Nuclisens easymag magnetic silica

HCV-RNA from DBS was extracted with either lysis buffer, protease K or plain, purified H2O. Plain water turned out to give the best yield with our RNA extraction procedure. One DBS was dissolved in 1mL H2O overnight. The samples were spun down at 10.800 G for 10 minutes.
We tested 500 and 100 μL of the supernatant, and 100 μL gave the best yield, whereas 500 μL resulted in some inhibition. Total RNA was isolated from 100 μL supernatant using NucliSENS easyMAG Magnetic Silica (bioMérieux, Marcy l’Etoile, France). Detection and quantification of viral nucleotide sequences were performed by In house real-time PCR using molecular beacons on a Mx3005P Real-Time PCR System (Stratagene, La Jolla, USA). We also tested our standard routine method Abbott RealTime HCV, but were not able to get meaningful results. Antibodies against HCV (anti-HCV) were tested using our routine chemiluminescence assay (anti-HCV, Architect; Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s recommendation only using supernatant instead of plasma.

Stool DNA was extracted with the NucliSENS^® easyMAG^® automated system (BioMérieux, Marcy-l’Etoile, France) following the protocol from Jeddi et al., 2013 [29 (link)]. Briefly, 400 mg of stool sample was homogenized with 1 mL of NucliSENS^® lysis buffer (BioMérieux, Marcy-l’Etoile, France) in a Lysing Matrix E tube (i.e., containing ceramic, silica and glass beads) (MP Biomedicals, Illkirch, France). It then underwent mechanical grinding using a FastPrep^®-24 (MP Biomedicals, Illkirch, France) at 6.0 m/s for 1 min. The stool suspension was then incubated at room temperature for 10 min before being centrifuged at 10,000× g for 10 min. Finally, 250 μL of supernatant was transferred in the DNA extraction NucliSENS^® easyMAG^® automated system (BioMérieux, Marcy-l’Etoile, France) with 50 µL of NucliSENS^® EasyMAG^® magnetic silica (Biomérieux, Marcy-l’Etoile, France). Elution was performed at RT with 100 μL of elution buffer. The eluted DNA volume obtained (100 μL) was then stored at −20 °C.

HBV DNA measurements, genotyping, mutation analysis including detection of pre-core mutation were performed at Section of Molecular Diagnostics, Aalborg University Hospital, Denmark. Total DNA was isolated from 500 µl plasma using NucliSENS easyMAG Magnetic Silica (bioMérieux, Marcy l'Etoile, France) and eluted with 125 µl H₂O. Detection and quantification of viral nucleotide sequences were performed by real-time PCR using molecular beacons on a Mx3005P Real-Time PCR System (Stratagene, La Jolla, USA). For HBV quantification, HBV-Ab and -Bb were selected from the pre-core region, amplifying a product of 189 bp. Limit of detection was 15 IU/ml (40 copies/ml). To detect pre-core mutations, sample DNA was added to each of two vials, each containing the reaction mix with 1 µmol/l of HBV 2N (wild type) or 1 µmol/l of HBV 2M (pre-core mutant). Genotype specific primer pairs and a common beacon probe from the pre-S region of the genome were used. Determination of HBV resistance was performed using direct sequencing (ABI PRISM 3130xl Genetic Analyzer, Applied Biosystems, Foster City, CA, USA) of the polymerase gene. These methods including selection of primers have been presented previously [30] (link), [31] (link).