Rnase free zirconium oxide beads

The urine samples were vortexed for a minimum of 10 s to ensure homogeneity before 500 µl of sample was transferred to a 2ml safe-lock tube (Eppendorf, Hamburg, Germany) containing approximately 100 µl of RNase-free zirconium oxide beads (Next Advance, Inc., Troy, NY, USA) along with 20 µl of proteinase K (Invitrogen, Waltham, MA, USA). Samples were lysed using a TissueLyser (Qiagen Inc., Hilden, Germany) at 30 Hz for 5 min. A 150-µl aliquot of the lysed sample was then combined with 50-µl internal control (IC) in a 96-well plate (Roche, Germany) and loaded into a MagNA Pure 96 System (Roche, Germany) programmed according to the manufacturer's guidelines using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Germany) with an elution volume of 100 µl. A synthetic DNA IC was spiked (5 µl) into each sample prior to DNA extraction, and successful extraction was confirmed by positive detection of IC by PCR amplification. Each sample was also spiked with T7 bacteriophage DNA (Microbiologics, St. Cloud, MN, USA), delivering a final concentration of 1.2 × 10 7 PFU/ml of the sample. Copies of T7 were used for computing the absolute concentration of the target copies detected by PM.