Magna pure dna tissue lysis buffer

Pure cultures of each strain were incubated in 750 μL lysis buffer (MagNA Pure DNA Tissue Lysis Buffer, Roche, Mannheim, Germany) and 75 μL proteinase K (proteinase K, lyophilisiert, ≥30 U/mg, Carl Roth GmbH & Co KG, Karlsruhe, Germany) for one hour at 65 °C. From this, 200 μL were utilized for automated nucleic acid (NA) extraction using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The resulting NAs were eluted in a volume of 100 μL. The isolated NAs were kept at −18 °C until the PCR tests were performed. The DNA used for the dilution series was extracted manually using the QIAamp^® DNA MicroKit (50) (Qiagen, Hilden, Germany), and the DNA concentration was measured with a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Inc., Wilmington, NC, USA).

All culture plates except MAC (regardless of visible bacterial growth) were washed with 3 mL sterile PBS and bacterial colonies were released by gently scraping agar surface with a sterile cell scraper. Bacteria from 1 mL of the resulting suspension were collected by centrifugation, resuspended in 0.2 mL MagNA Pure DNA Tissue Lysis Buffer (Roche) and stored at −80 °C until extraction. DNA was extracted from patient samples and plate washes using the QIAamp UCP Pathogen Mini Kit (Qiagen) with mechanical disruption of samples with 1.4-mm ceramic beads followed by enzymatic lysis via proteinase K. Next generation sequencing libraries were prepared and DNA sequencing was performed as previously described^{14 (link)}. Briefly, the 16S v1–v2 region was amplified using custom primers incorporating Illumina-compatible sequencing adaptors and a sample-specific 8-bp barcode sequence; paired-end sequencing was performed on an Illumina Miseq using a 500-cycle sequencing kit (version 2) to a minimum read depth of 50,000 reads per sample. Sequence data generated for this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession no. PRJNA555084.

Nasopharyngeal swabs (all dogs) and rectal swabs (sick dogs only) were collected and analyzed by Laboklin GmbH & Co. (Bad Kissingen, Germany) using conventional PCR or real-time PCR (qPCR and RT-qPCR). Swabs were incubated in 750 µL MagNA Pure DNA Tissue Lysis Buffer (Roche Diagnostics GmbH, DE-Mannheim, Germany) plus 75 µL Proteinase K (Carl Roth GmbH + Co. KG, DE-Karlsruhe, Germany) for 1 h at 65 °C. Automated isolation of nucleic acids (RNA and DNA) was performed with the MagNA Pure 96 system from Roche Diagnostics GmbH according to manufacturer's instructions. Nasopharyngeal swabs from sick dogs were tested for canine adenovirus type 2 (CAV-2) [16 (link)], Bordetella bronchiseptica [17 (link)], CDV [18 (link)], canine parainfluenza virus (CPIV) (in-house method), canine influenza A virus (CIV) [19 (link)] and canine herpesvirus-1 (Canid alphaherpesvirus-1: CaHV-1) [20 (link)] by Taqman real-time PCR on a LightCycler®96 (Roche Diagnostics, Basel, Switzerland) and for Mycoplasma spp. [21 (link)] by conventional PCR. Swabs from all sick dogs, and swabs from healthy dogs that presented α-SARS-CoV-2 IgG, were also tested for SARS-CoV-2 [22 ] by Taqman real-time PCR on a LightCycler®96 (Roche Diagnostics).