Magna pure compact instrument

Two hundred fifty to 500 mg of stool sample were suspended in 1 mL of PBS and frozen at -20°C before DNA extraction. Extraction was performed on 200 μL of stool suspension after bead-beating, using High Pure PCR Template Preparation Kit (Roche Diagnostics, Meylan, France) according to the manufacturer’s instructions.
For blood and urine, DNA extraction was performed using 1 mL of serum or 1 mL of urine pellet and eluted in a volume of 50 μL, using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche Diagnostics) on a MagNA Pure Compact Instrument (Roche Diagnostics).

Genomic DNA was extracted from peripheral blood samples. DNA was obtained out of 400 µL of whole blood in the MagNA Pure Compact Instrument (Roche Diagnostics, Mannheim, Germany). The DNA was diluted with nuclease-free water (Ambion, Austin, TX, USA) in every case at 12.5 ng/µL.

This protocol yielded total nucleic acids and was the only automated extraction method in this study that used the MagNA Pure Compact Instrument (Roche Diagnostics, Mannheim, Germany), which is a magnetic bead-based technology. Briefly, a pretreatment step was performed using MagNA Pure Bacteria Lysis Buffer (BLB) (Roche Diagnostics, Mannheim, Germany) following the product instructions with slight modifications to ensure complete lysis of the bacteria. First, 200 µL of the mock sample was mixed with 180 µL of BLB. Then, 10 µL of 100 mg/mL lysozyme was added, and the mixture was incubated at 37 °C for 30 min in a ThermoMixer (Eppendorf, Hamburg, Germany) with mixing at 700 rpm. After incubation, 20 µL of 10 mg/mL proteinase K was added and incubated at 65 °C for 10 min. The treated samples were cooled on ice and then extracted with the MagNA Pure Compact NA Isolation Kit I. The eluted samples were labeled “MagNA”.

Microbiological samples were obtained by tracheal aspiration (intubated infants) or nasopharyngeal swabs (non-intubated infants). In 2009 and 2010, 36 patient samples were tested with the Mycofast Screening Revolution (EliTech Diagnostic, Puteaux, France) assay based on liquid broth cultures performed according to the manufacturer’s instructions. The samples were further inoculated and cultured on a mycoplasma-selective A8 agar plate. After 24-48 hours of incubation, Ureaplasma colonies were observed with a stereomicroscope at 60 × magnification. In 2011, 13 (62%) samples were cultured as described, and six (29%) samples were tested with a specific multiplex real-time polymerase chain reaction (PCR, Allplex STI Essential Assay, Seegene, Seoul, South Korea). Two (9%) samples were both cultured and tested with PCR. DNA was extracted from the specimens by using the automated MagNA Pure Compact instrument and the MagNA Pure Compact Nucleic Acid Isolation Kit I with Bacteria Lysis Buffer and Proteinase K (all from Roche Diagnostics, Mannheim, Germany) pretreatment. Real-time PCR was performed on a CFX96 platform (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer´s instructions. In 2012 and afterwards, all the samples were tested with specific multiplex real-time PCR.