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Introduction: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. Methods: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. Results: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. Conclusion: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.
RESUMO
INTRODUCTION: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. METHODS: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. RESULTS: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. CONCLUSION: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Pandemias , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
INTRODUCTION: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and ribonuclease P genes. METHODS: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and Rnasa P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values ââ(Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. RESULTS: There were no significant differences in the Ct results between both techniques (P = .84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. CONCLUSIONS: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Reação em Cadeia da Polimerase , RNA Viral/análise , DNA Polimerase Dirigida por RNA/genética , Reprodutibilidade dos Testes , Ribonuclease P/genética , SARS-CoV-2/genéticaRESUMO
INTRODUCTION: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and RNase P genes. METHODS: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and RNase P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values (Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. RESULTS: There were no significant differences in the Ct results between both techniques (p = 0.84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. CONCLUSIONS: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.