Dataset Information

STANDARD M10 SARS-CoV-2 Assay for Rapid Detection of SARS-CoV-2: Comparison of Four Real-Time PCR Assays.

ABSTRACT: The demand for assays that can rapidly and accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains high. We evaluated the performance of two rapid real-time reverse transcription polymerase chain reaction (RT-qPCR) assays (STANDARD M10 SARS-CoV-2 and Xpert Xpress SARS-CoV-2) against conventional RT-qPCR assays (STANDARD M nCoV and Allplex SARS-CoV-2) for detecting SARS-CoV-2. A total of 225 swab samples were collected and tested using the four assays. The STANDARD M10 SARS-CoV-2 assay showed 97.4% positive percent agreement (PPA) and 100.0% negative percent agreement (NPA) compared to the STANDARD M nCoV assay and Allplex SARS-CoV-2 assay. STANDARD M10 exhibited high performance except in samples with low viral loads (cycle threshold (Ct) > 30). Xpert Xpress showed PPA and NPA of 100.0% compared to the two conventional RT-qPCR assays. The kappa coefficient (Κ) showed nearly almost perfect agreement between each assay and conventional RT-qPCR assays. The correlations of Ct values between the two rapid RT-qPCR and conventional RT-qPCR assays were >0.8, indicating strong correlations. All included assays could detect SARS-CoV-2 variants, such as the Alpha, Beta, and Gamma variants. The recently developed STANDARD M10 has a shorter turnaround time and random-access detection on automated devices, thereby facilitating efficient testing in emergency settings.

SUBMITTER: Jeong S

PROVIDER: S-EPMC9406901 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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STANDARD M10 SARS-CoV-2 Assay for Rapid Detection of SARS-CoV-2: Comparison of Four Real-Time PCR Assays.

Jeong Seri S Lee Nuri N Lee Su Kyung SK Cho Eun-Jung EJ Hyun Jungwon J Park Min-Jeong MJ Song Wonkeun W Kim Hyun Soo HS

Diagnostics (Basel, Switzerland) 20220818 8

The demand for assays that can rapidly and accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains high. We evaluated the performance of two rapid real-time reverse transcription polymerase chain reaction (RT-qPCR) assays (STANDARD M10 SARS-CoV-2 and Xpert Xpress SARS-CoV-2) against conventional RT-qPCR assays (STANDARD M nCoV and Allplex SARS-CoV-2) for detecting SARS-CoV-2. A total of 225 swab samples were collected and tested using the four assays. The STANDARD ...[more]

PMID: 36010348

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Project description:The coronavirus infectious disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. The pandemic has emerged as a global public health crisis, and the threat of fast-spreading of the latest variants of the coronavirus (such as omicron, delta) is rampant. Therefore, a fast and reliable diagnostic assay is needed to make the clinical decision for further treatment. The study aims to develop a Centers for Disease and Prevention (CDC)-modified qualitative real-time reverse transcriptase PCR (RT-qPCR) assay and parallel assessment of commercially available RT-qPCR assay (Altona, Seegene, BD, and GBC) to detect SARS-CoV-2. Two hundred nine samples were chosen randomly out of around two hundred thousand samples. The panel consisted of SARS-CoV-2-positive (n = 156) and SARS-CoV-2-negative (n = 52) nasopharyngeal swab specimens for a primary clinical evaluation. Furthermore, 29 positive samples were sequenced using Oxford Nanopore Minion technology. Two hundred nine patient sample data of the cycle threshold (Ct) readings for target genes of five assays are 100% sensitive for Ct values. Mean Ct values for N1, N2, RdRp, S, and E of the positive controls in CDC assay, RealStar®, Allplex, GBC, and SD Biosensor were 17.5 ± 0.49, 16.9 ± 0.51, 20 ± 0.49, 21.7 ± 0.38, and 23.1 ± 0.43, respectively. F test value shows ≥ 1, which was statistically significant. All assays showed an efficiency of < 120% and R squares were < 0.99, which is well above the required threshold value. Thus, when taking the CDC-modified assay as a gold standard, the other four assays demonstrated a p value of 0.0000, concordance at 100%, and a Kappa at 1.000. A maximum-likelihood (ML) tree was constructed and compared based on full-length SARS-CoV-2 with Wuhan isolate. These isolates are closely related to the B.1.617 lineage and reference sequences. Therefore, we conclude that all RT-PCR kits assessed in this study shall be used for routine diagnostics of COVID-19 in patients.Supplementary informationThe online version contains supplementary material available at 10.1007/s00580-022-03356-y.

Project description:BackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.MethodsSARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.ResultsA total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.ConclusionsThese data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.

Project description:Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.

Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

Project description:BackgroundReal-time polymerase chain reaction (RT-PCR) testing for the identification of viral nucleic acid is the current standard for the diagnosis of SARS-CoV-2 infection, but technical issues limit its utilization for large-scale screening. Serological immunoglobulin M (IgM)/IgG testing has been proposed as a useful tool for detecting SARS-CoV-2 exposure.ObjectiveThe objective of our study was to compare the results of the rapid serological VivaDiag test for SARS-CoV-2-related IgM/IgG detection with those of the standard RT-PCR laboratory test for identifying SARS-CoV-2 nucleic acid.MethodsWe simultaneously performed both serological and molecular tests with a consecutive series of 191 symptomatic patients. The results provided by a new rapid serological colorimetric test for analyzing IgM/IgG expression were compared with those of RT-PCR testing for SARS-CoV-2 detection.ResultsOf the 191 subjects, 70 (36.6%) tested positive for SARS-CoV-2 based on RT-PCR results, while 34 (17.3%) tested positive based on serological IgM/IgG expression. Additionally, 13 (6.8%) subjects tested positive based on serological test results, but also tested negative based on RT-PCR results. The rapid serological test had a sensitivity of 30% and a specificity of 89% compared to the standard RT-PCR assay. Interestingly, the performance of both assays improved 8 days after symptom appearance. After 10 days had passed since symptom appearance, the predictive value of the rapid serological test was higher than that of the standard molecular assay (proportion of positive results: 40% vs 20%). Multivariate analysis showed that age >58 years (P<.01) and period of >15 days after symptom onset (P<.02) were significant and independent factors associated with serological test positivity.ConclusionsThe rapid serological test analyzed in this study seems limited in terms of usefulness when diagnosing SARS-CoV-2 infection. However, it may be useful for providing relevant information on people's immunoreaction to COVID-19 exposure.

Project description:Background: The global COVID-19 outbreak relies on a quantitative real-time polymerase chain reaction (qRT-PCR) for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2), to facilitate the roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in our setting, where the supply of testing reagents is limited. Methods: Standards generated from a serially-diluted positive control and previously identified positive/negative samples were used to determine the optimal volumes of the qRT-PCR reagents and to evaluate the validity and performance of four assays: Charité Berlin and European Virus Archive - GLOBAL (EVAg) primer-probe sets, and DAAN and Beijing Genomics Institute (BGI) premixed commercial kits. A multiplex and singleplex RT-PCR kit was used with the two primer-probe sets and the recommended assay volumes of the two premixed kits were altered. Results: In comparison to the multiplex RT-PCR kit, the singleplex RT-PCR kit combined with the primer-probe sets yielded consistent cycle threshold (Ct) values across the different titrations tested. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit showed incomparable Ct values and inconsistent results between batches using the manufacturer's recommended volumes. Conclusion: We achieved a 2.5-fold and 4-fold increase in the number of tests/kit for the premixed kits and the primer-probe sets, respectively. The primer-probe set assays were reliable and consistent, and we preferred a combination of an EVAg and a Berlin target. Any inconclusive result was repeated by different individuals following the same protocol. DAAN was a consistent and reliable assay even at lower concentrations from the stated recommendations. BGI in contrast, required dilution to improve its performance and was hence an assay that was used in combination with EVAg or Berlin targets.

Dataset Information

STANDARD M10 SARS-CoV-2 Assay for Rapid Detection of SARS-CoV-2: Comparison of Four Real-Time PCR Assays.

Publications

STANDARD M10 SARS-CoV-2 Assay for Rapid Detection of SARS-CoV-2: Comparison of Four Real-Time PCR Assays.

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