Abstract The taxonomy of Burkholderia sensu lato (s.l.) has been revisited using genome-based tools, which have helped differentiate closely related species. Many species from this group are indistinguishable through phenotypic traits and 16S rRNA gene sequence analysis. Furthermore, they also exhibit whole-genome Average Nucleotide Identity (ANI) values in the twilight zone for species circumscription (95-96%), which may impair their correct classification. In this work, we provided an updated Burkholderia s.l. taxonomy focusing on closely related species and give other recommendations for those developing genome-based taxonomy studies. We showed that a combination of ANI and digital DNA-DNA hybridization (dDDH) applying the universal cutoff values of 95% and 70%, respectively, successfully discriminates Burkholderia s.l. species. Using genome metrics with this pragmatic criterion, we demonstrated that i) Paraburkholderia insulsa should be considered a later heterotypic synonym of Paraburkholderia fungorum; ii) Paraburkholderia steynii differs from P. terrae by harboring symbiotic genes; iii) some Paraburkholderia are indeed different species based on dDDH values, albeit sharing ANI values close to 95%; iv) some Burkholderia s.l. indeed represent new species from the genomic viewpoint; iv) some genome sequences should be evaluated with care due to quality concerns.

BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.

Abstract Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops.