Qiaamp dna stool extraction kit

DNA was isolated from 100 μL aliquots of the rectal swab samples using the QIAamp DNA Stool Extraction Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. Extracted DNA was precipitated with ethanol in a final elution volume of 200 μL. Each DNA sample was tested for Shigella by qPCR using the previously described SYBR green-based fluorescent dye method to detect the ipaH gene (81 (link)). The primers for ipaH were originally created by Vu et al. (82 (link)). Quantitation cycle (Cq) values of <35 detected in duplicate wells were required to consider a sample qPCR positive. Controls for the qPCR assay included extraction-negative, qPCR negative, and a known qPCR positive control. While this method cannot distinguish between Shigella and enteroinvasive Escherichia coli (EIEC), EIEC prevalence is minimal compared with Shigella in similar geographic settings (43 (link), 83 (link)). Therefore, all of the ipaH positive samples were assumed to be Shigella (79 (link)).

Total DNA was extracted from the thawed colonic contents samples using the QIAamp DNA stool extraction kit (Qiagen, Germany) following the manufacturer’s protocol. The genomic DNA was then examined by 1% agarose gel electrophoresis. All samples were quantified on a Qubit 2.0 Fluorometer (ThermoFisher Scientific, USA). Then, the V3-V4 hypervariable region of the 16S rRNA gene was amplified by PCR, which used 338F forward primer (5’-ACTCCTACGGGAGGCAGCA-3’) and the 806R reverse primer (5’-GGACTACHVGGGTWTCTAAT-3’). The PCR cycle was denaturation at 94°C for 3min (1 cycle); followed by 94°C for 45 s, annealing at 50°C for 60 s, and extension at 72°C for 90 s (25 cycles), and a final extension step of 72°C for 10min. The amplicon products were purified with AMPure XP beads (Beckman Coulter, USA). Sequencing libraries were generated using the TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations. The library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). The quality libraries were finally sequenced on an Illumina HiSeq 2500 platform with 250 bp of paired-end reads.

Stool specimens were handled according to the GEMS protocol (16 (link)). DNA was isolated from frozen stool specimens by using a bead beater with 3-mm diameter solid glass beads (Sigma-Aldrich, St. Louis, MO, USA) and, subsequently, with 0.1-mm zirconium beads (BioSpec Products, Inc., Bartlesville, OK, USA) to disrupt cells. The cell slurry was then centrifuged at 16,000 × g for 1 min, and the supernatant was processed by using QIAamp DNA Stool Extraction Kit (QIAGEN, Valencia, CA, USA). Extracted DNA was precipitated with ethanol and shipped to the United States.

Stool specimens were collected in sterile containers and examined within 24 h. Stools were stored at 2 to 8°C while in transit to the laboratory. Each fresh stool specimen was aliquoted into multiple tubes. All samples were analyzed by traditional microbiological tests for known bacterial, viral, and eukaryotic pathogens. Details of these methods can be found in Panchalingam et al. [43 (link)] DNA was isolated using a bead beater with 3 mm diameter solid glass beads (sigma Life Science), and subsequently 0.1 mm zirconium beads (BIO-SPEC Inc.) to disrupt cells. The cell slurry was then centrifuged at 16,000 g for 1 min, the supernatant removed and processed using the Qiagen QIAamp® DNA stool extraction kit. Extracted DNA was precipitated with 3 M sodium acetate and ethanol and the DNA shipped to the USA.

Stool DNA was extracted from 200 mg of stool using the QIAamp DNA stool extraction kit (Qiagen, UK) following the manufacturer’s protocol for pathogen detection. Extracted DNA was frozen and shipped to University of Edinburgh where samples were defrosted, quantified using Qubit 2 (Invitrogen), diluted and aliquoted. Aliquoted samples were shipped to the University of Warwick for high-throughput sequencing.

Genomic DNA was extracted from stool pellets using QIAamp stool DNA extraction kit (Qiagen, Valencia, CA) following manufacturer's instructions. Bacterial composition of the stool samples was assessed by automated ribosomal intergenic spacer analysis (ARISA) as described previously with some modifications [33] (link). Briefly, the intergenic regions between bacterial 16S and 23S rRNA genes were amplified using broad range primers 1406F (labeled at the 5′end with the phosphoramidite dye 5-FAM) and 23Sr. The intergenic lengths of 6-FAM labeled PCR products were determined on an ABI 3730 capillary sequencer (Applied biosystems, Grand Island, NY) using LIZ-1200 size standard and electropherograms were analyzed using peak scanner (Applied biosystems, Grand Island, NY). Real-time qPCR was performed on stool DNA using group-specific primers [34] (link) to determine the relative abundance of individual bacterial groups. A standard curve for each primer pair was plotted using plasmid with appropriate 16S rRNA gene sequence insert to quantify the qPCR values into 16S copy number/g of normalized stool.