Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.
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ABSTRACT:
SUBMITTER: Telwatte S
PROVIDER: S-EPMC8105137 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.HighlightsWe developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2Our panel targets nongenic and genic regions in genomic and subgenomic RNAsAll assays detect 1-10 copies and are linear over 3-4 orders of magnitudeAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load AssayClinical samples showed higher copy numbers for targets at the 3' end of the genome.
| S-EPMC7814816 | biostudies-literature
Project description:SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients. Oranger et al use digital droplet PCR to develop an assay that provides a dynamic snapshot of the levels of SARS-CoV-2 sub-genomic messanger RNA in infected individuals. This has the potential to improve diagnostic accuracy for COVID-19
| S-EPMC8536764 | biostudies-literature
Project description:A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.
| S-EPMC7923865 | biostudies-literature
Project description:We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.
| S-EPMC8015849 | biostudies-literature
Project description:Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. We combined bioinformatic and experimental analyses to improve the RT-LAMP assay performance for COVID-19 diagnosis. First, we developed an improved algorithm to design LAMP primers targeting the nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV-2. Next, we rigorously validated these new assays for their efficacy and specificity. Further, we demonstrated that multiplexed RT-LAMP assays could directly detect as low as a few copies of SARS-CoV-2 RNA in saliva, without the need of RNA isolation. Importantly, further testing using saliva samples from COVID-19 patients indicated that the new RT-LAMP assays were in total agreement in sensitivity and specificity with standard RT-qPCR. In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.
2021-04-22 | GSE173086 | GEO
Project description:Throughout the COVID-19 global pandemic there has been significant interest and investment in using Wastewater-Based Epidemiology (WBE) for surveillance of viral pathogen presence and infections at the community level. There has been a push for widescale implementation of standardized protocols to quantify viral loads in a range of wastewater systems. To address concerns regarding sensitivity, limits of quantification, and large-scale reproducibility, a comparison of two similar workflows using RT-qPCR and RT-ddPCR was conducted. Sixty raw wastewater influent samples were acquired from nine distinct wastewater treatment plants (WWTP's) served by the Hampton Roads Sanitation District (HRSD, Virginia Beach, Virginia) over a 6-month period beginning March 9th, 2020. Common reagents, controls, master mixes and nucleic acid extracts were shared between two individual processing groups based out of HRSD and the UNC Chapel Hill Institute of Marine Sciences (IMS, Morehead City, North Carolina). Samples were analyzed in parallel using One-Step RT-qPCR and One-Step RT-ddPCR with Nucleocapsid Protein 2 (N2) specific primers and probe. Influent SARS-CoV-2 N2 concentrations steadily increased over time spanning a range from non-detectable to 2.13E + 05 copies/L. Systematic dilution of the extracts indicated that inhibitory components in the wastewater matrices did not significantly impede the detection of a positive N2 signal for either workflow. The RT-ddPCR workflow had a greater analytical sensitivity with a lower Limit of Detection (LOD) at 0.066 copies/μl of template compared to RT-qPCR with a calculated LOD of 12.0 copies/μL of template. Interlaboratory comparisons using non-parametric correlation analysis demonstrated that there was a strong, significant, positive correlation between split extracts when employing RT-ddPCR for analysis with a ρ value of 0.86.
| S-EPMC8267102 | biostudies-literature
Project description:BackgroundSome mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in recently described Omicron subvariants. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals.ObjectiveTo develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment.Study designUsing three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant.ResultsMutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen.ConclusionWe describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.
| S-EPMC9212762 | biostudies-literature
Project description:Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.
| S-EPMC7784781 | biostudies-literature
Project description:The rapid scale-up of research on coronavirus disease 2019 (COVID-19) has spawned a large number of potential vaccines and immunotherapies, accompanied by a commensurately large number of in vitro assays and in vivo models to measure their effectiveness. These assays broadly have the same end-goal - to predict the clinical efficacy of prophylactic and therapeutic interventions in humans. However, the apparent potency of different interventions can vary considerably between assays and animal models, leading to very different predictions of clinical efficacy. Complete harmonization of experimental methods may be intractable at the current pace of research. However, here we analyse a selection of existing assays for measuring antibody-mediated virus neutralization and animal models of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a framework for comparing results between studies and reconciling observed differences in the effects of interventions. Finally, we propose how we might optimize these assays for better comparison of results from in vitro and animal studies to accelerate progress.
| S-EPMC7605490 | biostudies-literature
Project description:BackgroundDistinctive genotypes of SARS-CoV-2 have emerged that are or may be associated with increased transmission, pathogenicity, and/or antibody escape. In many countries, clinical and diagnostic laboratories are under mandate to identify and report these so-called variants of concern (VOC).ObjectivesWe used an external quality assessment scheme to determine the scope, accuracy, and reliability of laboratories using various molecular diagnostic assays to identify current VOC (03 March 2021).Study designParticipant laboratories were sent the same five patient-derived samples and were asked to provide their variant detection methods, variant detection results and interpretation of results.ResultsTwenty-five laboratories reported a range of RT-qPCR-based assays to identify specific variations in the SARS-CoV-2 spike protein that are characteristic of three VOC lineages. Laboratories that detected VOC-associated nucleotide mutations at four specific sites had the highest ratio of correct classification. Low template copy number and additional variation in target regions resulted in loss of confidence and accuracy in sample classification.ConclusionsMelting-curve-based assays to identify genomic variants are less time-consuming and require less bioinformatic analysis compared to partial or whole genome sequencing. However, our results suggest that correct classification of a given genotype/lineage (e.g., a VOC) relies on the ability to detect more than one variant site, adequate template in the sample (i.e., relatively high viral load/copy number) and results may be unclear in certain samples with additional genetic variations. These initial results suggest that some diagnostic laboratories may require additional training to interpret and report complex genetic information about a dynamic emerging virus.
| S-EPMC8262392 | biostudies-literature