ABSTRACT
In December 2019, the Chinese Center for Disease Control (CDC of China) reported an outbreak of pneumonia in the city of Wuhan (Hubei province, China) that haunted the world, resulting in a global pandemic. This outbreak was caused by a betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several of these cases have been observed in healthcare professionals working in hospitals and providing care on the pandemic's frontline. In the present study, nasopharyngeal swab samples of healthcare workers were used to assess the performance of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and subsequently compared with the real-time reverse-transcription quantitative PCR (RT-qPCR) method. Thus, in this study, we validated a method for detecting SARS-CoV-2 based on RT-LAMP that can be used to diagnose these workers. The methodology used was based on analyzing the sensitivity, specificity, evaluation of the detection limit, and cross-reaction with other respiratory viruses. The agreement was estimated using a dispersion diagram designed using the Bland-Altman method. A total of 100 clinical specimens of nasopharyngeal swabs were collected from symptomatic and asymptomatic healthcare workers in Pelotas, Brazil, during the SARS-CoV-2 outbreak. RT-LAMP assay, it was possible to detect SARS-CoV-2 in 96.7% of the healthcare professionals tested using the E gene and N gene primers approximately and 100% for the gene of human ß-actin. The observed agreement was considered excellent for the primer set of the E and N genes (k = 0.957 and k = 0.896), respectively. The sensitivity of the RT-LAMP assay was positive for the primer set of the E gene, detected to approximately 2 copies per reaction. For the primer set of the N gene, the assay was possible to verify an LoD of approximately 253 copies per reaction. After executing the RT-LAMP assay, no positive reactions were observed for any of the virus respiratory tested. Therefore, we conclude that RT-LAMP is effective for rapid molecular diagnosis during the COVID-19 outbreak period in healthcare professionals.
ABSTRACT
The severe acute respiratory syndrome originated by the new coronavirus (SARS-CoV-2) that emerged in late 2019, known to be a highly transmissible and pathogenic disease, has caused the COVID-19 global pandemic outbreak. Thus, diagnostic devices that help epidemiological public safety measures to reduce undetected cases and isolation of infected patients, in addition to significantly help to control the population's immune response to vaccine, are required. To address the negative issues of clinical research, we developed a Diagnostic on a Chip platform based on a disposable electrochemical biosensor containing laser-induced graphene and a protein (SARS-CoV-2 specific antigen) for the detection of SARS-CoV-2 antibodies. The biosensors were produced via direct laser writing using a CO2 infrared laser cutting machine on commercial polyimide sheets. The presence of specific antibodies reacting with the protein and the K3[Fe(CN)6] redox indicator produced characteristic and concentration-dependent electrochemical signals, with mean current values of 9.6757 and 8.1812 µA for reactive and non-reactive samples, respectively, proving the effectiveness of testing in clinical samples of serum from patients. Thus, the platform is being expanded to be measured in a portable microcontrolled potentiostat to be applied as a fast and reliable monitoring and mapping tool, aiming to assess the vaccinal immune response of the population.