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Journal of Clinical Microbiology, May 1999, p. 1454-1458, Vol. 37, No. 5
Department of Clinical Microbiology,
Received 3 September 1998/Returned for modification 7 December
1998/Accepted 13 February 1999
Mycobacterium malmoense is an opportunistic human
pathogen of increasing clinical importance. Since it is difficult to
detect and identify the organism by conventional techniques, it was
decided to seek a nucleic acid amplification method specific for
M. malmoense. The method was based on detection of a
conserved band in random amplified polymorphic DNA (RAPD) fingerprints
of 45 M. malmoense strains. This band was a 1,046-bp
product which was proven to be M. malmoense specific in dot
blot hybridization analysis with a panel of mycobacterial strains
belonging to 39 other species. The fragment was sequenced, and
oligonucleotide primers were synthesized to evaluate the specificity of
the PCR. Two primer pairs were found to be specific and sensitive in
the nested PCR that was developed. All 49 M. malmoense
strains analyzed produced a PCR product of the expected size. In
contrast, no strains belonging to the other mycobacterial species
tested produced amplicons with these primers under specified reaction
conditions. The results of the electrophoresis were confirmed by the
hybridization with the M. malmoense-specific
oligonucleotide probe. This method could be applied to the analysis of
clinical or environmental samples, permitting the rapid detection of
M. malmoense.
Mycobacterium malmoense
is a potential human pathogen which causes infections similar to those
caused by the Mycobacterium avium complex (9,
14). It has steadily increased in clinical importance in the
Nordic countries and Scotland (3, 7, 20), and recently, it
has also been isolated from clinical samples in other countries
(32). M. malmoense has also been detected in
natural environments (11, 23, 26). M. malmoense
is a difficult species to isolate (8, 12, 13), and its
identification may also be problematic by conventional identification
tests (34). Analytic techniques based on lipid profiles or
gene sequencing have been found to be more reliable for its
identification (21, 28, 33).
Several PCR-based methods have been developed for the detection and/or
identification of atypical mycobacteria. In these techniques, a number
of primers may be used (multiplex PCR [18]) or a
selected conserved region of the genome is amplified, and the
recognition of the species is based on the detection of differences in
some bases in the amplified sequence. In the latter case, the final species identification is based on the analysis of the amplicon by
nucleic acid sequencing (17, 28), restriction endonuclease digestion (4, 19, 35), or nucleic acid hybridization
(29, 30).
The genome of M. malmoense is poorly characterized. No
published information on M. malmoense-specific genes is
available. The data published so far comprise data for species-specific
portions of the genome that are only a few bases in length (6,
28). One useful way to discover longer species-specific parts in
a sequence of a microbe whose genomic data are unknown is to perform fingerprint profile analysis of several strains belonging to the same
species (22). We applied this method to the analysis of M. malmoense for species-specific sequences which could be
used as targets for an M. malmoense-specific PCR.
Strains.
The following Mycobacterium type strains
were included in the study: M. agri ATCC 27406 (American
Type Culture Collection [ATCC], Rockville, Md.), M. aichiense ATCC 27280, M. asiaticum ATCC 25276, M. austroafricanum ATCC 33464, M. avium ATCC 15769, M. branderi ATCC 51788 and ATCC 51789, M. celatum
I ATCC 51130, M. celatum II ATCC 51131, M. chitae
ATCC 19627, M. chubuense ATCC 27278, M. cookii
ATCC 49103, M. diernhoferi ATCC 19340, M. fallax ATCC 35219, M. flavescens ATCC 14474, M. fortuitum ATCC 35754, ATCC 6841, and ATCC 6842, M. gastri ATCC 15754, M. gordonae ATCC 14470, M. hiberniae ATCC 49874, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC
13950, M. kansasii ATCC 12478, M. komossense ATCC
33013, M. marinum ATCC 927, M. malmoense ATCC
29571, M. neoaurum ATCC 25795, M. nonchromogenicum ATCC 19530, ATCC 19533, and ATCC 35783, M. peregrinum ATCC 14467, M. phlei ATCC 12298, M. pulveris ATCC 35154, M. scrofulaceum ATCC 19981 and
ATCC 35792, M. shimoidei ATCC 27962, M. simiae
ATCC 25275, M. smegmatis ATCC 35797, M. sphagni
ATCC 33027, M. szulgai ATCC 35799, M. terrae ATCC
15755, M. triviale ATCC 23291, M. tuberculosis ATCC 25177 and ATCC 27294 (H37Rv), and M. xenopi ATCC 19250. M. bovis BCG 965/Gothenburg (vaccine strain) was a gift from
M. Riddel, University of Gothenburg, Gothenburg, Sweden. Two strains,
M. chelonae QC500/91 and M. haemophilum QC190/91,
that were distributed in an Australian quality control scheme were
gifts from A. Marzec, Austin Hospital, Melbourne, Australia. The
following clinical isolates from the culture collection of the
Department of Clinical Microbiology, Kuopio University Hospital, were
included: M. bovis BCG (one strain), M. chelonae
subsp. abscessus (one strain), and M. malmoense
(39 Finnish and 6 British strains) (10). In addition, three
Finnish environmental isolates of M. malmoense that
originated from stream water were included (11).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mycobacterium malmoense-Specific Nested
PCR Based on a Conserved Sequence Detected in Random Amplified
Polymorphic DNA Fingerprints
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C in Middlebrook 7H9 broth (Difco
Laboratories, Detroit, Mich.). For the present study, the strains were
grown on Middlebrook 7H11 agar with Middlebrook OADC (oleic acid,
albumin, dextrose, catalase) enrichment (Difco Laboratories). If
necessary, the identity of a strain was reconfirmed by fatty acid
analysis by gas-liquid chromatography complemented with a panel of
biochemical tests previously described in detail (10, 31).
20°C until they were
processed further.
DNA extraction.
The chromosomal DNA of the mycobacteria was
isolated as described previously (16). In brief, the
mycobacterial cells were lysed with Nagarse protease (Sigma, St. Louis,
Mo.) and lysozyme (Sigma). The lysis was followed by phenol-chloroform
extractions, RNase (Sigma) digestion, and ethanol precipitation. The
DNA concentrations were quantified both with the GeneQuant DNA/RNA
calculator (Pharmacia LKB Biochrom Ltd., Cambridge, United Kingdom) and
by agarose gel electrophoresis by comparison of a sample band to the
bands of HindIII-digested bacteriophage lambda DNA (New
England Biolabs, Inc., Beverly, Mass.). The DNA stock was stored at
4°C, and the diluted DNA was stored at 20°C.
RAPD-PCR. Random amplified polymorphic DNA PCR analysis (RAPD-PCR) of M. malmoense has been described in detail in a publication describing our previous study in which 45 strains were amplified with selected 10-mer primers (15). In the RAPD-PCR, the genomic DNA is amplified under optimized conditions by using a short, nonspecific oligonucleotide primer. In the present study, other Mycobacterium species were fingerprinted by the same method with the OPA2 primer (Operon Technologies, Inc., Alameda, Calif.).
Fragment extraction from agarose gel. A DNA product representing a 1,046-bp band that was present in all 45 M. malmoense strains was cut as a slice from the fingerprint of M. malmoense ATCC 29571 obtained in an agarose gel after electrophoresis. The DNA fragment was purified by a phenol extraction method described elsewhere in detail (27).
Cloning and sequencing. The fragment (named MA2/6) was cloned into SmaI-digested pBluescript II SK (Stratagene, La Jolla, Calif.) by the standard method (27). The insert was digested with restriction endonucleases SalI and PvuII, and the 557-bp SalI-PvuII fragment was recloned into SalI- and PvuII-digested pBluescript II SK. The cloned inserts were sequenced with an ALF automatic DNA sequencer with the Auto Read Sequencing kit (Pharmacia Biotech, Sollentuna, Sweden) with the primers for the T3 and T7 sequences of pBluescript II SK. The sequence segments were aligned to constitute the complete sequence. Analyses of the DNA sequences were performed with the GeneJockey II computer program (Biosoft, Cambridge, United Kingdom).
Dot blot hybridization. Dot blot hybridizations of mycobacterial chromosomal DNA were carried out by modifying the published protocol (2). One microgram of EcoRI-digested chromosomal DNA was applied onto each dot on an Immobilon-N membrane (Millipore, Bedford, Mass.) which had been prewetted for 2 s in ethanol and saturated in 3× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The DNA was denaturated by incubating the membrane for 10 min on two stacked blotting papers (Schleicher & Schuell GmbH, Dassel, Germany) saturated in an alkaline solution (0.5 M NaOH, 1 M NaCl). The neutralization was performed in the same way by using a neutralization solution (1 M Tris-HCl, 2 M NaCl [pH 7.5]). For DNA binding, the dried membrane was baked for 1 h at 80°C according to the instructions of the membrane manufacturer.
The MA2/6 fragment and the 16S rRNA gene (rDNA) probe (16) were chosen as probes for the dot blot hybridization analyses. The probes were 32P labelled with the Rediprime kit (Amersham, Buckinghamshire, United Kingdom) with Redivue [
-32P]dCTP AA 0005 (Amersham) according to the
instructions on the insert in the Rediprime package. The specific
activities of the probes were 4 × 108 cpm/µg for
the MA2/6 fragment and 2 × 108 cpm/µg for the 16S
rDNA probe.
The membrane was incubated in the prehybridization solution at 65°C
for 1 h before hybridization. There was 150 kcpm of labelled probe
per ml of hybridization solution, and the hybridization was allowed to
proceed for 18 h at 65°C. To visualize the label, the membrane
was exposed to Hyperfilm MP (Amersham). If the membrane was to be
reprobed, the attached probe was stripped off by incubating the
membrane in 0.1 M NaOH for 30 min and in 0.1× SSC-0.2 M Tris-HCl (pH
8.0) containing 0.5% sodium dodecyl sulfate for 45 min at room temperature.
PCRs. To prevent carryover contamination, treatment of the strains, extraction of DNA, preparation of PCR mixtures, amplification, and analysis of the amplicons were each done in separate areas. Filter-barrier pipette tips were used at all stages. In the amplification control reactions with 16S rRNA gene primers and nested amplifications with the MA2/6 primers, the PCR mixtures (total volume, 50 µl) contained 1× DynaZyme DNA polymerase reaction buffer (Finnzymes Oy, Espoo, Finland), 0.3 µM primers (synthesized by Medprobe, Oslo, Norway), 200 µM (each) dATP, dCTP, dGTP, and dTTP (Promega, Madison, Wis.), 1 to 2 ng of genomic DNA (or 1 µl from the product of the first reaction to the second reaction of the nested PCR), and 2 U of DynaZyme DNA polymerase (Finnzymes Oy).
The primers for the amplification control reactions were 16S-A (5'-GGATGACGGCCTTCGGGTTG-3'), which complements bases 366 to 385, and 16S-B (5'-GACAGCCATGCACCACCTGC-3'), which consists of bases 993 to 1012 in the M. malmoense 16S rRNA gene sequence (GenBank accession no. X52930) (25). The primers for the first reaction of the M. malmoense-specific nested PCR were MAL-1A (5'-CGACGAGACACCCCCAGC-3'), which complements bases 12 to 29, and MAL-1B (5'-TCAGCCAGCGGTGTAGC-3'), which consists of bases 988 to 1004; and the primers of the second reaction were MAL-2A (5'-CAGTCAGCGAGGAAGTCGGGAATC-3'), which complements bases 72 to 95, and 5'-end biotinylated primer MAL-2B-biot (5'-GGGTTCATCATTCCGCTGGGCTAC-3'), which consists of bases 482 to 505 in the M. malmoense MA2/6 sequence (GenBank accession no. AF085175). The amplification programs were optimized for a GeneAmp PCR System 9600 apparatus (Perkin-Elmer, Norwalk, Conn.). Control PCRs with 16S rRNA gene primers were performed by a program which included predenaturation at 98°C for 3 min, a first 15 cycles consisting of 96°C for 20 s, 54°C for 35 s, and 72°C for 50 s, and a subsequent 25 cycles consisting of 95°C for 25 s, 58°C for 35 s, and 72°C for 45 s; after these cycles, there was an extra extension step at 72°C for 10 min. In the M. malmoense-specific nested PCR, the first program included predenaturation at 98°C for 3 min and 25 cycles consisting of 96°C for 25 s, 63°C for 50 s, and 72°C for 1 min, and the second one included 25 cycles consisting of 96°C for 20 s, 65°C for 30 s, and 72°C for 40 s and, finally, an extra extension step at 72°C for 10 min. After the program was run, the reaction tubes were held at 4°C until agarose gel electrophoresis was run.Agarose gel electrophoresis. The PCR products were run on a 1.3% agarose (FMC Bioproducts, Rockland, Maine) gel containing 0.5 µg of ethidium bromide (Sigma) per ml and 1× Tris-acetate-EDTA buffer. The gel was photographed under UV light.
Hybridization of nested PCR product. The nested PCR products were hybridized with 5'-end digoxigenin-labelled oligonucleotide probe MAL-p-dig (5'-AGCTGTTCGTGGGTATCAGAGAGTTA-3'), which complements bases 160 to 185 (synthesized by Medprobe, Oslo, Norway) in the M. malmoense MA2/6 sequence, in microplate wells by a method described elsewhere (1), with slight modifications. After the amplification reaction with the M. malmoense-specific primers MAL-2A and MAL-2B-biot, 15 µl of the PCR product was applied to the well of a streptavidin-coated microplate (Labsystems, Helsinki, Finland) in 185 µl of working solution consisting of 1 M phosphate-buffered saline, 1% bovine serum albumin, and 0.1% Tween 20 (pH 7.4). The biotinylated amplification product was immobilized onto the well for 18 h at 4°C. For denaturation of the PCR product, the well was first washed once with 200 µl of 0.1 N NaOH, and after that, 200 µl of 0.1 N NaOH was added and the plate was incubated for 5 min at room temperature. After two washing steps with the working solution, the hybridization with the digoxigenin-labelled oligonucleotide probe MAL-p-dig was carried out in 150 µl of hybridization solution consisting of 6× SSC, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 10× Denhardt solution (which contains 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin) for 4 h at 56°C. After hybridization, the well was washed twice with working solution.
The hybridized digoxigenin-labelled oligonucleotide probe was detected by adding to each well 150 µl of anti-digoxin monoclonal antibody clone DI-22 conjugated with alkaline phosphatase (Sigma) at a dilution of 1:1,000 in working solution. After incubation for 1 h at room temperature, the well was washed two times. To detect the attached anti-digoxin antibody, 150 µl of substrate solution, prepared from Sigma Fast-pNPP substrate tablets (Sigma), was added. The substrate was incubated for 30 min at room temperature. The reaction was stopped by adding 40 µl of 3 N NaOH per well. The absorbance was measured at 405 nm with a Multiskan plus photometric reader (Labsystems).Nucleotide sequence accession number. The nucleotide sequence reported here has been submitted to the GenBank nucleotide sequence database under accession no. AF085175.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The 45 M. malmoense strains exhibited a variety of RAPD-PCR patterns when the strains were analyzed with the OPA2 10-mer primer. However, one band of 1,046 bp was conserved and was present in the fingerprint of every M. malmoense strain. The band of similar size was uncommon in the other mycobacterial species tested. An example of the fingerprint profiles is shown in Fig. 1.
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The existence of the MA2/6 sequence in the genomes of mycobacteria other than M. malmoense was estimated by dot blot hybridization analysis of the set of the mycobacterial strains selected. Only M. malmoense DNA exhibited a distinct positive result (Fig. 2). On the same membranes, all strains studied produced a positive result by the control dot blot hybridization assay, carried out with the labelled 16S rDNA fragment. On this basis, the MA2/6 fragment was regarded as a potential candidate for an M. malmoense-specific fragment. No high degrees of nucleotide sequence homologies were found in homology searches when the nucleotide sequence of MA2/6 was compared with those present in GenBank version 110.0.
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On the basis of the sequencing data for the MA2/6 fragment (Fig. 3), several primers were designed for PCR. The M. malmoense specificity of the primers were evaluated by combining the sense and antisense primers in different combinations. The primers found to be most suitable are described in Materials and Methods. Primers MAL-1A and MAL-1B produced a band of the expected size of 993 bp only from M. malmoense at 60°C or above. In contrast, a band was also obtained from M. tuberculosis, M. kansasii, and M. fortuitum if the annealing temperature was adjusted to below 56°C (data not shown). The band produced from M. tuberculosis and M. kansasii had a size of approximately 1.3 kbp, whereas the band from M. fortuitum was equal in size to the M. malmoense band. The PCR products amplified with primers MAL-1A and MAL-1B from M. tuberculosis, M. kansasii, and M. fortuitum were partially sequenced. No homologies with the M. malmoense sequence were observed (data not shown).
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Nested PCR, consisting of reactions first with primers MAL-1A and MAL-1B and second with primers MAL-2A and MAL-2B-biot, was highly specific for M. malmoense. Among the mycobacterial species included in this analysis, only the 48 M. malmoense strains produced a band of 434 bp in the nested PCR. No bands of other sizes were detected in the lanes containing M. malmoense. The lanes containing other Mycobacterium species were free of any bands. A subset of the results is presented in Fig. 4. In hybridization analysis of the nested PCR products with the digoxigenin-labelled oligonucleotide probe MAL-p-dig, all M. malmoense strains with the exception of one clinical isolate gave an absorbance value of 0.85 to 1.13; the absorbance value for the clinical isolate that was the exception was 0.58. In contrast, the absorbance values for the other Mycobacterium species were less than 0.09, indicating negative results. The mean value for the negative control reactions (PCRs without sample DNA) was 0.02.
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Control amplifications carried out with the primers for the 16S rRNA gene confirmed that the bacterial PCR templates also amplified those strains that remained negative with the M. malmoense-specific primers. All Mycobacterium strains produced a band of approximately 647 bp in the control reactions. The sensitivity of the nested PCR was estimated by using a dilution series of M. malmoense chromosomal DNA and the DNA of the plasmid containing the MA2/6 fragment. Both 4 fg of chromosomal DNA and 12 ag of plasmid DNA could be detected, indicating the limit of detection to represent one target molecule in a reaction tube. There was no difference in the detection limit by electrophoresis or hybridization. A slightly visible band by gel electrophoresis gave an absorbance reading of 0.50 by hybridization analysis.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The clinical significance of mycobacterial species such as M. malmoense is likely to be underestimated; its detection in clinical samples is particularly difficult due to its exceptionally slow rate of growth and the need for special culture conditions (8, 12, 13). We have previously confirmed that modifications to culture media increase the rates of detection of such species (12). Biochemical identification of M. malmoense is laborious, and some uncommon Mycobacterium species may easily be incorrectly identified as M. malmoense (34). Nucleic acid amplification methods offer an alternative approach to the detection and identification of microbes whose identities are difficult to confirm by conventional methods. Several PCR protocols have been used for the identification of atypical mycobacteria. In most cases, identification has been based on the detection of differences in conserved sequences after amplification with genus-specific primers. This also applies to M. malmoense, at least by selected methods (6, 24, 28). In the present study, we wanted to develop a rapid technique that was also potentially suitable for the direct detection of M. malmoense from clinical or environmental samples. The specific PCR method makes it possible to preliminarily identify M. malmoense DNA within a working day. With the hybridization of the amplicon, the method takes 2 days. All M. malmoense strains tested, including an ATCC strain and 48 clinical or environmental isolates, produced the expected PCR product. Furthermore, the results verified the high degree of species specificity of the MA2/6 fragment. None of the 43 other mycobacterial species tested produced amplicons if the specific PCR conditions were used. We performed the amplification reactions with the 16S rRNA gene primers as DNA or inhibition controls. The sensitivity of the nested PCR used was high under the experimental conditions used.
There are several ways to find species-specific areas in microbial genomic nucleic acids. The specific sites close to or within a conserved part of the genome, such as the 16S rRNA genes, are potential targets that are mostly found by sequencing genomes of several species. It is possible to use primers which anneal to the genomes of many species if further identification is done by analysis of the amplified sequences by restriction endonuclease digestion, oligonucleotide hybridization, or sequencing. All of these applications can reveal a difference of even a single base.
We used an approach in which the RAPD fingerprint profiles of M. malmoense strains and some closely related mycobacterial species were compared. The basis of the present study was the detection in the M. malmoense strains of conserved bands which were not present in the other species. One amplification product of this kind was found to be M. malmoense specific, and it was further analyzed as a suitable candidate for targeting by nested PCR analysis. To ensure the species specificity of fragment MA2/6, it was extracted from an agarose gel and used as a probe in a hybridization analysis in which the template DNA was derived from several M. malmoense strains and other closely related species. The primer candidates were selected from the sequence of the fragment by using the appropriate computer program, and the M. malmoense-specific primers were found experimentally.
Negative hybridization results with the other species tested suggested that the MA2/6 fragment was a potential candidate as an amplification target. The stringency in the dot blot hybridization was relatively high due to the hybridization temperature and the salt concentration used. Therefore, sequences closely related to the MA2/6 probe could have been detected. In addition, the MA2/6 sequence did not show a high degree of nucleotide sequence homology with any of the sequences in data banks. With the selected primers, some PCR products were obtained from mycobacterial species other than M. malmoense, but only at the low annealing temperatures that favor nonspecific binding. The specificity and sensitivity of the PCR were increased by using two nested primer pairs.
By combining the developed nested PCR with a sample processing method such as that of Eisenach et al. (5), it could be used not only for the identification of M. malmoense from cultures but also for the direct detection of M. malmoense from samples without the need for preceding isolation of the organism. This would allow the detection of the organism in both clinical samples and suspected environmental reservoirs. Since the identification is based on the detection of a specific sequence with species-specific primers instead of the detection of minor differences in the sequence flanked by the binding sites of the universal primers, the results can be interpreted quickly and easily. The sensitivity of the amplification reaction was excellent. When the sample collection and preparation are optimized, M. malmoense could be reliably detected in the course of a few days. This requires further evaluation with clinical and environmental specimens.
We conclude that the nested PCR described here is a specific method for the identification of M. malmoense and that the RAPD-PCR is a useful tool in searches for species-specific genomic fragments.
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ACKNOWLEDGMENTS |
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We thank Eila Pelkonen for technical assistance.
This study was supported by the Memorial Foundation of Maud Kuistila and the Finnish Antituberculosis Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Clinical Microbiology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Phone: 358-17-162708. Fax: 358-17-162705. E-mail: Juha.Kauppinen{at}uku.fi.
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Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, May 1999, p. 1454-1458, Vol. 37, No. 5
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