Qiamp dna stool kit

DNA was extracted from scat samples using a commercially available QIAmp DNA Stool Kit (QIAGEN Inc., Germany) following the manufacturer’s instructions. Species identification polymerase chain reaction (PCR) was performed using tiger-specific primers (TIF/TIR) (S1 Table) [41 (link)] that amplify a 162 bp mtDNA cytochrome b fragment. The genetically identified tiger samples were further processed for sex identification (S1 and S2 Tables) [42 ].

Fecal samples from challenged piglets were examined for ETEC K88 6 h after the challenge. Bacterial DNA was extracted using a QIAmp DNA stool kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. Adhesin gene F4 was detected by PCR using forward primer 5′-GCTGCATCTGCTGCATCTGGTATGG-3′ and reverse primer 5′-CCACTGAGTGCTGGTAGTTACAGCC-3′, as described previously [26 (link)].

DNA from stool and/or intestinal tissue was extracted from thawed samples using
the QIAmp DNA stool Kit (Qiagen) as previously described [18 (link)]. For detection of 16S rRNA genes,
modifications to enrich detection including additional steps of homogenization
in bead tubes (UltraClean fecal DNA bead tubes, Mo Bio Laboratories) in
400/360μL of ASL/ATL buffer using a Mini-Beadbeater for 1 minute, 30/40 μL of
Proteinase K for stool/tissue homogenates, and incubation of tissues at 56°C for
two hours [70 (link)].

Genomic DNA was extracted from fecal, blood and tissue samples using the QIAmp DNA Stool Kit (Qiagen, Valencia, CA) and the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), respectively, following the manufacturer’s protocol with modifications from [46 (link)]. Resulting extracts were quantified using a Qubit dsDNA BR Assay (Life Technologies, Carlsbad, CA). For each sample, a 5-10uL aliquot of the extract was included in 25uL PCR reactions that included the following reagents: 12.5uL of PCR Master Mix (Promega, Madison, WI), 0.8uL of the forward primer (10uM), 0.8uL of the reverse primer (10uM), 0.8uL BSA and finally PCR-grade water to bring the final volume to 25uL. Primers were designed from alignments of published platyrrhine sequences, with preference for primers to match the Cebus imitator genome from GenBank (Table 1). PCR cycling conditions for all exons were: (1) an initial denaturing at 94°C for 2 minutes followed by (2) 35 cycles of: 94°C for 30 seconds, 58°C for 40 seconds and 72°C for 1 minute, then (3) a final extension at 72°C for 7 minutes. The PCR product was visualized on a 1.5% agarose gel to confirm the presence of fragments of the expected size.

We extracted DNA from scat samples using a commercially available QIAmp DNA Stool Kit (QIAGEN Inc., Germany) following the manufacturer’s instructions. Each batch of DNA extraction included a negative extraction control. Extracted DNA was stored at -20°C. Tigers were identified using PCR assay that used tiger specific mtDNA Cytochrome-b (CYT-B) primers [63 (link)]. Individual tigers were identified by microsatellite analysis using a panel of eight microsatellite markers developed from the domestic cat (Felis catus) and tiger genomes [64 (link)–66 (link)] as described in Thapa et al. [32 (link)].

All collected samples were stored at −20 °C before DNA purification and the storage never exceeded 3 months, to minimise sample degradation. Each sample was homogenised using a MagnaLyser (Roche, Basel, Switzerland) and the homogenate was subjected to DNA purification. QIAmp DNA Stool kit (Qiagen, Hilden, Germany) was used for DNA purification from caecal contents, while DNeasy tissue kit (Qiagen, Hilden, Germany) was used for DNA purification from eggshell or bacterial mass collected from agar plates. Purified DNA was stored at −20 °C.