Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR.

ABSTRACT: Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex^®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3^® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution.

SUBMITTER: Boudet A

PROVIDER: S-EPMC8306735 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants.

Project description:BackgroundThe recent emergence of new SARS CoV-2 variants (variants of concern, VOC) that spread rapidly and may lead to immune escape has emphasized the urgent need to monitor and control their spread.MethodsWe analyzed 2018 SARS-CoV-2 positive specimens collected between February 9 and March 22, 2021 using the Thermofisher® TaqPath™ COVID-19 CE-IVD RT-PCR kit (TaqPath) and the ID solutions® ID™ SARS-CoV-2/UK/SA Variant Triplex RT-PCR (ID triplex) assay to screen for VOCs.ResultsThe ID triplex assay identified 62.8% of them as VOCs: 61.8% B.1.1.7 and 0.9% B.1.351/P.1. The agreement between the ID triplex results for B.1.1.7 and the TaqPath S gene target failure (SGTF)/ S gene target late detection (SGTL) profile for this variant agreed very well (k = 0.86). A low virus load was the main cause of discrepancies. Sequencing discordant results with both assays indicated that the TaqPath assay detected the B.1.1.7 lineage slightly better. Both assays suggested that the virus loads of B.1.1.7 variants were significantly higher than those of non-B.1.1.7 strains. Only 10/20 B1.351/P.1 strains detected with the ID triplex assay were confirmed by sequencing.ConclusionsWe conclude that the SGTF/SGTL profiles identified using the TaqPath assay and ID triplex results are suitable for detecting the B.1.1.7 lineage. The ID triplex assay, which rapidly determines all three current VOCs simultaneously, could be a valuable tool for limiting virus spread by supporting contact-tracing and isolation.

| S-EPMC8411575 | biostudies-literature

Sample pooling for SARS-CoV-2 RT-PCR screening.

Project description:ObjectiveTo evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid extraction and amplification.MethodsA total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place at each centre.ResultsWe found that 253 pools (2519 samples) were negative and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples), we found discordant results when compared to their correspondent individual samples, as follows: in 22 of 29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity and positive and negative predictive values for pooling were 97.10% (95% confidence interval (CI), 94.11-98.82), 100%, 100% and 99.79% (95% CI, 99.56-99.90) respectively; accuracy was 99.80% (95% CI, 99.59-99.92), and the kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted in a median loss of 2.87 (95% CI, 2.46-3.28) cycle threshold (Ct) for E gene, 3.36 (95% CI, 2.89-3.85) Ct for the RdRP gene and 2.99 (95% CI, 2.56-3.43) Ct for the N gene.ConclusionsWe found a high efficiency of pooling strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA testing across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity and positive and negative predictive values.

| S-EPMC7481316 | biostudies-literature

RT-PCR based SARS-CoV-2 variant screening assays require careful quality control.

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

Evaluation of RT-PCR assays for detection of SARS-CoV-2 variants of concern.

Project description:Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has been considered with great importance on correct screening procedure. The detection efficiency of recent variants of concern were observed by comparing 5 commercial RT-PCR kits and a SYBR-green method developed and validated in our laboratory. The RNA was extracted from nasopharyngeal samples from suspected COVID-19 patients and RT-PCR assay was performed according to the instruction of the respective manufacturers. The specificity and sensitivity of Maccura kit was 81.8% and 82.5%, A*Star kit was 100% and 75.4%, Da An Gene kit was 100% and 68.4%, Sansure kit was 54.5% and 91.2% and TaqPath kit was 100% and 70.2% respectively. Our in house SYBR-Green method showed a consistent detection result with 90.9% specificity and 91.2% sensitivity. We also found that detection kits targeting more genes showed better accuracy which facilitates less false positive results (< 20%). Our study found a significant difference (p < 0.005) in Ct value reported for common target genes shared by the RT-PCR kits in relation with different variants of COVID-19 infection. Recent variants of concerns contain more than 30 mutations in the spike proteins including 2 deletion and a unique insertion mutation by which makes detection of these variants difficult and these facilitates the variants to escape from being detected.

| S-EPMC9910272 | biostudies-literature

Rapid Base-Specific Calling of SARS-CoV-2 Variants of Concern Using Combined RT-PCR Melting Curve Screening and SIRPH Technology.

Project description:BackgroundThe emergence of novel variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands fast and reliable detection of such variants in local populations.MethodsHere we present a cost-efficient and fast workflow combining a prescreening of SARS-CoV-2-positive samples using reverse transcription polymerase chain reaction melting curve analysis with multiplexed IP-RP-HPLC-based single nucleotide primer extensions.ResultsThe entire workflow from positive SARS-CoV-2 testing to base-specific identification of variants requires about 24 hours.ConclusionsWe applied the sensitive method to monitor local variant of concern outbreaks in SARS-CoV-2-positive samples collected in a confined region of Germany.

| S-EPMC8364759 | biostudies-literature

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Project description:Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

| S-EPMC9863853 | biostudies-literature

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach.

Project description:The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

| S-EPMC8854350 | biostudies-literature

Rapid and accurate identification of SARS-CoV-2 Omicron variants using droplet digital PCR (RT-ddPCR).

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

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR).

Project description:Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.

| S-EPMC8776039 | biostudies-literature

Coronavirus antibody profiling in convalescent sera of RT-PCR SARS-CoV-2 confirmed hospitalised patients

Project description:Convalescent sera of RT-PCR SARS-CoV-2 confirmed hospitalised patients were tested on the protein array to profile IgG, IgM, and IgA antibody levels against human coronaviruses.

2022-03-08 | E-MTAB-11523 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data