More particularly, the present invention is directed to a method for discriminating Legionella species with each other by using a primer for the selective amplification of the specific portion of rpo B gene of Legionella species, rpo B DNA fragments of each species of which nucleotide sequences are different from each other and a restriction enzyme.

In addition, the present invention is directed to a method for the detection and identification of Legionella pneumophilla by amplifying rpo B DNA of pathogenic Legionella pneumophilla selectively, and directed to a primer used therein.

Background Art Legionella species has been found in natural environment such as river, lake, soil and so on as well as artificial environment such as hot-water supply systems, shower stalls, coolant of air-conditioning systems, evaporative condensers, humidifiers, medical sprayers, whirlpool spas, medical devices for respiratory disease, ornamental fountains, water supply systems and the like.

Legionella species are aerobic gram-negative rod and motile, which lack cysts and capsules. They are

grown well in BYE-A culture media containing L-cystein under 5% CO2. They usually show weak positive in the catalase test and oxidase test and show positive in the gelatinase test (Winn, W. C. Legionella, p. 572-582. In Murray, P. R. (ed. ), Mannual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D. C. , 1999. ). In addition, they are known to show resistance to rifampin in the case that mutations occur in the specific region of their rpoB gene (Severinov, K. , Soushko, M. , Goldfarb, A. and Nikiforov, V.

Rifampicin region revisited. J Biol Chem 268,14820- 14825,1993.).

Up to now, it has been known to the public that the genus Legionella has over 42 species and over 60 serogroups (Hookey, J. V. , Sauders, N. A. , Fry, N. K., Birtles, R. J. and Harrison, T. G. Phylogeny of Legionellaceae based on small-subunit ribosomal DNA sequences and proposal of Legionella lytica comb. nov. for Legionella-like amoebal pathogens. Int J Syst Bacteriol 46,526-531, 1996.; Lo Presti, F. , Riffard, S. , Meugnier, H. , Reyrolle, M. , Lasne, Y. , Grimont, P.

A. D. , Grimont, F. Vandenesch, F. , Etienne, J., Fleurette, J. and Freney, J. Legionella taurinensis sp. nov. , a new species antigenically similar to Legionella spiritensis. Int J Syst Bacteriol 49,397-403, 1999.; Swanson, M. S. and Hammer, B. K. Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54,567-613, 2000.; Winn, W. C. Legionella, p. 572-582. In Murray, P. R.

(ed. ), Mannual of clinical microbiology, 7th ed.

American Society for Microbiology, Washington, D. C., 1999. ), among which 21 species were isolated in the

pneumonia patients receiving immunosuppressive therapy (Fang, G. D. , Yu, V. L. and Vickers, R. M. Disease due to the Legionellaceae (other than Legionella pneumophila). Historical, microbiological, clinical, and epidemiological review. Medicine 68,116-132, 1989. ; Miyamoto, H. , Yamamoto, H. , Arima, K. , Fujii, J., Maruta, K. , Izu, K. , Shiomori, T. and Yoshida, S.-I.

Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water. Appl Environ Microbiol 63, 2489-2494, 1997. ).

It is known that such Legionella species invade alveolar cells of lungs, adhering to mist etc. , and cause disease. Legionella pneumophila is the most common bacteria responsible for legionellosis. 2% to 15% of infectious pneumonia which requires hospitalization in the U. S. A. are due to legionellosis (Marston, B. J. , Lipman, H. B. and Breiman, R. F.

Surveillance for Legionnairess disease. Arch Intern Med 154,2417-2422, 1994.; Swanson, M. S. and Hammer, B. K.

Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54, 567-613,2000.).

Meanwhile, several methods for identifying members of the genus Legionella have been known to the public. Some of the methods employ the detection of the difference between the components such as fatty acid and ubiquinone in the cell wall, (Waterer, G. W., Baselski, V. S. and Wunderink, R. G. Legionella and community-acquired pneumonia: a review of current diagnostic tests from a clinicians viewpoint. Amer. J.

Med. 110: 41-48.2001. ; Winn, W. C. Legionella, p. 572-

582. In Murray, P. R. (ed. ), Mannual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D. C. , 1999. ), others use phylogenetic analysis.

The biochemical tests for identifying bacteria include oxidase test, catalase test, hippurate hydrolysis test, autofluorescent test, urease test and so on ( (Fox, K. F. & Brown, A. Application of numerical systematics to the phenotypic differentiation of Legionellae. J Clin Microbiol 17,1952-1955. 1999.; Orrison, L. H. , Cherry, W. B. , Fliermans, C. B., McDougal, L. K. & Dodd, D. J. Characteristics of environmental isolates of Legionella pneumophila. Appl Environ Microbiol 42,109-115. 1981. ; Pine, L. , Hoffman, P. S. , Malcom, G. B. , Benson, R. F. & Gorman, G. W.

Whole-cell peroxidase test for identification of Legionella pneumophila. J Clin Microbiol 19,286-290.

1984.; Weaver, R. E. & Feeley, J. C. Cultural and biochemical characterization of Legionnaires disease bacterium. p. 20-25. In G. L. Jones and G. A. Hebert (ed.), Legionnaires, the disease, the bacterium, and methodology. Centers for Disease Control, Atlanta.

1979. ). CDC (Centers for Disease Control) of U. S. A and National Institute of Health of Korea have employed the method for identifying species and serogroups by using antiserum.

However, the species of the bacteria cannot be identified exactly through only the above methods because the results obtained even from the same species may be remarkably varied. In addition, only a few bacteria such as L. pneumophila serogroup 1-6, L. dumoffii, L. gormanii, L. micdadei and so on can be

identified by using rabbit immune serum or commercialized antiserum (Denka) (Winn, W. C. Legionella, p. 572-582. In Murray, P. R. (ed. ), Mannual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D. C. , 1999.).

Therefore, several novel methods based on gene analysis have been tried in order to overcome the above disadvantages of the prior arts for identification of bacteria species. At present, 16S rRNA gene is most frequently used as a marker for identification of each species (Woese, C. R. Bacterial evolution. Microbiol Rev 51,221-271, 1987. ; Woese, C. R. , Kandler, 0. and Wheelis, M. L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya.

Proc Natl Acad Sci USA 87,4576-4579, 1990. ).

However, the above marker is known to have some limitations led from the heterogenecity thereof (Wang, Y. , Zhang, Z. and Ramanan, N. The Actinomycete Thermobispora contains two distinct types of transcriptionally active 16S rRNA genes. J Bacteriol 179,3270-3276, 1997. ; Fogel, G. B. , Collins, C. R. , Li, J. and Brunk, C. F. Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microb Ecol 38,93- 113, 1999. ; Ueda, K. , Seki, T. , Kudo, T. , Yoshida, T. and Kataoka, M. Two distinct mechanisms cause heterogeneity of 16S rRNA. J Bacteriol 181,78-82, 1999. ; Dahllof, I., Baillie, H. and Kjelleberg, S. rpoB-based microbial community analysis avoids limitations inherent in 16S rDNA gene intraspecies heterogeneity. Appl Environ Microbiol 66,3376-3380, 2000.).

16S rRNA gene (Fry, N. K. , Warwick, S. , Saunders, N. A. and Embley, T. M. The use of 16S ribosomal RNA analyses to investigate the phylogeny of the family Legionellaceae. J Gen Microbiol 137,. 1215-1222, 1991. ; Birtles, R. J. , Rowbotham, T. J. , Raoult, D. and Harrison, T. G. Phylogenetic diversity of intra-amoebal legionellae as revealed by 16S rRNA gene sequence comparison. Microbiology 142,3525-3530, 1996. ; Hookey, J. V. , Sauders, N. A. , Fry, N. K. , Birtles, R. J. and Harrison, T. G. Phylogeny of Legionellaceae based on small-subunit ribosomal DNA sequences and proposal of Legionella lytica comb. nov. for Legionella-like amoebal pathogens. Int J Syst Bacteriol 46,526-531, 1996. ) or mip nucleotide sequences (Ratcliff, R. M., Lanser, J. A. , Manning, P. A. and Heuzenroeder, M. W.

Sequence-based classification scheme for the genus Legionella targeting the mip gene. J Clin Microbiol 36, 1560-1567,1998. ; Ratcliff, R. M. , Donnelan, S. C., Lanser, J. A. , Manning, P. A. and Heuzenroeder, M. W.

Interspecies sequence differences in the Mip protein from the genus Legionella : implication for function and evolutionary relatedness. Mol Microbiol 25,1149-1158, 1997. ) of Legionella species have also been used for identification of the species for phylogenetic analysis.

However, some discrepancies have been also reported in the case that the species are identified by using the above genes (Hookey, J. V. , Sauders, N. A. , Fry, N. K., Birtles, R. J. and Harrison, T. G. Phylogeny of Legionellaceae based on small-subunit ribosomal DNA sequences and proposal of Legionella lytica comb. nov. for Legionella-like amoebal pathogens. Int J Syst Bacteriol 46,526-531, 1996.; Ratcliff, R. M. , Donnelan,

S. C. , Lanser, J. A. , Manning, P. A. and Heuzenroeder, M. W. Interspecies sequence differences in the Mip protein from the genus Legionella : implication for function and evolutionary relatedness. Mol Microbiol 25, 1149-1158,1997.).

Therefore, the employment of only one gene for the phylogenetic analysis of specific bacteria species may lead to unreasonable and unreliable results (Woese, C. R. Reconstructing bacterial evolution with rRNA, p.

1-24. In Selander, R. K. , Clark, A. G. and Whittman, T.

S. (eds. ) Evolution at the Molecular Level. Sunderland, MA: Sinauer Associates, 1991. ; Ratcliff, R. M., Donnelan, S. C. , Lanser, J. A. , Manning, P. A. and Heuzenroeder, M. W. Interspecies sequence differences in the Mip protein from the genus Legionella : implication for function and evolutionary relatedness.

Mol Microbiol 25,1149-1158, 1997. ).

Several Legionella species have been reported to be pathogenic among more than 44 known Legionella species, among which Legionella pneumophila reported to cause pneumonia is most life-threatening pathogen.

Such Legionella pneumophila is known to have 15 serogroups, among which Legionella pneumophila serogroup 1 is most common pathogen causing pneumonia.

In addition, L. micdadei, L. bozemanii, L. longbeachae and so on have also been reported to cause pneumonia in the immunocompromised patient.

The most common disease caused by the above bacteria is Legionella pneumonia. Legionella pneumonia begins with loss of appetite, fatigue, muscle pain or headache. Symptoms include a high fever up to 39-40. 5 C accompanied by a chill, nonproductive cough,

diarrhea, anorexia, vomiting or abdominal pain.

Abnormalities in a chest X-ray show up after 3 days of infection, which begin with local or plaque infiltration, make progress in both of lungs and cause a lethal respiratory failure. In addition, hypotention occurs in the 17% of the patients infected with community-acquired Legionella pneumonia. Though mortality rate is low in the healthy person infected with a community-acquired pneumonia, it may reach 50% in the case of nosocomial infection.

Though antimicrobial drugs are effective, the pneumonia which show up in a chest X-ray may be aggravated because a chest X-ray view does not always coincide with clinical symptoms. It takes 1 to 2 months, occasionally 3 to 4 months after the improvement of symptoms for a chest X-ray view to return to normal condition. Other symptoms besides pneumonia, that is endocarditis, myocarditis and pericarditis after cardiac surgery, sinusitis, peritonitis and pyelonephritis may occur.

Therefore, the novel method for analyzing the nucleotide sequences of Legionella species which differ from one another depending on the species, has been needed for the rapid identification of the species and for the exact examination as to whether or not the infection occurred and if that was the case, as to what species cause the infection in the early stage of the infection.

Especially, the detection method for Legionella pneumophila and the identification method of specific serogroup such as Legionella pneumophila serogroup 1 based on the analysis of nucleotide sequences specific

for Legionella pneumophila, have been required for rapid diagnosis of Legionella pneumophila infection in the early stage of infection.

Disclosure of Invention Therefore, the object of the present invention is to provide RNA polymerase p-subunit (rpoB) gene fragments of Legionella species which differ from one another depending on the species in order to overcome the above problems of the conventional method for identifying Legionella species.

Yet another object of the present invention is to provide a method for discriminating Legionella species, comprising the steps of amplifying the above rpo B gene fragments, determining nucleotide sequences of said gene fragment amplified in the above step and comparing said nucleotide sequences determined in the above step.

Still another object of the present invention is to provide a method for discriminating Legionella species, comprising the steps of amplifying the above rpoB gene fragment, treating the amplified gene fragment with a restriction enzyme to check the cleavage of the specific restriction site existing in the gene fragment, analyzing the above gene fragment cleaved by the specific restriction site.

Still another object of the present invention is to provide a primer used for the selective amplification of a portion of rpoB gene of Legionella species and the discrimination and identification method of Legionella species by using the above primer.

Still another object of the present invention is to provide a primer used for the selective

amplification of rpoB gene of Legionella pneumophila and the discrimination and identification method of Legionella species by using the above primer.

The above object of the present invention can be achieved by providing a method comprising the steps of amplifying selectively a portion of the rpoB gene of 38 species belonging to the genus Legionella by using a pair of primer of SEQ ID NO : 42 and 43 used for the selective amplification of a portion of the rpoB gene of Legionella species (5 GATGATATCGATCAYCTDGG3, TTCVGGCGTT CAATNGGAC'), determining nucleotide sequences of the above amplified rpoB gene fragment and discriminating each species belonging to the genus Legionella by comparing said nucleotide sequences determined in the above step.

The mixture of a forward primer (5 GATGATATCGATCAYCTDGG3) and a reverse primer ('TTCVGGCGTTCAATNGGAC') may be employed in the step of amplifying selectively a portion of the rpoB gene of Legionella species. In addition, the above primers may be employed for the analysis of nucleotide sequences of the selectively amplified rpoB gene fragment. The mixture of the forward primer and the reverse primer may be also used in the analysis of nucleotide sequences.

Nucleotide sequences of each fragment obtained by the above method are compared with those of SEQ ID NO: 1 to 42 contained in the specification to discriminate the species of bacteria for analysis.

Another object of the present invention can also be achieved by providing a diagnostic reagent for the selective discrimination of Legionella species

characterized by comprising a pair of primers of SEQ ID NO : 43 and SEQ ID NO : 44.

Another object of the present invention can be achieved by providing nucleotide sequences of SEQ ID NO: 1 to 42.

The above nucleotide sequences of SEQ ID NO : 1 to 42 consist of 34 rpoB gene fragments of 34 species belonging to the genus Legionella and 8 rpoB gene fragments of total 8 subspecies belonging to other 4 species. Phylogenetic relationship among the Legionella species for the analysis may be analyzed based on such 42 nucleotide sequences.

Another object of the present invention can be achieved by providing a method for discriminating Legionella species characterized by comprising the steps of amplifying a portion of rpo B gene of Legionella species for analysis by using the above primers, treating the amplified rpoB gene fragment of Legionella species having the specific restriction site with the specific restriction enzyme and confirming whether or not the above gene fragment is cleaved by the above specific restriction enzyme.

The mixture of the forward primer and the reverse primer of SEQ ID NO : 43 and 44 may be also used for amplifying a portion of rpoB gene of Legionella species for analysis in the above method for discriminating the species. In addition, the species of Legionella bacteria for analysis can be determined by confirming whether or not the fragment is cleaved by the specific restriction enzyme when the amplified fragment is treated by the restriction enzyme specific for each species.

When the amplified fragment is treated by the specific restriction enzyme (RFLP-Restriction Fragment Length Polymorphism), the resultant mixture is electrophoresed, the Legionella species may be discriminated based on the size of the cleaved fragment and the number of electrophoresed bands obtained by analyzing the result of electrophoresis, because the cleavage of the fragment by the restriction enzyme and the restriction site thereof depend on each Legionella species.

Another object of the present invention can be achieved by providing a pair of primers of SEQ ID NO : 44 and 45 used for the selective amplification of rpoB DNA of Legionella pneumophila.

5'TCAGTTTAGAGTAGGTCTT3, SCCCAAGRGCCGATACACG3' A pair of primers of SEQ ID NO : 44 and 45 used for the selective amplification of rpoB DNA of Legionella pneumophila may identify their characteristic nucleotide sequences from the nucleotide sequences of Legionella pneumophila of SEQ ID NO : 32 to amplify selectively rpoB gene fragment of Legionella pneumonia.

Another object of the present invention can be achieved by providing a diagnostic reagent for the selective discrimination of Legionella pneumophila, comprising a pair of primers of SEQ ID NO : 45 and SEQ ID NO : 46.

Another object of the present invention can be achieved by providing a method for the detection and identification of Legionella pneumophila, comprising the step of amplifying rpoB gene fragment of Legionella pneumophila.

The mixture of the forward primer (5 TCAGTTTAGAGTAGGTCTT3) and the reverse primer (5 CCCAAGRGCCGATACACG3') of SEQ ID NO : 44 and 45 may be used in the step of amplifying rpoB gene fragment of Legionella pneumophila selectively.

Legionella pneumophila may be selectively detected and identified from other Legionella species because rpoB gene fragment of Legionella pneumophila can be selectively amplified by using the above primers.

That is, Legionella pneumophila may be selectively detected and identified because only rpoB DNA fragment of Legionella pneumophila is amplified and that of other Legionella species is not amplified when PCR is performed by using the above primers.

Another object of the present invention can be achieved by providing a method for discriminating Legionella pneumophila subspecies, comprising the steps of amplifying rpoB gene fragment of Legionella pneumophila, treating said amplified rpoB gene fragment of Legionella pneumophila with a restriction enzyme to confirm the cleavage of the fragment at the specific restriction site existing in rpoB DNA fragment of Legionella pneumophila and analyzing the cleavage of the above gene fragment.

The mixture of the forward primer (5 TCAGTTTAGAGTAGGTCTT3) and the reverse primer (5 CCCAAGRGCCGATACACG3') of SEQ ID NO : 44 and 45 may be used in the step of amplifying rpoB gene fragment of Legionella pneumophila selectively.

In addition, when the above amplified fragment is treated by the specific restriction enzyme (RFLP- Restriction Fragment Length Polymorphism) and the

resultant mixture is electrophoresed, the Legionella pneumophila subspecies may be discriminated by analyzing the results of electrophoresis, because the cleavage of the fragment by the restriction enzyme and the restriction site thereof depend on each Legionella pneumophila subspecies.

When 217bp of rpoB DNA fragment is amplified through PCR using the above primers specific for Legionella pneumophila and then treated by BamHI restriction enzyme, rpoB DNA fragments of other subspecies are cleaved into 136bp and 81bp of DNA fragment whereas those of serogroup 4,5 and 15 are not cleaved by BamHI. The above results show that these three strains belong to the subspecies fraseri and the others belong to the subspecies pneumophila.

Brief Description of the Drawings The objects and other advantages of the present invention will become more apparent by describing in detail a preferred embodiment thereof with reference to the attached drawings, in which: FIG. 1 is a schematic view of the primer used for the amplification of rpoB gene of Legionella species; FIG. 2 shows the results obtained by performing PCR with rpoB gene fragments of the species belonging to the genus Legionella and the species belonging to other genuses; FIG 3. shows the results of comparison of the distances among rpoB gene, rRNA gene and mip gene; FIG 4. shows a phylogenetic tree of Legionella species based on the nucleotide sequences of the rpoB gene fragment;

FIG 5. shows a phylogenetic tree of Kpl, Kp2 and Kp3 isolated from the patients, which are prepared by comparing their nucleotide sequences with those of reference strains by the NJ method.

FIG 6. shows the results obtained by performing PCR with Legionella pneumophila and other species belonging to the genus Legionella by using Legionella pneumophila-specific primer; and FIG 7. shows the results obtained by performing PCR with reference strains of Legionella pneumophila by using Legionella pneumophila-specific primer.

Best Mode for Carrying Out the Invention Hereinafter, the present invention will be described in greater detail with reference to the following example. The example are given for illustration of the invention and not intended to be limiting the present invention.

Example 1 : preparation of strains Total 38 species (54 strain) were used concerning the genus Legionella (Table 1). 16S rRNA gene and nucletide sequences of mip registered in GenBank, were used for analyzing the phylogenetic relationship among the species. The isolated strains were isolated from the Korean patients (3 weeks; Kpl, Kp2, Kp3) or the environment and identified by biochemical tests.

The species which might cause pneumonia such as Legionella species or might be isolated from human respiratory organ or Gastrointestinal tract (Bacillus subtilis, Bacteroides fragilis, Branhamella catarrhalis, Corynebacterium diphtheriae, Escherichia coli, Haemophilus influenzae, Helicobacter pylori,

Mycobacterium fortuitum, Neisseria sicca, Staphylococcus aureus, Streptococcus faecalis, Streptococcus pyogenes) were used as reference species for examining the specificity of PCR.

Example 2: Isolation of DNA from each species DNAs of each species were prepared by the Bead beater/Phenol extraction method (Kim et al. , 1999).

100p1 of bacteria suspension was placed in a 2. Oml screw-cap microcentrifuge tube and 1001 of phenol: chloroform: isopropylalcohol (50: 49: 1) and 1001 (packed volume) of glass bead was added.

The bacteria was disrupted by oscillating the tube on a Mini-Bead beater for 30 seconds (5, OOOrpm).

The tube was centrifuged (15, 000rpmxlOmin) and the supernatant was transferred into another clean tube.

The same volume of isopropylalcohol was added to precipitate DNAs. DNAs were solubilized with zu distilled water and used for PCR.

Example 3: PCR amplification of rpoB gene The primers specific for Legionella (5 GATGATATCGATCAYCTDGG3 5 TTCVGGCGTTCAATNGGAC3) were prepared to amplify DNA (369-bp) fragment including rifampin-resistant region (rifr) (Severinov et al., 1993 ; Kim et al. , 1999; Nielsen et al. , 2000) from nucleotide sequences of rpoB gene of Escherichia coli (Acession no.

V00340), Coxiella burnetii (U86688) and L. pneumophila (AF087812) and so on which are registered in GenBank.

As is already known, rpoB genes of B. subtilis and E. coli consist of 4 conserved domains (C1-C4) and 3

variable domains (V1-V3).

The forward primer and the reverse primer were located in C2 highly conserved resion (HCR5i 444~454, HCR6 ; 547-577) including rifr related to rifampin- resistance of E. coli and bacteria causing turberculosis (codon 507-533, E. coli numbering) (Table 1).

50ng of template DNA and 20pmole of each primer were added to a PCR mixture tube (AccuPower PCR Premix ; Bioneer, Korea) containing 1 unit of Taq DNA Polymerase, 250µ M of each deoxynucleoside triphosphate, 50mM of Tris-HC1 (pH 8.3), 40mM of KCl, 1.5mM of MgCl2 and gel loading dye. And then, distilled water was added in a final volume of 20p 1, and PCR was performed. In this connection, denaturation (95C, 30 seconds), annealing (55C, 30 seconds), extension (72°C, 1 minute) and final extension (72°C, 5 minutes) were carried out for 30 cycles, respectively, using a thermocycler (Model 9700, Perkin-Elmer Cetus, USA).

The PCR products thus amplified were electrophoresed on 1. 5% agarose gel and purified for the determination of nucleotide sequences by using QIAEX II gel extraction kit (QIAGEN, Hilden, Germany).

Example 4: Determination of Nucleotide Sequences of rpoB Gene Fragments Nucleotide sequences of purified PCR products were determined using the forward primer and the reverse primer by employing Applied Biosystems model 377 automated sequencer and BigDye Terminator Cycler Sequencing kit (Perkin-Elmer Applied Biosystems, Warrington, United Kingdom).

For the sequencing reaction, 30ng of purified PCR product, 2. 5pmol of each primer and 4p l of BigDye Terminator RR mix (Perkin-Elmer Applied Biosystems part no. 4303153) were mixed and distilled water was added in a final volume of 10 p 1. The reaction was carried out using 5% (v/v) dimethylsulfoxide for 30 cycles of 15 seconds at 95'C, 5 seconds at 50C and 4 minutes at 60C.

As a result, 71.0% (L. geestiana-L. spiritensis)-98. 7% (L. jametownensis-L. lodiniensis) homogeneity was observed among the nucleotide sequences of rpoB DNA of each Legionella species.

Each nucleotide sequence must be well conserved, however on the other side, some variation depending on each species are needed in order that they may reflect closely the phylogenetic relationship among the species and thus nucleotide sequences of rpoB gene may be used for discriminating the species.

Therefore, nucleotide sequences of rpoB DNA were compared with those of 16S rRNA gene and mip for the investigation of pairwise distance (Fig 3).

Divergence of nucleotide sequences of rpoB gene was 3.5 times as much as that of 16S rRNA gene, which meant that rpoB gene showed larger variation than 16S rRNA gene. In addition, divergence of nucleotide sequences of mip was 1.5 times as much as that of rpoB gene. Therefore, the above results showed that thenucleotide sequence of rpoB gene had appropriate resolving power for discriminating Legionella species.

Though the strains identified by biochemical tests were used, their 400-bp nucleotide sequences at 3'end of 16S rRNA gene were amplified and sequenced to confirm that they were Legionella species by comparing

with the nucleotide sequences registered in GenBank (Hookey et al. , 1996). The determined sequences were illustrated in SEQ ID NO : 1 to 42.

Example 5: Phylogenetic Analysis The Phylogenetic relationship among Legionella species was analyzed by using the nucleotide sequences corresponding to 300-bp from 369-bp of rpoB DNA fragment amplified through PCR, in order to confirm that nucleotide sequences of rpoB gene were useful to classify and identify Legionella species. Such nucleotide sequences were registered in GenBank (Table 1).

The above nucleotide sequences were arrayed by using CLUSTAL X (Thompson et al. , 1997) as multiple array program and then a phylogenetic tree was constructed in accordance with neighbor-joining (NJ) method (Saitou & Nei, 1987) and parsimony method of PAUP program.

Pairwise distance in the NJ method was calculated as maximum likelihood option. In addition, heuristic search was conducted in the parsiomony analysis, selecting tree bisection reconstruction (TBR) branch swapping option.

Branch supporting value was calculated through 1000 bootstrap replication (Felsenstein, 1985 ; Hillis &Bull, 1993), selecting Coxiella burnetii causing Q fever as a outgroup.

As a result, each species was well branched off.

In addition, similar species were closely clustered with each other, compared with the conventional methods using the phenotypes such as autofluorescence, fatty

acids in the cell wall, ubiquinone group and so on (Fig 4). A scale located in the bottom of fig 4 indicates the number of inferred substitutions per site.

Autofluorescence of each species (BW, brown-white; YG, yellow-green; R, red), ubiquinone groups (A to F) and fatty acid groups (I to IV) were illustrated in the right side of corresponding Legionella species.

All nucleotide sequences of rpoB genes of Kpl, Kp2 and Kp3 isolated from the Korean patients were clustered with those of L. pneumophila subsp.

Pneumophila, and thus all the bacteria were identified as L. pneumophila subsp. Pneumophila (Fig 5).

In addition, data sets of rpoB gene, 16S rRNA gene and mip were compared with each other. In Fig 3, dots in the graph indicates pairwise distance between each Legionella species and Coxiella burnetii as a outgroup.

(A) rpoB gene and 16S rRNA gene, (B) mip and 16S rRNA gene, and (C) rpoB gene and mip were compared with each other. (B) mip and 16S rRNA gene showed linear relationship, whereas (A) rpoB gene and 16S rRNA gene and (C) rpoB gene and mip showed non-linear relationship. The above result meant that rpoB gene was frequently mutated at the level of DNA. In this connection, pairwise distance was calculated as maximum likelihood option of PAUP (Swofford, 1999) and the analysis was conducted by employing PAUP program (Swofford, 1999).

Table 1. Reference strains used for the present invention.

Species Abbreviation Serogroup Strain L. pneumophila pn sg-1 ATCC 33152 (Philadelphia 1) sg-1 ATCC 33153 (Knoxville 1) sg-1 SF9 sg-1 ATCC 43109 (OLDA) sg-2 ATCC 33154 (Togus 1) sg-3 ATCC 33155 (Bloomington 2) sg-4 ATCC 33156 (Los Angeles 1) sg-5 ATCC 33216 (Dallas 1 E) sg-6 ATCC 33215 (Chicago 2) sg-7 ATCC 33823 (Chicago 8) sg-8 ATCC 35096 (Concord 3) sg-9 ATCC 35289 (International 23) sg-10 ATCC 43283 (Leiden 1) sg-11 ATCC 43130 (797-PA-H) sg-12 ATCC 43290 (570-CO-H) sg-13 ATCC 43736 (82A3105) sg-14 ATCC 43703 (1169-MN-H) sg-15 ATCC 35351 (Lansing 3) L. adelaidensis ad UOEH 13562 (1762-AUS-E) L. anisa* an ATCC 35292 (WA-316-C3) L. birminghamensis* bi UOEH 11749 (1407-AL-H) L. bozemanii* bo sg-1 ATCC 33217 (WIGA) sg-2 ATCC 35545 (Toronto 3) L. brunensis br UOEH 12655 (444-1) L. cherrii* ch UOEH 10742 (ORW) L. cincinnatiensis* ci UOEH 12201 (72-OH-H) L. dumoffi* du ATCC 33279 (NY-23) L. erythra er sg-1 ATCC 35303 (SE-32-A-C8) L. fairfieldensis fa UOEH 13563 (1725-AUS-E) L. feeleii* fe sg-1 ATCC 35072 (OW-44C) sg-2 UOEH 10744 (691-WI-H) L. geestiana ge ATCC 49504 (1308) L. gormanii* go ATCC 33297 (LS-13) L. gratina gr ATCC 49413 (Lyon 8420412) L. hackeliae* ha sg-1 ATCC 35250 (Lansing 2) sg-2 UOEH 11368 (798-PA-H) L. israelensis* is ATCC 43119 (Bercovier 4) L. jametowniensis ja ATCC 35298 (JA-26-Gi-E2) L. jordanis* jo HM 7000 (BL-540) L. Iansingensis* la ATCC49751 (167-MI-H) L. londiniensis Id ATCC 49505 (1477) L, longbeachae* lo sg-1 ATCC 33462 (Long Beach 4)

sg-2 ATCC 33484 (Tucker 1) L. maceachernii* ma ATCC 35300 (PX-1-G2-E2) L. micdadei* mi ATCC 33218 (TATLOCK) L. moravica mo ATCC 43877 (316-36) L. nautarium na ATCC 49506 (1224) L. oakridgensis* oa HM 7002 (Oak Ridge 10) L. parisiensis* pa UOEH 11745 (PF-209C-C2) L. quinlivanii qu sg-1 ATCC 43830 (1442-AUS-E) L. rubrilucens ru ATCC 35304 (WA-270-C2) L. sainthelensi* sa sg-1 ATCC 35248 (Mt St Helens) L. santicrucis sc UOEH 11746 (SC-63-C7) L. shakespearei sh ATCC 49655 (214) L. spiritensis sp sg-1 UOEH 11199 (Mt St Helens 9) L. steigerwaltii st UOEH 11199 (Mt St Helens 9) L. tucsonensis* tu ATCC 49180 (1087-AZ-H) L. wadsworthii* wa ATCC 33877 (81-716A) L. worsleiensis wo ATCC 49508 (1347) * pathogenic to human.

L. pneumophila subsp. pneumophila was represented as Lpn-p, and subsp. fraseri as Lpn-f.

Example 6: PCR by using primers specific to Legionella species The nucleotide sequences are useful because a probe, which may be used for PCR restriction analysis for discriminating the species, for PCR or for hybridization, may be prepared based on the characteristic nucleotide sequences existing in each species, as well as because the species may be identified by comparison of homogeneity of nucleotide sequences.

The signature nucleotide sequences used for the above purpose were identified (Table 2). That is, signature nucleotides existing the species commonly causing human infection such as Legionella pneumophila were identified from the nucleotide sequences of rpoB gene of Legionella species.

Based on the above nucleotides, primers for amplifying seletively Legionella species was prepared and PCR was performed using the primers (30 cycle; denaturation (95 C, 30 seconds), annealing (58C, 30 seconds), extension (72C, 30 seconds) and final extension (72C, 5 minutes) ). PCR products were electrophoresed on the 3% agarose gel.

As a result, Legionella species showed 374bp of PCR products (Fig 2A), whereas other species showed no PCR products (Fig 2B).

In Fig 2A, each abbreviation is as follows: M1, 100 bp DNA ladder; M2, ou174 RF DNA/HaeIII marker. pn, L. pneumophila ; an, L. anisa; bi, L. birminghamensis ; ci, L. cincinnatiensis ; du, L. dumoffii ; go, L. gormanii ; is, L. israelensis ; jo, L. jordanis ; la, L. lansingensis ; lo, L. longbeachae ; mi, L. micdadei; pa, L. parisiensis ; sa, L. sainthelensi ; tu, L. tucsonensis.

In Fig 2B, each abbreviation is as follows: M, 100 bp DNA ladder; 1, L. pneumophila ; 2, L. longbeachae ; 3, S. aureus; 4, E. coli ; 5, B. subtilis ; 6, C. diphtheriae; 7, M. fortuitum; 8, N. sicca; 9, B. catahalis ; 10, S. feacalis ; 11, H. influenzae ; 12, S. pyogens; 13, B. fragilis ; 14, Norcardia sp.; 15, H. pylori.

Table 2. signature nucleotides of rpoB gene existing the species commonly causing human infection

Species Position Nucleotides L. pneumophila subsp. pneumophila Serogroup 1, 2,3, 6,7, 8,36 A 9,10, 11,12, 13,14 159 A 192 G (Lpn9, C) 213 G 220 A L. pneumophila subsp. fraseri Serogroup 4, 5, 15 6 C 45 C 66 G 105 C 246 T 258 C L. micdadei 42 A 48 G 57 C 117 A 159 G 180 C 222 G 267 G L. Iongbeachae 129 G 213 G Example 7: A Primer specific to Legionella pneumophila A probe, which may be used for PCR restriction analysis for discriminating the species, for PCR or for hybridization, was prepared based on the characteristic nucleotide sequences existing in each species.

The signature nucleotide sequences used for the above purpose were identified (Table 2). That is, the signature nucleotide sequences were identified from the nucleotide sequences of rpoB gene of Legionell pneumophila. Based on them, the forward primer (5 TCAGTTTAGAGTAGGTCTT3) and the reverse primer

(5 CCCAAGRGCCGATACACG3) for amplifying selectively Legionella pneumophila were prepared.

Example 8: Selective detection method for Legionella pneumophila The nucleotide sequences of Legionella pneumophila and other species belonging to the genus Legionella were amplified through PCR by using the primer based on the signature nucleotide of Legionella pneumophila (30 cycle; denaturation 95C 30 seconds, annealing 58C 30 seconds, extension 72 C 30 seconds, final extension 72°C 5 minutes).

The mixture of the forward primer (5 TCAGTTTAGAGTAGGTCTT3) and the reverse primer (5 CCCAAGRGCCGATACACG3) was used in the step of amplifying selectively a portion of rpoB DNA of Legionella pneumophila.

As a result, only the DNA fragments (217-bp) of Legionella pneumophila (lanes 1,2, 5,6, 15) were amplified, whereas those of other species (lanes bo, du, go, jo, lo, mi, pa) were not amplified (Fig 1).

Three strains (Kpl, Kp2, Kp3) isolated from the Korean patients had the same PCR products and thus, they were identified as Legionella pneumophila. In addition, other species belonging to the genus Legionella had no PCR products (Fig 1, Ml, 100-bp DNA ladder; M2, Ou174 RF DNA/HaeIII digest; Lanes (1, 2,5, 6,15), L. pneumophila serogroup 1,2, 5,6, 15 ; bo, L. bozemanii ; du, L. dumoffii; go, L. gormanii; jo, L. jordanis; lo, L. longbeachae ; mi, L. micdadei; pa, L. parisiensis ; Kpl, Kp2, and Kp3, bacteria isolated from the Korean patients).

Example 9: Discrimination of Legionella pneumophila subspecies BamHI restriction enzyme which acts on the specific restriction site located on the nucleotide sequences searched by employing MapDraw program, was used for discriminating Legionella pneumophila subspecies.

217-bp DNA was amplified by using the primer specific to Legionella pneumophila and then, cleaved by BamHI. Reference strains of Legionella pneumophila were amplified through PCR specific to Legionella pneumophila and then cleaved by BamHI.

As a result, 217-bp of rpoB DNA fragments were amplified in all reference strains through the above PCR. In addition, the DNA fragments of serogroup 4,5 and 15 were not cleaved by BamHI, whereas those of the other strains were cleaved into 136-bp and 81-bp of DNA fragments by BamHI. Therefore, the above three serogroups belonged to subsp. fraseri and the others belonged to subsp. pneumophila (Fig 7, Ml, 100-bp DNA ladder; M2, OX174 RF DNA/HaeIII digest; Lanes 1-15, L. pneumophila serogroup 1~15).

Industrial Applicability As illustrated and demonstrated in the above, nucleotide sequences of rpoB gene of each Legionella species can be obtained by the present invention. In addition, a primer specific to rpoB DNA fragments of Legionella species can be prepared. Finally, the present invention can provide a method for discriminating the species based on rpoB DNA fragment

amplified by using the above primer. That is, the discrimination method for Legionella species using RELP can be provided.

In addition, a primer specific to rpoB DNA fragments of Legionella pneumophila can be prepared by the present invention. Legionella pneumophila can be detected and identified by amplifying selectively rpoB DNA of Legionella pneumophila using the above primer.

Furthermore, Legionella pneumophila subspecies such as serogroup 1, the most representative pathogen among Legionella pneumophila subspecies can be discriminated exactly through the RELP analysis.

Therefore, when unknown bacteria suspected as Legionella species is isolated from a pneumonia patient or the environment, the nucleotide sequences of the bacteria is determined and compared with those of reference strains to discriminate the species more exactly than the conventional methods. Therefore, therapeutic policy for the patient infected with Legionella species can be determined more promptly.

Furthermore, Legionella pneumophila can be more exactly identified and discriminated from other species belonging to the genus Legionella by using a primer specific to rpoB gene fragments of Legionella pneumophila. Therefore, therapeutic policy for the patient infected with Legionella pneumophila can be determined more promptly. In addition, Legionella pneumophila subspecies can be simply discriminated by the present invention.