A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2

Chan, Chloe Toi-Mei; Leung, Jake Siu-Lun; Lee, Lam-Kwong; Lo, Hazel Wing-Hei; Wong, Evelyn; Wong, Denise; Ng, Timothy Ting-Leung; Lao, Hiu-Yin; Jim, Stephanie Hoi-Ching; Yau, Miranda Chong-Yee; Lam, Jimmy Yiu-Wing; Ho, Alex Yat-Man; Luk, Kristine Shik; Yip, Kam-Tong; Que, Tak-Lun; To, Kelvin Kai‐Wang; Siu, Gilman Kit-Hang

doi:10.1016/j.jviromet.2021.114333

Cited by 12 publications

(9 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Commercial PCR kits for variant detection are currently available. However, these kits also target a few mutation markers originating from SARS-CoV-2 lineages of interest ( 23 – 25 ). As we showed above, targeting a single mutation might make the assay less accurate in certain regions due to the presence of other lineages that have the same mutation.…”

Section: Discussionmentioning

confidence: 99%

Design of SARS-CoV-2 Variant-Specific PCR Assays Considering Regional and Temporal Characteristics

Sashittal

Zhou

et al. 2022

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 99%

Design of SARS-CoV-2 Variant-Specific PCR Assays Considering Regional and Temporal Characteristics

Sashittal

Zhou

et al. 2022

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Additional confidence in prevalence of VOCs is provided by the fact that the mutation and Universal signals can be quantified using the same standard. In comparison, other VOC qPCR assays estimate prevalence by comparing a mutant allele to a genetic marker of SARS-COV-2 in another amplicon, /genetic locus such as the CDC_N1 (Vogels et al, 2021; Wolfe et al, 2021), or by comparing the frequencies of the mutant and “wild-type” (e.g., ancestral/endemic allele) at the same locus (Chan et al, 2022; Graber et al, 2021; Lee et al, 2021; Peterson et al, 2022). While these assays can produce accurate data (from both analytical and clinical perspectives), there are additional controls needed to account for variations in efficiencies of variation (inter- assay variability and matching standard quantities), that are eliminated by the N200 assay.…”

Section: Discussionmentioning

confidence: 99%

“…or by comparing the frequencies of the mutant and "wild-type" (e.g., ancestral/endemic allele) at the same locus (Chan et al, 2022;Graber et al, 2021;Lee et al, 2021;Peterson et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater

Fuzzen

Harper

Dhiyebi

et al. 2022

Preprint

View full text Add to dashboard Cite

Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named “N200” focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.

show abstract

“…Alternative approaches to NGS for surveillance of VOCs and/or epidemiologically important mutations is a rapidly expanding area: commercially available molecular assays for SARS-CoV-2 VOCs have been developed, including the TIB MolBio ViRSNiP (26) tests, however these tests only identify a single SNP or deletion per assay, potentially requiring a panel of tests for comprehensive surveillance. Elsewhere, academic groups have developed assays for particular epidemiologically important mutations using either hydrolysis probes (27), molecular beacons (28) or isothermal methods (29). However, the HRM approach we present is the rst development of a comprehensive tool kit of primers that can be combined in response to local epidemiology, and furthermore is readily adaptable to the emergence of new VOCs, as demonstrated by the incorporation of primers for detection of the Omicron VOC in our assay.…”

Section: Discussionmentioning

confidence: 99%

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

Fraser

Greenland-Bews

Kelly

et al. 2022

Preprint

View full text Add to dashboard Cite

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high resolution melting (HRM) assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs, assay based on HRM analysis, an endpoint RT-qPCR detection method. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected at varying points of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities.

show abstract

A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2

Cited by 12 publications

References 13 publications

Design of SARS-CoV-2 Variant-Specific PCR Assays Considering Regional and Temporal Characteristics

Design of SARS-CoV-2 Variant-Specific PCR Assays Considering Regional and Temporal Characteristics

Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

Contact Info

Product

Resources

About