Truncated human cytidylate-phosphate-deoxyguanylate-binding protein for improved nucleic acid amplification technique-based detection of bacterial species in human samples

Abstract

A trunk of human cytidylate-phosphate-deoxyguanylate-binding protein/CXXC finger protein 1 (CFP1), immobilized onto an aminohexyl-Sepharose column, can be used as a preanalytical tool for the selective enrichment of bacterial DNA from mixed solutions with high amounts of human background DNA for nucleic acid amplification technique-based detection of pathogens. The transcriptional activator protein exhibits a high affinity for nonmethylated CpG dinucleotide motifs, which are differentially distributed in prokaryotic and higher eukaryotic genomes. The feasibility of the affinity chromatography (AC) step was tested with DNA from severely septic patients. AC using 16S rRNA gene primers substantially increased PCR sensitivity. Approximately 90% of eukaryotic DNA was removed, which significantly increased the signal-to-noise ratio. Threshold cycle values revealed that sensitivity was elevated at least 10-fold. The change in the ratio of bacterial DNA to human DNA increased from 26% to 74% the likelihood of culture-independent PCR-based identification of bacterial presence. Compared to the results seen with blood culture (which is the clinical gold standard for systemic infections, exhibiting 28% positives), the combination of AC and PCR achieves a significant increase in sensitivity and contributes to shortening the time to results for the initiation of guided antibiotic therapy.

FIG. 1.

Characteristics of recombinant P181. (A) P181 comprises amino acids 106 to 286, including the CXXC zinc finger domain of mature human DNA-binding protein 1 (hCGBP/CFP1) (left box, PHD type zinc finger; middle [dark gray] box, CXXC zinc finger; right box, coiled-coil region). (B) SDS-PAGE analysis of 0.11 μg of recombinant P181 (Coomassie blue staining). M, molecular mass marker.

FIG. 2.

Binding of P181 to prokaryotic DNA as assessed by electrophoretic mobility shift analysis and lack of prokaryotic DNA retardation by HSA-Sepharose. (A) Retarded electrophoretic migration of nonmethylated plasmid pUC18emmC (second and fourth lanes) containing the gene encoding the M protein of the group C streptococcal strain 25287 in the presence of P181. Plasmid DNA (0.15 μg) was incubated in each binding reaction with either 10 μg (first, second, and fifth lanes) or 5 μg (third and fourth lanes) of P181 at room temperature for 30 min and was subjected to electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Any retardation that occurred was observed upon methylation of the plasmid (first and third lanes), as is consistent with maintained recognition of nonmethylated CpG motifs by the truncated protein. The fifth lane presents results for nonmethylated pUC18emmC DNA in the absence of P181 (control); the sixth lane represents the molecular mass marker. (B) Proof of nonbinding of prokaryotic DNA on HSA-Sepharose columns (in the absence of P181). A mixture of 25 μg of human DNA spiked with 0.02 μg of S. aureus chromosomal DNA in 100 μl of water was applied to 100 μl of HSA-Sepharose. PCR with the obtained fractions was performed to detect the 16S rRNA gene. Lane 1, flowthrough; lanes 2 and 3, fractions occurring after washing with buffer devoid of NaCl (10 mM Tris, 10 mM EDTA, pH 7.5); lanes 4 and 5, fractions occurring after washing with buffer containing 1 M NaCl; lane 6, total DNA loaded onto HSA-Sepharose column; lane 7, positive control with S. aureus DNA; lane 8, negative control; lane M, pGEM molecular mass standard.

FIG. 3.

Separation of nonmethylated plasmid DNA from an excess of eukaryotic DNA by P181-AC. A molar excess of 2 μg of calf thymus DNA spiked with 0.025 μg of pUC18emmC plasmid DNA was loaded onto P181-Sepharose columns and eluted by incremental increases of [NaCl] in 10 mM Tris-HCl elution buffer (pH 7.0). The majority of calf thymus DNA in the eluate appeared at concentrations below 0.3 M NaCl. Elution of plasmid DNA started at 0.4 M NaCl, as shown by a peak of A₂₅₄ in fraction 21, which was confirmed by PCR using plasmid-specific primers M13fw and M13rv as outlined in Materials and Methods.

FIG. 4.

Binding of 0.02 μg of ³²P-labeled S. pyogenes BK 42440 DNA to 200 μl of P181-Sepharose in competition with 50 μg of human DNA. bact., bacterial.

FIG. 5.

Detection of E. coli PCR targets within a molar excess of human DNA via qPCR using irp2-specific primers (Table 1). A mixture of genomic E. coli (0.02 μg) and human DNA (50 μg) was used as a template. Flowthrough and starting material (the DNA mixture prior to P181-AC) showed high C_T values due to low target concentrations and high background DNA charges. The AC elution fraction exhibited a significantly lower C_T value indicative of an improved signal-to-noise ratio. The calculated C_T values showed >10-fold-higher sensitivity after P181-AC.

FIG. 6.

Application of P181-AC for the improvement of PCR-based identification of bacterial presence in buffy coats from 3.5 ml of EDTA whole-blood samples from severely septic patients. (A) Positive PCR results with 16S rRNA gene primers (see Table 1) rose from 26% to 74%. (B) PCR analysis after enrichment of bacterial DNA via P181-AC. Both selected patients had consistently negative BCs. Bacterial presence in the enriched DNA fractions was analyzed by 16S rRNA gene qPCR. Lanes 1 and 7, flowthrough; lanes 2 and 8, washing fractions (buffer without NaCl); lanes 3 and 9, washing fractions (buffer containing 0.5 M NaCl); lanes 4 and 10, washing fractions (buffer containing 1.0 M NaCl); lanes 5 and 11, original samples applied to the P181-AC column; lanes 6 and 12, inhibition controls (sample DNA [without P181-AC] plus S. aureus DNA; lane 13, positive control (S. aureus DNA); lane 14, washing buffer-negative control (no-template control); lane M, pGEM molecular mass marker. Patient I tested positive for P. aeruginosa by sequencing of the 16S rRNA gene amplicons and rpoD-specific PCR; patient II tested positive for S. aureus (confirmed by hla-specific PCR as outlined in Results).