Powerfood microbial dna isolation kit

Total microbial DNA was extracted directly from the insect samples using PowerFood Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) as described by Osimani et al. (2017a (link)). The extracted DNA was quantified and checked for the purity using a NanoDrop ND 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) and then standardized to 2 ng μL⁻¹ for qPCR assays and to 25 ng μL⁻¹ for PCR-DGGE analysis. The effective extraction of bacterial DNA was confirmed by conventional PCR amplification of 2 μL (50 ng) of extracted DNA suspensions in a My Cycler Thermal Cycler (BioRad Laboratories, Hercules, CA, USA) using universal prokaryotic primers 27F and 1495R as described by Osimani et al. (2015 (link)).

DNA was extracted from mice fecal samples using the Powerfood Microbial DNA Isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA, United States) according to the manufacturer’s protocol. Intestinal bacterial community compositions were analyzed using 16S rRNA gene sequencing, which was performed according to a previous report (Ryu et al., 2022 (link)). The obtained data were analyzed using Mothur (v. 1.45.3). To visualize differences in the microbiota composition between groups, nonmetric multidimensional scaling plots were generated on the basis of unweighted and weighted UniFrac distances. The α-diversity and relative abundance of OTUs at family and genus levels were analyzed using the MicrobiomeAnalyst web-based platform (Chong et al., 2020 (link)).

A 1.5 mL aliquot of each sample homogenate (10⁻¹ dilution) was processed for direct extraction of bacterial DNA using a commercial kit suitable to the sample type under examination. In detail, bacterial DNA from raw and cured meat, kidney, and liver was extracted using a PowerFood microbial DNA isolation kit (MoBio Laboratories, Carlsbad, California, USA); for animal faeces and feed samples an E.Z.N.A. soil DNA kit (Omega Bio-tek, Norcross, Georgia, USA) and DNeasy® PowerSoil® Pro kit (Qiagen, Hilden, Germany) were used, respectively, following the manufacturers’ instructions. DNA purity and yield were assessed spectrophotometrically (Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA) and fluorometrically (Qubit, Thermo Fisher Scientific, Waltham, MA, USA).

DNA was extracted from duplicate 1 g samples collected from each of three regions (just under the rind, core, inside) for each of the 15 Gouda cheeses. Extractions were conducted using the PowerFood Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA) according to the manufacturer’s instructions. DNA was quantified using the Qubit dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and stored at − 20 °C prior to PCR.

According to the manufacturer’s instruction, the microbial DNA was extracted from the hams with PowerFood Microbial DNA Isolation kit (MO BIO Laboratories, Inc., USA). The V3 hypervariable region of the 16S rDNA was PCR amplified from the microbial genomic DNA using universal primer (forward primers: 5′-ACTCCTACGGGAGGCAGCAG-3′, reverse primers: 5′-TTACCGCGGCTGCTGGCAC-3′). PCR was subjected to 1 cycle of 98 °C for 5 min, followed by 25 cycles of denaturation at 98 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s, and finally extension at 72 °C for 5 min. Barcoded V3 amplicon was sequenced by Illumina Miseq at Personal Biotechnology Co., Ltd (Shanghai, China) using the pair-end method. All related sequence data have been deposited in the National Center for Biotechnology Information (NCBI) as a bio-project (BioProject ID: PRJNA354505, Accession Number: SRP093702). The samples were given the accessions numbers as SAMN 06046958 -SAMN06046966.

A 1.5 mL aliquot of each sample was processed for direct extraction of bacterial DNA with a commercial kit suitable to the type of sample under examination. In particular, bacterial DNA was extracted from raw meat using a PowerFood microbial DNA isolation kit—MoBio Laboratories; from human stool samples using a QIAamp fast DNA stool ini kit (Qiagen, Hilden, Germany); from animal stool samples using an E.Z.N.A. soil DNA kit—VWR and DNeasy^® PowerSoil^® Pro kit (Qiagen, Hilden, Germany), following the manufacturers’ instructions. For the bivalve molluscs (clams), bacterial DNA was extracted from the digestive gland or from clam homogenates. Nuclisens^® lysis buffer (Biomerieux, Lyon, France) and a Nuclisens^® magnetic extraction kit (Biomerieux, Lyon, France) were used respectively for extraction and purification of DNA from the digestive gland, according to the manufacturer’s instructions. From the whole clam homogenates, DNA was extracted using the Tissue Lyser II (Qiagen, Hilden, Germany) and the DNeasy^® PowerSoil^® Pro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA purity and yield were assessed spectrophotometrically (Qubit, Thermo Fisher Scientific, Waltham, MA, USA) and fluorometrically (Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA).