Nuclisens minimag

Total RNA was extracted from 500 µl of shellfish homogenate using a NucliSENS^® miniMAG extraction machine and NucliSENS^® magnetic extraction reagents (BioMerieux) following the manufacturer’s instructions (eluting in 100 µl elution buffer). A negative (water only) extraction control sample was also prepared and tested in parallel with each set of samples extracted. Eluted RNA was stored at − 20 °C until required.

DNA was extracted from blood or urine samples using NucliSens® miniMAG™ (BioMérieux Inc., Durham, NC, USA) according to the manufacturer’s instructions. Urine samples were centrifuged (6.500 G-force, 25 °C, 25 min) and pellets were resuspended in 1 ml sterile phosphate-buffered saline (PBS – pH 7.2) prior to DNA extraction. Extracted DNA was subjected to PCR amplification using a previously reported Leptospira genus-specific protocol and primers targeting a 331 bp fragment of the 16S rRNA gene [18 (link)]. Cycling conditions were carried out as follows: 94 °C for 5 min, 40 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and a final extension at 72 °C for 5 min. Pure L. interrogans serovar Canicola (strain Hond Utrecht IV) genomic DNA was used as a positive control and DNase-free water as a negative control in all PCR runs. The amplified products were separated by electrophoresis on a 2% agarose gel stained with SYBR Safe DNA gel stain (Invitrogen, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and analyzed under UV transillumination.

RNA was extracted from 200 μl of samples by NucliSENS Lysis Buffer and NucliSENS® miniMAG® (bioMérieux, Marcy l’Etoile, France) according to manufacturer’s instructions.

A propidium monoazide (PMA) viability RTqPCR assay was performed as previously described [22 (link)], with minor modifications. Briefly, saliva samples were incubated with 50 μM PMAxx (Biotinum) and 0.5% Triton X-100 in the dark at room temperature (RT) for 10 min at 150 rpm and exposed to light for 15 min using a photoactivation system (Led-Active Blue, Geniul). Nucleic acid extraction was performed using the NucliSens^® miniMAG magnetic kit (BioMérieux) according to the manufacturer’s instructions, and viral genomes were quantified according to the ISO 15216-1:2017 method [18 ]. An aliquot of saliva which had not been treated with PMAxx was extracted simultaneously.

The concentrated sample underwent viral RNA extraction using the NucliSENS miniMAG semi-automated extraction system with magnetic silica, following the manufacturer’s instructions (bioMerieux, Marcy l’Etoile, France) with some modifications. In particular, the lysis phase was prolonged from 10 to 20 min, and a short centrifugation (2000× g, 1 min) was used to pellet the sediment. Additionally, instead of 50 μL, 100 μL of magnetic silica beads was added to each sample. The extracted nucleic acids were further purified by polymerase chain reaction (PCR) inhibitors using the OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine, CA, USA) and stored at −20 °C until molecular analysis.
After the RNA extraction, a portion of each sample was processed in the EnLab laboratory (Bari, Italy) for qualitative assessment, and a portion was transferred to the Istituto Superiore di Sanità laboratory (Rome, Italy, hereafter referred to as ISS) for quantitative assessment.

All strains were cultivated in the CTMA laboratory except for B. anthracis; cultivated strains were extracted using the fully automated EZ1 system (Qiagen, Venlo, The Netherlands). DNA was extracted from powder samples and swabs using the NucliSens miniMag semiautomated apparatus (bioMérieux, Boxtel, The Netherlands). DNA from spiked environmental soil samples was extracted using the PowerMax Soil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA). Extraction was carried out according to the manufacturer's instructions; the resulting DNA was eluted in Tris-HCl buffer (pH 8) and stored at −20°C. The DNA extractions performed at the Institute of Veterinary Bacteriology in Bern were performed with the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Lysates were carried out as described previously (38 (link)).