral variants through result patterns of 3-9 molecular targets across multiple reaction wells (20)(21)(22)(23)(24)(25)(26)(27)(28), which are constrained to distinguishing current circulating variants but may not be sufficient to distinguish nascent, increasingly divergent variants.…”

Section: Introductionmentioning

confidence: 99%

A robust, highly multiplexed mass spectrometry assay to identify SARS-CoV-2 variants

Hernandez

Banu

Shrestha

et al. 2022

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are characterized by differences in transmissibility and response to therapeutics. Therefore, discriminating among them is vital for surveillance, infection prevention, and patient care. While whole viral genome sequencing (WGS) is the "gold standard" for variant identification, molecular variant panels have become increasingly available. Most, however, are based on limited targets and have not undergone comprehensive evaluation. We assessed the diagnostic performance of the highly multiplexed Agena MassARRAY® SARS-CoV-2 Variant Panel v3 to identify variants in a diverse set of 391 SARS-CoV-2 clinical RNA specimens collected across our health systems in New York City, USA as well as in Bogota, Colombia (September 2, 2020 - March 2, 2022). We demonstrate almost perfect levels of interrater agreement between this assay and WGS for 9 of 11 variant calls (κ ≥ 0.856) and 25 of 30 targets (κ ≥ 0.820) tested on the panel. The assay had a high diagnostic sensitivity (≥93.67%) for contemporary variants (e.g., Iota, Alpha, Delta, Omicron [BA.1 sublineage]) and a high diagnostic specificity for all 11 variants (≥96.15%) and all 30 targets (≥94.34%) tested. Moreover, we highlight distinct target patterns that can be utilized to identify variants not yet defined on the panel including the Omicron BA.2 and other sublineages. These findings exemplify the power of highly multiplexed diagnostic panels to accurately call variants and the potential for target result signatures to elucidate new ones.

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Comparative Analysis of Five Multiplex RT-PCR Assays in the Screening of SARS-CoV-2 Variants

Cited by 22 publications

References 25 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

An updated review of SARS‐CoV‐2 detection methods in the context of a novel coronavirus pandemic

A robust, highly multiplexed mass spectrometry assay to identify SARS-CoV-2 variants

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