Hybrid Opto‐Thermocycler for RT‐qPCR Detects SARS‐CoV‐2

Although real‐time quantitative reverse transcription polymerase chain reaction (RT‐qPCR) is the gold standard for detecting the virus severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) and other pathogens, the coronavirus disease 2019 (COVID‐19) pandemic has highlighted the scarcity of instruments, devices, and reagents for polymerase chain reaction (PCR) testing in constrained settings. At least for under‐resourced countries, it has become critical to deploy instruments that can be rapidly constructed and satisfy this demand. Instead of separating the optical system from the thermal module (typical of qPCR thermocyclers), we report a portable Hybrid Opto‐Thermocycler—dubbed HybOT Cycler—that takes advantage of the high‐temperature tolerances (>100 °C) of electronic and optical components to combine thermal control, illumination, and fluorescence detection into a highly integrated hybrid module. This simple configuration allowed us to reduce the overall number of components, thus simplifying its assembly and reducing the instrument size. The HybOT Cycler is wirelessly controlled from an application installed in a tablet. PCR assays are carried out in a bubble‐free microfluidic device that can be easily replicated from an acrylic mold. Using the HybOT Cycler, down to 100 copies/µL of genetic material of the virus SARS‐CoV‐2 with 95% sensitivity and 100% specificity is detected. The HybOT Cycler can assist in diagnosing SARS‐CoV‐2 and other pathogens in resource‐poor settings. [ FROM AUTHOR] Copyright of Advanced Materials Technologies is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.

Hybrid Opto‐Thermocycler for RT‐qPCR Detects SARS‐CoV‐2

Although real‐time quantitative reverse transcription polymerase chain reaction (RT‐qPCR) is the gold standard for detecting the virus severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) and other pathogens, the coronavirus disease 2019 (COVID‐19) pandemic has highlighted the scarcity of instruments, devices, and reagents for polymerase chain reaction (PCR) testing in constrained settings. At least for under‐resourced countries, it has become critical to deploy instruments that can be rapidly constructed and satisfy this demand. Instead of separating the optical system from the thermal module (typical of qPCR thermocyclers), we report a portable Hybrid Opto‐Thermocycler—dubbed HybOT Cycler—that takes advantage of the high‐temperature tolerances (>100 °C) of electronic and optical components to combine thermal control, illumination, and fluorescence detection into a highly integrated hybrid module. This simple configuration allowed us to reduce the overall number of components, thus simplifying its assembly and reducing the instrument size. The HybOT Cycler is wirelessly controlled from an application installed in a tablet. PCR assays are carried out in a bubble‐free microfluidic device that can be easily replicated from an acrylic mold. Using the HybOT Cycler, down to 100 copies/µL of genetic material of the virus SARS‐CoV‐2 with 95% sensitivity and 100% specificity is detected. The HybOT Cycler can assist in diagnosing SARS‐CoV‐2 and other pathogens in resource‐poor settings. [ FROM AUTHOR]

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

Recombinant Antigens Based on Non-Glycosylated Regions from RBD SARS-CoV-2 as Potential Vaccine Candidates against COVID-19.

The Receptor-Binding Domain (RBD) of the Spike (S) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has glycosylation sites which can limit the production of reliable antigens expressed in prokaryotic platforms, due to glycan-mediated evasion of the host immune response. However, protein regions without glycosylated residues capable of inducing neutralizing antibodies could be useful for antigen production in systems that do not carry the glycosylation machinery. To test this hypothesis, the potential antigens NG06 and NG19, located within the non-glycosylated S-RBD region, were selected and expressed in Escherichia coli, purified by FPLC and employed to determine their immunogenic potential through detection of antibodies in serum from immunized rabbits, mice, and COVID-19 patients. IgG antibodies from sera of COVID-19-recovered patients detected the recombinant antigens NG06 and NG19 (A450 nm = 0.80 ± 0.33; 1.13 ± 0.33; and 0.11 ± 0.08 for and negatives controls, respectively). Also, the purified antigens were able to raise polyclonal antibodies in animal models evoking a strong immune response with neutralizing activity in mice model. This research highlights the usefulness of antigens based on the non-N-glycosylated region of RBD from SARS-CoV-2 for candidate vaccine development.