Allprep powerviral dna rna kit

Q-PCR using strain-specific primers against the single copy RpoB gene were used to quantify the composition of the GUT-103 and GUT-108 consortia after gavage in gnotobiotic mice. RpoB primers were validated to ensure there was no significant cross-reactivity between their target bacterium and other bacteria that were present in the consortia (Supplementary Fig. 6). The strain-specific RpoB primers are provided in Supplementary Data 7. The genomic DNA was extracted from fecal or culture media using AllPrep PowerViral DNA/RNA Kit (Qiagen). Q-PCR were performed with QuantStudio3 (Thermo Fisher Scientific, PA, USA) using SYBR No-ROX reagents (Bioline, TN, USA) and 10 ng of genomic DNA per strain with the following PCR settings: 95 °C, 3 min; 40 cycles of (95 °C, 5 sec; 60 °C, 20 sec); melting curve analysis: 95 °C, 15 sec; 60 °C, 15 sec; 95 °C, 15 sec. The data were created by comparative Ct method (2^-Ct). Melting curve analysis confirmed the presence of single products with expected melting temperatures.

On 6/12/17, 7/17/17, 8/8/17, 8/21/17, 9/11/17, 9/25/17, 10/30/17, and 5/7/18 the iron chloride procedure was used on the pond water after sequential 1 μm and 0.2 μm filtration, with the 0.2 μm a common pore size used in virome generation to prevent cellular contamination [33 (link), 37 (link), 42 (link)]. A 1 mL solution of FeCl₃ (4.83 g FeCl₃ into 100 ml H₂O) was added to the filtered pond water and incubated in the dark for 1 h. The samples were then filtered onto 142-mm 1 μm polycarbonate filters (Sigma-Aldrich, MO, United States) to capture flocculated viral particles [43 (link)]. Filters were stored at 4 °C in the dark until resuspension. For resuspension, filters were rocked overnight at 4 °C in 10 mL of 0.1 M EDTA - 0.2 M MgCl₂₋ 0.2 M Ascorbate Buffer, described in detail elsewhere [43 (link)]. Resuspended viral particles were then subjected to a DNase I (Sigma-Aldrich, MO, United States) treatment for 1 h and passed through a 33-mm diameter sterile syringe filter with a 0.2 μm pore size (Millipore Corporation, MA, United States). DNA was extracted from 500 μl of the viral concentrate using the AllPrep PowerViral DNA/RNA Kit (Qiagen, CA, United States) per the manufacturer’s instructions. Prior to sequencing, viral DNA was tested for the presence of bacterial contamination via 16S rRNA gene PCR.

Samples of filtered and unfiltered water were collected from three of the irrigation events in 50 mL volumes, each in triplicate (n = 18). The samples were concentrated using Centricon 100 kDa filters (Millipore Sigma-Aldrich) via centrifugal ultrafiltration. Viral RNA was extracted (AllPrep PowerViral DNA/RNA Kit; Qiagen; Hilden, Germany) and detection was performed using a real-time quantitative PCR (RT-qPCR) molecular assay. The primer-probe set [27 (link),28 (link)] was selected for use with the Rotor-Gene Q apparatus (Qiagen), based on the available research of successful PMMoV detection in environmental waters.