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

Oh, Chamteut; Sashittal, Palash; Zhou, Aiguo; Wang, Leyi; El-Kebir, Mohammed; Nguyen, Thanh H.

doi:10.1128/aem.02289-21

Cited by 14 publications

(17 citation statements)

References 39 publications

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“…First, we analyzed the SARS-CoV-2 N gene, S:A570D mutation, and S:P681R mutation. These two mutations are confirmed by in silico analysis and in vitro experiments to be exclusive to the Alpha and Delta variants circulating in IL, USA, during our monitoring period (Oh et al, 2022b). Then, we plotted these data onto the clinical COVID-19 testing data obtained from the neighborhood-scale sewersheds, allowing us to estimate high COVID-19 incidence with WBE data.…”

Section: Introductionsupporting

confidence: 68%

“…We determined the COVID- 19 incidence rate for each sewershed by normalizing the COVID-19 occurrence (cases/day) to the conresponding populations. We analyzed 138 sequences from the Global Initiative on Sharing All Influenza Data (GISAID), which were reported from Champaign County in 2021 using an algorithm, PRIMES developed by Oh et al (2022b) to determine the temporal prevalence of SARS-CoV-2 variants in Champaign County.…”

Section: Methodsmentioning

confidence: 99%

“…We analyzed 138 sequences from the Global Initiative on Sharing All Influenza Data (GISAID), which were reported from Champaign County in 2021 using an algorithm, PRIMES developed by Oh et al (2022b), to determine the temporal prevalence of SARS-CoV-2 variants in Champaign County.…”

Section: Methodsmentioning

confidence: 99%

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Application of neighborhood-scale wastewater-based epidemiology in low COVID-19 incidence situations

Zhou

O’Brien

et al. 2022

Preprint

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Wastewater-based epidemiology (WBE) is an emerging approach for community-wide COVID- 19 surveillance. WBE was characterized mostly at large sewersheds such as wastewater treatment plants serving a large population. Although informed public health measures can be implemented in a small population, WBE for neighborhood-scale sewersheds is less studied and not fully understood. This study applied WBE to seven neighborhood-scale sewersheds (average population 1,471) from January to November 2021. Community testing data showed an average of 0.004% incidence rate in these sewersheds (97% of monitoring periods reported two or fewer daily infections). In 92% of sewage samples, SARS-CoV-2 N gene fragments were below the limit of quantification. COVID-19 cases poorly correlated with SARS-CoV-2 N gene concentrations except for one location where frequent COVID-19 testing was required. Thus, we developed a COVID-19 warning system for high COVID-19 risk when the SARS-CoV-2 N gene concentration normalized to pepper mild mottle virus (PMMOV) in wastewater is higher than 10-2. This COVID- 19 warning system now identified neighborhood-scale outbreaks (COVID-19 incidence rate higher than 0.5%) with 73% sensitivity and 71% specificity. Importantly, it can discern neighborhood- level outbreaks that would not otherwise be identified by county-scale WBE. We also demonstrated that SARS-CoV-2 variant-specific RT-qPCR assays could be combined with the COVID-19 warning system to identify the introduction of SARS-CoV-2 variants into sewersheds with a low COVID-19 incidence. This new COVID-19 warning system will contribute to effective community-wide disease surveillance when COVID-19 incidence may be maintained at a low level.Graphical abstract

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Section: Introductionsupporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

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Application of neighborhood-scale wastewater-based epidemiology in low COVID-19 incidence situations

Zhou

O’Brien

et al. 2022

Preprint

Self Cite

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Section: Methodsmentioning

confidence: 99%

“…In these cases, if the difference between Ct values was greater than 10 Ct, then the target with the higher value was called as not determined. This was likely caused by non-specific amplification typically observed in mutation-specific qPCR assays [27][28][29] . Amplicon Library Generation, Next Generation Sequencing, Phylogenetic Analysis Viral cDNA was generated using LunaScript RT SuperMix, followed by incubation using the thermal profile: 25°C for 2 minutes, 55°C for 10 minutes, and 95°C for 2 minutes.…”

Section: Data Interpretationmentioning

confidence: 99%

Efficient Tracing of the SARS-CoV-2 Omicron Variants in Santa Barbara County Using a Rapid Quantitative Reverse Transcription PCR Assay

Aralis

Comer

Ansorg

et al. 2022

Preprint

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The recent emergence of the SARS-CoV-2 Omicron variant is associated with a dramatic surge of cases around the globe in late 2021 and early 2022. The numerous mutations in this variant, particularly in the Spike protein, enhance its transmission, increase immune evasion, and limit treatment with monoclonal antibodies. Identifying a community's introduction to a novel SARS-CoV-2 variant with new clinical features related to treatment options and infection control needs is imperative to inform decisions by clinicians and public health officials, and traditional sequencing techniques often take weeks to result. Here, we describe a quantitative reverse transcription PCR assay (RT-qPCR) to accurately and precisely detect the presence of the Omicron sublineages BA.1/BA1.1 and BA.2 viral RNA from patient samples in less than four hours. The assay uses primers targeting the BA.1/BA1.1 unique mutations N211del, L212I, and L214 insertion EPE in the Spike protein gene, and the BA.2 specific mutations T19I and L24/P25/P26 deletion in the Spike protein gene. Using this assay, we detected 169 cases of Omicron, 164 BA.1/BA1.1 and 5 BA.2, from 270 residual SARS-CoV-2 positive samples collected for diagnostic purposes from Santa Barbara County (SBC) between December 2021 to February 2022. The RT-qPCR results show concordance with whole viral genome sequencing. Our observations indicate that Omicron was the dominant variant in SB County and is likely responsible for the surge of cases in the area during the sampling period. Using this inexpensive and accurate test, the rapid detection of Omicron in patient samples allowed clinicians to modify treatment strategies and public health officers to enhance contact tracing strategies. This RT-qPCR assay offers an alternative to current variant-specific detection approaches, provides a template for the fast design of similar assays, and allows the rapid, accurate, and inexpensive detection of Omicron variants in patient samples. It can also be readily adapted to new variants as they emerge in the future.

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CRISPR‐Cas system: A promising tool for rapid detection of SARS‐CoV‐2 variants

Li,

Zhang,

Lin

et al. 2024

Journal of Medical Virology

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COVID‐19, caused by SARS‐CoV‐2, remains a global health crisis. The emergence of multiple variants with enhanced characteristics necessitates their detection and monitoring. Genome sequencing, the gold standard, faces implementation challenges due to complexity, cost, and limited throughput. The CRISPR‐Cas system offers promising potential for rapid variant detection, with advantages such as speed, sensitivity, specificity, and programmability. This review provides an in‐depth examination of the applications of CRISPR‐Cas in mutation detection specifically for SARS‐CoV‐2. It begins by introducing SARS‐CoV‐2 and existing variant detection platforms. The principles of the CRISPR‐Cas system are then clarified, followed by an exploration of three CRISPR‐Cas‐based mutation detection platforms, which are evaluated from different perspectives. The review discusses strategies for mutation site selection and the utilization of CRISPR‐Cas, offering valuable insights for the development of mutation detection methods. Furthermore, a critical analysis of the clinical applications, advantages, disadvantages, challenges, and prospects of the CRISPR‐Cas system is provided.

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

Cited by 14 publications

References 39 publications

Application of neighborhood-scale wastewater-based epidemiology in low COVID-19 incidence situations

Application of neighborhood-scale wastewater-based epidemiology in low COVID-19 incidence situations

Efficient Tracing of the SARS-CoV-2 Omicron Variants in Santa Barbara County Using a Rapid Quantitative Reverse Transcription PCR Assay

CRISPR‐Cas system: A promising tool for rapid detection of SARS‐CoV‐2 variants

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