Dna and viral na small volume kit

Total nucleic acid (NA) was extracted from respiratory samples using the DNA and Viral NA Small Volume Kit and the MagNA Pure 96 instrument (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Extracted NA were tested with a commercially available test (NxTAG RPP, Luminex Corporation, Austin, TX, USA) for respiratory viruses including EVs and rhinoviruses. According to the manufacturer, the limit of detection for rhinovirus/enterovirus in the Luminex assay was determined to be 1.1 × 10³ copies/mL sample.
The presence of EV RNA was confirmed by a RT-qPCR targeting the highly conserved 5′UTR [18 (link)] with a cycle threshold value ≤ 40. In case of EV-positive results, partial VP1 viral capsid gene region was amplified using primers AN88 and AN89 [4 (link),18 (link)]. DNA amplicons were gel-purified (Wizard SV Gel and PCR Clean-Up System, Promega Corporation, Madison, WI, USA) and sequenced using BigDyeTerminator v1.1 Cycle Sequencing Kit on a 3500 Genetic Analyzer (both, Thermo Fisher Scientific, Applied Biosystem, Waltham, MA, USA). Sequences were analysed (Geneious Prime Biomatters, Auckland, New Zealand) and genotyped (Enterovirus Genotyping Tool Version 0.1, National Institute for Public Health and the Environment, Bilthoven, The Netherlands) [19 (link)]. Identified sequences were submitted to GenBank (accession no. MW731790 to MW732022).

Total NA was extracted from 200 μL of each respiratory sample using the DNA and Viral NA Small Volume Kit with a MagNA Pure 96 instrument (both, Roche, Mannheim, Germany) according to the manufacturer’s instructions. Nucleic acids were stored in aliquots at −80 °C until further use. The presence of genomes of common respiratory viruses was assessed using a multiplex panel assay (NxTAG RPP, Luminex corporation, Austin, TX, USA) according to the manufacturer’s instructions. The panel included influenza viruses A and B, respiratory syncytial viruses A and B, parainfluenza viruses 1 to 4, human coronaviruses (including 229E, NL63, OC43, and HKU1), human metapneumoviruses, adenoviruses, human bocaviruses, rhinoviruses, and enteroviruses. Samples that reacted to the combined enterovirus/rhinovirus target of the assay were further analyzed to determine the presence of rhinovirus-specific RNA. In brief, one-step real-time RT-PCR was performed using the QuantiFast^® Multiplex kit (Qiagen, Hilden, Germany) based on a previously proposed protocol and a modified forward primer, which provides increased binding strength due to the incorporation of locked nucleic acids (5’-CY+AGCCTGCGTGGC-3′) [27 (link)]. For a detailed description of the reaction conditions, see Supplementary Tables S1–S3.

DNA was extracted from swabs using an enzymatic prelysis step (30 min incubation at 37 °C with an enzyme solution containing 4 U lysostaphine (SAE0091), 25 U mutanolysin (sae0092), and 3 mg lysozyme (L4919) (Sigma-Aldrich, St. Louis, MO, USA); then 30 min incubation at 56 °C with 20 µL protein kinase K (RPROTKSOL-RO, Sigma-Aldrich, St. Louis, MO, USA), followed by DNA extraction on a MagNa-Pure 96 robot using a DNA and Viral NA Small Volume Kit (Roche, Mannheim, Germany).
The V3-V4 region of the 16S rRNA gene and that of the tuf gene were amplified in two separate PCRs (95 °C for 3 min; 25 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 45 s; 72 °C for 5 min), using primers (16SrRNA: 341F: 5′- CCTACGGGNGGCWGCAG -3′; 805R: 5′- GACTACHVGGGTATCTAATC-3′; tuf: F: 5′- CAGAAGAAAAAGAACGTGG-3′; R: 5′- GTCCTCAACWGGCATCA-3′) with preceding heterogeneity spacers [23 (link),24 (link)]. Amplicon libraries were constructed using nextera indexing primers (Illumina Inc., San Diego, CA, USA) (PCR program used: 95 °C for 3 min; 20 cycles of 98 °C for 20 s, 55 °C for 15 s, and 72 °C for 45 s; 72 °C for 5 min) and sequenced on a MiSeq instrument using a 600 cycle V3 kit (Illumina Inc., San Diego, CA, USA).