Immediately before DNA isolation, samples were defrosted at room temperature. For a quantitative analysis of microbial characteristics, genomic bacterial DNA was isolated from faecal samples using a commercial kit designated for human faecal specimens (GeneMATRIX Stool DNA Purification Kit, EURx, Gdańsk, Poland) according to the manufacturer’s protocol using the bead-beating method and a Gyrator UNIPREP 3D vortex (UniEquip, Planegg, Germany) as described in [22 (link)].
Genematrix stool dna purification kit
Manufactured by EURx
Sourced in Poland
The GeneMATRIX Stool DNA Purification Kit is a laboratory equipment designed to efficiently extract and purify DNA from stool samples. It is a tool intended for use in research and diagnostic applications that require high-quality DNA from fecal material.
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Qiaquick pcr purification kit, by Qiagen (1 mentions)
Midori green, by Nippon Genetics (1 mentions)
Mp fastprep 24 instrument, by MP Biomedicals (1 mentions)
Seakem le agarose gel, by Lonza (1 mentions)
Genelute bacterial genomic dna kit, by Merck Group (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
12 protocols using genematrix stool dna purification kit
1
Bacterial DNA Isolation from Fecal Samples
2
Cecal DNA Extraction Protocol
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Genomic DNA from each sample was isolated from 200 mg of cecal content using GeneMatrix Stool DNA Purification Kit (EURx, Gdansk, Poland). All procedures were performed according to the manufacturer's instructions. The quantity of DNA was measured spectrophotometrically at 260 nm using Epoch Microplate Spectrophotometer (Biotek, VT, USA).
3
Fecal Microbiome DNA Isolation and Amplification
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DNA was isolated from all 49 fecal samples using GeneMATRIX Stool DNA Purification Kit (EURx, Gdańsk, Poland) according to the manufacturer’s instructions. Obtained DNA was stored at −20 °C until further use.
PCR amplification was performed on a sets of nested primers amplifying the ITS region of the ribosomal ribonucleic acid (rRNA) gene, i.e., EBITS3, EBITS4 and EBITS1, EBITS2.4 for E. bieneusi (Buckholt et al. 2002 (link)) and INT580F, INT580R and Msp3, Msp4a for Encephalitozoon spp. (Katzwinkel-Wladarsch et al. 1996 (link)). A fragment of Cryptosporidium 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes were amplified (Pedraza-Diaz et al. 2001 (link); Spano et al. 1997 (link); Xiao et al. 1999 (link)). For the amplification of actin genes, we used cycling parameters elaborated by Sulaiman et al. (2002 (link)). For all PCR reactions, negative and positive controls were performed with sterile water and reference DNA, respectively. Secondary PCR products were subjected to electrophoresis on a 1.0 % agarose gel and stained with Midori Green (Nippon Genetics Europe GmbH). Products of expected size were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and stored at 4 °C until sequencing.
PCR amplification was performed on a sets of nested primers amplifying the ITS region of the ribosomal ribonucleic acid (rRNA) gene, i.e., EBITS3, EBITS4 and EBITS1, EBITS2.4 for E. bieneusi (Buckholt et al. 2002 (link)) and INT580F, INT580R and Msp3, Msp4a for Encephalitozoon spp. (Katzwinkel-Wladarsch et al. 1996 (link)). A fragment of Cryptosporidium 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes were amplified (Pedraza-Diaz et al. 2001 (link); Spano et al. 1997 (link); Xiao et al. 1999 (link)). For the amplification of actin genes, we used cycling parameters elaborated by Sulaiman et al. (2002 (link)). For all PCR reactions, negative and positive controls were performed with sterile water and reference DNA, respectively. Secondary PCR products were subjected to electrophoresis on a 1.0 % agarose gel and stained with Midori Green (Nippon Genetics Europe GmbH). Products of expected size were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and stored at 4 °C until sequencing.
4
Fecal DNA Extraction Protocol
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Extraction of DNA from feces samples and luminal content of the ceca was performed using the GeneMATRIX Stool DNA Purification Kit (E3575, EURx, Gdańsk, Poland) optimizing the manufacturer's protocol. The quality and quantity of the extracted DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Warsaw, Poland). The integrity of the DNA was confirmed by performing electrophoresis on a 2% agarose gel. The DNA samples were stored at −80˚C until use.
5
Fecal DNA Extraction Protocol
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After thawing, samples were homogenized using the MP FastPrep-24 Instrument (MP Biomedicals Inc.). Nucleic acids were extracted from 200 mg of feces by the GeneMATRIX Stool DNA Purification Kit (Eurx Ltd.) and stored at −20°C before further use. To avoid cross contamination of samples, the whole process was performed using sterile equipment. The quantity and quality of the extracted DNA were evaluated by using a Nano Drop spectrophotometer and agarose gel electrophoresis.
6
Enhanced DNA Extraction from Stool Samples
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Positive samples kept at 4 °C by adding distilled water were vortexed. Then, 1 ml of the samples was taken, and 1 ml of phosphate-buffered saline (PBS) was added to it. The mixture was centrifuged at 5000 xg for 5 min, and the supernatant was discarded. DNA isolation was performed from the pellet using a DNA extraction kit (EURx GeneMATRIX Stool DNA Purification Kit, Poland). The method was performed with some modifications to the manufacturer’s instructions. After incubation at 70 °C for 5 min according to the manufacturer’s instructions, incubation was performed at 95 °C for 45 min in a dry block heater. During the incubation, vortexing was done at 5-min intervals. After incubation, the tubes were placed in a horizontal vortex and vortexed at maximum speed for 30 min. Afterwards, the manufacturer’s instructions were applied.
7
Fecal Microbiome Profiling via 16S rRNA Sequencing
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Genomic DNA samples were extracted from about 15 mg fecal samples using the GeneMATRIX Stool DNA Purification Kit (EurX, Gdańsk, Poland) and following the manufacturer’s protocol. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using primers 341F/785R 29 [28 (link)]. Details were congruent with methodology described in Kaczmarczyk-Ziemba et al. [29 (link)] and Szewczyk et al. [30 (link)]. Raw NGS data are deposited and fully available in the Sequence Read Archive (accession number PRJNA914996).
8
Extracting and Purifying E. coli rDNA
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The total rDNA was extracted from faecal and lagoon samples with the GeneMATRIX Stool DNA Purification Kit (E3575, EURx Ltd, Gdańsk, Poland). The rDNA concentration and purity were determined with NanoDrop 1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) by migration in 0.8% SeaKem® LE Agarose gels (50004L, Lonza Group Ltd, Basel, Switzerland) in a 1× TBE buffer. The 16S rDNA from the confirmed E. coli was isolated with the GenEluteTM Bacterial Genomic DNA Kit (NA2120, Merck (Sigma-Aldrich, St. Louis, MO, USA).
9
Fecal DNA Extraction Protocol
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DNA was extracted from the fecal samples using the GeneMATRIX Stool DNA Purification Kit (EURx, Gdańsk, Poland) according to the manufacturer’s protocols. The DNA isolates obtained were stored at −20 °C until molecular analysis was completed.
10
Blastocystis Molecular Analysis Protocol
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For molecular analysis of Blastocystis, DNA extraction was performed on frozen samples using the GeneMATRIX Stool DNA Purification Kit (EURx, Gdansk, Poland) according to the instructions of the manufacturer. Briefly, a total of 70 mg of fecal sample was used in each DNA extraction with a final elution volume of 100 μl. For detection of Blastocystis, small subunit rDNA (SSU-rDNA), and the primers BhRDr (5′-GAGCTTTTTAACTGCAACAACG-3′) and RD5 (5′-ATCTGGTTGATCCTGCCAGT-3′) were used in a touchdown PCR (12 (link)). Two microliters of DNA template were added to the standard PCR mixture yielding a total reaction volume of 25μl.
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