RESUMO
SARS-CoV-2 variants surveillance is a worldwide task that has been approached with techniques such as Next Generation Sequencing (NGS); however, this technology is not widely available in developing countries because of the lack of equipment and limited funding in science. An option is to deploy a RT-qPCR screening test which aids in the analysis of a higher number of samples, in a shorter time and at a lower cost. In this study, variants present in samples positive for SARS-CoV-2 were identified with a RT-qPCR mutation screening kit and were later confirmed by NGS. A sample with an abnormal result was found with the screening test, suggesting the simultaneous presence of two viral populations with different mutations. The DRAGEN Lineage analysis identified the Delta variant, but there was no information about the other three mutations previously detected. When the sequenced data was deeply analyzed, there were reads with differential mutation patterns, that could be identified and classified in terms of relative abundance, whereas only the dominant population was reported by DRAGEN software. Since most of the software developed to analyze SARS-CoV-2 sequences was aimed at obtaining the consensus sequence quickly, the information about viral populations within a sample is scarce. Here, we present a faster and deeper SARS-CoV-2 surveillance method, from RT-qPCR screening to NGS analysis.
Assuntos
COVID-19/diagnóstico , Análise Mutacional de DNA/métodos , Genoma Viral/genética , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , Reprodutibilidade dos Testes , SARS-CoV-2/fisiologia , Sensibilidade e EspecificidadeRESUMO
Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.
Assuntos
Cobre/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Ferro/metabolismo , Lacase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fusarium/química , Fusarium/genética , Lacase/química , Lacase/genética , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologiaRESUMO
ABSTRACT Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.
Assuntos
Proteínas Fúngicas/metabolismo , Cobre/metabolismo , Lacase/metabolismo , Fusarium/enzimologia , Ferro/metabolismo , Doenças das Plantas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Solanum lycopersicum/microbiologia , Lacase/genética , Lacase/química , Fusarium/genética , Fusarium/químicaRESUMO
ABSTRACT Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.
RESUMO
Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.(AU)