Article Figures & Data
Figures
- Fig. 1.
Specificity of the PCR-based identification of O. formigenes. PCRs were carried out with 1 ng of genomic DNA isolated from each of the following: Bacteroides ovatus,Proteus vulgaris, Enterobacter cloacae,Escherichia coli, Enterobacter aerogenes, Clostridium perfringens, Clostridium sordellii,Veillonella parvula, Streptococcus pneumoniae,Staphylococcus aureus, Pseudomonas aeruginosa,Streptococcus bovis, Enterococcus faecalis,Staphylococcus epidermidis, Moorella thermoautotrophica, Shigella boydii, Proteus mirabilis, Salmonella typhimurium, Oxalobacter formigenes, Citrobacter brakii, Lactobacillus acidophilus, Moraxella osloensis, Moorella thermoacetica, Alcaligenes sp., Klebsiella oxytoca, and Oxalobacter formigenes (top panel, lanes 1 to 26, respectively). Southern blots of the gels were carried out with an Oxalobacter genus-specific DNA oligonucleotide probe (middle panel). To test the ability of each genomic-DNA preparation to support a PCR, PCRs were carried out with 1 ng of genomic DNA isolated from O. formigenes or from one of the 24 other bacterial strains and a set of universal primers for bacterial 16S rRNA (bottom panel). Arrows indicate lanes containing O. formigenesDNA.
- Fig. 2.
Synthesis pathway of a competitive DNA template for quantifying the PCR. A truncated form of the 416-bp fragment of theoxc gene containing both the 5′ and 3′ primer sites was synthesized by PCR by using a modified 3′ primer. This truncated and modified sequence was ligated into the pCR2.1 vector system in order to produce high copy numbers.
- Fig. 3.
Quantification of the number of O. formigenesgenomes in a sample by using the QC-PCR. Dilutions, ranging from 50 to 250,000 molecules of purified plasmid containing the competitive template, were used either as DNA templates in PCR to establish standard curves (top, center panel) or as competitive DNA by mixing with 2 × 104 (left panel) or 2 × 102 (right panel) copies of purified O. formigenes OxB genomes. PCR band intensities were scanned and normalized for molecular mass, and the log ratios of O. formigenes to template band intensities were graphed to determine log equivalence (bottom panels). QCT, quantitative competitive template.
- Fig. 4.
Comparison of the numbers of O. formigenesCFU in a bacterial culture detected by culture and QC-PCR. An overnight culture of O. formigenes OxB containing approximately 0.7 × 108 CFU/ml, as determined by optical density at 600 nm, was serially diluted 10-fold. DNA isolated from each dilution was used in QC-PCR. Log equivalences of O. formigenes to template band intensities were determined and compared to the estimated number of CFU for each dilution.
- Fig. 5.
Testing clinical samples for stability and reproducibility in detecting O. formigenes. Four fecal swabs collected and transported in the Cary-Blair culture transport system were processed on days 2, 3, 4, and 6 following collection to analyze the sample stability and the reproducibility of the QC-PCR assay system. DNA was isolated from approximately 20 mg of fecal specimen eluted from each swab, and the presence of O. formigenesgenomes was determined by QC-PCR (insert). Log equivalences of O. formigenes to template band intensities were graphed to quantify the number of genomes present in each sample. The number of CFU detected on days 2, 3, and 4 was 8 × 108/g (wet weight), while the number of CFU detected on day 6 was 2.8 × 108/g (wet weight). QCT, quantitative competitive template.
- Fig. 6.
Quantifying temporal changes in O. formigenescolonization induced by changes in diet. Detection of O. formigenes in fecal samples obtained from a subject on a normal diet who was then placed on a diet low in oxalate-containing foods followed by a diet high in oxalate-containing foods. Amplifications were carried out with 1 μl of sample DNA alone (lanes 6) and in the presence of 42 (lanes 1), 83 (lanes 2), 415 (lanes 3), 830 (lanes 4), or 8,300 (lanes 5) copies of the competitive template.
Tables
- Table 1.
Correlation of CFU quantification by standard culturing and QC-PCR
Clinical sample no.a CFU/g of feces (wet weight) quantified by: Culture method QC-PCR 1 0 0 2 0 0 3 0 0 4 2.3 × 106 2.5 × 106 5 5.5 × 106 3.0 × 106 6 2.1 × 107 3.5 × 107 7 3.1 × 107 3.1 × 107 8 1.0 × 107 1.6 × 107 ↵a Specimens were collected in North Carolina, where CFU were quantified by culture; samples were mailed to Florida, where CFU were quantified by QC-PCR.
