Collaborative Equilibrium Coupling of Catalytic DNA Nanostructures Enables Programmable Detection of SARS-CoV-2.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID-19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS-CoV-2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme-DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi-enzyme catalysis; through system-wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single-copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS-CoV-2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID-19 patients.

Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2.

Despite the importance of nucleic acid testing in managing the COVID-19 pandemic, current detection approaches remain limited due to their high complexity and extensive processing. Here, we describe a molecular nanotechnology that enables direct and sensitive detection of viral RNA targets in native clinical samples. The technology, termed catalytic amplification by transition-state molecular switch (CATCH), leverages DNA-enzyme hybrid complexes to form a molecular switch. By ratiometric tuning of its constituents, the multicomponent molecular switch is prepared in a hyperresponsive state-the transition state-that can be readily activated upon the binding of sparse RNA targets to turn on substantial enzymatic activity. CATCH thus achieves superior performance (~8 RNA copies/µl), direct fluorescence detection that bypasses all steps of PCR (<1 hour at room temperature), and versatile implementation (high-throughput 96-well format and portable microfluidic assay). When applied for clinical COVID-19 diagnostics, CATCH demonstrated direct and accurate detection in minimally processed patient swab samples.

Head-to-head evaluation on diagnostic accuracies of six SARS-CoV-2 serological assays.

In this study, we evaluated and compared six SARS-CoV-2 serology kits including the Abbott SARS-CoV-2 IgG assay, Beckman Access SARS-CoV-2 IgG assay, OCD Vitros OCD Anti-SARS-CoV-2 Total antibody assay, Roche Elecsys Anti SARS-CoV-2 assay, Siemens SARS-CoV-2 Total assay, and cPass surrogate viral neutralising antibody assay. A total of 336 non-duplicated residual serum samples that were obtained from COVID-19 confirmed patients (n=173) on PCR and negative controls (n=163) obtained pre-December 2019 before the COVID-19 pandemic were used for the study. These were concurrently analysed on the different immunoassay platforms and correlated with clinical characteristics. Our results showed all assays had specificity ranging from 99.3% to 100.0%. Overall sensitivity across all days of symptoms, in descending order were OCD (49.1%, 95% CI 41.8-56.5%), cPass (44.8%, 95% CI 37.5-52.3%), Roche (41.6%, 95% CI 34.5-49.0%), Siemens (39.9%, 95% CI 32.9-47.3%), Abbott (39.8%, 95% CI 32.9-47.3%) and Beckman (39.6%, 95% CI 32.5-47.3%). Testing after at least 14 days from symptom onset is required to achieve AUCs greater than 0.80. OCD and cPass performed the best in terms of sensitivity for >21 days symptoms with 93.3% (95% CI, 73.5-99.2%) and 96.7% (95% CI, 82.8-99.9%), respectively. Both also shared the greatest concordance, kappa 0.963 (95% CI 0.885-1.0), p<0.001, and had the lowest false negative rates. Serology results should be interpreted with caution in certain cases. False negatives were observed in a small number of individuals with COVID-19 on immunosuppressive therapy, pauci-symptomatic or who received antiretroviral therapy. In conclusion, all assays exhibited excellent specificity and total antibody assays with spike protein configurations generally outperformed nucleocapsid configurations and IgG assays in terms of diagnostic sensitivity.

Comparative Clinical Evaluation of the Roche Elecsys and Abbott Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Serology Assays for Coronavirus Disease 2019 (COVID-19).

CONTEXT.­: The use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic tests detects antibodies in the host, contributing to the identification of individuals who have been exposed to coronavirus disease 2019 (COVID-19). OBJECTIVE.­: To critically evaluate 2 commercially available SARS-CoV-2 serology tests. DESIGN.­: A total of 333 unique, nonduplicated serum samples obtained from COVID-19 patients (n = 170) and negative controls (n = 163) obtained before December 2019 were used in the study. Samples were tested on the Roche E411 and Abbott Architect i4000SR platforms, and results were correlated to reverse transcription polymerase chain reaction (PCR) results and clinical symptoms. RESULTS.­: There was a strong level of agreement in the qualitative results between both assays, with a Cohen κ value of .840, P < .001. The specificity for both Roche and Abbott were excellent at 100%. Roche exhibited marginally better performance in the 21 days or more group with a sensitivity of 90.6% (95% CI, 75.8%-96.8%) versus an Abbott sensitivity of 84.4% (95% CI, 68.3%-93.1%), as well as in the 14- to 20-day group with a sensitivity of 85.7% (95% CI, 65.4%-95.0%) versus an Abbott sensitivity of 81.0% (95% CI, 60.0%-92.3%). Less than 14 days of symptoms groups exhibited poor sensitivity at less than 50% for both assays. The areas under curve (± standard error) for Roche (0.894 ± 0.025, P < .001) and Abbott (0.884 ± 0.026, P < .001) were very similar. Potential confounders for negative serologic results include antiretroviral medication use and pauci-symptomatic patients. CONCLUSIONS.­: Specificities for high-throughput Roche and Abbott immunoassays are excellent, but users need to be cautious to interpret serologic test results after 14 days of symptoms to avoid false negatives.