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Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2.

ABSTRACT: Accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great importance to control the COVID-19 pandemic. The gold standard assays for COVID-19 diagnostics are mainly based on separately detecting open reading frame 1ab (ORF1ab) and nucleoprotein (N) genes by RT-PCR. However, the current approaches often obtain false positive-misdiagnose caused by cross-contamination or undesired amplification. To address this issue, herein, we proposed a dumbbell-type triplex molecular switch (DTMS)-based, logic-gated strategy for high-fidelity SARS-CoV-2 RNA detection. The DTMS consists of a triple-helical stem region and two-loop regions for recognizing the ORF1ab and N genes of SARS-CoV-2. Only when the ORF1ab and N gene are concurrent, DTMS experiences a structural rearrangement, thus, bringing the two pyrenes into spacer proximity and leading to a new signal readout. This strategy allows detecting SARS-CoV-2 RNA with a detection limit of 1.3 nM, independent of nucleic acid amplification, holding great potential as an indicator probe for screening of COVID-19 and other population-wide epidemics.

SUBMITTER: Chen T

PROVIDER: S-EPMC9420052 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2.

Chen Ting T Liu Pengfei P Wang Huanxiang H Su Yue Y Li Sheng S Ma Shimeng S Xu Xuan X Wen Jie J Zou Zhen Z

Sensors and actuators. B, Chemical 20220828

Accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great importance to control the COVID-19 pandemic. The gold standard assays for COVID-19 diagnostics are mainly based on separately detecting open reading frame 1ab (ORF1ab) and nucleoprotein (N) genes by RT-PCR. However, the current approaches often obtain false positive-misdiagnose caused by cross-contamination or undesired amplification. To address this issue, herein, we proposed a dumbbell-type triplex m ...[more]

PMID: 36059586

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Project description:Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Project description:There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance along with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the probable causes of false-positive reactions were determined. The assays were evaluated using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of false positive results from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, assays lacked reproducibility. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with the same antigen that was used for coating ELISAs prior to performing the assay), suggesting positive reaction could be attributed to the presence of antibodies against these antigens. In other cases, reactivity was consistently detected but not abrogated by the spiking, suggesting positive reaction was not attributed to the presence of antibodies against these antigens. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the corresponding antigen, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

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Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2.

Publications

Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2.

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