Journal of virological methods
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Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and affordable. Many assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or are expensive with suboptimal specificity (e.g. commercial ELISAs and RDTs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies against multiple antigens and against other pathogens. ⋯ While we show that neutralizing antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of the recombinant nucleocapsid protein (NP) and receptor-binding domain (RBD) results in highly specific (99 %) IgG antibody detection five months after infection in 96 % of cases. Although most severe Covid-19 cases developed a clear IgM and IgA response, titers fell below the detection threshold in more than 20 % of mild cases in our bead-based assay. In conclusion, our data supports the use of RBD and NP for the development of SARS-CoV-2 serological IgG bead-based assays.
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There is an ongoing need for reliable antigen assays for timely and easy detection of individuals with acute SARS-CoV-2 infection. Using 75 swabs from patients previously tested positive by SARS-CoV-2 PCR and 75 swabs from patients previously tested negative by SARS-CoV-2 PCR, we investigated the sensitivity and specificity of the SARS-CoV-2 Rapid Antigen Test (Roche). ⋯ We conclude that sensitivity and specificity of the antigen assay is inferior to the PCR assay. However, the antigen assay may be a quick and easy to perform alternative for differentiation of individuals contagious for SARS-CoV-2 from non-contagious individuals.
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Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the "gold standard" for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. ⋯ The analytical sensitivity of the assay was about 7.15-9 copies of vRNA/μL, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 μL final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RT-qPCR is not available.
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Comparative Study
SARS-CoV-2: Comparative analysis of different RNA extraction methods.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. ⋯ It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.
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The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. ⋯ Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.