KR101372175B1 - Primer and probe for detection of Halomonas sp. and method for detection of Halomonas sp. using the same - Google Patents

Abstract

The present invention relates to a method for detecting or quantifying bacteria of the genus Halomonas . Specifically, the present invention relates to a method that can detect or quantify bacteria of the genus Halomonas by using a specific primer pair, the method of the present invention halomonas acts as a etiological factor in various species It can be used to detect genus bacteria more simply and efficiently.

Description

Primers and probes for the detection of bacteria of the genus Halomonas and methods for detecting bacteria of the genus halomonas using the same {Primer and probe for detection of Halomonas sp. and method for detection of Halomonas sp. using the same}

The present invention relates to a method for detecting or quantifying bacteria of the genus Halomonas by using a primer pair specific to the bacteria of the genus Halomonas.

Gram-negative rod-shaped halo of Pseudomonas spp (Halomonas sp . ) Were mainly found in the salinity of the ocean with high ecosystem (Vreeland et al., 1980) , halo Pseudomonas spp found suspected contaminated bacteria while trying to dialysis in kidney treatment center in the United States Santa Clara Valley Medical Center To humans as a pathogenic microorganism (David A et al., 2009).

Halo identification of bacteria belonging to the genus Pseudomonas have been carried out on the basis of biochemical and antigenic properties (Holt et al ., 1994). In the reported to date halo Pseudomonas bacteria H. alimentaria, H. alkaliphila, H. almeriensis , H. anticariensis, H. aquamarina, H. axialensis, H. boliviensis, H. campaniensis, H. campisalis, H. canadensis, H . cupida, H. denitrificans, H. desiderata , H. elongata, H. eurihalina, H. gomseomensis, H. halmophila, H. halocynthiae, H. halodenitrificans, H. halodurans, H. halophila, H. hamiltonii, H. hydrothermalis , H. indalinina, H. israelensis, H. janggokensis, H. johnsoniae, H. koreensis, H. magadiensis, H. marisflavi, H. maura, H. meridiana, H. muralis, H. neptunia, H. nitroreducens, H . organivorans, H. pacifica, H. pantelleriensis , H. salaria, H. salina, H. stevensii, H. subglaciescola, H. sulfidaeris, H. taeanensis, H. titanicae, H. variabilis, H. ventosae, H. venusta comprising the 48 bacteria, such as, most Halo Pseudomonas spp was found in the sea and mudflats, of the kind found in humans H. hamiltonii, H. johnsoniae, H. stevensii Three kinds of these are included.

The present inventors have confirmed that the halo Pseudomonas spp are predominantly found in patients with urinary disorders, which halo Monastir It is suggested that the genus bacteria may play an important role in urinary or prostatitis in men.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made intensive studies to develop a halo Pseudomonas genus (Halomonas sp.) Bacteria in a more specific way and can be easily detected or quantified. As a result, the present inventors have specifically a halo Pseudomonas genus in the various samples, in particular male sample of urinary disease to produce a pair of primers capable of detecting 16S rRNA gene in the halo Pseudomonas specifically, and using the procedure timer pair and The present invention has been completed by confirming that it can be easily detected.

Accordingly, it is an object of this invention to provide a halo such as Pseudomonas genus (Halomonas sp.) Detection method to detect bacteria using a specific primer, probe, and this.

Another object of the present invention is to provide a kit for detecting or quantifying bacteria of the genus Halomonas .

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a halo Pseudomonas genus (Halomonas sp.) Detecting or assaying a pair of primers for the bacteria comprising the primers having the base sequences of primers and SEQ ID NO: 2 having the base sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a method for detecting or assaying a halo Pseudomonas genus (Halomonas sp.) Bacteria, using the primer pairs of the present invention.

In another aspect, the present invention provides a comprising the primer pair of the invention, halo Pseudomonas genus (Halomonas sp.) Bacterial detection or quantification kit of.

In another aspect, the present invention provides a halo Pseudomonas genus (Halomonas sp.) Detecting or assaying bacterial probe comprising all or a portion of the amplified using the primer pairs of the present invention the gene product.

In another aspect, the present invention provides a halo Pseudomonas genus (Halomonas sp.) Bacterial detection microarray for such a manner that the probe is integrated on the substrate.

The present invention provides a pair of primers designed to detect the bacterial genus Pseudomonas halo specifically, a halo Pseudomonas genus in a sample by using the above primer pair can be very simply and effectively be detected or quantified. In addition, the method of the present invention has the advantage of accurately detecting the bacteria of Halomonas using a very small amount of DNA as a template.

Thus, the method of the present invention halomonas acts as a etiological factor in various species It is expected that it may be used to detect and / or quantify genus bacteria more simply and efficiently.

Figure 1 shows the result of the PCR for the genomic DNA extracted from the 22 bacteria, including 3 halo Pseudomonas sp using CAU-HalF and CAU-HalR primer ssangreul [Lanes: M, 100 bp DNA ladder (Bioneer Corporation , Daejeon, Korea); Lane 1: Halomonas hamiltoni KCTC 22154 ^T , Lane 2: Halomonas stevensi KCTC 22148 ^T , Lane 3: Halomonas johnsaniae KCTC 22157 ^T , Lane 4: Shigella flexneri KCTC 2008 ^T , Lane 5: Salomonella chloraesuis ACTC 13312 ^T , Lane 6: Salomonella pullorum ACTC 9120 ^T , Lane 7: Burkholdaria ambifaria KCTC 12943 ^T , Lane 8: Burkholdaria vietnamiensis KCTC 2974 ^T , Lane 9: Pseudomonas aeruginosa KCTC 1637 ^T , Lane 10: Proteus Vulgaris KCTC 2512 ^T , Lane 11: Pseudomonas aeruginosa KCTC 2592 ^T , Lane 12: Escherichia coli KCTC 2597 ^T , Lane 13: Vagococcus fluvialis LMG 9464 ^T , Lane 14: S almonella typhimuroum ATCC 6994 ^T , Lane 15: Pseudomonas aeruginosa KCTC 1750 ^T , Lane 16: Enterococcus villorum KCTC 13904 ^T , lane 17: Enterococcus cecorum KCTC 3642 ^T , Lane 18: Enterococcus raffinosus KCTC 5189 ^T , Lane 19: Enterococcus hirae KCTC 3616 ^T , Lane 20: Enterococcus munatti KCTC 3630 ^T , Lane 21: Enterococcus caseliflavus KCTC 3638 ^T , Lane 22: Enterococcus faecalis KCTC 3206 ^T ].
FIG. 2 shows the results of PCR using CAU-HalF and CAU-HalR primer pairs for samples collected from 17 patients with urological diseases collected from the Department of Urology, Chung-Ang University Hospital.

The present inventors halo Pseudomonas genus (Halomonas sp.), By seeking to develop a method that the bacteria can be detected making the sought results, halo Pseudomonas in the 16S rRNA primer pair capable of detecting the gene specifically to carry out an amplification reaction, food It has been confirmed that bacteria from Halomonas can be detected and / or quantified in a variety of samples, including natural environmental substances and patient secretions.

According to the invention, it is possible to detect or quantify highly effective and halo Pseudomonas genus (Halomonas sp.) Bacteria easily from a variety of samples.

The present invention provides a primer for detecting genus Halomonas .

The primer of the present invention may have a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a base sequence complementary thereto. In addition, the primers of the present invention may be modified (eg, added, deleted, substituted) within a range that does not affect specific detection of genus Halomonas .

Primers of the present invention can effectively detect the genus Pseudomonas halo, because only a specific characteristic, the non-specific and other bacteria in the Pseudomonas halo halo except in Pseudomonas. The inventors have confirmed that in the halo Pseudomonas 16S rRNA gene is specific genes that can be distinguished from other sokreul halo Pseudomonas strain other than the genus Pseudomonas halo (see Example 1 and Figure 1).

As used herein, "primer" refers to a short nucleic acid sequence that can form base pairs with complementary templates and that serves as a starting point for template strand copying.

The primers may include restriction fragment length polymerphism (RFLP), randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), arbitrarily primed PCR (AP-PCR), sequence tagged site (STS), expressed sequence tag (EST), SCAR (sequence characterized amplified regions), inter-simple sequence repeat amplication (ISSR), amplified fragment length polymorphism (AFLP), cleaved amplified polymorphic sequence (CAPS), single-strand conformation polymorphism (PCR-SSCP), and the like. It can be designed to suit a variety of DNA analysis.

According to one embodiment of the present invention, the primers of the present invention are used for amplification reactions.

As used herein, “amplification reaction” means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), real-time PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Methods of ligase chain reaction (LCR) (17, 18), Gap-HI (WO 89/06700) and Davey, C. et al (EP 329,822) (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) 19 (WO 88/10315), self sustained sequence replication) 20 (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence-based amplification acid sequence based amplification (NASBA) (U.S. Patent Nos. 5,130,238, 5,4 09,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification (LAMP) (23) It is not limited. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

In a preferred embodiment of the present invention, the amplification process is carried out according to the polymerase chain reaction (PCR) disclosed in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

There is no restriction on the PCR method used in the present invention.

In another embodiment of the present invention, the detection method using the primer pair of the present invention may include detecting a 16S rRNA gene of Halomonas in a sample to be analyzed, for example, a urine disease patient sample. Specifically, the present invention performs a gene amplification reaction using a primer pair that binds to cDNA synthesized from mRNA in the sample. In other words, the total RNA of the bacteria is isolated and then 16S rRNA gene is detected. Therefore, the present invention performs a gene amplification reaction using a primer that binds to cDNA synthesized from mRNA in the sample.

To obtain the mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al . , Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al . , Plant Mol . Biol . Rep . 9: 242 (1991); Ausubel, FM et al . , Current Protocols in Molecular Biology , John Willey & Sons (1987); And Chomczynski, P. et al . , Anal . Biochem . 162: 156 (1987). For example, Trizol can be used to easily isolate total RNA in cells. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. When total RNA is isolated from a human sample, the end of the mRNA has a poly-A tail, and cDNA can be easily synthesized using oligo dT primer and reverse transcriptase using this sequence characteristic ( PNAS USA , 85). : 8998 (1988); Libert F, et al . , Science , 244: 569 (1989); And Sambrook, J. et al . , Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the "Clenow" fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from various bacterial species, which is Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu) and the like.

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. So that the amplification degree to be obtained the joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is preferred. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

Annealing in the present invention is carried out under stringent conditions that allow for specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The amplified 16S rRNA gene is analyzed by a suitable method to detect or quantify the genus Halomonas. For example, 16S rRNA gene can be detected by gel electrophoresis of the above-described amplification reaction product and observing and analyzing the resulting band.

Therefore, when the method of the present invention is carried out based on an amplification reaction using cDNA, specifically (i) performing an amplification reaction using primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2; And (ii) analyzing the product of said amplification reaction, thereby Halomonas Genus bacteria can be detected or quantified.

In another aspect, the present invention provides a halo Pseudomonas genus (Halomonas sp.) Detecting or assaying bacterial probe comprising all or a portion of the amplified using the primer pairs of the present invention the gene product. The probe of the present invention is halomonas Modifications (eg, additions, deletions, substitutions) can be made without affecting the specific detection of the genus bacteria.

As used herein, the term "probe" refers to a nucleic acid fragment corresponding to a few to several hundred bases, which is capable of specific binding with DNA or RNA, and is labeled to confirm the presence or absence of a specific DNA or RNA. have. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like, and are labeled with biotin, FITC, rhodamine, DIG, etc. Or radiolabeled isotopes.

The present invention also provides a microarray for detecting bacteria of the genus Halomonas , wherein the probe is integrated on a substrate.

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods of preparing microarrays by immobilizing probe polynucleotides on substrates are well known in the art. The term “probe polynucleotide” refers to a polynucleotide capable of hybridization, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid.

Halomonas of the present invention The process of immobilizing a probe polynucleotide associated with a genus on a substrate can also be readily prepared using this prior art. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

Halomonas using the primer or probe of the present invention Methods for detecting the genus include polymerase chain reaction (PCR) analysis, DNA sequencing, hybridization by microarrays, PCR-RFLP (restriction fragment length polymerphism) analysis, and RAPD (randomly amplified polymorphic). DNA (DNA), DNA amplification fingerprinting (DAF), arbitrarily primed PCR (AP-PCR), sequence tagged site (STS), expressed sequence tag (EST), sequence characterized amplified regions (SCAR), inter-simple sequence repeat amplication (ISSR) , AFLP (amplified fragment length polymorphism), cleaved amplified polymorphic sequence (CAPS), single-strand conformation polymorphism (PCR-SSCP), Northern hybridization and Southern hybridization It may be performed using various known DNA polymorphism assays, but is not limited thereto.

In addition, PCR products amplified using the primer pair of the present invention were inserted into pCR 2.1 cloning vectors, respectively, and transformed into E. coli TOP10F (Invitrogen, Carlsbad, CA, USA), followed by Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and ABIPRISM 3730 automated DNA sequencer. Nucleotide sequence homology was determined using the BLAST suite of programs (Altschul et al., 1997) against the NCBI GenBank non-redundant database ( http://www.ncbi.nlm.nih.gov ).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Bacterial strains

The bacterial strains used in the present invention are shown in Table 1 below.

Tested bacterial strains (n = 22) number Bell One Halomonas hamiltoni KCTC 22154 ^T 2 Halomonas stevensi KCTC 22148 ^T 3 Halomonas johnsaniae KCTC 22157 ^T 4 Shigella flexneri KCTC 2008 ^T 5 Salomonella chloraesuis ACTC 13312 ^T 6 Salomonella pullorum ACTC 9120 ^T 7 Burkholdaria ambifaria KCTC 12943 ^T 8 Burkholdaria vietnamiensis KCTC 2974 ^T 9 Pseudomonas aeruginosa KCTC 1637 ^T 10 Proteus Vulgaris KCTC 2512 ^T 11 Pseudomonas aeruginosa KCTC 2592 ^T 12 Escherichia coli KCTC 2597 ^T 13 Vagococcus fluvialis LMG 9464 ^T 14 Salmonella typhimuroum ATCC 6994 ^T 15 Pseudomonas aeruginosa KCTC 1750 ^T 16 Enterococcus villorum KCTC 13904 ^T 17 Enterococcus cecorum KCTC 3642 ^T 18 Enterococcus raffinosus KCTC 5189 ^T 19 Enterococcus hirae KCTC 3616^T 20 Enterococcus munatti KCTC 3630 ^T 21 Enterococcus caseliflavus KCTC 3638 ^T 22 Enterococcus faecalis KCTC 3206 ^T

All strains were purchased from the Korea Biotechnology Research Institute, Korea Collection for Type Cultures (KCTC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) or Belgian Co-ordinated Collections of Micro-organisms (BCCM / LMG). Bacteria were cultured in aerobic environment at 37 ° C. in trypticase soy yeast extract (TSYE), Brain Heart Infusion (BHI) or nutrient broth.

Genome DNA  detach

Bacterial genomic DNA was isolated according to Ausubel et al. (Ausubel et al., Current protocols in molecular biology, section 22.4, John Wiely and Sons, New York, NY, 1994).

Extraction of DNA was transferred to 1.5 ml microtubes of the strain incubated in 3 ml of nutrient broth (Difco) and centrifuged at 15,000 rpm for 2 minutes to precipitate the cells. The precipitated cells were well suspended by adding 100 μl of TE buffer (pH 8.0), and 50 μl of 20 μl of 10% sodium dodecyl sulfate (SDS) and 10 μl of protease K (10 mg / ml, Sigma). It was made to react at 1 degreeC. The reacted cells were mixed well by adding 200 μl of 10% CTAB (cetyltrimethylammonium bromide) /0.7 M NaCl solution, followed by reaction at 65 ° C. for 10 minutes, and the same amount of chloroform / isoamyl alcohol (24: 1) solution was added thereto. After shaking for 5 minutes and centrifuged for 5 minutes at 4 ℃, only the supernatant was transferred to a new tube. To this was added 1/10 fold 3 M sodium acetate and 2 fold 100% ethanol to precipitate gDNA. The precipitated gDNA was washed with 70% ethanol, dissolved in 50 μl of TE buffer, RNase (Sigma) was added to a final concentration of 20 μg / ml, and reacted at 42 ° C. for 30 minutes to remove RNA. The purified DNA was quantitated at 260 nm using Infinite 200 NanoQuant (Tecan, Switzerland) and used in the following experiment while preserving at -20 ° C.

Halomonas Genus specific Primer  making

In order to detect specific genes of the genus Halomonas, 16S rRNA gene sequences of a group of bacteria included in the genus Halomonas obtained from the GenBank database were aligned using the Clustal X program (Thompson et al., 1997), and then 16S rRNA gene sequences Primers were designed using primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) program (Table 2). The designed primer was commissioned by Genotech Co., Ltd. (Taejon, Korea) on a 200 pM scale and purified by PAGE. The synthesized primers were used in this experiment while diluted to 10 μM concentration and stored at -20 ℃.

SEQ ID NO: primer Base sequence Amplification size (bp) One CAU-HalF (forward primer) 5'-ATCACYCRCAGAAGAAGCACC-3 ' 195 2 CAU-HalR (reverse primer) 5'-AATTCTACCTTCCTCTCCTGC-3 '

Polymerase chain reaction  Used Halomonas  genus  Specific gene detection

Carried out to determine whether there is a primer produced in Example 2 can be detected by specific only halo Pseudomonas genus genetically, by using a pair of primers of the synthesized SEQ ID NO: 1 and SEQ ID NO: 2 was carried out the polymerase chain reaction (PCR) . First, the reaction solution (1 μl (10 ng) of DNA of the isolated and purified halomonas genus and the control strains disclosed in Table 1 above (2 μl of 10 × Taq buffer, 1 μl of 2.5 mM dNTPs, DW 13.7, 10 uM forward primer) 1 μl, 1 μl of 10 uM reverse primer and 0.3 μl of Taq polymerase were added to each of 19 μl, followed by GeneAmp PCR system 9700 (Applied Biosystem) once at 94 ° C for 5 minutes, 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 μl. The reaction was repeated 30 times for 40 seconds at 40 ° C. and amplified for 10 minutes at 72 ° C. The PCR products were electrophoresed on 2% SeaKem LE agarose gel (FMC Bioproducts, Philadelphia, PA, USA) and then subjected to EtBr staining. Visualization is shown in Figure 1 the results.

As shown in Figure 1, it was confirmed that only the gene product (195 bp) amplified by the primer pair of the present invention can be obtained only in Halomonas genus strain of lanes 1-3.

In urinary patient samples Halomonas Genus strain identification

In order to confirm the detectability of the genus Halomonas, PCR was performed in the same manner as in Example 3 in patients with urinary disease and the results are shown in FIG. 2. The urinary patient sample was specifically collected by the urine and prostate glands secretion secreted by the urine and prostate gland for patients diagnosed with prostatitis in the Department of Urology, Chung-Ang University Hospital using a sampling tool Quick Swab (3M, USA).

As shown in Figure 2, was identified that the gene product (195 bp) amplified by the primer pair of the present invention in a sample of 15 patients 17 patients samples, which means that there is a halo Pseudomonas sp. Patients whose gene products were not identified (patient samples 2 and 4) are considered to be the result of the degree of disease and / or the degree of infection.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. Therefore, it is to be understood that the embodiments described above are exemplary in all respects and not restrictive.

[references]

Altschul, SF, Madden, TL, Schaffer, AA, Zhang, J., Zhang, Z., Miller, W. & Lipman, DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res , 25 , 33893402.

Ausubel FM, Brent R, Kingston R E, Moore D D, Seidman JG & Struhl K (1993) Miniprep of bacterial genomic DNA. Current protocols in molecular biology, vol. Pp. 2.4. John Wiely & Sons, New York.

Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T. and Williams, S. T. (eds.). (1994) In Bergeys manual of determinative bacteriology. Williams & Wilkins, Baltimore, Maryland, 9th edition pp. 527-558.

Rozen, S. & Skaletsky, H. (2000) Primer 3 on the WWW for general users and for biologist programmers. Methods Mol Biol , 132 , 365386

Thompson, JD, Gibson, TJ, Plewniak, F., Jeanmougin, F. & Higgins, DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res , 25 , 48764882.

Toranzo, AE, Devesa, S, Heinen, P, Riaza, A, Nunez, S, Barja, JL (1994) Streptococcosis in cultured turbot caused by an Enterococcus-like bacterium. Bull Eur Assoc Fish Pathol , 14 , 1923.

Wallbanks, S., Martnez-Murcia, AJ, Fryer, L., Phillips, BA & Collins, MD (1990) 16S rRNA sequence determination for members of genus Carnobacterium and related lactic acid bacteria and description of Vagococcus salmoninarum sp. nov. Int J Syst Bacteriol , 40 , 224230.

Walter, J., Hertel, C., Tannock, G. W., Lis, C. M., Munro, K. & Hammes, W. P.

Yoon, JH, Lee, KC, Kho, YH, Kang, KH, Kim, CJ & Park, YH (2002) Halomonas alimentaria sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol , 52 , 123130.

David A. Stevens, MD, John R. Hamilton, PhD, Nancy Johnson, RN, Kwang Kyu Kim, PhD, and Jung-Sook Lee. (2009) Halomonas, a Newly Recognized Human Pathogen Causing Infections and Contamination in a Dialysis Center. Medicine 88 : 244-9.

R. H. VREELAND, C. D. LITCHFIELD, E. L. MARTIN AND E. ELLIOT. (2009) Halomonas elongata, a New Genus and Species of Extremely Salt-Tolerant Bacteri. Int J Syst Evol Microbiol. 30: 485-495.

Attach an electronic file to a sequence list

Claims (3)

Composition for diagnosing urinary disease comprising a primer pair consisting of a forward primer having a nucleotide sequence of SEQ ID NO: 1 and a reverse primer having a nucleotide sequence of SEQ ID NO: 2. The halo in Pseudomonas, using the composition of claim 1 (Halomonas sp.) Bacteria, comprising the step of detecting or quantitatively, to provide information to diagnose urinary disorders. A kit for diagnosing urinary disease, comprising the composition of claim 1.

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