Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14).

ABSTRACT: The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.

SUBMITTER: Pierri B

PROVIDER: S-EPMC8660663 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs.

Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples.MethodsThe RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy).ResultsThe present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration.ConclusionDuring the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

| S-EPMC7934789 | biostudies-literature

DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP.

Project description:The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.

| S-EPMC7239058 | biostudies-literature

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

Project description:The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

| S-EPMC7556528 | biostudies-literature

Self-Collection of Saliva Specimens as a Suitable Alternative to Nasopharyngeal Swabs for the Diagnosis of SARS-CoV-2 by RT-qPCR.

Project description:A nasopharyngeal swab is a sample used for the diagnosis of SARS-CoV-2 infection. Saliva is a sample easier to obtain and the risk of contagion for the professional is lower. This study aimed to evaluate the utility of saliva for the diagnosis of SARS-CoV-2 infection. This prospective study involved 674 patients with suspected SARS-CoV-2 infection. Paired nasopharyngeal and saliva samples were processed by RT-qPCR. Sensitivity, specificity, and kappa coefficient were used to evaluate the results from both samples. We considered the influence of age, symptoms, chronic conditions, and sample processing with lysis buffer. Of the 674 patients, 636 (94.4%) had valid results from both samples. The virus detection in saliva compared to a nasopharyngeal sample (gold standard) was 51.9% (95% CI: 46.3%-57.4%) and increased to 91.6% (95% CI: 86.7%-96.5%) when the cycle threshold (Ct) was ≤ 30. The specificity of the saliva sample was 99.1% (95% CI: 97.0%-99.8%). The concordance between samples was 75% (κ = 0.50; 95% CI: 0.45-0.56). The Ct values were significantly higher in saliva. In conclusion, saliva sample utility is limited for clinical diagnosis, but could be a useful alternative for the detection of SARS-CoV-2 in massive screening studies, when the availability of trained professionals for sampling or personal protection equipment is limited.

| S-EPMC7830328 | biostudies-literature

Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach.

Project description:The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).

| S-EPMC7547948 | biostudies-literature

Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients.

Project description:Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.

| S-EPMC7478621 | biostudies-literature

SARS-CoV-2 RNA Quantification Using Droplet Digital RT-PCR.

Project description:Quantitative viral load assays have transformed our understanding of viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time RT-PCR, yield semiquantitative results only. Droplet digital RT-PCR (RT-ddPCR) offers an attractive platform for SARS-CoV-2 RNA quantification. Eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests were evaluated for use in RT-ddPCR; three were identified as the most efficient, precise, and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. For example, the analytical efficiency for the E-Sarbeco primer/probe set was approximately 83%, whereas assay precision, measured as the coefficient of variation, was approximately 2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. RT-ddPCR-derived SARS-CoV-2 E gene copy numbers were further calibrated against cycle threshold values from a commercial real-time RT-PCR diagnostic platform. This log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from cycle threshold values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

| S-EPMC8164350 | biostudies-literature

Cotton-Tipped Plastic Swabs for SARS-CoV-2 RT-qPCR Diagnosis to Prevent Supply Shortages.

Project description:CDC and WHO guidelines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis only recommend synthetic fiber swabs for nasopharyngeal (NP) sampling. We show that cotton-tipped plastic swabs do not inhibit PCR and have equivalent performance to rayon swabs. Cotton-tipped plastic swabs are massively produced worldwide and would prevent swab supply shortages under the current high SARS-CoV-2 testing demands, particularly in developing countries.

| S-EPMC7324669 | biostudies-literature

Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs

Project description:The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.

| S-EPMC8016541 | biostudies-literature

Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples.

Project description:ObjectivesThe gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection.MethodsWe evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before the RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, c) in a RNAsnapTM buffer.ResultsAlthough with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min.ConclusionsOur findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.

| S-EPMC7833505 | biostudies-literature

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