7500 fast sequence detection system

To quantify Hh colonization levels, DNA was prepared from the cecal content using a High Pure PCR Template kit according to the manufacturer’s protocol (Roche Applied Science). Levels of the Hh 16s rRNA gene were measured by qPCR in the 7500 Fast Sequence Detection System (Applied Biosystems) as described previously ^{23 (link)}. The colonization levels of Hh were quantified by normalizing to µg of mouse chromosomal DNA measured by qPCR using 18S rRNA gene-based primers and probe mixture (Life Technology).

DNA was extracted from the stomach using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, IN). As previously published, samples were probed with 18S rRNA-based primers for quantifying mouse DNA (Applied Biosystems) and with H. pylori DNA-specific primers and probe based on the H. pylori ureB gene [8 (link)]. ASF copy numbers were measured using SyBr-based qPCR using the 7500 Fast Sequence Detection System (Applied Biosystems) [30 (link), 31 (link)]. Plasmid DNA containing the 16S rDNA of each of the 8 ASF species was used to generate standard curves of six 10-fold dilutions, ranging from 10⁶ to 10 copies. Subsequently, the bacterial DNA quantity was converted into copy number of each ASF genome based on the number of 16S rRNA gene copies. ASF copy numbers were then normalized to μg of mouse chromosomal DNA.

DNA was extracted from fecal pellets using the UltraClean Fecal DNA Kit (MO BIO Laboratories). Amplicons were generated using oligonucleotide primers that target approximately 300 bp of the V4 variable region of the 16S rRNA gene (primers 515F and 806R) ^{40 (link)} and also were barcoded and pooled to construct the sequencing library, followed by sequencing with an Illumina MiSeq instrument to generate paired-end 150×150 reads. The software package QIIME 1.7.0 was used to analyze, display, and generate figures of microbiome data using a previously defined method^{41 (link)}. Bacterial DNA of Enterobacteriaceae and Bacteriodetes from fecal content was quantified using six-point standard curves constructed with reference bacteria specific for each bacterial group measured by qPCR in the 7500 Fast Sequence Detection System (Applied Biosystems). All the reactions were set up in Fast SYBR Green Master Mix (Applied Biosystems) at 20 µL total volume. Copy number of Enterobacteriaceae and Bacteriodetes was normalized to copy number of universal bacteria. The initialization step was 95°C for 10 minutes, the amplification step had 40 cycles of 95°C for 10 seconds followed by optimal annealing temperature for 45 seconds. The primers and reaction conditions are listed in Table S1.