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Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.
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The appearance of new viruses and diseases has made the development of rapid and reliable diagnostic tests crucial. In light of it, we proposed a new method for assembling an electrochemical immunosensor, based on a one-step approach for selective layer formation. For this purpose, a mixture containing the immobilizing agent (polyxydroxybutyrate, PHB) and the recognition element (antibodies against SARS-CoV-2 nucleocapsid protein) was prepared and used to modify a screen-printed carbon electrode with electrodeposited graphene oxide, for the detection of SARS-CoV-2 nucleocapsid protein (N-protein). Under optimum conditions, N-protein was successfully detected in three different matrixes - saliva, serum, and nasal swab, with the lowest detectable values of 50 pg mL-1, 1.0 ng mL-1, and 50 pg mL-1, respectively. Selectivity was assessed against SARS-CoV-2 receptor-binding domain protein (RBD) and antibodies against yellow fever (YF), and no significant response was observed in presence of interferents, reinforcing the suitability of the proposed one-step approach for selective layer formation. The proposed biosensor was stable for up to 14 days, and the mixture was suitable for immunosensor preparation even after 60 days of preparation. The proposed assembly strategy reduces the cost, analysis time, and waste generation. This reduction is achieved through miniaturization, which results in the decreased use of reagents and sample volumes. Additionally, this approach enables healthcare diagnostics to be conducted in developing regions with limited resources. Therefore, the proposed one-step approach for selective layer formation is a suitable, simpler, and a reliable alternative for electrochemical immunosensing.
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Técnicas Biossensoriais , COVID-19 , Humanos , COVID-19/diagnóstico , Imunoensaio , SARS-CoV-2 , Anticorpos , Proteínas do NucleocapsídeoRESUMO
Accurate, self-collected, and non-invasive diagnostics are critical to perform mass-screening diagnostic tests for COVID-19. This systematic review with meta-analysis evaluated the accuracy, sensitivity, and specificity of salivary diagnostics for COVID-19 based on SARS-CoV-2 RNA compared with the current reference tests using a nasopharyngeal swab (NPS) and/or oropharyngeal swab (OPS). An electronic search was performed in seven databases to find COVID-19 diagnostic studies simultaneously using saliva and NPS/OPS tests to detect SARS-CoV-2 by RT-PCR. The search resulted in 10,902 records, of which 44 studies were considered eligible. The total sample consisted of 14,043 participants from 21 countries. The accuracy, specificity, and sensitivity for saliva compared with the NPS/OPS was 94.3% (95%CI= 92.1;95.9), 96.4% (95%CI= 96.1;96.7), and 89.2% (95%CI= 85.5;92.0), respectively. Besides, the sensitivity of NPS/OPS was 90.3% (95%CI= 86.4;93.2) and saliva was 86.4% (95%CI= 82.1;89.8) compared to the combination of saliva and NPS/OPS as the gold standard. These findings suggest a similarity in SARS-CoV-2 RNA detection between NPS/OPS swabs and saliva, and the association of both testing approaches as a reference standard can increase by 3.6% the SARS-CoV-2 detection compared with NPS/OPS alone. This study supports saliva as an attractive alternative for diagnostic platforms to provide a non-invasive detection of SARS-CoV-2.
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The SARS-CoV-2 worldwide outbreak prompted the development of several tools to detect and treat the disease. Among the new detection proposals, the use of peptides mimetics has surged as an alternative to avoid the use of antibodies, of which there has been a shortage during the COVID-19 pandemic. However, the use of peptides in detection systems still presents some questions to be answered, mainly referring to their stability under different environmental conditions. In this work, we synthesized an ACE2 peptide mimic and evaluated its stability in different pH, salinity, polarity, and temperature conditions. Further, the same conditions were assessed when using the ability of the peptide mimic to detect the recombinant SARS-CoV-2 spike protein in a biotin-streptavidin-enzyme-linked assay. Finally, we also tested the capacity of the peptide to detect SARS-CoV-2 from patients' samples. The results indicate that the peptide is structurally sensitive to the medium conditions, with relevance to the pH, where basic pH favored its performance when used as a SARS-CoV-2 detector. Further, the proposed peptide mimic was able to detect SARS-CoV-2 comparably to RT-qPCR results. Therefore, the present study promotes knowledge advancement, particularly in terms of stability considerations, in the application of peptide mimics as a replacement for antibodies in detection systems.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , RNA Viral , Pandemias , Peptídeos , Ligação ProteicaRESUMO
Fast, sensitive, simple, and cheap sensors are highly desirable to be applied in the health system because they improve point-of-care diagnostics, which can reduce the number of cases of infection or even deaths. In this context, here we report the development of a label-free genosensor using a screen-printed electrode modified with 2D-carbonylated graphitic carbon nitride (c-g-C3N4), poly(diallyldimethylammonium) chloride (PDDA), and glutathione-protected gold nanoparticles (GSH-AuNPs) for photoelectrochemical (PEC) detection of SARS-CoV-2. We also made use of Arduino and 3D printing to miniaturize the sensor device. The electrode surface was characterized by AFM and SEM techniques, and the gold nanoparticles by UV-Vis spectrophotometry. For SARS-CoV-2 detection, capture probe DNA was immobilized on the electrode surface. The hybridization of the final genosensor was tested with a synthetic single-strand DNA target and with natural saliva samples using the photoelectrochemistry method. The device presented a linear range from 1 to 10,000 fmol L-1 and a limit of detection of 2.2 and 3.4 fmol L-1 using cpDNA 1A and 3A respectively. The sensibility and accuracy found for the genosensor using cpDNA 1A using biological samples were 93.3 and 80% respectively, indicating the potential of the label-free and portable genosensor to detect SARS-CoV-2 RNA in saliva samples.
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Introduction: The COVID-19 pandemic is still in force, causing global public health challenges and threats. Although vaccination and herd immunity have proven to be the most efficient way to control the pandemic, massive and early testing of patients using the RT-qPCR technique is crucial for constant genomic surveillance. The appearance of variants of SARS-CoV-2 with new mutations can reduce the efficiency of diagnostic detection. In this sense, several commercial RT-qPCR kits have been the target of extensive analysis because low assay performance could lead to false-negative diagnoses. Methods: In this study, we evaluated the performance of three commercial RT-qPCR kits; Thermo Fisher (TaqMan 2019-nCoV Assay Kit v1), BGI and Roche (LightCycler® Multiplex RNA Virus Master) used for the diagnosis of COVID-19 throughout the pandemic in Santiago de Chile. Results: Under our best assay conditions, we found significant differences in Cq amplification values for control and viral probes, against the same nasopharyngeal swab samples (NPSs). In addition, in some cases, the sensitivity of the RT-qPCR kits decreased against viral variants. Conclusion: Our study suggests evaluating the RT-qPCR kits used to detect SARS-CoV-2 because variants such as Omicron, which has several mutations, can compromise their detection and underestimate viral circulation.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Pandemias , COVID-19/diagnóstico , Chile , Nasofaringe , RNA Viral/genética , RNA Viral/análise , Sensibilidade e EspecificidadeRESUMO
The COVID-19 pandemic has been monitored by applying different strategies, including SARS-CoV-2 detection with clinical testing or through wastewater-based epidemiology (WBE). We used the latter approach to follow SARS-CoV-2 dispersion in Tapachula city, located in Mexico's tropical southern border region. Tapachula is a dynamic entry point for people seeking asylum in Mexico or traveling to the USA. Clinical testing facilities for SARS-CoV-2 monitoring are limited in the city. A total of eighty water samples were collected from urban and suburban rivers and sewage and a wastewater treatment plant over 4 months in Tapachula. We concentrated viral particles with a PEG-8000-based method, performed RNA extraction, and detected SARS-CoV-2 particles through RT-PCR. We considered the pepper mild mottle virus as a fecal water pollution biomarker and analytical control. SARS-CoV-2 viral loads (N1 and N2 markers) were quantified and correlated with official regional statistics of COVID-19 bed occupancy and confirmed cases (r > 91%). Our results concluded that WBE proved a valuable tool for tracing and tracking the COVID-19 pandemic in tropical countries with similar water temperatures (21-29 °C). Monitoring SARS-CoV-2 through urban and suburban river water sampling would be helpful in places lacking a wastewater treatment plant or water bodies with sewage discharges.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Humanos , México/epidemiologia , Pandemias , RNA Viral/genética , Rios , SARS-CoV-2/genética , Esgotos , Águas Residuárias , ÁguaRESUMO
Timely detection of severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been the gold- strategy for identifying positive cases during the current pandemic. However, faster and less expensive methodologies are also applied for the massive diagnosis of COVID-19. In this way, the rapid antigen test (RAT) is widely used. However, it is necessary to evaluate its detection efficiency considering the current pandemic context with the circulation of new viral variants. In this study, we evaluated the sensitivity and specificity of RAT (SD BIOSENSOR, South Korea), widely used for testing and SARS-CoV-2 diagnosis in Santiago of Chile. The RAT showed a 90% (amplification range of 20 ≤ Cq <25) and 10% (amplification range of 25 ≤ Cq <30) of positive SARS-CoV-2 cases identified previously by RT-qPCR. Importantly, a 0% detection was obtained for samples within a Cq value>30. In SARS-CoV-2 variant detection, RAT had a 42.8% detection sensitivity in samples with RT-qPCR amplification range 20 ≤ Cq <25 containing the single nucleotide polymorphisms (SNP) K417N/T, N501Y and E484K, associated with beta or gamma SARS-CoV-2 variants. This study alerts for the special attention that must be paid for the use of RAT at a massive diagnosis level, especially in the current scenario of appearance of several new SARS-CoV-2 variants which could generate false negatives and the compromise of possible viral outbreaks.