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Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center,1 Veterans Administration Medical Center, Baylor College of Medicine, Houston, Texas2
Received 4 March 2002/ Returned for modification 16 June 2002/ Accepted 12 July 2002
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ABSTRACT |
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INTRODUCTION |
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At present, there are at least seven diagnostic assays for H. pylori: bacterial culture, a rapid urease test, a urea breath test, histology, PCR, serology, and a stool antigen test (14, 15, 20, 22, 26, 28, 33). The sensitivities of bacterial culture, the rapid urease test, the urea breath test, histology, and the stool antigen test are limited when few organisms are present or when patients are taking acid-suppressing agents (proton pump inhibitors) (29). More importantly, none of these techniques accurately quantifies the number of H. pylori present in test samples. Because H. pylori is a fastidious, slow-growing bacterium, it requires 4 to 5 days to grow in rich media and requires specific culture conditions (24). Because normal stomach flora can cause serious interference with the culture of H. pylori, highly restricted culture plates containing several antibiotics are required. Generally, however, the different types of antibiotics used in agar culture plates suppress the growth not only of normal stomach flora but also of H. pylori (35, 36).
All of these characteristics of H. pylori culture make accurate quantification of the bacteria unreliable. The urease assays are not sensitive (12, 49, 52) and may not be specific in the presence of other urease-positive bacteria (37, 38). Serology may not differentiate active from past infection and cannot be used to indicate the clearance of H. pylori from the stomach because antibodies may stay at the same level even after eradication of the bacteria. Like these techniques, PCR also has drawbacks. PCR detects only specific gene fragments, not viable bacteria. However, all of the methods mentioned here, except for PCR, are qualitative assays. To monitor the effectiveness of therapy or a vaccine, a quantitative assay is required.
Presently, the only available quantitative assays for H. pylori are based on either competitive or noncompetitive PCR. The PCR products can be measured by the intensity of ethidium bromide luminescence on agarose gels (18), an enzyme-linked immunosorbent assay (ELISA) after capturing biotin-labeled probe-amplicon complexes on streptavidin-coated microtiter plates, or an ELISA-based bioluminescence assay in which a calcium-dependent flash-type bioluminescent tag (aequorin) is used to detect the product at the attomolar level (1, 2). The last two detection techniques have been used in our laboratory (44, 46). Furthermore, coamplification methods involving competitive and noncompetitive PCR use endpoint analysis, which is adversely affected by variable amplification efficiency (13, 43). Not knowing if amplification efficiencies are equal between the internal or external control and the target amplicon sacrifices the sensitivity and accuracy of these assays. Also, coamplification methods are not suitable for multigene analysis and clinical application because of low throughput and a narrow dynamic range of the products.
A more accurate quantitative assay for detecting H. pylori could facilitate monitoring of therapy, enable more accurate epidemiological studies on the acquisition and spread of H. pylori infection, and provide a standard by which to measure the effectiveness of vaccines against H. pylori. The present study is the first to use real-time quantitative (Q)-PCR to detect the number of H. pylori in clinical samples. With the LightCycler, a temperature-controlled microvolume fluorimeter that provides rapid real-time Q-PCR and product analysis in a single closed-tube system (58), we have demonstrated the specificity, sensitivity, and reproducibility of this method for detecting H. pylori in clinical samples.
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MATERIALS AND METHODS |
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Clinical biopsy samples. One hundred human gastric biopsy samples that had been obtained within the last 2 years were tested. These samples were collected from patients seen in the Digestive Disease Section, Veterans Affairs Medical Center, Houston, Tex. The samples were not collected specifically for the current study. Histological and microbiological data were available for all samples tested. The frozen samples were thawed at room temperature and homogenized in 1 ml of transport medium (Brucella broth with 20% glycerol). A 100-µl aliquot of each homogenized tissue was used for DNA isolation with the revised proteinase K-phenol method.
The clinical biopsy samples were divided into five groups: high H. pylori infection with high contamination, high H. pylori infection with low contamination, high H. pylori infection without contamination, low H. pylori infection without contamination, and H. pylori negative. These were established on the basis of results from bacterial culture and histopathological assays, as follows: (i) negative samples were defined as those that were negative for H. pylori by histology and culture; (ii) positive samples were those positive for H. pylori by both histology and culture; (iii) contaminated samples were those that had bacteria besides H. pylori recovered on culture; (iv) noncontaminated samples were those that had only H. pylori isolates recovered on culture; (v) low H. pylori infection was defined as less than 500 colonies; and (vi) high H. pylori infection was defined as more than 1,000 colonies. Quantitation of bacteria was based on the number of colonies growing on test plates (viable numbers). Laboratory records indicated the level of growth as 1 to 10 colonies, 25 to 50 colonies, 100 colonies, 500 colonies, or more than 1,000 colonies (semiquantitative). No actual counting of colonies was performed.
Template preparation for standard curve. Total DNA from H. pylori SS1 was isolated with the Easy-DNA kit (Invitrogen, Inc.) according to the manufacturer's instructions. PCR amplification of the ureC gene fragment of H. pylori was performed with the primer pair HP-FOR-out (5'-TCTGTCTGATTCGCTTTTCTG-3') and HP-REV-out (5'-AAGCTCGCTAAAAACGACC-3') (Integrated DNA Technologies, Inc., Coralville, Iowa). Total H. pylori DNA (25 ng) was added to a final 50-µl reaction mixture containing 20 mM Tris-Cl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each of the four deoxynucleotides, and 0.5 µM each of the oligonucleotides. The Taq DNA polymerase (2.5 U; Roche Molecular Biochemicals, Indianapolis, Ind.) was added, and the reaction mixture was overlaid with approximately 40 µl of mineral oil to prevent evaporation. Amplification consisted of an initial denaturation of target DNA at 95°C for 10 min, followed by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 54°C for 30 s, and extension at 72°C for 1 min. The final cycle included extension for 10 min at 72°C to ensure full extension of the product. Samples were amplified with a Perkin-Elmer Cetus DNA thermal cycler.
The PCR amplicon (820 bp) was cloned into pUni/V5-His-TOPO and transformed with the Echo cloning system, (Invitrogen Inc.) following the manufacturer's instructions. Automated DNA sequencing further confirmed that the expected ureC gene fragment had been cloned into pUni/V5-His-TOPO; the resulting construct was called pUni-Uc-out3. The circular plasmid was dissolved in 10 mM Tris-CDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid) buffer and linearized with SacI (Boehringer, Mannheim, Germany) at 37°C for at least 16 h. The absorbance of the DNA solution was measured three times at 260 nm on a DU 530 Life Science UV/Vis spectrophotometer (Beckman), and the mean value was taken as the actual absorbance.
The copy number of the ureC gene was calculated as copy number of ureC gene = [(concentration of linearized plasmid)/(mass of pUni-UreC)] x (6.023 x 1023) (formula 1).
LightCycler Q-PCR. LightCycler (Roche Diagnostics, Indianapolis, Ind.) Q-PCR was performed (operating system version 3.0) in 10-µl mixtures containing 1 µl of Faststart DNA Cybergreen I (Roche Molecular Biochemicals), 1.5 mM MgCl2, 0.5 mM each primer (HP-FOR, 5'-TTATCGGTAAAGACACCAGAAA-3', and HP-REV, 5'-ATCACAGCGCATGTCTTC-3'), and 5 µl of extracted DNA (1 to 25 ng). The reaction was performed with preliminary denaturation for 10 min at 95°C (slope, 20°C/s), followed by 40 cycles of denaturation at 94°C for 10 s (slope, 20°C/s), annealing at 54°C for 5 s (slope, 20°C/s), primer extension at 72°C for 8 s (slope 20°C/s), and product detection at 77°C for 5 s (slope, 20°C/s). A final cooling step was performed at 4°C for 1 min (slope, 20°C/s). A 132-bp product resulted from the reaction.
Determination of ureC gene copy number in H. pylori SS1. Genomic DNA from H. pylori SS1 was extracted by the revised proteinase K-phenol protocol and dissolved in 1x TC buffer (10 mM Tris and 1 mM CDTA [pH 8.0]). The absorbance of the DNA solution was measured at 260 nm three times, and the mean value was recorded as the actual absorbance. The number of bacteria was calculated as number of H. pylori per microliter = [(concentration of DNA solution)/(mean mass of H. pylori genome)] (formula 2). The mean mass of the H. pylori genome was calculated from the mean size of the genome, which was assumed to be 1.6 Mb. The dilution series of H. pylori as calculated from formula 2 was coamplified with the dilution series of the standards, as calculated from formula 1.
There is one copy of the ureC gene per H. pylori organism (54), and there is also one copy of the ureC gene in pUni-UreC; thus, pUni-UreC was used to create a standard curve to determine the number of H. pylori in an unknown sample.
Quantitation of H. pylori in clinical samples. Eighty-five randomly selected clinical biopsy samples were assayed by Q-PCR one time each. The sample with the highest copy number and the sample with the lowest copy number (<1,000) from each PCR were repeated in the next Q-PCR assay.
Detection of H. pylori by PCR amplification of other genes. Twenty additional clinical biopsy samples that were negative for H. pylori by bacterial culture were subjected to PCR for four different H. pylori genes: hpaA, ureA, vacA, and ureC. A DNA fragment of the pUni vector was also amplified. All oligonucleotide primers were purchased from Integrated DNA Technologies, Inc. The hpaA gene was amplified with primers hpaA-FOR (5'-CAATCAAGGATAGAACGATG-3') and hpaA-REV (5'-CTAACGCTTGAACTTTCTC-3') for 35 cycles of denaturation at 94°C for 30 s, primer annealing at 49°C for 1 min, and extension at 72°C for 1 min to produce a 179-bp product. The ureA gene was amplified with primers ureA-FOR (5'-GAGAATGAGATGAAACTCACCC-3') and ureA-REV (5'-TTGTCTGCTTGTCTATCAACC-3') for 35 cycles of 94°C for 30 s, 54°C for 1 min, and 72°C for 1 min to produce a 627-bp product. The vacA gene was amplified with primers vacA-FOR (5'-ATGGAAATACAACAAACACAC-3') and vacA-REV (5'-CTGCTTGAATGCGCCAAAC-3') for 35 cycles of 94°C for 30 s, 51°C for 1 min, and 72°C for 1 min to produce a 259-bp product. The DNA fragment of pUni-Uc was amplified with primers pUni-FOR (5'-AGCCATCATCACCATCACC-3') and pUni-REV (5'-GCCAACTCAGCTTCCTTTC-3') for 35 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min to produce a 299-bp product.
All amplifications began with an initial denaturation of target DNA at 95°C for 10 min, and the final cycles all concluded with extension for 10 min at 72°C to ensure full extension of the product. These four amplifications were performed with the Perkin-Elmer Cetus DNA thermal cycler. The amplified products were characterized by ethidium bromide staining of electrophoresed agarose gels. PCR for the ureC gene was performed with the LightCycler as previously described. The ureC PCR products were removed from the capillary tubes and analyzed by the same procedure as the other amplified samples.
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RESULTS |
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LightCycler Q-PCR. The specificity of Q-PCR amplification of this fragment of the ureC gene has been tested previously in our laboratory (44). No PCR occurred with the ureC primers with DNA extracts from normal mouse stomachs (not infected with H. pylori), DNA extracts from normal mouse stomach flora grown on Luria-Bertani (LB) agar plates (44), or human genomic placental DNA. Additionally, the sequencing results from two human stomach biopsy samples matched the H. pylori ureC gene but no genes from any other organism, as shown by Blast analysis on the National Center for Biotechnology Information web site.
ureC gene copy numbers for the standard curve were calculated with formula 1. As shown in Fig. 1A, the values calculated by Q-PCR for each dilution from 109 to 103 copies were very close to their standard values. Linear regression analysis, plotting the cycle number versus the log concentration of the amplicon, gave a straight-line plot and a correlation coefficient of -1.0, which indicated that the threshold cycle number from 109 to 103 ureC gene copies varied linearly. The mean curve shift value was approximately three cycles. The amplification efficiencies for each dilution of the amplicon were the same (approximately two) between Q-PCR and the standard curve, which indicated that under optimal PCR conditions, 109 to 103 ureC gene copies could be amplified accurately, providing the ideal conditions for Q-PCR.
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The reproducibility of Light Cycler Q-PCR was also evaluated in 15 clinical biopsy samples (Table 1). Analysis of variance of 12 samples (80%) indicated that the reproducibility was acceptable, with no significant difference within or between runs. However, results from three samples (3103, 3114, and 3116) showed that a quantitative reaction had not occurred.
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Although 24 (89%) of 27 samples that were negative for H. pylori by culture had a high ureC gene copy number in the Q-PCR assay (the mean ureC gene copy number in a 100-µl aliquot of all the negative samples combined was 107), 3 (4.1%) of 73 samples that were positive for H. pylori by culture also had fewer than 1,000 copies of the ureC gene.
Detection of H. pylori by PCR amplification of other genes. To further examine the discordance between Q-PCR and the culture assay, three other H. pylori genes (hpaA, ureA, and vacA) were examined by traditional PCR on DNA from 20 clinical samples. A DNA fragment of the pUni vector was also amplified by PCR to determine whether the biopsy samples were contaminated with pUni.
The PCR results showed that 9 (45%) of 20 samples were hpaA positive, 14 (70%) were ureA positive, and all 20 (100%) were vacA positive. All 20 (100%) were also negative for the pUni DNA fragment (data not shown). Although some of the samples were positive for both hpaA and ureA, others were positive for only hpaA or ureA. All 20 of the biopsy samples were ureC positive (data not shown). LightCycler analysis showed that the samples each contained between 3.3 x 106 and 2.9 x 107 copies of the ureC gene.
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DISCUSSION |
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The classical protocol for isolating nucleic acid from relatively large amounts of tissue (200 mg to 1 g) is the proteinase K-phenol method. For isolating nucleic acids from small amounts of tissue, there are no established protocols. However, there is one publication describing DNA isolation from small tumor biopsies (47). To isolate H. pylori DNA from fresh or frozen biopsy samples of the stomach, three methods have been used: kits from commercial companies, the proteinase K-phenol method, and a boiling-water method. The best of the three methods for obtaining a high yield or purity of H. pylori DNA has been shown to be the classical proteinase K-phenol method (10, 18, 19, 25, 44, 51, 56), which is supported by our results. Although we showed little difference in DNA degradation between DNA isolated with the commercial kit and that isolated with the revised proteinase K-phenol method, the revised proteinase K-phenol method produced a much higher yield. Thus, this method of isolation was used in our subsequent experiments to guarantee the accuracy and reliability of Q-PCR.
Presently, there are two types of Q-PCR: kinetic PCR and coamplification PCR. Although the kinetic methods have several advantages over the coamplification methods (13, 43), the few published studies of quantitation of H. pylori in biopsy samples (18, 39, 44, 45) have used the coamplification method. This is the first report of real-time Q-PCR to detect H. pylori.
The specificity of our Q-PCR assay was verified by PCR with normal human placental DNA as a control and DNA sequence analysis of the PCR products from two clinical samples. When optimal PCR conditions were used, the quantitative sensitivity of the assay was 103 gene copies. Fewer than 103 copies could not be accurately quantified, although specific products were detected for 101 and 102 copies, indicating that as few as 10 copies could be amplified. This is in agreement with published reports showing sensitivities of between 10 and 60 gene copies (3, 34, 42).
There are several possible reasons why 103 to 101 copies were not quantitatively detected with our assay. First, the PCR conditions may not have been optimal, resulting in the formation of primer dimers and nonspecific products. However, this is unlikely, because no primer dimers or nonspecific products were formed during the amplification of 101 to 109 copies. Second, the PCR could have been random because of the intrinsic nature of the very diluted amplicon. This reason seems more convincing. Physically, PCR is a thermodynamic reaction, which requires the collision of the amplicon and primer molecules. When sufficient primer molecules are used in the reaction and the amount of amplicon is large enough, the chance of collision between the amplicon and primer molecules does not vary much between cycles. However, if the amount of amplicon is low, the chance of collision varies, which leads to random product formation and causes a random threshold cycle number.
Fifteen clinical biopsy samples were repeatedly tested to assess the reproducibility of the Q-PCR assay. Only 3 (20%) of the 15 samples produced results that were not quantifiable. Thus, the LightCycler technique has good reproducibility, as previously reported (27, 34, 41, 42, 57), and can be used in the quantification of H. pylori in clinical samples.
Presently, two methods are available to calculate the number of H. pylori in a sample: optical density measurement and a measurement based on the mass of H. pylori genomic DNA. The former measures the optical density (turbidity) of the culture medium at 600 nm and is based on the assumption that an absorbance of 1.0 equals 1.6 x 109 organisms/ml. The latter method is based on the fact that there is only a slight variation in the size of genomic DNA between H. pylori organisms, and 1.6 to 1.8 Mb is used as the mean size of the chromosomal DNA of one organism. This method is inaccurate, mainly because different strains have different chromosome sizes and because the presence of plasmid DNA also changes the total DNA quantity.
The SS1 strain of H. pylori contains plasmids (16), which was confirmed in our study (data not shown). Therefore, our strategy for calculating the bacterial number was to use the relationship that the number of ureC genes equals the number of H. pylori in a sample, because we knew that one H. pylori organism has only one copy of the ureC gene. We clearly showed the feasibility of our strategy by demonstrating that the bacterial number first calculated from the mean mass and then recalculated with the ureC gene copy number based on the standard was approximately 50% less than the initial value calculated from the mean mass. This suggested that there was at most one copy of the ureC gene per H. pylori. The reason the recalculated value was 50% of the initial value calculated from the mean mass was partially explained by the existence of plasmids in H. pylori SS1. Also, our calculation strategy was quick, easier than the optical density measurement, and more accurate than the mean mass method.
Eighty-five randomly selected clinical biopsy samples were assayed by LightCycler Q-PCR, and the results were compared with those from bacterial culture and histopathological assays to determine whether Q-PCR could be used clinically. Based on our dot-plot graph of the log number of bacteria for each group and on analysis of variance, we conclude that the clinical groups we established according to the bacterial culture assay were not suitable based on our Q-PCR results. The reason may have been that the Q-PCR and bacterial culture assays measured different parameters within the same clinical samples.
Q-PCR measures the presence of a specific DNA fragment, while bacterial cultures measure the presence of whole, viable bacterial cells. Contamination with dead H. pylori could affect the results of Q-PCR but not the results of bacterial culture. Stomach flora had no effect on our Q-PCR results but could have adversely affected the culture assay. There was a significant difference between the clinical H. pylori infection group and the H. pylori-negative group, but there was no significant difference between the high H. pylori infection and low H. pylori infection groups. The differences we detected may have been due to the effects of stomach flora or antibiotics used in bacterial culture.
The most striking result of our examination of clinical biopsy samples was that many that were negative for H. pylori by culture contained large amounts of H. pylori DNA when analyzed by Q-PCR. A possible explanation for this is that there is another form of H. pylori that is difficult to detect by bacterial culture and histopathologic assays but can be detected by PCR. H. pylori does exist in two forms, an actively dividing spiral form and a coccoid form (8). The coccoid was first identified in the human stomach in 1993 by Chan et al. (9), who also showed that it could be distinguished from pathogenic and nonpathogenic bacterial cocci, fungal spores, and cryptosporidia. Growing evidence supports the concept that the coccoid form of H. pylori is not simply a degenerate morphological manifestation but is alive and metabolically active, although it cannot be cultured (6, 9, 17, 23, 30-32, 53). The coccoid form may result from antibiotic treatment, as in the case of H. pylori gastritis.
Another possible explanation for our results is that there were no viable bacteria in the stomach biopsy samples but only dead organisms or chromosomal DNA left over after cell death. However, how long dead bacteria or leftover chromosomal DNA can remain in the stomach is unknown. Therefore, at this time, there is no solid evidence for this explanation. Contamination must also be considered because the tissue homogenization step, bacterial culture step, and DNA isolation step of Q-PCR were all performed in a single, crowded laboratory. In addition, H. pylori or genetic material from H. pylori could have entered the humans from which samples were taken via contaminated water or food (7).
Because of the conflicting findings between the bacterial culture assay and Q-PCR, we examined 20 additional clinical biopsy samples, all of which were confirmed to be negative for H. pylori by bacterial culture. In these samples, the number of H. pylori genes tested was increased from one (ureC) to four (hpaA, vacA, ureA, and ureC). Furthermore, to check whether the isolated DNA from bacterium-negative culture samples was contaminated with pUni, PCR for a fragment of the pUni vector was performed. The results showed that 9 (45%) of the samples were hpaA positive, 14 (70%) were ureA positive, 20 (100%) were vacA positive, and 20 (100%) were negative for the pUni DNA fragment (data not shown).
The reason some samples were hpaA and ureA negative by PCR may have been that the specificity of the two primer sets used to detect H. pylori was relatively low. The formation of nonspecific PCR products could also have affected the formation of specific products. Also, based on the LightCycler results, there were 3.3 x 106 to 2.9 x 107 ureC gene copies in the samples. Also, only the expected PCR product (
132 bp) was detected, which indicated that PCR for the ureC gene has a higher specificity than that for the hpaA, vacA, and ureA genes in the clinical biopsy samples. Thus, the quantification method with the LightCycler Q-PCR can be applied to clinical samples. A PCR amplicon in H. pylori-negative culture samples could indicate the presence of a coccoid form of H. pylori or contamination with H. pylori genetic material.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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