Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis

Abstract

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 μg, thereby avoiding false-negative results.

PCR is used to diagnose many infectious diseases (6–9, 11). PCR has to be very sensitive and specific to detect small amounts of microbial DNA in eukaryotic gene material. The optimum result from most PCRs is a single strongly stained gel band representing the desired product. Frequently, however, weak, multiple bands or a complete absence of bands is observed. Many different parameters can influence the outcome of a PCR amplification. In clinical samples it is important to control for the presence of potential PCR-inhibitory compounds, such as EDTA, heparin, porphyrins, and H_XPO₄ⁿ⁻ (12). EDTA and heparin may be present in blood samples as anticoagulants. Moreover, the heme compounds from hemolized erythrocytes are often copurified with DNA in the extraction process, and this interferes with PCR (1, 2). Most of the protocols used for the extraction and precipitation of DNA unavoidably require various washing steps: a first washing of blood with erythrocyte lysis solution to remove most of the hemoglobin derivatives and various other washings of the leukocyte pellet with water, for lysis and for eliminating other substances that can interfere with the PCR process.

On the other hand, the diagnostic PCR signal can be inhibited if the amount of host DNA exceeds a certain threshold value (5). Since the particular threshold for each system may not be known, the phenomenon may be ignored altogether, and this may lead to false negatives.

We evaluated the effects of washing and studied the acceptable concentrations of total DNA (bacterial plus host leukocytic) to obtain an optimal yield and to ensure the reliability of PCR signals for the diagnosis of Brucella spp. in human peripheral-blood samples.

MATERIALS AND METHODS

Clinical specimens.

Peripheral-blood samples from 32 patients with active brucellosis diagnosed in the Infectious Diseases Unit of “Carlos Haya” Regional Hospital, Malaga, Spain, over a period of 12 months were taken before starting appropriate antibiotic treatment. In 20 cases the diagnosis of brucellosis was established by the isolation of Brucella cells in blood culture, and in the other 12 cases diagnosis was based on a compatible clinical picture together with the presence of high titers of antibrucella antibodies or a fourfold or greater increase in titers between two paired serum samples drawn 2 to 3 weeks apart. High titers were considered to be ≥1/160 for Wright’s seroagglutination test and ≥1/320 for Coomb’s antibrucella test. The clinical characteristics of the patients included in this study have been described previously (11). Control blood samples were obtained from 25 healthy subjects with no history of brucellosis or exposure to Brucella spp.

Isolation of DNA from clinical blood samples.

A modification of the method described by Miller et al. (10) was used. Briefly, 0.5 ml of the blood collected in sodium citrate and stored at −20°C was resuspended in 1 ml of erythrocyte lysis solution (320 mM saccharose, 5 mM Mg₂Cl, 1% Triton X-100, and 10 mM Tris-HCl [pH 7.5]) and the suspension was mixed and centrifuged at 15,000 × g for 2 min. The supernatant was discarded, and the leukocyte pellet was washed with Milli-Q water to lyse the cells. This washing with water was carried out by two different methods.

(i) Method A.

Treatment with 1 ml of water was performed twice, and samples were centrifuged as described above. The pellets were agitated vigorously and remained whole without breaking. After the two washings the supernatant was transparent, having lost all reddish coloring, but the leukocyte pellets maintained a light reddish color. Template DNA was obtained from the leukocytes as follows. Four hundred microliters of nucleic lysis buffer (60 mM NH₄Cl and 24 mM Na₂-EDTA [pH 8.0]) containing proteinase K (1 mg/ml) and sodium dodecyl sulfate (1%) was added to the pellet, and the solution was mixed and incubated for 30 min at 55°C. After digestion the samples were cooled at room temperature, and 100 μl of 7.5 M ammonium acetate was added to each tube. The tubes were shaken for 30 s, followed by centrifugation at 15,000 × g for 10 min. The supernatant containing total DNA was transferred to a fresh tube. Two volumes of absolute ethanol were added, and the tubes were inverted several times until the DNA precipitated. DNA was recovered by centrifuging the samples at 15,000 × g for 10 min. The pellets were rinsed with 1 ml of 70% ethanol, dried, and resuspended in 30 μl of water. A template mixture of 20 μl was used for amplification. The concentration and purity of DNA were measured after PCR but not before.

(ii) Method B.

The leukocyte pellets were washed and homogenized four or five times until all reddish coloring disappeared, and a DNA preparation was made as described for method A. The concentration and purity of the DNA were then determined spectrophotometrically by readings of A₂₆₀ and A₂₈₀. To amplify the target sequence, 2 to 4 μg of total DNA was used, although very occasionally not even 2 μg was available (although this still proved to be sufficient to give a good amplification band), but never more than 4 μg.

The hemoglobin solution from erythrocyte lysates was prepared with peripheral blood. Briefly, 1 ml of fresh whole blood (150 mg of human hemoglobin per ml) was added to 500 ml of double-distilled water. The mixture was shaken vigorously, and after 30 min of sedimentation the solution was aliquoted and centrifuged for 15 min at 13,000 × g to remove cellular membranes. The absorbance spectra at 350 to 680 nm from 1 ml of this aqueous mixture containing 0.3 mg of human hemoglobin and from the product of the DNA purification step with 7.5 M ammonium acetate were recorded with a Shimazu UV-160A double-beam spectrophotometer.

All the peripheral blood samples from the patients were compared with samples from healthy control subjects to control for any possible contamination during the process of extraction and DNA purification.

DNA amplification.

A 223-base-pair PCR target sequence present in a gene encoding a 31-kDa Brucella abortus antigen was selected for amplification. The PCR primers used in this study were those described by Baily et al. (4) and had been utilized by us previously (11). PCR was performed with a 50-μl mixture containing template DNA, PCR buffer (10 mM Tris-HCL [pH 8.4], 50 mM KCl, 1.0 mM MgCl₂), a 100 nM concentration of each PCR primer (Pharmacia LKB, Barcelona, Spain), a 200 μM concentration of each deoxyribonucleoside triphosphate (Boehringer, Mannheim, Germany), and 1.25 U of Taq polymerase (Boehringer). The reaction was performed in a DNA thermal cycler without mineral oil (model 2400; Perkin-Elmer, Norwalk, Conn.). PCR consisted of a preheating at 93°C for 5 min, 35 cycles of 90°C for 1 min, 60°C for 30 s, and 72°C for 1 min, and an incubation at 72°C for 7 min. The PCR products were loaded on a 2% agarose gel with 2 μg of ethidium bromide per ml to determine the sizes of the amplified products.

Negative controls containing all of the reagents but lacking template DNA were routinely processed exactly as described above to monitor for contamination with Brucella DNA and were negative in all experiments. Positive controls with 100 ng of genomic DNA isolated from a suspension of B. abortus B-19 were included in each experiment. All PCRs were carried out in duplicate.

RESULTS

The effects of the number of washing steps and the total DNA concentration on the PCR results for 5 of the 32 peripheral blood samples from patients with brucellosis are shown in Fig. 1. Table 1 shows the mean DNA concentrations and purities for the samples from all 32 patients, as well as the mean amounts of DNA per PCR. When the leukocyte pellet was washed twice and the concentration and purity of template DNA in the amplification reaction mixture were unknown, the five samples performed in duplicate showed complete inhibition of the 223-bp amplification product (Fig. 1A). On the other hand, when the number of washing steps was increased from two to four or five and the amount of total DNA per PCR was reduced to 2 to 4 μg, a clear visualization of PCR-amplified fragments was possible in all cases after electrophoresis with agarose gel (Fig. 1B).

FIG. 1.

(A) Agarose gel electrophoresis and ethidium bromide staining. Lane MW, DNA ladder; lane 1, no DNA added; lane 2, positive control (B. abortus B-19 DNA); lanes 3 to 12, DNAs from five patients with brucellosis for which PCR was carried out in duplicate, with two washings and high concentrations of total DNA. (B) Results for DNA in samples from the same five patients, with the number of washing steps increased to four or five and the amount of total DNA decreased to 2 to 4 μg. The photocomposition of the figure was obtained from the original Polaroid films with a ScanJed IIcx scanner (Hewlett-Packard, Corvallis, Oreg.). After the initial image was scanned and saved as a TIFF file, the file was opened in Adobe Photoshop, version 3.0 (Adobe System, Inc., Seattle, Wash.).

TABLE 1.

Concentrations, purities, and amounts of template DNA under the two sets of experimental conditions for samples from patients with brucellosis^a

Exptl conditions	DNA concn (ng of DNA/μl)	A260/280	Amt of template DNA (μg)/PCR
2 washings; DNA concn measured after PCRb	520.8 ± 14.1	1.6 ± 0.02	10.4 ± 0.28
4 washings; DNA concn measured prior to PCRc	347.7 ± 34.6	1.7 ± 0.03	3.1 ± 0.14

^aResults are expressed as the means ± standard errors of the means for samples from 32 patients. 

^bThe sample volume added to the PCR mixture was always 20 μl. 

^cThe sample volume was variable and depended on the concentration of DNA obtained in the extraction process. 

To examine the effects of washing, the absorption spectrum of a solution of hemoglobin from erythrocyte lysis was recorded together with the absorption spectra obtained in the DNA purification step with ammonium acetate. The hemoglobin solution and its derivatives showed specific absorbances at 409, 540, and 576 nm in the spectrum. After the DNA purification step with ammonium acetate and two washings the mixture was yellowish and showed specific absorbance at 409 nm, the absorbances at 540 and 576 nm having disappeared. After four or five washings the yellowish solution became transparent and the specific absorbance at 409 nm showed a significant decrease (Fig. 2).

FIG. 2.

Spectra at 350 to 680 nm of hemoglobin from an erythrocyte lysis solution (a), after two washings of the leukocyte pellet with ammonium acetate in the DNA purification step (b), and after four washings of the leukocyte pellet under the same conditions (c). The spectral peaks at 540 and 576 nm are characteristic of oxyhemoglobin. The spectral peak at 409 nm (Soret band) is characteristic of porphyrins and heme compound derivatives.

We then examined in combination the two possible factors that could cause inhibition of the amplification signal. First, to check whether the heme compound and its derivatives were responsible for the inhibition, we washed the leukocyte pellet twice but we kept the total DNA amount within the correct range of 2 to 4 μg. The resultant PCR products were inhibited (Fig. 3, lane 4). Second, to test whether the large amount of total DNA was responsible for inhibiting the amplification, we washed the leukocyte pellet four or five times, thereby eliminating the potential interference by the heme compounds, but added a large amount of total DNA (from 8 to 11 μg). In this case, the signal was also inhibited (Fig. 3, lane 8). Finally, when we washed the leukocyte pellet four or five times and added 2 to 4 μg of total DNA to the PCR mixture, the presence of an amplification signal was detected (lanes 5 to 9). All these experiments were repeated with the healthy control subjects, and the results were negative in all cases (Fig. 3, lanes 6, 7, 10, and 11).

FIG. 3.

Agarose gel electrophoresis and ethidium bromide staining. Lane MW, DNA ladder; lane 2, no DNA added; lane 3, positive control (B. abortus B-19 DNA); lanes 4 and 6, DNAs from a patient with brucellosis and a healthy subject, respectively, after two washings and with total DNA amounts within the correct range (2 to 4 μg); lanes 5 and 7, DNAs from a patient with brucellosis and a healthy subject, respectively, after four washings and with total DNA amounts within the correct range (2 to 4 μg); lanes 8 and 10, DNAs from a patient with brucellosis and a healthy subject, respectively, after four washings and with large amounts of total DNA (8 to 11 μg); lanes 9 and 11, DNAs from a patient with brucellosis and a healthy subject, respectively, after four washings and with total DNA amounts within the correct range (2 to 4 μg).

DISCUSSION

The conditions of each PCR and the handling of the reagents for this technique are very important to maintain the sensitivity and guarantee the quality of the results. False negatives have been detected in PCR assays, suggesting the possible existence of inhibitors in the clinical samples which can compromise the sensitivity of the assay and which are as important as false positives caused by contamination.

Serious interference with PCR has been described in forensic studies of bloodstains in which the heme compounds were often copurified with DNA by phenol-chloroform extraction. These heme compounds are likely to be the products of the proteinase K digest of some heme blood protein complex, such as ferrous or ferric heme and serum albumin, known as hemalbumin or metahemalbumin, which interfered with the amplification reaction (1, 3). In our study, to eliminate the possibility of PCR inhibitors such as heparin (which binds Taq polymerase) and EDTA (which chelates Mg²⁺ ions from the PCR mixture), we used sodium citrate as the anticoagulant. The possible cause for false negatives in Fig. 1A could, therefore, be the presence of porphyrins or the heme compounds, which remain in the DNA sample due to the lack of sufficient washings and to the high concentrations of DNA in the PCR mixture. Bearing in mind the results shown in Fig. 2, the contaminants were probably heme compound precursors or derivatives (porphyrins, hematin, or a heme-blood protein complex).

On the other hand, Cogswell et al. (5) demonstrated that host DNA can interfere with PCR detection of Borrelia burgdorferi in skin biopsy specimens. In this case the problem was solved by diluting the host DNA sample to a level below the threshold value. Likewise, an increase in PCR sensitivity was noticed by von Stedingk et al. (13) at higher dilutions of skin DNA samples, a result achieved empirically by these authors but not investigated further. When the total DNA used as a template is present at high concentrations, it is able to inhibit competitively primer-template hybridization. Moreover, the inhibition may occur as a simple consequence of a decrease in the rate of diffusion of all macromolecular components of the reaction mixture in the presence of large amounts of long strands of host DNA (5). Matar et al. (9) recently described the necessity of using nested PCR to visualize correctly the amplification product of 223 bp in samples from patients with brucellosis. However, if the DNA concentration from peripheral mononuclear cells was not measured, a high DNA concentration in the first amplification mixture could explain the failure to visualize the amplified product. In this case, the heme compounds would not have interfered with the amplification because the peripheral mononuclear cells were separated from the erythrocytes with Ficoll-Hypaque.

In our case, the results obtained seem to show that high concentrations of total DNA inhibit the amplification signal, but when the amount (2 to 4 μg) and purity of template DNA are known before amplification, a clear visualization of PCR-amplified fragments is possible. In conclusion, based on these findings we recommend that blood specimens to be analyzed by PCR be washed five times after lysis and that no more than 4 μg of total DNA be permitted in the aliquot to be analyzed.