New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

Bacterial strains and growth conditions.The strains used in this study are shown in Table 1. Strains were grown in GAM broth or agar (Nissui Pharmaceutical Co, Tokyo, Japan) and incubated at 37°C in a Concept 400 anaerobic chamber (Ruskinn Technology, Leeds, United Kingdom) with the exception of E. coli K-12, which was grown in Trypticase soy broth (Pronadisa, Madrid, Spain) and incubated at 37°C in aerobic conditions.

Fecal samples.A total of 24 human fecal samples, 14 from 1-year-old infants, four from adults, and six from elderly (>65 years old) persons, were included in this study. Fecal specimens were cooled to 4°C immediately after collection, delivered within 24 h, and frozen at −75°C directly on receipt until analysis.

Oligonucleotides.The oligonucleotide primers and probes used for quantitative real-time PCR (Table 2) were purchased from Thermo Electron Corporation (Thermo Biosciences, Ulm, Germany). To check for specificity, the sequences of the selected PCR primers and probes were compared to the sequences available at both the BLAST database search program (www.ncbi.nlm.nih.gov/BLAST ) (1) and with the Probe Match application at the Ribosomal Database Project II (www.rdp.cme.msu.edu/html ) (4). Two bases that do not hybridize with the target sequence were added to the 3′ end of the real-time PCR probe to make sure that it could not act as a primer during the PCR. The bifidobacteria detection probe (Bifidoprobe) was labeled postsynthetically with an isothiocyanate-modified, stable, fluorescent europium chelate as described by Nurmi and coworkers (26).

Testing of the oligonucleotide primers by qualitative PCR.In order to test the specificity of the primers, DNA was extracted from pure cultures of the different intestinal and food microorganisms indicated in Table 1 as follows: 1 ml of culture (A₆₀₀ ≈ 1) was harvested, washed twice with TE buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8]), resuspended on 50 μl of TE saline buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA [pH 8], 0.9% [wt/vol] NaCl), heated at 98°C for 10 min, immediately cooled, and centrifuged at 16,000 × g for 10 min, and the supernatant was stored at −20°C until its use.

Amplification of the DNA was performed with primers Bifido5 and Bifido3 in a PCR iCycler apparatus (Bio-Rad, Espoo, Finland). The total volume of each reaction mixture was 50 μl, employing 4 μl of DNA extract as a template. The PCR mixture was composed of 1× PCR buffer II (Applied Biosystems, Foster City, Calif.) with 2.5 mM MgCl₂, 0.2 μM each primer, 200 μM each of the four deoxynucleoside triphosphates (Amersham Biosciences, Helsinki, Finland), and 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems). The thermal cycle program consisted of an initial cycle of 95°C for 10 min for denaturation and polymerase activation, 30 cycles of 95°C for 15 s, 60°C for 1 min, and 72°C for 45 s, and a final extension step of 10 min at 72°C. Amplified products were subjected to gel electrophoresis in 1% agarose gels and visualized by ethidium bromide staining.

Quantitative real-time PCR.We weighed 1 g of fecal material and homogenized it with 9 ml of phosphate-buffered saline buffer in a Stomacher 400 (Seward Ltd., London, United Kingdom) at full speed for 2 min; 200 μl of the homogenate was used for the DNA extraction with the QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. For standard curves, different dilutions (200 μl) of microbial cultures of three predominant human intestinal bifidobacteria, such as B. infantis, B. longum, and B. adolescentis (cell numbers ranging from 1.5 × 10⁴ to 3.2 × 10⁹ cells/ml), were used for the DNA extraction with the QIAamp DNA stool mini kit (Qiagen). To test the different strains by real-time PCR, 200 μl of a culture (A₆₀₀ ≈ 1) of the microorganism was used. The DNA extracts were frozen at −20°C until analysis.

Samples (1 μl) were analyzed in 50-μl amplification reactions consisting of 1× PCR buffer II, 3.5 mM MgCl₂, 0.2 μM each primer, 200 μM each deoxynucleoside triphosphate, 0.024 μM europium-labeled Bifidoprobe, 0.166 μM quencher probe, and 1.25 U of AmpliTaq Gold DNA polymerase. All reactions were performed on MicroAmp optical plates sealed with MicroAmp optical caps (Applied Biosystems). Thermal cycling (iCycler) consisted of an initial cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 1 min, and 61°C for 45 s, and 35°C for 15 s. Europium fluorescence measurements were performed in real time at the end of each cycle with a Wallac Victor 1420 multilabel counter (Perkin Elmer, Turku, Finland) at 35°C to determine the threshold cycles (C_t) of individual reactions. The C_t was defined as the PCR cycle at which the europium signal-to-noise ratio crosses a threshold value of 1.5. Standard curves were made by plotting the C_t values obtained for the standard cultures (different dilutions from cultures of B. longum JCM 1217^T, B. infantis DSM 20088^T, and B. adolescentis JCM 1275^T, 25 reactions in total) as a linear function of the base 10 logarithm of the initial number of bifidobacteria in the culture determined by plate counting. The number of cells of bifidobacteria in the fecal samples was determined by comparing the C_t values obtained to the standard curve. DNA extracts from the different samples were analyzed in duplicate in each PCR in two independent PCR runs.

FISH.FISH was performed as previously described (15) with the fluorophore indocarbocyanine (Cy3)-labeled oligonucleotide probe BIF164 (5′-CATCCGGCATTACCACCC) (18). Bifidobacteria were counted visually with an Olympus BX51 epifluorescence microscope.

Specificity of the assay.Table 1 shows the results of the test for the specificity of the primers used in this work and the results obtained by analyzing the different strains with the quantitative real-time PCR procedure. In addition to this specificity test, the sequences of the primers and probe were compared with the target sequences of the nonbifidobacteria test species included in this study (Table 1) showing one mismatch of the primer Bifido5′ with species of the genus Eubacterium, Lactobacillus casei, and Lactobacillus paracasei and two or more mismatches with the rest of the test organisms. With regard to the primer Bifido3′ and the probe (Bifidoprobe), all the nonbifidobacteria test species had at least four or five mismatches, respectively (data not shown).

The PCR primers (Bifido5′and Bifido3′) and the real-time PCR conditions were specific for the bifidobacteria species tested. All nonbifidobacterial strains tested showed no amplification during PCR and no increase in fluorescence during real-time PCR.

Quantification of bifidobacteria in feces.Standard curves obtained for the strains B. infantis DSM 20088^T, B. longum JCM 1217^T, and B. adolescentis JCM 1275^T were in good agreement (data not shown). For the standard curve, the results obtained (C_t values) for the three strains were plotted together against the initial number of cells in the cultures (Fig. 1). Results were found to be linear over the range of cell concentrations tested (10⁴ to 10⁹ cells/ml).

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FIG. 1.

Calibration curve obtained by plotting C_t values as a linear function of the base 10 logarithm of the initial number of bifidobacteria (B. infantis DSM 20088^T, B. longum JCM 1217^T, and B. adolescentis JCM 1275^T) in the culture determined by plate counting.

To evaluate the applicability of the assay to the quantification of bifidobacteria in fecal samples, 24 samples were analyzed by quantitative real-time PCR and FISH. The results showed, in general, a good correlation between the two methods (Table 3), but in samples with small numbers of bifidobacteria (samples I6, I12, and A3), the results of FISH appear to overestimate the number of bifidobacteria with respect to the real-time PCR by about two logarithmic units. The presence of PCR inhibitors in those samples was tested by conducting a real-time PCR with a control sample in the presence of 1 μl of DNA extract from the problem sample; the fluorescent signal was the same for the control sample in the presence or absence of DNA extract from the problem samples, indicating that there were no PCR inhibitors in those DNA extracts.

The inter-PCR reproducibility of the quantification was found to be very high based on the C_t values obtained for two DNA extracts (a pure culture of B. infantis DSM 20088^T and a fecal specimen) in five replicate runs. The average C_t values obtained for those samples were 23.35 (range, 23.0 to 23.5) and 26.9 (26.8 to 27.0). Similar results were observed with other samples. On the other hand, by analyzing duplicate DNA extracts from five fecal samples, the interextract variability was found to be less than 10% (mean, 9.82%; standard deviation, 5.3%) (data not shown).

Quantification of bifidobacteria in spiked germ-free feces.In order to determine the detection limit of the real-time PCR method and to compare the accuracy of real-time PCR and FISH, feces from germfree Agus rats were spiked with different amounts of B. infantis DSM 20088^T and analyzed by the two methods (Fig. 2). Both methods led to accurate quantification in the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate than FISH for samples with low levels of analyte.

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FIG. 2.

Results of the analysis of germfree rat feces spiked with known amounts of B. infantis DSM 20088. □, cells added; ○, cells detected by FISH; ×, cells detected by real-time PCR.

On the other hand, the detection limit of the real-time PCR procedure with the DNA extract from feces and the PCR conditions described in this paper was found to be about 5 × 10⁴ cells/g of feces.