Nuclisens easymag

RNA extraction from plasma samples were performed using the NucliSENS® easyMAG™ (Biomerieux, Durham, NC) automated nucleic acid extraction system according to the manufacturer's recommendations (NucliSENS easyMAG user manual, v 1.1; BioMérieux, Boxtel, Netherlands). Five hundred microlitres of each sample was placed in the disposable sample vessel and the sample vessel was loaded onto the extractor. After the initial lysis incubation for 10 min, 50 µl of magnetic silica was added to each sample and the extractor was restarted. The samples were eluted in 50 µl of extraction buffer 3. All samples were transferred to a 1.5-ml microcentrifuge tube and stored at −70°C.

Total nucleic acids were extracted from 400 µL of serum using NucliSENS easyMag (bioMérieux, Marcy-l'Étoile, France). Total nucleic acids extractions in concentrated sewage were preliminarily prepared with omnicleave endonuclease (epicenter, Lucigen, Middleton, US) to reduce the load of free nucleic acids in the samples. Then 300 µL concentrated sewage were extracted using NucliSENS easyMag (bioMérieux). RealStar HAV real-time PCR assay (version 1.0, Altona diagnostics, Hamburg, Germany) or in-house PCR assay (described below for sequencing) were used for molecular confirmation of HAV infection.

A volume of 150 µL of the clinical specimen or virus isolate material was inactivated in an equal volume of lysis buffer [NucliSENS easyMAG Lysis Buffer (1,000 mL) ref.280134; bioMérieux] that was spiked with a fragment of exogenous Soil-borne cereal mosaic virus (SBCMV) (gb:AJ298069) (18 (link)) synthetic transcript for internal process control purposes. The inactivated specimen preparations underwent nucleic acid extraction on either NucliSENS easyMAG (bioMérieux) or chemagic 360 (Perkin Elmer) extraction platform using NucliSENS easyMAG (bioMérieux) extraction reagents with a “Generic 2.0.1” protocol or Chemagic Viral DNA/RNA 300 Kit H96 (Perkin Elmer, CMG-1033-S), with default total nucleic acid elution volume of 100 µL per extracted specimen from any automated extraction platform.

Extraction of nucleic acids in the 29 anti-HEV IgM positive samples sent to the CMVL in Sweden was performed from a 250 μL serum mixed with 2 mL of lysis buffer (NucliSENSeasyMAG, bioMérieux, Marcy-l’Étoile, France). The mixture was incubated for 10 min at room temperature before the addition of 50 μL of NucliSENSeasyMAG Magnetic Silica and thereafter incubated for an additional 10 min. RNA was eluted in 110 μL of distilled water using the NucliSENSeasyMAG instrument according to the manufacturer’s instructions (bioMérieux).
cDNA synthesis and PCR amplification of the partial open reading frame (ORF) 1 region were performed as previously described [27 ,28 (link)]. The partial ORF2 region (778 nucleotides) was also amplified using 5 μL of cDNA and primers gt1-ORF2-S1: 5’-GCGGCCTACCGACAGAATTGATTTCGTC-3’ (at position 6247 of AY204877) and gt1-ORF2-AS1: 5’-TCCCGAGTTTTACCCACCTTCATYTTAAG-3’ (at position 7053). This product was semi-nested with primers gt1-ORF2-S2: 5’-ACGCCCAGTCGTCTCAGCCAATGG-3’ (at position 6299) and gt1-ORF2-AS1: 5’-TCCCGAGTTTTACCCACCTTCATYTTAAG-3’ (at position 7053) to yield a 778 bp fragment. The amplified fragments were purified and sequenced in both directions with the primers used in the PCR amplification, as previously described [28 (link)]. The sequences obtained in this study are deposited in GenBank with accession number KX879758-KX879765.

P. jirovecii DNA was extracted using the NucliSens^TM EasyMAG^TM technology (BioMérieux, Marcy-l’Étoile, France). PCR amplification targeting the mitochondrial large subunit ribosomal RNA gene using the forward primer pAZ102-H (5′-GTGTACGTTGCAAAGTACTC-3′), the reverse primer pAZ102-E (5′-GATGGCTGTTTCCAAGCCCA-3′) and an in-house probe (6FAM-TCTGGGCTGTTTCCCTTTCGACT) [8 (link)] was carried out using the LightCycler^® 480 Probes Master kit (Roche Diagnostics, Meylan, France) according to the manufacturer’s recommendations. A negative control (water), positive control (DNA from plasmids containing the target sequence), and extraction controls (albumin gene) were included in each run.

All C. glabrata isolates were typed with eight microsatellite markers (GLA2, GLA3, GLA4, GLA5, GLA6, GLA7, GLA8 and GLA9) as previously described by Brisse et al.37 (link). Genomic DNA was extracted using the NucliSENS^TM EasyMAG^TM (bioMérieux) system38 , eluted in 50 μl and stored at −20 °C. Amplification reactions were performed using a Lightcycler^TM 480 (Roche Diagnostics, GmbH, Germany) instrument with Lightcycler^TM 480 Probes Master (Roche Diagnostics, GmbH, Germany). The loci, primer sequences, fluorophores and hybridization temperatures are described in Table 4. The PCR products were visualized using 2% agarose gel electrophoresis in 1X of Tris borate EDTA buffer (Euromedex, France) with SYBR^TM safe DNA gel stain (Invitrogen, USA). Next, 1 μl of 1:100 diluted PCR products was mixed with a solution containing 25 μl HiDi formamide (Life Technologies, France) and 0.5 μl Gene Scan^TM 500 LIZ^TM size standard (Applied Biosystems, UK). The fragment length was determined via capillary electrophoresis using an ABI 3130 Genetic Analyzer (Applied Biosystems, France) and analyzed using GeneMapper software v4.0 (Applied Biosystems, France).