Qiaamp minelute virus spin kit

Nucleic acids were extracted from samples stored in 200 μl of universal transport medium and eluted in 60 μl of elution buffer using QiaAmp MinElute virus spin kits (Qiagen, France) according to the manufacturer's instructions. An internal control (T4 and MS2 phages) was added to each extraction tube to assess the quality of the extraction at the end of the amplification [13 (link)].

Supernatants from control and Dox-induced iSLK-BAC16 cells were collected, filtered, and extracted for viral DNA using QIAamp MinElute Virus Spin kits (Qiagen, 57704) following the manufacturer’s protocol. Quantification was performed by TaqMan quantitative PCR targeting Orf73 (5’-FAM/TCA GAA CAT CAC CAC CCC ACA GAC/BHQ-3’) [37 (link)].

Viral nucleic acid was extracted from 200 μL of sample using QIAamp MinElute Virus Spin Kits (Qiagen, Germany). cDNA was synthesized using an AMV reverse transcriptase and random hexamer primers (Promega, United States), as described previously (Lu et al., 2012 (link)). Nested RT-PCR targeting of the 5′-UTR was employed for HRV screening, and of the VP4–VP2 regions for genotyping. All (totally nine, except HEV IS only) of HRV or HRV/HEV primers from 5-UTR to VP4–VP2 were used to detect HRV, as described previously (Wisdom et al., 2009 (link)). Specimens from which amplification of the VP4–VP2 regions failed were defined as untyped. PCR products were confirmed by sequencing. Phylogenetic analysis was conducted using Molecular Evolutionary Genetics Analysis (MEGA) software (ver. 7).

Sputum was collected within 72 hours of patient admission. Nebulisation was used for younger children. Induced sputum samples from the inhalation of hypertonic saline solution were collected by trained personnel according to standard operating procedures, as published previously (Lahti et al., 2009 (link); Honkinen et al., 2012 (link)).
A total of 2 mL of sputum was obtained from each patient with sterility sputum aspiratory tubes and stored at -80°C. The nucleic acids of the viruses were extracted using QIAamp MinElute Virus Spin kits (QIAGEN, Hilden, Germany), and cDNA was synthesized using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To extract bacterial DNA, 200 μL of sputum were centrifuged at 5000 × g for 10 min, and pellets resuspended in 150 μL enzyme cocktail containing 6 mg lysozyme, 30 U lysostaphin, 37.5 U mutanolysin, and 30U lyticase in lysis buffer of 20 mM Tris-HCl (pH 8), 2 mM EDTA, and 1.2% Triton. The mixture was incubated at 37°C for 30 min to lyse the cell walls of Gram-positive bacteria. DNA was then purified using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purified DNA was eluted in 200 μL nuclease-free water.