Qiamp dna stool mini kit

All 386 subjects underwent standard colonoscopy examinations at Prince of Wales Hospital, the Chinese University of Hong Kong, including 118 patients with CRC, 140 patients with CRA and 128 normal control participants. The average age of NC group was 64.03 years, 65.84 years for CRA group and 73.21 years for CRC group (Table S1). The distribution of gender and obesity among NC, CRA and CRC groups are shown in Table S1. All CRA and CRC subjects had intact colonic lesions at the time of stool collection. Stool samples were collected and stored at − 20 °C within 4 h and at − 80 °C within 24 h for long-term storage. Qiagen QIAmp DNA Stool Mini Kit (Qiagen) was used for DNA extraction according to the manufacturers’ instructions. All patients provided written informed consent for participation in this study. The study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.

For enterobacteria and coliform counts, samples were serially diluted in Ringer’s lactate solution (Sigma-Aldrich, Madrid, Spain) and proper dilutions seeded in MacConkey agar (Oxoid; Madrid, Spain) and eosin methylene blue agar (Scharlab; Barcelona, Spain). Plaques were incubated for 24 h at 37°C and colonies were manually counted. From colon content and ileal mucosal scrapings, the total bacterial DNA was extracted using QIAmp DNA Stool Mini Kit (Qiagen; West Sussex, United Kingdom) following the manufacturer’s instructions. The ETEC F4 concentration was than assessed by qPCR targeting the gene coding the F4 fimbria of E. coli, according to the procedure described by Hermes et al. (2013) (link), using SYBR green dye with the ABI 7900 HT Sequence Detection System (PE Biosystems, Warrington, United Kingdom) with optical grade 96-well plates. The results were scored in five levels according to the number of gene copies per gram of fresh matter (FM). Scores were defined as following: negative = less than 4 logarithmic units of gene copies per gram FM; low = 4–5.5 logarithmic units of gene copies per gram FM; medium = 5.5–7 logarithmic units of gene copies per gram FM; high = 7–8.5 logarithmic units of gene copies per gram FM; and very high = more than 8.5 logarithmic units of gene copies per gram FM.

The genomic DNA extraction was carried out using the modified CTAB-Chloroform method [7 (link)] and QIAmp DNA stool mini kit (Qiagen, Germany) as per manufacturer’s instructions from the liver aspirates and stool samples respectively. The extracted DNA was stored at -20°C for further processing.

Prior to DNA extraction, 1 g of each stool sample was resuspended in 8 mL of saline solution and concentrated using Bioparapred-Midi columns (Leti Diagnostics, Barcelona, Spain). Then, the supernatants were discarded and 200 mg of the pellets obtained were used for total DNA extraction using the QIAmp DNA Stool Mini kit (QIAGEN, Hilden, Germany) following the manufacturer´s instructions. Purified DNA samples were stored at -20°C until use in RT-PCR at ISCIII, Madrid, Spain, and afterwards in PCR and LAMP assays at CIETUS, Salamanca, Spain.

For positive controls, cultivated Blastocystis hominis organisms were isolated from cultures and washed 2 times in fresh Locke's solution. Harvested organisms were added to 0.2 grams of Blastocystis-negative dog feces to produce a spiked positive control. DNA was extracted using a commercial DNA extraction kit (QIAmp DNA Stool Mini Kit, Qiagen, Valencia, CA) following the manufacturer's protocol, with DNA extracts eluted in 100 µl of AE buffer. Fecal samples previously tested negative for Blastocystis spp were used as negative controls. DNA was stored at −70°C until used. Each DNA extraction batch included a spiked canine fecal sample as a positive control.
For shelter-resident and client-owned canine and feline samples, DNA from 0.20 grams of feces was extracted as described above. The resulting filtrates containing DNA were aliquoted and stored at −70°C until analyzed.

Total community DNA was extracted from each faecal samples using QIAmp DNA Stool Mini kit (Qiagen, Madison USA) as per manufacturer’s protocol. The concentration of resulting DNA was measured using Nanodrop-1000, (Thermo Scientific, USA). DNA concentration was normalised to 100 ng/μl and used as template for amplification bacterial genes.