RESUMO
BACKGROUND: The novel coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has quickly spread worldwide since its outbreak in December 2019. One of the primary measures for controlling the spread of SARS-CoV-2 infection is an accurate assay for its diagnosis. SARS-CoV-2 real-time PCR kits suffer from some limitations, including false-negative results in the clinic. Therefore, there is an urgent need for the development of a rapid antibody test kit for COVID-19 diagnosis. METHODS: The nuclear capsid protein (N) and spike protein 1 (S1) fragments of SARS-CoV-2 were expressed in Escherichia coli, and rapid antibody-based tests for the diagnosis of SARS-CoV-2 infection were developed. To evaluate their clinical applications, the serum from COVID-19 patients, suspected COVID-19 patients, recovering COVID-19 patients, patients with general fever or pulmonary infection, doctors and nurses who worked at the fever clinic, and health professionals was analyzed by the rapid antibody test kits. The serum from patients infected with Mycoplasma pneumoniae and patients with respiratory tract infection was further analyzed to test its cross-reactivity with other respiratory pathogens. RESULTS: A 47 kDa N protein and 67 kDa S1 fragment of SARS-CoV-2 were successfully expressed, purified, and renatured. The rapid antibody test with recombinant N protein showed higher positive rate than the rapid IgM antibody test with recombinant S1 protein. Clinical evaluation showed that the rapid antibody test kit with recombinant N protein had 88.56 % analytical sensitivity and 97.42 % specificity for COVID-19 patients, 53.48 % positive rate for suspected COVID-19 patients, 57.14 % positive rate for recovering COVID-19 patients, and 0.5-0.8 % cross-reactivity with other respiratory pathogens. The analytical sensitivity of the kit did not significantly differ in COVID-19 patients with different disease courses (p < 0.01). CONCLUSIONS: The rapid antibody test kit with recombinant N protein has high specificity and analytical sensitivity, and can be used for the diagnosis of SARS-CoV-2 infection combined with RT-PCR.
Assuntos
Anticorpos Antivirais , Teste Sorológico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2 , Teste para COVID-19 , Humanos , Proteínas Recombinantes , SARS-CoV-2/imunologiaRESUMO
The in vivo functions of Hje and Hjc, two Holliday junction resolvases in Sulfolobus islandicus were investigated. We found that deletion of either hje or hjc had no effect on normal cell growth, while deletion of both hje and hjc is lethal. Although Hjc is the conserved resolvase in all archaea, the hje deletion rather than hjc deletion rendered cells more sensitive to DNA-damaging agents such as hydroxyurea, cisplatin, and methyl methanesulfonate than the wild type (WT). Intriguingly, the sensitivity of Δhje could not be rescued by ectopic expression of Hje from a plasmid and Hje overexpression slowed growth and large cells appeared with more than two genome equivalents. We showed that Hje was maintained at a low level in WT cells. Furthermore, transcriptomic microarray analysis revealed that the abundance of transcripts of many genes including those involved in DNA replication, repair, transcription regulation, and cell division changed drastically in the Hje-overexpressed strain. However, only limited genes were up- or downregulated in the hje deletion strain. Our findings collectively suggest that Hje is the primary resolvase involved in DNA repair and its expression must be tightly controlled in cells.