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Journal of Clinical Microbiology, November 2002, p. 4211-4217, Vol. 40, No. 11
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.11.4211-4217.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Medical Microbiology, University of Turku,1 National Public Health Institute, Department in Turku, Turku, Finland2
Received 29 April 2002/ Returned for modification 21 June 2002/ Accepted 17 August 2002
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ABSTRACT |
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INTRODUCTION |
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In general, an optimal sample processing method should concentrate the DNA, especially that derived from the target organism, and wash out inhibitory factors commonly present in biological fluids. To be applicable for routine diagnostic use, the process should be suitable for use with an array of clinical specimens, simple or preferably at least semiautomatic, reproducible, and safe for the staff handling the specimens. Furthermore, design of the process should prevent cross-contamination between the samples; i.e., samples with large amounts of target microbial cells or their nucleic acids should not contaminate other specimens in the batch.
In contrast to species-specific PCR assays, the possible target organisms of a clinical broad-range bacterial PCR test can be highly variable in their capability to resist chemical and physical treatments. In other words, an optimal sample preparation procedure should efficiently break very resistant bacterial cell walls, like those of streptococci and mycobacteria, without being too harsh for the DNA released from cells that are easily lysed. Another aspect specific for broad-range bacterial PCR is the danger of introducing bacterial DNA from the reagents used in various phases of sample processing and amplification (9), which may result in false-positive PCR results. Elimination of this background bacterial DNA from the reactions has proved very difficult (1). Thus, aiming at maximal analytical sensitivity in the broad-range bacterial PCR assay may eventually result in impaired clinical performance of the test in diagnosing true infectious conditions.
In the study described here we studied the use of bead beating and sonication to enhance the lysis of bacterial cell walls prior to standard DNA extraction and 23S ribosomal DNA (rDNA)-targeted PCR. These lysis methods were then combined with some DNA purification protocols available in kit format, and the analytical sensitivities were compared by using dilution series of some model bacteria. Finally, selected processing methods were applied to clinical specimens in parallel with the previously used sample preparation procedures, and the results obtained by bacterial PCR and sequencing were evaluated.
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MATERIALS AND METHODS |
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Lysis of bacterial cells. A total of 100 µl of S. pyogenes or H. influenzae cell suspensions (containing a total of 200,000 to 2 CFU) was transferred into sterile screw-cap Eppendorf tubes with 0.3 g of zirconia-silica beads (diameter, 0.1 mm; Biospec Products, Bartlesville, Okla.), and 100 µl of sterile UV-irradiated water (two times for 90 s each time with UV Stratalinker [Stratagene, La Jolla, Calif.]) was added. Before addition of the samples the tubes with the beads had been UV irradiated as described above for the water. The dilution series were homogenized with a Mini-beadbeater (Biospec Products) for 30 s or 1, 2, 3, or 5 min, after which the DNA was extracted with two phenol-chloroform-isoamyl alcohol extractions followed by one ether wash (3). Similarly, dilution series of bacterial cells were subjected to sonication (51 kHz; Branson 5200; Branson Cleaning Equipment Company, Shelton, Conn.), with or without glass beads, for 1, 3, 5, or 10 min prior to standard DNA isolation as described above.
DNA isolation kits and instruments. A mixture of bacterial dilution series (100 µl containing 200,000 to 2 CFU) and human monocytes (106 human monocytes in 100 µl of PBS to simulate the excess of human cells in clinical specimens) was used as a sample in the testing of various DNA isolation methods. The commercial DNA isolation kits used in this study were the Masterpure DNA purification kit (Epicentre Technologies, Madison, Wis.), DNA-Pure Yeast Genomic kit (CPG Inc., Lincoln Park, N.J.), Qiamp DNA Mini kit (Qiagen GmbH, Hilden, Germany), High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany), and Magna Pure LC DNA isolation kit III (bacteria, fungi) with the Magna Pure instrument (Roche Diagnostics). In the first two kits, lysis of cells and precipitation of proteins are followed by precipitation of DNA with isopropanol, drying, and resuspension of the DNA pellet. In the Qiamp and High Pure kits, the released DNA is bound on a silica gel membrane and a glass fiber filter, respectively, and washed before elution. In the Magna Pure application, the DNA released by use of lysis buffer is bound on glass-covered magnetic beads, which the instrument transfers through several washing steps. Finally, the DNA is eluted and the beads are discarded. All the kits were used according to the manufacturers' instructions, with the exception that the final volume of the DNA preparation was always adjusted to 200 µl with the elution or resuspension buffer provided with the kit. According to the manufacturers, the capacities of the Qiagen and High Pure kits are at least 60 and 10 µg of DNA, respectively. A total of 200,000 genomes of our model bacteria, S. pyogenes and H. influenzae, are estimated to contain about 0.0004 µg of DNA, and a million human cells are estimated to contain about 3.2 µg of DNA. When used in combination with the kits, the bead-beating and sonication steps were performed after incubation with the lysis buffer provided with the kit. A total of 5 µl of purified template DNA was used in the PCR.
Bacterial PCR. An 850-bp sequence of bacterial 23S rDNA was amplified with primers MS37 and MS38, which have been described previously (4). The 30-cycle amplification was performed in a DNA Thermal Cycler 480 (Applied Biosystems, Foster City, Calif.) or a PTC-200 Thermal Cycler (MJ Research, Watertown, Mass.) in 50-µl reaction mixtures with the reagents, cycling times, and temperatures described earlier (4). For detection of PCR products, 10 µl of the amplified DNA was run on a 1.5% SeaKem agarose gel (FMC BioProducts, Rockland, Maine), stained with ethidium bromide, and visualized under UV light.
Before addition of the template DNA, all reaction mixtures were UV irradiated for 3 min to degrade endogenous bacterial DNA. A positive control (50 ng of DNA from Bacillus subtilis ATCC 6051), a negative isolation control (PBS without added bacteria for bacterial dilution series and sterile UV-irradiated distilled water for patient samples), and negative reagent controls (for which sterile UV-irradiated distilled water instead of DNA template was added to the reaction mixture) were included in all runs. The precautions taken to avoid carryover contamination have been described earlier (7). In the case of clinical specimens, inhibition of PCR was assessed by amplification of a fragment of the human growth hormone gene from all template DNA preparations, as described previously (7).
Clinical specimens. A total of 137 clinical specimens sent to our laboratory for routine bacterial PCR were divided in two. One half of each specimen was processed by the routine proteinase K-phenol-ether protocol (7), and the other half was processed by one of the selected test protocols, i.e., with the Masterpure kit (69 samples) or the High Pure kit with an additional 5-min sonication step after the lysis buffer-proteinase K treatment (68 samples). In the case of the liquid samples, two 1-ml aliquots were concentrated by centrifugation (8,000 x g, 5 min) and 800 µl of the supernatant was removed from each tube. The remaining 200 µl was used for DNA isolation by the standard method (aliquot 1) or one of the test methods (aliquot 2). Apart from the additional sonication step in the High Pure protocol, the kits were used according to the manufacturers' instructions.
The 23S rDNA-targeted PCR described above was used to screen for the presence of bacteria in the samples. The bacterial DNA present in a 23S rDNA PCR-positive sample was identified by sequencing the 16S and/or 23S rDNA by our routine laboratory procedure (7). The 16S rDNA was preferably used for sequencing, and 23S rDNA was sequenced if sequencing of 16S rDNA failed. The 16S rDNA PCR primers and conditions have been described previously (4).
The PCR product was purified by use of a GFX PCR and Gel Band Purification kit (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). Eleven microliters of the purified product was sequenced by use of the ABI Prism DNA sequencing kit, Big Dye Terminator Cycle Sequencing (version 2.0 or 3.0), and ABI Prism 310 genetic analyzer (Applied Biosystems). Sequencing primer 533f targets bases 515 to 533 in the bacterial 16S rDNA sequence (Escherichia coli numbering), and primer JJ04 targets bases 1601 to 1629 in the bacterial 23S rDNA sequence (4). Additional sequencing primers (3) were used if they were considered necessary for clinical purposes. Sequence comparisons were done by using an in-house algorithm in a local database (4) and the FastA program (5) in the European Molecular Biology Laboratory (EMBL) prokaryote database (8).
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RESULTS |
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Clinical specimens. On the basis of the results presented above and the ease of integration in the routine work flow, the Masterpure DNA purification kit without additional lysis steps and the High Pure PCR template purification kit with an additional sonication step were selected for comparison with the standard phenol-ether extraction method (which included no additional lysis steps). Sixty-nine consecutive specimens (12 tissue biopsy specimens [including 3 cardiac valve specimens], 15 synovial fluid specimens, 11 pleural fluid specimens, 10 cerebrospinal fluid specimens, 7 amniotic fluid specimens, 3 pericardial fluid specimens, 1 ascitic fluid specimen, and 10 pus samples) were treated by standard phenol-ether extraction and purified with the Masterpure kit. Of these, 48 samples were negative by the broad-range bacterial PCR after both DNA isolation protocols. Eleven samples were positive for bacterial DNA by both methods, two were positive by the standard method only, and one was positive only by the Masterpure kit (Table 2). A biopsy specimen from an aortic valve was inhibitory to the PCR after purification with the Masterpure kit, whereas streptococcal DNA was amplified from the phenol-purified half (Table 2, sample 12). A cerebrospinal fluid sample was inhibitory to the PCR after routine purification, as judged by the failure to detect human DNA also after 90 ng was spiked into the sample (7); the aliquot purified with the Masterpure kit was negative by bacterial PCR. Four samples purified with the Masterpure kit yielded weak bands in the PCR and were classified as contaminated on the basis of a weak band from the negative isolation control of the batch. All remained negative by PCR with the phenol-purified aliquot.
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DISCUSSION |
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In the early diagnostic applications of PCR, classic phenol extraction was successfully used to prepare DNA for amplification. A major advantage of phenol is that it inactivates microbes very efficiently, including, e.g., spores of Bacillus anthracis, which are very resistant to inactivation by other methods (unpublished observations). Unfortunately, phenol is corrosive and toxic; and ether, which is used to remove it from the samples, is explosive. Although the reagents are inexpensive, the classic organic extraction is relatively laborious and unpractical for the processing of large numbers of samples that arrive in the laboratory at different times during the day. Residual phenol may also inhibit amplification of the extracted DNA. An array of commercially available DNA isolation systems has been developed to circumvent these drawbacks. Ideally, use of DNA isolation kits offers standardized, quality-controlled reagents with optimized compositions for all steps of the process. However, the kits are often designed for isolation of human DNA from human tissue or microbial DNA from cultivated cells rather than for detection of a minor amount of microbial DNA among an abundance of human DNA. Preliminary tests by use of several commercial DNA isolation kits failed to detect the relatively low numbers of bacterial cells in our experimental setting.
Hendolin et al. (2) demonstrated the problem of finding a DNA isolation procedure for clinical specimens that would produce DNA from both gram-positive and gram-negative bacteria with equal efficiencies. They analyzed middle ear effusions for the presence of H. influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis by multiplex PCR after processing the samples either by the classic phenol-ether method or a modified Qiamp DNA extraction protocol with an additional boiling step in sodium dodecyl sulfate-NaOH-chaotropic salt. The phenol method yielded more positive PCR results (48 of 49 specimens; 98%) than the Qiamp method (20 of 24 specimens; 83%). Interestingly, the proportion of specimens positive for gram-negative bacteria (H. influenzae, M. catarrhalis) was significantly higher (P < 0.001) among phenol-extracted effusions, while the Qiagen protocol produced a higher proportion of samples positive for gram-positive organisms (S. pneumoniae and A. otitidis) with an equal statistical significance (P < 0.001).
The cell walls of gram-positive bacteria can be efficiently broken by use of the peptidoglycan-degrading enzymes lysozyme and mutanolysin. However, to minimize the number of reagents (and possible sources of bacterial DNA) in the PCR and to find a method equally efficient for cell walls of streptococci, staphylococci, and mycobacteria, we preferred the use of physical disintegration methods, i.e., bead beating and sonication. We aimed at determining a treatment time window that would enhance the release of DNA from difficult-to-lyse bacterial cells without severely compromising the detection of gram-negative organisms. A short bead beating was the most efficient lysis method for this purpose, but use of glass beads in combination with the membrane-formatted DNA purification methods proved difficult, as a small number of beads tended to end up in the purification column with the lysate and block the column. A sonication step was also integrated into the routine work flow more easily than bead beating. The sonicator used in these experiments is originally a washing sonicator; i.e., the sample tubes are placed in a water bath, which mediates the oscillation produced by the ultrasound crystal. As the tubes remain closed during and immediately after the sonication, there is no risk of cross-contamination.
The variability of clinical specimen types adds to the complexity of diagnostic bacterial PCR. This is reflected by the difficulty of extrapolating the experimentally determined detection limits to the true sensitivity in finding bacterial DNA in the clinical samples. The only DNA purification kit (Masterpure) which detected bacteria in lower numbers than the standard method without additional lysis treatments (Table 1) did not prove to be superior to the standard method when it was applied to clinical specimens. In comparison to the results obtained by the phenol-ether extraction procedure, the gram-positive bacteria in two samples remained unidentified, and those in one sample were not detected from the aliquot purified by the kit. Identifiable bacterial DNA was not detected after Masterpure purification in any sample that remained negative by the standard method. Modification of the downstream steps, i.e., amplification and sequencing reactions, might have improved the performance of this and some other kits, but these steps were not optimized for analysis of the template DNA produced by each purification method. According to our experience, amplification of the same template DNA by use of different enzymes and amplification systems may result in highly variable detection limits (6; unpublished observations). The Masterpure kit is inexpensive in comparison to the other kits tested, rapid, and applicable to an array of adequate sample types, including biological fluids, cells, and fresh and paraffin-embedded tissues. The contamination observed for four samples was probably cross-contamination. As the isolation control of this run was positive and the reagent control was negative, the contamination is likely to have occurred in the DNA isolation process. The run included a sample (Table 2, sample 7) with a very large amount of bacteria (as judged from the bands on the agarose gel), which could have been a source of cross-contamination. Unfortunately, sequencing was unsuccessful and insufficient quantities of the original samples were left for reextraction to verify this hypothesis. The isolation controls of the other runs remained negative.
The High Pure kit produced DNA suitable for our downstream applications, as the PCR and sequencing results were well in accord with those obtained by the routine method (Table 3). Four additional samples were found to be PCR positive by the High Pure method, three of which contained DNA of gram-positive organisms; among those were the important pathogens S. pyogenes and Staphylococcus aureus. Three samples were positive by the standard method but negative after purification by the High Pure method; Streptococcus peroris in a pleural fluid specimen and Staphylococcus warneri in a synovial fluid specimen would have remained undetected if only purification by the High Pure method had been used. By use of the standard method, bacterial DNA was also detected in a mandibular bone biopsy specimen, but sequencing of the PCR product failed. These findings indicate that no single method results in optimal recovery of all bacteria possibly present in the samples and that at least two different methods should be used, whenever possible.
The frequent detection of sequences related to the anginosus group of streptococci among the clinical specimens tested in the present study raised some concern about the validity of the findings. The results of bacterial cultures for these samples were clarified in order to rule out the possibility of PCR artifacts. Except for the pleural fluid specimen positive by the phenol purification method only (Table 3, sample 12), microaerophilic or viridans group streptococci had been isolated from the same specimens (Table 3, samples 1, 2, and 15). Thus, these streptococcal DNAs were most likely derived from the samples rather than reagents. Use of kit-form DNA isolation systems does not necessarily diminish the problem of reagent-derived bacterial DNA, as contamination of commercial purification columns with Legionella DNA was recently reported (10).
Regarding the removal of inhibitors, the three methods tested seemed equally efficient for the samples analyzed here. In a previous study we found the High Pure purification procedure to be superior to phenol-ether purification in preparing simulated sputum and bronchoalveolar lavage specimens for detection of Legionella pneumophila by PCR (6). These findings were associated with the possibility of efficient washing of DNA bound to the glass fiber filter. An additional buffer that removes inhibitors has been included in the new versions of the High Pure kit but was not used in this study.
In recent years, several instruments have been introduced for automated DNA extraction. We had the possibility to perform some isolations with the Magna Pure instrument. The preliminary results by use of the kit intended for bacteria were rather promising, and the process is applicable to an array of clinical specimens. However, the need for manual pretreatment steps, the relatively long time required to load the reagents on the instrument, and the high cost of the plastic ware that it uses make isolation with this instrument uneconomical if less than several tens of samples are processed daily.
The preanalytical phases are crucial for the success of any diagnostic laboratory method, and this is no less the case for tests applying molecular biology methods; but often, little attention is paid to their optimization in relation to the efforts made to polish other stages of the assays. Here we evaluated the use of physical lysis methods and commercial DNA purification kits to prepare DNA from clinical specimens for a broad-range bacterial PCR assay, which has previously proved to be a valid diagnostic test for the detection of bacterial DNA in normally sterile anatomical sites (7). We succeeded in finding a DNA isolation method which released and purified bacterial DNA, especially that from streptococci and staphylococci, at least as efficiently as the standard phenol-ether method and avoided the use of toxic and explosive components. Furthermore, sequencing of the PCR products revealed that the amplicons were derived from clinically relevant bacteria, and the results were well concordant with the results obtained by the validated standard method. However, our results also indicate that no single method is optimal for the detection of all the bacteria that might be present in tissues and biological fluids, and the use of more than one method is recommended, at least in research settings.
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ACKNOWLEDGMENTS |
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Matti Viljanen is acknowledged for useful comments on the manuscript. Kirsi Sundholm, Tiina Haarala, Kaisa Arvonen, Tarja Laine, and Merja Mikkola are thanked for excellent technical assistance; and Tuula Närä is thanked for processing of the figures.
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FOOTNOTES |
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REFERENCES |
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