Nuclisens easymag platform

Upon arrival in the laboratory, all specimens were processed for DNA automated extraction. Twenty mg of fresh tissue from tonsils and adenoids were diced into small pieces, suspended for digestion in a mixture composed by 400 μL of easyMAG lysis buffer (bioMérieux, France) and 40 μL of proteinase K (200 μg/mL), and incubated at 56 °C overnight. Stool specimens were dissolved in sterile water, vortexed and clarified by centrifugation at 6000 g for 10 min (min), and the supernatant recovered for extraction. Total available urines were collected in a 50 mL tube, centrifuged at 4500 g for 20 min and the pellet suspended in sterile water. Whole EDTA blood (100 μL) was diluted in easyMAG lysis buffer (900 μL) at room temperature for 10 min before extraction.
DNA isolation was performed using the NucliSenseasyMAG platform (bioMérieux, France), according to the manufacturer’s protocol. DNA was eluted to a final volume of 50 μL and stored at −80 °C until analysis.

Viral RNA was isolated from plasma samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) or by automatic extraction with the NucliSENS easyMAG platform (Biomerieux, Nürtingen, Germany). HIV-1 genotyping was performed on extracted RNA using the ViroSeq HIV-1 Genotyping System (Abbott, Wiesbaden, Germany) with subsequent population-based Sanger sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Dreieich, Germany) or an inhouse HIV-1 genotyping assay covering the protease (PR) and reverse transcriptase (RT) with subsequent population-based Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Dreieich, Germany) [26 (link)]. Sequence analysis was performed on an ABI Prism 310 capillary sequencer (Thermo Fisher Scientific, Germany). SeqMan Pro (Lasergene v10.0.1, DNASTAR, USA) was used to generate the consensus sequence. In addition, some HIV-1 sequences were provided by participating study centers. HIV-1 subtypes were assessed with the REGA HIV-1 subtyping tool Version 3.0 [27 (link)].
Sequences are available at GenBank with accession numbers MH470511 to MH472562 and as published previously [18 (link), 28 (link)].

Approximately 0.2 g of stool samples was added to a 5 mL eppendorf tube, which contained 2 mL pH 7.2 phosphate buffered saline (PBS). After being vortexed for 30 sec, samples were clarified by centrifugation at 8,000 × g for 5 min at room temperature. Nucleic acid was extracted from the suspension. Nucleic acid was extracted from a 250 μl suspension using the NucliSens easyMAG platform with the NucliSens magnetic extraction reagents (bioMe´rieux, The Netherlands) according to the manufacturer’s instruction. The elution volume of nucleic acid was 50 μl.

This study enrolled, all B. melitensis strains that were sent to the reference laboratory for human Brucellosis at the Portuguese National Institute of Health during the last nine years, comprising 37 isolates. Genotyping and demographic data are summarized in Table 1. For genomic comparative purposes, it also included 18 strains isolated in Spain, Germany, Hungary and Belgium, which were kindly provided to our lab and that were subjected to all laboratory procedures and analysis (described below). For bioinformatics analysis, all B. melitensis genome sequences available at NCBI until January 2019 (n = 217) were also included yielding a total of 272 B. melitensis genomes to be analyzed.
All samples were handled in a BLS-3 biocontainment laboratory at the Portuguese National Institute of Health. Brucella isolates were cultured on blood agar for 3 to 5 days at 37° C under 5% CO2 and total DNA was extracted from fresh cultures on the NucliSens easyMAG platform (Biomerieux), according to the manufacturer’s instructions. All isolates had previously been confirmed as Brucella spp. by real-time PCR detecting the Brucella specific gene IS711, BME and Brab [20 ]. For simplification purposes, all Brucella isolates from the Portuguese collection that are enrolled in this study are designated with the prefix “PT” throughout the text.