Magna pure lc total nucleic acid isolation kit large volume

Fecal samples for the study of the gut microbiota were collected and frozen −80 °C until their processing for the analysis.
For the isolation of bacterial DNA from fecal samples, the MagNA Pure 2.0 LC robot and the MagNA Pure LC Total Nucleic Acid Isolation Kit - Large Volume were used (Roche Diagnostics, Mannheim, Germany). To assess a correct extraction, we performed the quantification of the bacterial DNA obtained from each sample using the Qubit Fluorometric Quantitation reader using the Qubit 1x dsDNA HS Assay kit (Thermofisher, Massachusetts, MA, USA).
To determine the composition of the gut microbiota, we performed high-throughput sequencing of the 16S rDNA gene. The bacterial 16S rDNA gene amplicons were obtained following the 16S rDNA gene Metagenomic Sequencing Library Preparation Illumina protocol (Illumina, San Diego, CA, USA). The specific amplified sequences are in the V3 and V4 regions (459 bp) of the 16S rDNA gene. The specific primers were selected from the current bibliography in this regard [17 (link)]:
Sequence forward primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG.
Sequence reverse primer: GTCTCGTGGGCTGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.
The amplicon libraries were sequenced in a MiSeq Sequencer (Illumina, San Diego, CA, USA) following the manufacturer’s specifications.

The specimens were vortexed for 1 min and centrifuged at 4000×g for 20 min before nucleotide extraction. Total nucleotide extraction was performed with MagNA Pure LC Total Nucleic Acid Isolation Kit-Large Volume (ROCHE, Co, USA) by a Roche MagNA Pure LC 2.0 nucleic extraction system (ROCHE, Co, USA) according to the manufacturer’s instructions. EV, EV-A71 and CVA16 were identified using real-time RT-PCR Kit (DAAN Gene, Guangzhou, China) [10 ]. Samples of non-EV-A71 and non-CVA16 were further identified by detecting the VP1 region according to previously described method [11 (link)]. Complete nucleotide sequences of VP1 genes were amplified using previously described specific primers [12 (link)]. Polymerase Chain Reaction (PCR) products of complete VP1 genes were purified and sequenced using ABI PRISM310 Genetic Analyzer.

Total nucleotide extraction was carried out with a Roche MagNA Pure LC 2.0 nucleic extraction system (ROCHE, Co, United States) using MagNA Pure LC Total Nucleic Acid Isolation Kit–Large Volume (ROCHE, Co, United States), according to the manufacturer’s instructions.
Complete nucleotide sequences of the VP1 gene were amplified using specific primers as previously described (Mirand et al., 2010 (link)). PCR products of completeVP1 genes were purified and sequenced using ABI PRISM 310 Genetic Analyzer.
Human rhabdomyosarcoma (RD) cells were used to isolate enterovirus from the supernatant of the throat swab specimen. The RD cells were cultured using MEM with 10% fetal bovine serum till 75% of flask bottom was covered by monolayer RD cell. Then the cell culture medium was removed and the supernatant of the specimen was inoculated on the RD cells. After 1 h of incubation at 36°C, the specimen supernatant was replaced by MEM with 2% fetal bovine serum. Cytopathic effect of the enterovirus infected RD cells was observed every day. Cells were collected when 75–90% covered cells showed cytopathic effect.