KR20030087913A - DNA Probe Derived from 23S rRNA Gene for Detection of Infectious Bacteria - Google Patents

Abstract

The present invention relates to a DNA probe that is useful for detecting the causative agent of bacterial infectious diseases in a biological sample, each DNA probe is composed of 15 or more nucleotides derived from each of the bacteria 23S rRNA targets derived from each bacteria It hybridizes specifically with DNA and not with nucleic acid molecules derived from other bacteria. The present invention also relates to a DNA chip useful for diagnosing an infectious disease caused by bacteria causing at least one bacterial infectious disease by integrating at least one of the DNA probes onto a substrate.

Description

DNA probe for detection of bacterial infectious diseases causing bacteria derived from 23S rRNA gene {DNA Probe Derived from 23S rRNA Gene for Detection of Infectious Bacteria}

The present invention relates to probes useful for the detection of the causative agent of infectious diseases and the diagnosis of related diseases. Specifically, the present invention relates to a DNA probe derived from the 23S rRNA gene of bacteria causing bacterial infectious diseases and useful for detecting the bacteria, and a DNA chip in which the DNA probe is integrated on a substrate.

Bacterial infectious diseases are diseases that occur when bacteria start appearing and inhabiting blood, body fluids and tissues, and can lose life if the correct identification and proper treatment of bacteria are not performed. More recently, the misuse of antibiotics has resulted in a reduction in bacterial culture rates. As a result, a variety of causative strains have resulted from increased use of immunosuppressants following transplantation and increased drug administration due to chemotherapy. Diagnostic methods for diagnosing infectious diseases are increasingly difficult. In particular, the specific bacteria, including anaerobic bacteria cause a very fatal and serious disease in the human body, so it is important to diagnose them as soon as possible and identify the type of causative bacteria in the treatment. Due to this necessity, diagnostic methods for identifying infectious agents for a long time have been researched and developed. Significant advances have been made in detecting a large number of microorganisms over the past decade, but the diagnostic methods currently in use still require a lot of labor and are low in sensitivity and specificity.

Many of these problems could be solved by using nucleic acid probe techniques involving polymerase chain reaction (PCR). Except for viruses, all prokaryotic organisms contain rRNA genes that encode homologs of prokaryotic 5S, 16S and 23S rRNA molecules. In the case of eukaryotic cells, these rRNA molecules are 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA, which are substantially similar to prokaryotic molecules. Many probes have been published for the specific targeting of rRNA subsequences of specific organisms or groups of organisms in biological samples. By using such nucleic acid probes with PCR, many of these problems in conventional diagnostic methods can be solved. In nucleic acid probe technology, the selection of target genes to be amplified is very important. In general, 23S rRNA genes and Internal Transcribed Spacer Regions (hereinafter referred to as "ITS") are used, and these probe sequences are advantageously different under appropriate tightening conditions. It is known that the probability of cross-reaction with nucleic acids derived from bacterial species or infectious organisms is low (P. Wattiau et. Al., Appl. Microbiol. Biotechnol. , 56, 816-819, 2001; D, A. Stahlm et. Al. ., J. Bacteriol ., 172, 116-124, 1990; Boddinghaus. Et.al., J. Clin., Microbiol ., 28, 1751-1759, 1990; T. Rogall et al., J. Gen. Microbiol ., 136, 1915-1920, 1990; ..... T. Rogall, et al, Int J. System Bacteriol, 40, 323-330, 1990;.. K. Rantakokko-Jalava et al, J. Clin. , Mirobiol, 38 (1), 32-39, 2000;. Park et al, J. Clin, Mirobiol, 38 (11), 4080-4085, 2000;...... A. Schmalenberger et al, Appl Microbiol Biotechnol. , 67 (8), 3557-3563, 2001; WO98 / 55646; Jannes et al. US Patent No. 6025132; and US Patent No. 6,277,577 to Rossau et al.).

Therefore, there is still a need to develop a probe having a high specificity derived from the sequence of the 23S rRNA gene of each bacteria for many bacteria.

An object of the present invention is to develop a probe specific for each bacterium derived from the 23S rRNA gene of each of the following bacteria, and use the probe for the diagnosis of an infectious disease caused by each bacterium:

Actinomyces israelii ;

Staphylococcus epidrmidis ;

Burkholderia cepacia ;

Salmonella spp. ( Enteritidis );

Escherichia coli ;

Klebsiella pneumoniae ;

Proteus mirabilis ;

Streptococcus pneumoniae ;

Vibrio vulnificus ;

Shigella flexneri ;

Pseudomonas aeruginosa ;

Aeromonas hydrohila ;

Listeria monocytogenes ;

Enterococcus faecium ;

Staphylococcus aureus ;

Neisseria meningitidis ;

Serratia marcesens;

Citrobacter freundii ; And

Legionella pneumophila .

The present inventors have developed probes having base sequences that specifically hybridize with each of the above-mentioned 23S rRNA genes and do not cross-react with nucleic acids of other organisms, and have also fabricated DNA chips incorporating these probes through clinical trials. The object of the present invention was achieved by identifying each specificity of the probe.

In one aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from the fungus 23S rRNA, in which Actinomyces Israel is:

AACCTGGCTGGTGGC (Acii1, SEQ ID NO: 1).

In another aspect, the present invention provides a nucleic acid probe for the detection of bacteria of the actinomyces Israel comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting the bacterium of Extinomyses Israel comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention (a) composition comprising a nucleic acid probe for detecting the actinomyces Israeli bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying bacteria by actinomyces Israel in biological samples.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) appropriate hybridization and washing of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of the bacterium Extinomyse Israel comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 Contacting under conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying bacteria in said biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from the 23S rRNA of Staphylococcus epidermidis bacterium:

GATAGATAACAGGTG (SeM01, SEQ ID NO: 2); And

AGGGTTCACGCCCAG (SeM02, SEQ ID NO: 3).

In another aspect, the present invention provides a nucleic acid probe for detecting Staphylococcus epidermidis bacteria, comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 2 and 3.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Staphylococcus epidermidis bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 2 and 3.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Staphylococcus epidermidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 2 and 3; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, to provide a kit for detecting and identifying Staphylococcus epidermidis bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising at least one nucleic acid molecule comprising the base sequence of any one of SEQ ID NO: 2 and 3.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a composition comprising a nucleic acid probe for the detection of Staphylococcus epidermidis bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 2 and 3 in the polynucleic acid of steps (a) and (b) Contacting with appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Staphylococcus epidermidis bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from 23S rRNA of the bacterium Sephacia strain:

TTGTTAGCCGAACGC (Bur23, SEQ ID NO: 4);

GCCAGGAGGGTGAAG (Bur001, SEQ ID NO: 5); And

GGGTGTGGCGCGAGC (Bur01, SEQ ID NO: 6).

In another aspect, the present invention provides a nucleic acid probe for the detection of Bulcoderia sefacia bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 4 to 6.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for the detection of Bulcoderia cephacia bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 4 to 6.

In another aspect, the present invention (a) composition comprising a nucleic acid probe for the detection of Bulcoderia Sephacia bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 4 to 6; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Bulcoderia cephacia bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NOs: 4 to 6.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of step (a) and (b) comprises a composition comprising a nucleic acid probe for the detection of Bulcoderia sefacia bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 4 and 6 and Contacting under appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Bulcoderia cephacia bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from Salmonella enteritidis bacteria 23S rRNA:

GCCTGAATCAGCATG (Styp23, SEQ ID NO: 7).

In another aspect, the present invention provides a nucleic acid probe for detecting Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7.

In another aspect, the present invention (a) composition comprising a nucleic acid probe for the detection of Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Salmonella enteritidis bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of steps (a) and (b) under appropriate hybridization and washing conditions with a composition comprising a nucleic acid probe for detecting Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7 Contact; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Salmonella enteritidis bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising the following base sequence derived from 23S rRNA of Escherichia coli bacterium:

GAGCCTGAATCAGTG (Eco23, SEQ ID NO: 8);

GTTAGCGGTAACGCG (E coli 001, SEQ ID NO: 9); And

CTGAAGCGACAAATG (E coli 003, SEQ ID NO: 10).

In another aspect, the present invention provides a nucleic acid probe for detecting Escherichia coli, characterized in that it comprises a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 8 to 10.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Escherichia coli, including a nucleic acid molecule comprising any one of the nucleotide sequences of SEQ ID NOs: 8 to 10.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Escherichia coli bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 8 to 10; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Escherichia coli bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising a nucleic acid molecule comprising the base sequence of any one of SEQ ID NOs: 8 to 10.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of step (a) and (b) is suitable for a composition comprising a nucleic acid probe for detecting Escherichia coli, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 8-10. Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Escherichia coli bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising the following base sequence derived from the 23S rRNA of the Crevciella pneumoniae bacterium:

GTACACCAAAATGCA (Kpneu 23, SEQ ID NO: 11);

ACGCTGGTGTGTAGG (K.pneu001, SEQ ID NO: 12);

GCTGAGACCAGTCGA (K.pneu002, SEQ ID NO: 13); And

ACCTTCGGGTGTGAC (Kpneu01, SEQ ID NO: 14).

In another aspect, the present invention provides a nucleic acid probe for the detection of Crevciella pneumoniae bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NOs: 11-14.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Crebesciella pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Creebsiella pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying CrebSiela pneumoniae in a biological sample.

In another aspect, the present invention provides a DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a nucleic acid probe for detecting Crebsiella pneumoniae comprising the nucleic acid molecule of any one of SEQ ID NOs: 11 to 14 in the polynucleic acid of step (a) and (b) Contacting the composition under appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strains present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying the Crebeciella pneumoniae bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from 23S rRNA of Proteus mirabilis:

GTTACCAACAATCGT (Pm, SEQ ID NO: 15);

AAGGCTAGGTTGTCC (Pm001, SEQ ID NO: 16);

GGCGACGGTCGTCCC (Pm002, SEQ ID NO: 17);

GATGACGAACCACCA (Pm003, SEQ ID NO: 18);

TGAAGCAATTGATGC (Pm004, SEQ ID NO: 19);

TAAAGTCCCTCGCGG (Pm005, SEQ ID NO: 20); And

AGGCAGAGTGATTAG (Pm01, SEQ ID NO: 21).

In another aspect, the present invention provides a nucleic acid probe for detecting Proteus mirabilis bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 15 to 21.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Proteus mirabilis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 15 to 21.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Proteus mirabilis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 15 to 21; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Proteus mirabilis bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising at least one nucleic acid molecule comprising the base sequence of any one of SEQ ID NO: 15 to 21.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of step (a) and (b) is suitable for a composition comprising a nucleic acid probe for detecting proteus mirabilis comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOS: 15-21. Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Proteus mirabilis bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from the Streptococcus pneumoniae 23S rRNA:

TAGGACTGCAATGTG (StreppM, SEQ ID NO: 22).

In another aspect, the present invention provides a nucleic acid probe for detecting Streptococcus pneumoniae bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22.

In another aspect, the present invention (a) composition comprising a nucleic acid probe for the detection of Streptococcus pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Streptococcus pneumoniae bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 22.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) appropriate hybridization and washing conditions with a composition comprising a nucleic acid probe for detecting Streptococcus pneumoniae comprising the polynucleic acid of step (a) and (b) comprising the nucleic acid molecule of SEQ ID NO: 22; Contacting under; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Streptococcus pneumoniae bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from the 23S rRNA of Vibrio Bernique Picus:

AGTAACAGCCACTTG (VvulM, SEQ ID NO: 23); And

GTTGACGATGCATGT (Vvul02, SEQ ID NO: 24).

In another aspect, the present invention provides a nucleic acid probe for the detection of Vibrio Bernickus bacteria, comprising a nucleic acid molecule comprising any one of the nucleotide sequences of SEQ ID NOs: 23 and 24.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for the detection of Vibrio Bernickus bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 23 and 24.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Vibrio Bernique Picus bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 23 and 24; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Vibrio Bernoullican bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NOs: 23 and 24.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of step (a) and (b) is suitable for a composition comprising a nucleic acid probe for the detection of Vibrio Bernique Picus bacterium comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 23 and 24. Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Vibrio Bernoullican bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from Shigella flexneri sp. 23S rRNA:

AATCAAGGCCGAGGC (Sflexneri01, SEQ ID NO: 25); And

GCCGAGGCGTGATGA (Sflexneri04, SEQ ID NO: 26).

In another aspect, the present invention provides a nucleic acid probe for the detection of Shigella flexneri bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 25 and 26.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for the detection of Shigella flexneri bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 25 and 26.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Shigella flexneri bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 25 and 26; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Shigella flexneri bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising at least one nucleic acid molecule comprising the base sequence of any one of SEQ ID NO: 25 and 26.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of step (a) and (b) is suitable for a composition comprising a nucleic acid probe for the detection of Shigella flexneri bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 25 and 26. Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Shigella flexneri bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from Pseudomonas aeruginosa 23S rRNA:

GAAGTGCCGAGCATG (P.aeru001, SEQ ID NO: 27);

GTGTCACGTAAGTGA (P.aeru002, SEQ ID NO: 28); And

GGATCTTTGAAGTGA (Pa03, SEQ ID NO: 29).

In another aspect, the present invention provides a nucleic acid probe for detecting Pseudomonas aeruginosa, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 27 to 29.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Pseudomonas aeruginosa bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 27 to 29.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for detecting Pseudomonas aeruginosa bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 27 to 29; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Pseudomonas aeruginosa bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NO: 27 to 29.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a composition comprising a nucleic acid probe for detecting Pseudomonas aeruginosa comprising the polynucleic acid of step (a) and (b) comprising a nucleic acid molecule of any one of SEQ ID NOs: 27-29 Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Pseudomonas aeruginosa bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from the 23S rRNA of Eromonas hydrophila:

GGCGCCTCGGTAGGG (Ah, SEQ ID NO: 30).

In another aspect, the present invention provides a nucleic acid probe for detection of the E. monas hydrophila bacterium, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for the detection of E. hydronas bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Eromonas hydrophila bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying the E. hydromonas bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO: 30.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of step (a) and (b) under appropriate hybridization and washing conditions with a composition comprising a nucleic acid probe for the detection of Eromonas hydrophila comprising the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30; Contact; (d) detecting the hybrid formed in step (c); (e) identifying the strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying the E. hydronas bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from 23S rRNA of Listeria monocytogenes fungus:

GGGTGCAAGCCCGAG (LM, SEQ ID NO: 31).

In another aspect, the present invention provides a nucleic acid probe for detecting Listeria monocytogenes bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Listeria monocytogenes bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) the polynucleic acid of steps (a) and (b) under appropriate hybridization and washing conditions with a composition comprising a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31. Contact; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Listeria monocytogenes bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising the following base sequence derived from the 23S rRNA of Enterococcus pesium bacterium:

GTTCTTTCAGATAGT (Enfcium1 (Enfaeci23), SEQ ID NO: 32);

CTGAAGAGGAGTCAA (Enfcium2 (Enfaeci M), SEQ ID NO: 33);

TTACGATTGTGTGAA (E.faecium002, SEQ ID NO: 34); And

ATAGCACATTCGAGG (E.faecium003, SEQ ID NO: 35).

In still another aspect, the present invention provides a nucleic acid probe for detecting enterococcus fungus comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35.

In still another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting enterococcus fungus comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of enterococcus fungus bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 32 to 35; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying enterococcus bacterium in a biological sample.

In another aspect, the present invention provides a DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 32 to 35.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) appropriate hybridization of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of Enterococcus pesium bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Enterococcus pesium bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from 23S rRNA of Staphylococcus aureus:

AGGACGACATTAGAC (S.aureus 001, SEQ ID NO: 36);

CGAAGCGTGCGATTG (S. aureus 003, SEQ ID NO: 37);

GATTGCACGTCTAAG (S.aureus 004, SEQ ID NO: 38);

AATCCGGTACTCGTT (S.aureus 005, SEQ ID NO: 39); And

TCTTCGAGTCGTTGA (Saure03 (Saureus03), SEQ ID NO: 40).

In another aspect, the present invention provides a nucleic acid probe for detecting Staphylococcus aureus bacteria, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 36 to 40.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for the detection of Staphylococcus aureus bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 36 to 40.

In another aspect, the present invention (a) composition comprising a nucleic acid probe for the detection of Staphylococcus aureus bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 36 to 40; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Staphylococcus aureus bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NOs: 36 to 40.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a composition comprising a nucleic acid probe for detecting Staphylococcus aureus, wherein the polynucleic acid of steps (a) and (b) comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 36 to 40 Contacting with appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Staphylococcus aureus bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from 23S rRNA of Neisseria meningitidis bacteria:

GTTTACTGGCATGGT (Nm001, SEQ ID NO: 41);

AGATGTGAGAGCATC (Nm002, SEQ ID NO: 42); And

CCGGGTCTTCTTAAC (Nm02, SEQ ID NO: 43).

In still another aspect, the present invention provides a nucleic acid probe for detecting a strain of meningia meningitidis, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 41 to 43.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 41 to 43.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 41 to 43; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Neisseria meningitidis bacteria in biological samples.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NO: 41 to 43.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a composition comprising a nucleic acid probe for detection of Neisseria meningitidis bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 41 to 43 as the polynucleic acid of steps (a) and (b) Contacting with appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Neisseria meningitidis bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from the 23S rRNA of Seracia malcesense:

ATCGAACTCACGACC (Seme01, SEQ ID NO: 44); And

CTGTGTATTTTAGTG (Seme02, SEQ ID NO: 45).

In another aspect, the present invention provides a nucleic acid probe for the detection of Seracia malcesense bacteria, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 44 and 45.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for the detection of Seracia malcesense bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 44 and 45.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Ceracia malcesense bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 44 and 45; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Seracia malcesense bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NO: 44 and 45.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a composition comprising a nucleic acid probe for detecting the Seracia malcesense bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 44 and 45, wherein the polynucleic acid of step (a) and (b) Contacting under appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Seracia malcesense bacteria in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising the following base sequence derived from Citrobacter prundi or the 23S rRNA of the bacterium:

CGGAGGTTCCAGGTA (Citf001, SEQ ID NO: 46).

In another aspect, the present invention provides a nucleic acid probe for detecting the strain of Citrobacter prundi, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Citrobacter prundi bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for detecting the strain of Citrobacter prundi comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Citrobacter prundi bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) appropriate hybridization and washing conditions of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for detecting the strain of Citrobacter prundi comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46 Contacting under; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Citrobacter prundi in the biological sample.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising the following base sequence derived from the 23S rRNA of Legionella pneumophila:

TGGAGAGCATTTTAT (L.pneu011, SEQ ID NO: 47);

GTGATTTTGAGGTGA (L.pneu012, SEQ ID NO: 48); And

AGATGGTAAAGAAGA (L.pneu013, SEQ ID NO: 49).

In another aspect, the present invention provides a nucleic acid probe for detecting Legionella pneumophila, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 47 to 49.

In another aspect, the present invention provides a composition comprising a nucleic acid probe for detecting Legionella pneumophila, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 47 to 49.

In another aspect, the present invention (a) a composition comprising a nucleic acid probe for the detection of Legionella pneumophila bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 47 to 49; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying Legionella pneumophila in a biological sample.

In another aspect, the present invention provides a DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 47 to 49.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) appropriate hybridization of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for detecting Legionella pneumophila comprising the nucleic acid molecule comprising any of the nucleotide sequences of SEQ ID NOs: 47 to 49 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying Legionella pneumophila in the biological sample.

In still another aspect, the present invention provides a nucleic acid probe for detecting a bacterium Actinomyces Israel comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of any one of SEQ ID NOs: 2 and 3 Detecting at least one of the nucleic acid probes for detecting Staphylococcus epidermidis bacteria comprising a nucleic acid molecule comprising a nucleic acid molecule comprising any one of the nucleotide sequences of SEQ ID NOs: 4 to 6. At least one nucleic acid probe for use, a nucleic acid probe for detecting Salmonella enteritidis comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7, and a nucleic acid molecule comprising any one of SEQ ID NOs: 8 to 10 One or more of the nucleic acid probe for detecting Escherichia coli, comprising the nucleotide sequence of any one of SEQ ID NOs: 11 to 14. One or more nucleic acid probes for detecting Crebciella pneumoniae comprising a nucleic acid molecule, and a nucleic acid for detecting Proteus mirabilis comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 15 to 21 Probe, Nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22, Vibrio bunched blood containing a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 23 and 24 One or more of the nucleic acid probes for detecting the bacterium, one or more of the nucleic acid probes for the detection of Shigella flexneri bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 to 29 One of the nucleic acid probes for detecting Pseudomonas aeruginosa containing a nucleic acid molecule comprising a nucleotide sequence of any one of In addition, a nucleic acid probe for detection of E. monona hydrophilic bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30, a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31 At least one of nucleic acid probes for detecting enterococcus bacterium comprising a nucleic acid molecule having any one of SEQ ID NOs: 32 to 35, and a nucleic acid molecule having any one of SEQ ID NOs: 36 to 40 In one or more of the nucleic acid probe for the detection of Staphylococcus aureus, comprising the nucleic acid probe for detection of Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 41 to 43 At least one nucleic acid molecule comprising a nucleotide sequence of any one of SEQ ID NOs: 44 and 45 One or more of the nucleic acid probes for detecting the Ceracia malcesense bacteria, the nucleic acid probes for detecting the strain of Citrobacter prundi comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46 and the bases of SEQ ID NOs: 47 to 49 A composition comprising one or more probes selected from the group consisting of one or more of the nucleic acid probes for detecting Legionella pneumophila comprising a nucleic acid molecule comprising a sequence; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, thereby providing a kit for detecting and identifying one or more species of said bacteria in a biological sample.

In another aspect, the present invention provides a DNA chip comprising one or more nucleic acid molecules comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 49.

In another aspect, the present invention provides a method for the preparation of (a) isolating and / or concentrating polynucleic acid, optionally present in a biological sample, and (b) optionally amplifying the polynucleic acid with an appropriate forward and reverse primer pair; (c) a nucleic acid probe for detecting the actinomyces israel bacterium, wherein the polynucleic acid of steps (a) and (b) comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, wherein Vulcoderia comprising a nucleic acid molecule comprising any one of the nucleotide sequence of any one of SEQ ID NO: 4 to 6 of the nucleic acid probe for the detection of Staphylococcus epidermidis bacteria comprising a nucleic acid molecule comprising any one nucleotide sequence A nucleic acid probe for detecting Salmonella enteritidis, comprising one or more of the nucleic acid probes for detecting Sephacia bacteria, a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7, and the nucleotide sequence of any one of SEQ ID NOs: 8 to 10 One or more of the nucleic acid probes for detecting Escherichia coli, including the nucleic acid molecule, comprising the nucleotide sequence of any one of SEQ ID NOs: 11 to 14. For the detection of Proteus mirabilis, including one or more of the nucleic acid probes for detecting Crebciella pneumoniae containing one nucleic acid molecule, and the nucleic acid molecule comprising any one of the nucleotide sequences of SEQ ID NOS: 15-21. A nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid probe comprising the nucleotide sequence of SEQ ID NO: 22, and a vibrio bee comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 23 and 24 At least one of the nucleic acid probes for detecting the Lactucas, at least one of the nucleic acid probes for the Shigella flexneri bacterium comprising a nucleic acid molecule comprising any one of the nucleotide sequences of SEQ ID NOs: 25 and 26; Under nucleic acid probe for detecting Pseudomonas aeruginosa containing a nucleic acid molecule containing any one of the nucleotide sequences of 29 Herein, the nucleic acid probe for detecting E. monona hydrophilic bacteria comprising the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30, the nucleic acid probe for detecting Listeria monocytogenes bacteria comprising the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31. At least one of nucleic acid probes for detecting enterococcus bacterium comprising a nucleic acid molecule having any one of SEQ ID NOs: 32 to 35, and a nucleic acid molecule having any one of SEQ ID NOs: 36 to 40 In one or more of the nucleic acid probe for the detection of Staphylococcus aureus, comprising the nucleic acid probe for detection of Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 41 to 43 At least one nucleic acid molecule comprising a nucleotide sequence of any one of SEQ ID NOs: 44 and 45; One or more of the nucleic acid probes for detecting the Ceracia malcesense bacteria, including the nucleic acid probes for detecting the strain of Citrobacter prundai, comprising the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46; Contacting under appropriate hybridization and washing conditions a composition comprising at least one probe selected from the group consisting of at least one of a nucleic acid probe for detection of Legionella pneumophila comprising a nucleic acid molecule comprising a base sequence; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), thereby providing a method for detecting and identifying one or more species of said bacteria in said biological sample.

1 shows the location of the primers used to amplify polynucleic acid in a biological sample. (16S: 16S rRNA; ITS: Internal Transcribed Spacer Region; 23S: 23S rRNA; 1585F and 23BR, 37F and 38R and 238F and 37R: Base Sequences Present in 16S rRNA to 23S rRNA Common to Bacteria part).

FIG. 2 shows a design of a DNA chip in which a probe candidate group for 37 standard strains is integrated, including a probe for a bacterium Actinomyces Israel according to an embodiment of the present invention.

Figure 3 shows the results of hybridization of genomic DNA extracted from Actinomyces Israeli bacteria in the DNA chip of Figure 2 retrieved using Scanarray (Scanarray) 5000.

4 shows a design of a DNA chip in which probe candidate groups for 15 standard strains are integrated, including a probe for Staphylococcus epidermidis bacteria according to another embodiment of the present invention.

FIG. 5 shows hybridization results of genomic DNA extracted from Staphylococcus epidermidis bacteria in the DNA chip of FIG. 4 retrieved using ScanAray 5000.

FIG. 6 shows the results of hybridization of genomic DNA extracted from Vulcoderia cephacia bacteria in the DNA chip of FIG. 4 retrieved using ScanAray 5000.

FIG. 7 shows the results of hybridization of genomic DNA extracted from Salmonella enteritidis bacteria in the DNA chip of FIG. 4 retrieved using ScanAray 5000.

Figure 8 shows the results of hybridization of genomic DNA extracted from Escherichia coli bacteria in the DNA chip of Figure 4 retrieved using ScanAray 5000.

FIG. 9 shows a design of a DNA chip in which probe candidate groups for 17 standard strains are integrated, including probes for Crevciella pneumoniae bacteria of the present invention.

FIG. 10 shows the results of hybridization of genomic DNA extracted from Crebesciella pneumoniae bacteria in the DNA chip of FIG. 9 retrieved using ScanAray 5000.

FIG. 11 shows the results of hybridization of genomic DNA extracted from Proteus mirabilis bacteria in the DNA chip of FIG. 7 retrieved using ScanAray 5000.

FIG. 12 shows hybridization results of genomic DNA extracted from Streptococcus pneumoniae bacteria in the DNA chip of FIG. 7 retrieved using ScanAray 5000.

FIG. 13 shows the results of hybridization of genomic DNA extracted from Vibrio Bernickicus bacteria in the DNA chip of FIG. 9 retrieved using ScanAray 5000.

FIG. 14 shows hybridization results of genomic DNA extracted from Shigella flexneri bacteria in the DNA chip of FIG. 9 retrieved using ScanAray 5000.

FIG. 15 shows hybridization results of genomic DNA extracted from Pseudomonas aeruginosa bacteria in the DNA chip of FIG. 9 retrieved using ScanAray 5000.

FIG. 16 shows the results of hybridization of genomic DNA extracted from the Eromonas hydrophila bacteria in the DNA chip of FIG. 9 retrieved using ScanAray 5000.

Figure 17 shows the results of hybridization of genomic DNA extracted from the Listeria monocytogenes bacteria in the DNA chip of Figure 9 retrieved using ScanAray 5000.

FIG. 18 shows the results of hybridization of genomic DNA extracted from Enterococcus pesium bacteria in the DNA chip of FIG. 7 retrieved using ScanAray 5000.

Terms

A "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term “isolated” includes nucleic acid molecules isolated from chromosomes to which genomic DNA is naturally bound.

"Probe" refers to a single chain-specific oligonucleotide having a sequence that is sufficiently complementary to the target sequence to be detected and hybridizes.

"Target" means a nucleic acid molecule from a biological sample having a sequence complementary to any of the probes according to the invention. The target nucleic acid may be DNA (optionally obtained after amplification) or RNA.

A "biological sample" is a sample of a clinical sample (eg, juice, saliva, blood, urine, etc.), environmental sample, bacterial colony, contaminated or pure culture, purified nucleic acid, etc., for which a target sequence of interest is to be detected. Point to.

"Polynucleic acid" refers to whole nucleic acid or ribosomal RNA extracted from a biological sample, in particular 23S rRNA, or DNA obtained from reverse transcription of genomic DNA or 23S ribosomal RNA.

An "oligonucleotide" refers to a chain of nucleotide units characterized by information sequences of natural (or optionally modified) nucleic acids that can hybridize, such as natural nucleic acids, with nucleotide fragments that are complementary or substantially complementary under predetermined conditions. This chain may contain nucleotide units of a structure different from that of the natural nucleic acid and may contain from 15 to 100 monomers. Oligonucleotides are generally obtainable by natural nucleic acid molecules and / or gene recombination and / or chemical synthesis.

A “nucleotide” is the basic unit of nucleic acid consisting of phosphate groups, 5-carbosaccharides and nitrogen bases. The 5-saccharide found in RNA is ribose. The 5-saccharide in DNA is 2-deoxyribose. In the case of 5-nucleotides, the sugars contain hydroxyl groups (-OH) at 5-carbon-2. The term also includes analogs of these basic units, such as methoxy groups at the 2 position of ribose.

By "hybridization" is meant the process by which two nucleotide fragments with sufficiently complementary sequences can be joined under stable conditions and through a specific hydrogen bond to form a double strand.

"Primer" refers to an oligonucleotide that can act as an initiation point for synthesis under conditions predetermined for the initiation of enzymatic polymerization in amplification techniques such as PCR. Oligonucleotide primers may be natural or synthetically prepared, such as purified restriction enzyme digests.

"Tension" is used to describe the temperature and solvent composition during hybridization and subsequent processing.

“Complementarity” is provided by the base sequence of the Japanese chain of DNA or RNA capable of forming hybrid or double-stranded DNA: DNA, RNA: RNA or DNA: RNA via hydrogen bonding between individual stranded Watson-Crick base pairs. It indicates the nature of being. Adenine (A) is usually complementary to thymine (T) or uracil (U) and guranine (G) is usually complementary to cytosine (C).

“Fully complementary” or “substantially complementary” refers to a nucleic acid having a sufficient amount of combustive complementary nucleotides to form a stable hybrid for detection under high constriction hybridization conditions.

"Matched" refers to all pairs of two nucleotides that do not form normal Watson-Crick hydrogen bonds in a hybrid.

A "label" refers to any atom or molecule that provides a detectable (preferably quantifiable) signal and can be bound to a nucleic acid or protein. Labels can provide signals detectable by fluorescence, radioactivity, chromaticity, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.

A "hybrid" or "duplex" is a complex formed between two single-stranded nucleic acid sequences by Watson-Crick base pairs or nonstandard base pairs between complementary bases.

"Probe specificity" refers to the characteristics of a probe to describe its ability to distinguish between target and nontarget sequences.

Sympathy

Actinomyces Israeli, Staphylococcus epidermis, Vulcoderia sepacia, Salmonella enteritidis, Escherichia coli, Crevciella pneumoniae, Proteus mirabilis, Streptococcus pneumoniae, Vibrio Bournepickers, Shigella Flexneri, Pseudomonas aeruginosa, Eromonas hydrophila, Listeria monocytogenes, Enterococcus pesium, Staphylococcus aureus, Neisseria meningitidis, Cerasia malcesense, sheet In order to fabricate a probe for the detection of Lobacter Prundi or Legionella pneumophila, it is necessary to have specificity only for each bacterium. The specific probe candidate group sorts and compares the sequences of all possible bacteria in a line through multiple alignments to separately classify specific sequences present only in each of the bacteria to prepare and select a probe candidate group present therein. Probe candidates are determined within the 23S rRNA gene region in each bacterium. The specificity of the probe candidate group is first confirmed by comparing the similarities between the bacterial strains through BLAST search, and the strains are applied to the hybridization reaction, and the probes that react only with each bacteria are selected for identification in the candidate group. Test strains applied to the hybridization reaction include, but are not limited to:

Porphylomonas gingivalis

Peptostreptococcus prevotii ;

Cardiobacterium hominis ;

Chryseobacterium meningosepticum ;

Ochrobactrum anthropi ;

Actinomyces israelii ;

Comamonas acid ovorans ;

Bacteroides fragilis ;

Unaerobiospyroom Succinsi Prodersense

Anaerobiospirillum succiniproducens ;

Acinetobacter baumanii ;

Klebsiella oxytoca ;

Stomatococcus mucilaginosus (Rothia );

Shigella sonnei ;

Morganella morganii ;

Staphylococcus epidermidis ;

Streptococcus pyogenes ;

Burkholderia cepacia ;

Vibrio cholerae ;

Salmonella spp. (Enteritidis );

Haemophilus influenza;

Escherichia coli ;

Streptococcus viridans ;

Klebsiella pneumoniae ;

Proteus mirabilis ;

Strentrophomonas maltophila ;

Streptococcus pneumoniae ;

Vibrio vulnificus ;

Shigella flexneri ;

Pseudomonas aeruginosa ;

Aeromonas hydrophila ;

Listeria monocytogenes ;

Enterococcus faecium ;

Enterococcus faecalis ;

Staphylococcus aureus ;

Neisseria meningitidis ;

Serratia marcescens ;

Citrobacter freundii ;

Sutterella wadsworthii ;

Clostidium ramosum ;

Peptostreptococcus aerobius ;

Peptostreptococcus magnus ;

Fusobacterium necrophorum ;

Shigella dysenteriae ;

Proteus vulgaris (Proteus vulgaris);

Enterobacter aerogenes ;

Streptococcus mutans ;

Corynebacterium diphtheria ;

Kingella kingae ;

Bacteroides vulgatus ;

Bacteroides ovatus ;

Haemophilus actinomycetem comitans;

Bacteroides thetaiotaomicron ;

Clostridium diffcile ;

Enterobacter cloacae ;

Haemohilus aphrophilas ;

Neisseria gonorrhea ;

Branhamella catarrhalis ;

Eikenella corrodens ; And

Legionella pneumophila .

Hereinafter, the probes identified according to the present invention will be described by bacteria. The probe of the present invention comprises a mutant oligonucleotide having 15 oligonucleotides derived from each bacterium 23S rRNA gene or one or more nucleotides added to both ends thereof.

Actinomyces Israel Probes from Bacillus

Probes of the present invention suitable for the detection and identification of Actinomyces Israeli bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 1 below. These probes show specificity that Actinomyces Israel is derived from the fungal 23S rRNA and that Actinomyces Israel hybridizes only with the target DNA derived from the bacterium and not with nucleic acids of other organisms. Preferred probe is Acii1.

designation Sequence location SEQ ID NO: Specificity Acii1 AACCTGGCTGGTGGC 23S rRNA One Of the 59, 57 species except Rothia, porphylomonas did not cross-react. Acii2 GACACTTTTGTGTCA 23S rRNA 50 No signal when applying genomic gene of standard strain ATCC 12102

Standard strain ATCC 12102: Actinomyces Israel

Probes derived from Staphylococcus epidermidis bacteria

Probes of the invention suitable for the detection and identification of Staphylococcus epidermidis bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 2 below. These probes are derived from the 23S rRNA of Staphylococcus epidermidis bacteria and exhibit specificity that hybridizes only with target DNA derived from Staphylococcus epidermidis bacteria and not with nucleic acids of other organisms. Preferred probes are SeM01 and SeM02.

designation Base sequence location SEQ ID NO: Specificity SeM01 GATAGATAACAGGTG 23S rRNA 2 No cross-reaction in 16 of 21 clinical samples (76.19% efficiency). SeM02 AGGGTTCACGCCCAG 23S rRNA 3 No cross-reactions in 15 of 21 clinical samples (71.43% efficiency) Se1 TTCTCTCTTGAGTGG 23S rRNA 51 No signal when applying genomic gene of standard strain KCTC 1917 (ATCC 1228) Se2 CGTGCTGTTGGAGTG 23S rRNA 52 Cross-reaction occurs when applying genomic gene of standard strain KCTC 1917 (ATCC 1228)

Standard strain KCTC 1917 (ATCC 1228): Staphylococcus Epidermis

Probe Derived from Vulcoderia Sephacia

Probes of the present invention suitable for the detection and identification of Bulcoderia cephacia bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 3 below. These probes are derived from 23S rRNA of the bacterium Sephacia bacterium and exhibit specificity that hybridizes only with the target DNA derived from the bacterium Sephacia bacterium and not with nucleic acids of other organisms. Preferred probes are Bur23, Bur001 and Bur01.

designation Base sequence location SEQ ID NO: Specificity Bur23 TTGTTAGCCGAACGC 23S rRNA 4 No cross reaction with any target DNA derived from the 59 strains Bur001 GCCAGGAGGGTGAAG 23S rRNA 5 None of the 59 strains except Cardiobacterium, E.coli, K.pneumoniae, K.oxytoca, Burkholderia, Salmonella spp., P.mirabilis, E.facium, S.marcescens Bur01 GGGTGTGGCGCGAGC 23S rRNA 6 No cross reaction with any target DNA derived from the 59 strains

Standard strain ATCC 25416: Vulcoderia Sephacia

Probes derived from Salmonella enteritidis

Probes of the invention suitable for the detection and identification of Salmonella enteritidis bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 4 below. These probes are derived from Salmonella enteritidis bacteria 23S rRNA and exhibit specificity that hybridize only with target DNA derived from Salmonella enteritidis bacteria and not with nucleic acids of other organisms. Preferred probe is Styp23.

designation Base sequence location SEQ ID NO: Specificity Styp (Styp23) GCCTGAATCAGCATG 23S rRNA 7 No cross-reaction occurred in four of five clinical samples. (80% efficiency).

Standard strain KCCM 12021: Salmonella enteritidis

Probes derived from Escherichia coli

Probes of the invention suitable for the detection and identification of Escherichia coli bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 5 below. These probes are derived from 23S rRNA of Escherichia coli and exhibit specificity that hybridizes only with target DNA derived from Escherichia coli and not with nucleic acids of other organisms. Preferred probes are Eco1 (Eco23), E coli 001 and E coli 003.

designation Base sequence location SEQ ID NO: Specificity Eco1 (Eco23) GAGCCTGAATCAGTG 23S rRNA 8 Cross reaction did not occur in 58 of 59 species except S. flexneri. E coli 001 GTTAGCGGTAACGCG 23S rRNA 9 No cross-reactions in 4 of 8 clinical samples (50%), cross-reactions in S. maltophila E coli 003 CTGAAGCGACAAATG 23S rRNA 10 Cross-reaction did not occur in 10 of 25 clinical samples (40% efficiency). Eco2 (Ecoli) GTTAGCGGTAACGCG 23S rRNA 53 No signal when applying genomic gene of standard strain ATCC 25922 E coli 002 ATGCACATATTGTGA 23S rRNA 54 No signal when applying genomic gene of standard strain ATCC 25922

Standard strain ATCC 25922: Escherichia coli

Probes Derived from Crevciella pneumoniae

Probes of the present invention suitable for the detection and identification of Crevsciella pneumoniae strains include nucleic acid molecules having the nucleotide sequences set forth in Table 6 below. This probe is derived from the 23S rRNA of Crebciella pneumoniae and shows specificity that hybridizes only with the target DNA derived from Crebciella pneumoniae and not with nucleic acids of other organisms. Preferred probes are Kpneu 23, K.pneu001 and K.pneu002.

designation Sequence location Sequence Listing Specificity Kpneu 23 GTACACCAAAATGCA 23S rRNA 11 No cross-reaction occurred in 14 of 17 clinical samples (82.35% efficiency). K.pneu001 ACGCTGGTGTGTAGG 23S rRNA 12 None of the 59 species except C. hominis, A, israelii, Rothia, H. influenza, E. coli, P.mirabilis did not cross-react K.pneu002 GCTGAGACCAGTCGA 23S rRNA 13 No cross-reaction occurred in 15 of 17 clinical samples (88.24% efficiency). Kpneu01 ACCTTCGGGTGTGAC 23S rRNA 14 No one cross-reaction occurred when applying genomic gene 7 of the standard strain ATCC 700603 (14.3%)

Standard strain ATCC 700603: Crevciela New Monie

Probes Derived from Proteus Mirabilis

Probes of the invention suitable for the detection and identification of Proteus mirabilis bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 7 below. These probes are derived from the 23S rRNA of Proteus mirabilis and exhibit specificity that hybridizes only with the target DNA derived from the Proteus mirabilis and not with nucleic acids of other organisms. Preferred probes are Pm, Pm001, Pm002, Pm003, Pm004, Pm005 and Pm01.

designation Sequence location SEQ ID NO: Specificity Pm GTTACCAACAATCGT 23S rRNA 15 No cross-reaction occurred in 19 of 22 clinical samples (86.36% efficiency). Pm001 AAGGCTAGGTTGTCC 23S rRNA 16 6 cross-reactions (86%) with genomic gene 7 of the standard strain KCCM 11381 Pm002 GGCGACGGTCGTCCC 23S rRNA 17 No cross-reaction occurred in 19 of 22 clinical samples (86.36% efficiency) Pm003 GATGACGAACCACCA 23S rRNA 18 No cross-reaction occurred in 21 of 22 clinical samples (95.45% efficiency). Pm004 TGAAGCAATTGATGC 23S rRNA 19 No cross-reaction occurred in 19 of 22 clinical samples (86.36% efficiency) Pm005 TAAAGTCCCTCGCGG 23S rRNA 20 Application of the genomic gene 7 of the standard strain KCCM 11381 did not result in 4 cross reactions (57%). Pm01 AGGCAGAGTGATTAG 23S rRNA 21 No 7 cross reactions occurred when applying genomic gene 7 of the standard strain KCCM 11381 (57%)

KCCM 11381 (ATCC 21100): Proteus Mirabilis

Probes derived from Streptococcus pneumoniae

Probes of the invention suitable for the detection and identification of Streptococcus pneumoniae bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 8 below. This probe is derived from the Streptococcus pneumoniae 23S rRNA and shows specificity that hybridizes only with the target DNA derived from Streptococcus pneumoniae but not with other nucleic acids of other organisms. Preferred probe is StreppM.

designation Sequence location SEQ ID NO: Specificity StreppM TAGGACTGCAATGTG 23S rRNA 22 No cross-reaction occurred in nine of 10 clinical samples (90% efficiency). Strepp01 ATGTGGTACAGACAC 23S rRNA 55 No signal when applying genomic genes of standard strains KCCM 40410, 41568, 41569 and 41570 Strepp02 GGTTAAACGCTAGAA 23S rRNA 56 No signal when applying genomic genes of standard strains KCCM 40410, 41568, 41569 and 41570 Strepo03 CAGGATACTGCTAAG 23S rRNA 57 No signal when applying genomic genes of standard strains KCCM 40410, 41568, 41569 and 41570 Strepp04 GAGTAAACTCTTCGG 23S rRNA 58 No signal when applying genomic genes of standard strains KCCM 40410, 41568, 41569 and 41570

Standard strain KCCM 40410, 41568, 41569, 41570: Streptococcus pneumoniae

Probes Derived from Vibrio Bernique Pics

Probes of the invention suitable for the detection and identification of Vibrio Bernoullican bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 9 below. This probe is derived from the 23S rRNA of Vibrio Bulcancus and shows specificity that hybridizes only with target DNA derived from Vibrio Bulcancus and does not hybridize with nucleic acids of other organisms. Preferred probes are VvulM and Vvul02.

designation Sequence location SEQ ID NO: Specificity VvulM AGTAACAGCCACTTG 23S rRNA 23 No cross-reaction occurs once with genome 4 of the standard strain KCTC 2962 (ATCC 3385) (25% efficiency) Vvul02 GTTGACGATGCATGT 23S rRNA 24 No cross reaction with any target DNA derived from the 59 strains V.vul001 ATAGCTCAATGAAGC 23S rRNA 59 No signal when applying genomic gene of standard strain KCTC 2962 (ATCC 3385) V.vul002 GGCGCCATAGTCTCT 23S rRNA 60 No signal when applying genomic gene of standard strain KCTC 2962 (ATCC 3385)

Standard strain KCTC 2962 (ATCC 3385)

Probes derived from Shigella flexneri

Probes of the invention suitable for the detection and identification of Shigella flexneri bacteria include nucleic acid molecules having the base sequences set forth in Table 10 below. These probes are derived from Shigella flexneri strains and exhibit specificity that hybridizes only with target DNA derived from Shigella flexneri strains but not with nucleic acids of other organisms. Preferred probes are Sflexneri01 and Sflexneri04.

designation Sequence location SEQ ID NO: Specificity Sflexneri01 AATCAAGGCCGAGGC 23S rRNA 25 Of the 59 species, 58 species except Shigella sonnei did not cross-react. Sflexneri04 GCCGAGGCGTGATGA 23S rRNA 26 Cross reaction did not occur in 55 species except Rothia, Shigella sonnei, Pseudomonas aeruginosa, Serratia. Sflex GCTGATACGTAGGTG 23S rRNA 61 Cross-reaction occurs when applying genomic gene of standard strain ATCC 11836 S.flex001 TTCGGGTCCGTAATT 23S rRNA 62 No signal when applying genomic gene of standard strain ATCC 11836.

Standard strain ATCC 11836: Shigella flexneri

Probes derived from Pseudomonas aeruginosa

Probes of the invention suitable for the detection and identification of Pseudomonas aeruginosa bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 11 below. These probes are derived from Pseudomonas aeruginosa strains and exhibit specificity that hybridize only with target DNA derived from Pseudomonas aeruginosa strains but not with nucleic acids of other organisms. Preferred probes are P.aeru001, P.aeru002 and Pa03.

designation Sequence location SEQ ID NO: Specificity P.aeru001 GAAGTGCCGAGCATG 23S rRNA 27 No cross-reaction occurs with all 18 clinical samples (100% efficiency). P.aeru002 GTGTCACGTAAGTGA 23S rRNA 28 Cross reaction does not occur in 58 species except M.morganii among 59 species Pa03 GGATCTTTGAAGTGA 23S rRNA 29 No cross-reaction occurs with all 18 clinical samples (100% efficiency). P.aeru003 AGTCGTCTTTTAGAT 23S rRNA 63 No signal when genome gene of the standard strain KTCT 1636 is applied. P.aeru004 ACTCCGTAAGCTCTG 23S rRNA 64 No signal when genome gene of the standard strain KTCT 1636 is applied. Pa01 TAGGATAACCTAGGT 23S rRNA 65 No signal when genome gene of the standard strain KTCT 1636 is applied. Pa02 TAAGCTTCATTGATT 23S rRNA 66 No signal when genome gene of the standard strain KTCT 1636 is applied. Pa GTTTCCCGTGAAGGC 23S rRNA 67 Cross-reaction occurs when applying genomic gene of standard strain KTCT 1636

Standard strain KTCT 1636,1637, 1750, 2004, 2198, 2450, 2513, 2592, 2641, 2651, 2695, 2742: Pseudomonas aeruginosa

Probes Derived from Eromonas Hydrophila

Probes of the present invention suitable for the detection and identification of E. monas hydrophila bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 12 below. These probes are derived from the 23S rRNA of Eromonas hydrophila and exhibit specificity that hybridizes only with target DNA derived from Eromonas hydrophila and does not hybridize with nucleic acids of other organisms. Preferred probe is Ah.

designation Sequence location SEQ ID NO: Specificity Ah GGCGCCTCGGTAGGG 23S rRNA 30 No cross reaction with any target DNA derived from the 59 strains. Ae.hy 001 TAAGCCGTGAGCAGT 23S rRNA 68 No signal when applying genomic gene of standard strain KCCM 32586 (ATCC 11163). Ae.hy 002 CATCTTGGAAGTTAG 23S rRNA 69 No signal when applying genomic gene of standard strain KCCM 32586 (ATCC 11163). Ae.hy 003 TCAAACCAGGCACCG 23S rRNA 70 No signal when applying genomic gene of standard strain KCCM 32586 (ATCC 11163). Ae.hy 004 GATTCACGCTAAGCG 23S rRNA 71 No signal when applying genomic gene of standard strain KCCM 32586 (ATCC 11163). Ae.hy 005 ACGGTGCGGAAGCCA 23S rRNA 72 No signal when applying genomic gene of standard strain KCCM 32586 (ATCC 11163). Ah01 CACGAAAACAACCTT 23S rRNA 73 No signal when applying genomic gene of standard strain KCCM 32586 (ATCC 11163).

Standard strain KCCM 32586 (ATCC 11163): Eromonas hydrophila

Probes derived from Listeria monocytogenes bacteria

Probes of the invention suitable for the detection and identification of Listeria monocytogenes bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 13 below. These probes are derived from 23S rRNA of Listeria monocytogenes bacteria and exhibit specificity that hybridizes only to target DNA derived from Listeria monocytogenes bacteria and not to nucleic acids of other organisms. The probable probe is LM.

designation Sequence location SEQ ID NO: Specificity LM GGGTGCAAGCCCGAG 23S rRNA 31 No cross reaction with any target DNA derived from the 59 strains. Lm01 AGTATCCTTCGTGA 23S rRNA 74 No signal when applying genomic gene of standard strain ATCC 700603. Lm02 GTGAGGAAGGCAGAC 23S rRNA 75 No signal when applying genomic gene of standard strain ATCC 700603. Lm03 GGCTTTCCCTCCAGA 23S rRNA 76 No signal when applying genomic gene of standard strain ATCC 700603. Lm04 CCGCTTCTCACGAAG 23S rRNA 77 No signal when applying genomic gene of standard strain ATCC 700603.

Standard strain ATCC 700603: Listeria monocytogenes

Probes Derived from Enterococcus Phenium

Probes of the present invention suitable for the detection and identification of Enterococcus fungus bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 14 below. These probes are derived from the 23S rRNA of Enterococcus pesium and exhibit specificity that hybridizes only with target DNA derived from Enterococcus pesium and not with nucleic acids of other organisms. Preferred probes are Enfaeci23, EnfaeciM, E.faecium002 and E.faecium003.

designation Sequence location SEQ ID NO: Specificity Enfaeci23 GTTCTTTCAGATAGT 23S rRNA 32 Can be used as an Enterococcus genus specific probe EnfaeciM CTGAAGAGGAGTCAA 23S rRNA 33 Can be used as an Enterococcus genus specific probe E.faecium002 TTACGATTGTGTGAA 23S rRNA 34 No cross-reaction occurred in four of nine clinical samples (44.44% efficiency). E.faecium003 ATAGCACATTCGAGG 23S rRNA 35 No cross-reaction occurred in 6 of 9 clinical samples (66.67% efficiency). E.faecium001 GCTGATCATACGATC 23S rRNA 78 No signal when applying genomic gene of standard strain ATCC 19434 E.faecium004 CTTCTTTTCTTAAGG 23S rRNA 79 No signal when applying genomic gene of standard strain ATCC 19434

Standard strain ATCC 19434: Enterococcus pesium

Probes derived from Staphylococcus aureus

Probes of the present invention suitable for the detection and identification of Staphylococcus aureus bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 15 below. These probes are derived from the 23S rRNA of Staphylococcus aureus and exhibit specificity that hybridizes only with target DNA derived from Staphylococcus aureus and not with nucleic acids of other organisms. Preferred probes are S.aureus001, S.aureus003, S.aureus004, S.aureus005 and Saure03.

Ching Sequence location SEQ ID NO: Specificity S.aureus001 AGGACGACATTAGAC 23S rRNA 36 No cross-reaction occurred in three of eight clinical samples (38%) S.aureus003 CGAAGCGTGCGATTG 23S rRNA 37 Cross-reaction did not occur in 4 of 12 clinical samples (33.3%) S.aureus004 GATTGCACGTCTAAG 23S rRNA 38 No cross-reaction occurred in 13 of 21 clinical samples (61.90% efficiency). S.aureus005 AATCCGGTACTCGTT 23S rRNA 39 No cross-reaction occurred in 11 of 21 clinical samples (52.38% efficiency). Saure03 TCTTCGAGTCGTTGA 23S rRNA 40 No signal was found in 13 of 21 clinical specimens (61.90% efficiency). Saure01 AAGCAGTAAATGTGG 23S rRNA 80 No signal when applying genomic gene of standard strain KCTC 1621 Saure02 GAGAAGACATTGTGT 23S rRNA 81 No signal when applying genomic gene of standard strain KCTC 1621 Saureus01 ATATCAGAAGGCACA 23S rRNA 82 No signal when applying genomic gene of standard strain KCTC 1621 Saureus02 ACAAAGGACGACATT 23S rRNA 83 No signal when applying genomic gene of standard strain KCTC 1621 Saur GTTAACGCCCAGAAG 23S rRNA 84 No signal when applying genomic gene of standard strain KCTC 1621 S.aureus002 AAAATGTTGTCTCTC 23S rRNA 85 No signal when applying genomic gene of standard strain KCTC 1621

Standard strain KCTC 1621, 1916, 1928, 2199, 1927: Staphylococcus aureus

Probes derived from Neisseria meningitidis

Probes of the invention suitable for the detection and identification of Neisseria meningitidis bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 16 below. These probes are derived from 23S rRNAs of Neisseria meningitidis bacteria and exhibit specificity that hybridizes only with target DNA derived from Neisseria meningitis bacteria and does not hybridize with nucleic acids of other organisms. Preferred probes are Nm001, Nm002 and Nm02.

designation Sequence location SEQ ID NO: Specificity Nm001 GTTTACTGGCATGGT 23S rRNA 41 Of the 59 species, 58 species except S. seonnei do not cross-reaction Nm002 AGATGTGAGAGCATC 23S rRNA 42 No cross-reaction with any target DNA derived from the 59 strains Nm02 CCGGGTCTTCTTAAC 23S rRNA 43 Of the 59 species except for N.gonorrhoea 58 species do not cross-reaction Nm01 TAAAGCAATGATCCC 23S rRNA 86 No signal when applying genomic gene of standard strain ATCC 13100 Nm CCCTGGAGGGTCGCA 23S rRNA 87 No signal when applying genomic gene of standard strain ATCC 13100 Nm-1 TTTGAATTGAACCGT 23S rRNA 88 No signal when applying genomic gene of standard strain ATCC 13100

Standard strain ATCC 13100: Neisseria meningitidis

Probe derived from Seracia Malcesense

Probes of the invention suitable for the detection and identification of Seracia malcesense bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 17 below. These probes are derived from the 23S rRNA of Cerasia malcesense bacteria and exhibit specificity that hybridizes only to target DNA derived from Cerasia malcesense bacteria and not to nucleic acids of other organisms. Preferred probes are Seme01 and Seme02.

designation Sequence location SEQ ID NO: Specificity Seme01 ATCGAACTCACGACC 23S rRNA 44 Cross reaction did not occur in 58 of 59 species except P.mirabilis Seme02 CTGTGTATTTTAGTG 23S rRNA 45 Cross reaction did not occur in 58 of 59 species except P.mirabilis

Standard strain KCTC 1299, 2197, 2216, 2354, 2355, 2356, 2516, 2798, 2799, 2800,2801: Cerasia Malcesense

Probes Derived from Citrobacter Furundai

Probes of the invention suitable for the detection and identification of Citrobacter furundai bacteria include nucleic acid molecules having the nucleotide sequences set forth in Table 18 below. These probes are derived from the 23S rRNA of Citrobacter Purundi and exhibit specificity that hybridizes only with target DNA derived from Citrobacter Purundi and does not hybridize with nucleic acids of other organisms. Preferred probe is Citf001.

designation Sequence location SEQ ID NO: Specificity Citf001 CGGAGGTTCCAGGTA 23S rRNA 46 No cross-reaction occurred in two (7%) of 7 clinical trials Citf (Citf M) CCGGTACCTTTTTAA 23S rRNA 89 No signal when applying genomic gene of standard strain ATCC 8090 Citf002 AGTGGTTTACTCATG 23S rRNA 90 No signal when applying genomic gene of standard strain ATCC 8090 Citf 01 ACGTGATACATAGCG 23S rRNA 91 No signal when applying genomic gene of standard strain ATCC 8090 Citf02 GGAGGTTCCAGGTAA 23S rRNA 92 No signal when applying genomic gene of standard strain ATCC 8090 Citf03 GGTACCTTTTTAACG 23S rRNA 93 No signal when applying genomic gene of standard strain ATCC 8090

Standard strain (ATCC 8090), KCTC 2006, 2195, 2209, 2359, 2509, 2951: Citrobacter Prundai

Probes derived from Legionella pneumophila

Probes of the invention suitable for the detection and identification of Legionella pneumophila include nucleic acid molecules having the nucleotide sequences set forth in Table 19 below. These probes are derived from the 23S rRNA of Legionella pneumophila and exhibit specificity that hybridizes only with target DNA derived from Legionella pneumophila and does not hybridize with nucleic acids of other organisms. Preferred probes are L.pneu011, L.pneu012 and L.pneu013.

designation Sequence location SEQ ID NO: Specificity L.pneu011 TGGAGAGCATTTTAT 23S rRNA 47 No cross reaction for 22 strains including legionella pneumonia, legionella sainthelensi and legionella gormanii L.pneu012 GTGATTTTGAGGTGA 23S rRNA 48 Reaction with Legionella pneumonia, no cross reaction for 22 strains including legionella sainthelensi and legionella gormanii L.pneu013 AGATGGTAAAGAAGA 23S rRNA 49 No cross reaction for 22 strains including legionella pneumonia, legionella sainthelensi and legionella gormanii L.pneu001 CCTCAAGATGAGTTT 23S rRNA 94 No signal when applying genomic gene of standard strain L.pneu002 GAAGCCCGTTGAAGA 23S rRNA 95 No signal when applying genomic gene of standard strain L.pneu003 GCAGTAATGCGTGAA 23S rRNA 96 No signal when applying genomic gene of standard strain L.pneu004 TTGTCTTGACCATAT 23S rRNA 97 No signal when applying genomic gene of standard strain L.pneu005 ACCATATAATCTGAG 23S rRNA 98 No signal when applying genomic gene of standard strain L.pneu006 TGCCCACACAGTTTG 23S rRNA 99 No signal when applying genomic gene of standard strain L.pneu007 TGATTTTGAGGTGAT 23S rRNA 100 No signal when applying genomic gene of standard strain L.pneu008 CCACCATTTAATGAT 23S rRNA 101 No signal when applying genomic gene of standard strain L.pneu009 CCACCATTTAATGAT 23S rRNA 102 No signal when applying genomic gene of standard strain L.pneu010 AGCATTTTATTCTGG 23S rRNA 103 No signal when applying genomic gene of standard strain

Probe

The probes of the present invention can be used for all known hybridization techniques for testing for the presence of target nucleic acids in biological samples for diagnostic purposes, such as point deposition techniques on filters called “dot-blot” (MANIATIS et al., Molecular Cloning, Cold Spring Harbor, 1982), DNA delivery technology called Southern blot (SOUTHERN, EM, J. Mol. Biol., 98, 503 (1975)), and RNA delivery technology called "Northone blot".

The probes of the invention can also be used in sandwich hybridization systems that enhance the specificity of nucleic acid probe-based assays. The principles and uses of sandwich hybridization in nucleic acid probe-based assays are known (eg, DUNN and HASSEL, Cell, 12: 23-36, 1977; and RNAKI et al., Gene, 21: 77-85, 1983). The sandwich hybridization technique uses a capture probe and / or a detection probe, which can hybridize with two different regions of the target nucleic acid and at least one of them (generally the detection probe) is specific for the desired bacterium or group of fungi. May hybridize with the region of the target. Capture probes and detection probes should have at least partially different nucleotide sequences. While direct hybridization assays offer advantageous kinetics, sandwich hybridization is advantageous in that it has a high signal-to-noise ratio. In addition, sandwich hybridization can increase the specificity of nucleic acid probe-based assays. Incubation and subsequent washing steps, which constitute the main step of the sandwich hybridization process, are carried out at constant temperature, about 20-65 ° C., respectively. Nucleic acid hybrids are dependent on the number of hybridized bases (the temperature increases in proportion to the size of the hybrid) and also have dissociation temperatures that are dependent on the nature of the hybridized base and the nature of the adjacent base for each hybridized base. It is known. The hybridization temperature used in the sandwich hybridization technique should be selected below the half-dissociation temperature of the hybrid formed between the given probe and the target of the complementary sequence. This semi-dissociation temperature can be determined by simple routine experimentation.

In addition, the probes of the present invention can be used in a competitive hybridization protocol. In competitive hybridization, the target molecule competes with hybrid formation between a particular probe and its complement. The more targets are present, the less hybrid is formed between the probe and its complement. Positive signals indicating the presence of a specific target appear to decrease the hybridization response compared to a system without a target added. In certain embodiments, conveniently labeled specific oligonucleotide probes are hybridized with a target molecule. Subsequently, the mixture is placed in a microtiter dish well immobilized with oligonucleotides complementary to the specific probe, allowing the hybridization to continue. After washing, the hybrid between the complementary oligonucleotide and the probe is preferably quantified according to the label used.

The probe of the present invention can also be used for reversed hybridization (Proc. Natl. Acad. Sci. USA, 86: 6230-6234, 1989). In this case, first, the target sequence can be enzymatically amplified by performing PCR using 5 biotinylated primers. The product amplified in the second step is detected by hybridization with specific oligonucleotides immobilized on a solid support. Reverse hybridization can be performed without amplification. In this case, nucleic acids present in the biological sample must be labeled or modified specifically or nonspecifically by chemical or specific dye addition prior to hybridization.

Probe production

The probe of the present invention can be manufactured according to a conventional method, and there are two main methods. First, a single stranded probe is obtained. As the most representative example, a deprotection reaction may be performed by synthesizing by an automatic chemical synthesizer by DMT (dimethoxytrityl) off method, and then a probe having a desired number of bases may be synthesized. The probe thus prepared can be attached to a fluorescent substance such as fluorescein isothiocyanate (FITC) at one end of the strand to confirm the presence of a specific nucleic acid. Alternatively, single-stranded DNA is used as a template to make a probe having a base sequence complementary thereto. The primer is hybridized to the DNA of the template, and the Klenow enzyme and the fluorescent material are attached from the primer. Base (dNTP) will be used to synthesize the probe. Since the fluorescent material is attached to the inside of the DNA, it is possible to synthesize a probe having high sensitivity and specificity. Second, there is a way to get a double stranded probe. Genomic DNA or plasmid DNA can be cut with specific restriction enzymes to obtain probes containing genes or bases of desired portions. Random priming is a method of hybridizing six random hexamers and template DNA, and then synthesizing probes of various lengths to which fluorescent substances are attached. ³² P at the end of the T4 polynucleotide kinase (polynucleotide kinase) (transfer) can be synthesized by fluorescence-provided probe, DNA cleavage (DNase) I using a DNA strand (Dase) to make a cleavage section, Using this DNA as a template, a probe can be synthesized using DNA polymerase I and a base (dNTP) to which a fluorescent substance is attached. This double-stranded probe is used for hybridization after denaturation to make it single-stranded.

Probes of the invention can be advantageously labeled. Any conventional label can be used. Probes can be labeled using radioactive isotopes such as ³² P, ³⁵ S, ¹²⁵ I, ³ H and ¹⁴ C. Radiolabeling can be carried out according to conventional methods such as end labeling the 3 or 5 position with radiolabeled nucleotides, polynucleotide kinases, terminal transferases or ligases. Another method of radiolabeling is to chemically iodide the probe of the present invention, which results in the binding of several ¹²⁵ I atoms onto the probe. As such, when one of the probes of the present invention is labeled as radioactive, radiation is generally detected using autoradiography, liquid scintillation, gamma coefficients, or other conventional methods. In addition, the probes of the present invention may include residues with immune properties (eg, antigens or haptens), residues with specific affinity for some reagents (eg ligands), residues that provide a detectable enzymatic reaction (eg enzymes, Coenzymes, enzyme substrates or substrates participating in enzymatic reactions) or in combination with residues which provide physical properties of fluorescence, luminescence or absorption at certain wavelengths. Moreover, the antibody which specifically detects the hybrid formed by a probe and a target can be used. Non-radioactive labels can be provided when chemically synthesizing the probe of the present invention. Adenosine, guanosine, cytidine, thymidine and uracil residues readily bind to other chemical residues and allow detection of hybrids formed between the probe or probe and complementary DNA or RNA fragments.

DNA chip

The probe of the present invention is also used as a gene chip. A preferred embodiment of the present invention provides a DNA chip comprising the probe of the present invention. A DNA chip is a high density accumulation of fragments of several nucleotide sequences on a small substrate and is used to detect DNA of an unknown sample by hybridization between DNA immobilized on a substrate and an unknown DNA sample complementary thereto. The substrate on which the probe is to be fixed is made of an inorganic material such as glass or silicone, or a polymer material such as acrylic, polyethylene terephtalate (PET), polystylene, polycarbonate, polypropylene, and the like. Flat or multiple holes can be used. The probe is fixed either covalently to the substrate at either 5 'or 3'. The immobilization method is a conventional technique, for example using an electrostatic force or by attaching an amine group on the synthesized oligo to an aldehyde coated slide to induce bonding between the two, or on an amine group coated slide or L-lysine It can be done by integrating on this coated slide or on a slide with a nitrocellulose membrane coated. In one embodiment of the present invention, when the probe is synthesized, a base having an amino residue in the 3 'position is inserted to covalently bond to a glass plate coated with an aldehyde residue.

Fixing and arranging different probes on a substrate surface uses methods such as pin microarrays, inkjets, photolithography, electrical arrays, and the like. In one embodiment of the present invention (Yoon et al, J. Microbiol. Biotechnol., 10 (1), 21-) using a microarrayer prepared according to a known method while the probe is dissolved in a buffer solution . 26, 2000). The principle of microarrays is that the microfabricated pins carry DNA from the plate and transfer it to the same location specified by the computer on the substrate. Between the amine groups on the 3 'position of the probe and the aldehyde groups coated on the glass plate, under conditions where the humidity is maintained between 45% and 65%, preferably between 50% and 55%, for fixing the probe transferred by the microarray. In order to facilitate the reaction, the reaction is induced for at least 1 hour, and left for at least 6 hours to fix the DNA probe.

If necessary to detect cells derived from a living organism or the organism itself, these cells may be partially or completely lysed by chemical and / or physical methods to facilitate access to the RNA and / or DNA of those cells and Contact. The contact can be carried out on a suitable support such as nitrocellulose, cellulose or nylon filters in a liquid medium or solution. Such contact may occur under optimal conditions, suboptimal conditions or constraint conditions. These conditions reduce the temperature, reactant concentrations, the presence of substances that lower the base pair optimal temperature of the nucleic acid (eg formamide, dimethylsulfoxide and urea) and those that reduce the reaction volume and / or accelerate hybrid formation (eg dextran). Sulfates, polyethylene glycols or phenols).

Target

The nucleic acid is extracted from the sample to provide a nucleic acid substrate for use in detecting and identifying bacteria in the biological sample. Nucleic acids can be extracted from various samples using standard techniques or commercially available kits. For example, kits for separating RNA or DNA from tissue samples may be found in Qiagen, Inc. (California, USA) and Stratagene (California, USA). The QIAAMP Blood Kit allows for the separation of DNA from blood as well as bone marrow, body fluids or cell suspensions. The QIAAMP Tissue Kit allows for the separation of DNA from muscles and organs. As a preferred method of determining whether a biological sample contains rRNA or rDNA indicating the presence of each bacterium, nucleic acid is sonicated from bacterial cells, for example according to the method described in US Pat. No. 5,374,522 to Murphy et al. Can be released. Other known methods of crushing cells include enzyme use, osmotic shock, chemical treatment, and vortexing with glass beads. Other suitable methods for releasing nucleic acids from strains are described in US Pat. No. 5,837,452 to Clark et al. And US Pat. No. 5,364,763 to Kacian et al. Labeled probes may be added after or concurrent with release of the rRNA in the presence of a hybridization promoter and incubated for the time necessary to reach a significant hybridization reaction at the optimal hybridization temperature.

If the target nucleic acid is double stranded, it is preferable to perform denaturation before performing the detection process. Modification of the double-stranded nucleic acid can be carried out by known methods of chemical, physical or enzymatic modification, in particular by heating to an appropriate temperature, a temperature of at least 80 ℃.

In addition, the target DNA that hybridizes with the probe may be prepared by two kinds of methods. First, the method used in Southern blot or Northern blot, genomic DNA or plasmid DNA is cut with a suitable restriction enzyme and subjected to electrophoresis on an agarose gel to separate the DNA fragments by size. The second method is to amplify and use DNA of a desired portion using a PCR method. At this time, if you look at the PCR method used, asymmetric amplification method that can obtain a double strand and a single strand at the same time by asymmetrically adding the most popular PCR and primer asymmetrically to amplify the forward and reverse primer using the same amount (Asymmetric PCR), multiplex amplification method (multiplex PCR) that can be amplified at the same time by inserting several primers, ligase that amplifies by using a specific four primers and ligase (Ligase) and then fluorescence by enzyme immunoassay Ligase Chain Reaction (LCR) method, as well as hot start PCR, nest PCR, modified oligonucleotide primer PCR, reverse transcriptase PCR, semi-quantitative reverse transcriptase PCR, real time PCR, Rapid Amplification of cDNA Ends (RACE), Competitive PCR, Short Tandem Repeats (STR), Single Strand Polymorphization (Single) Strand Conformation Polymorphism (SSCP), in situ PCR, and DDRT-PCR (Differential Display Reverse Transcriptase PCR).

As a template for the amplification of certain PCR products, relatively homogeneous samples (e.g. blood, bacterial colonies or cerebrospinal fluid) are known to be more suitable than more complex samples (e.g. urine, saliva or feces) (Shibata in PCR: The Polymerase). Chain Reaction, Mullis et al., Eds., Birkhauser, Boston (1994), pp. 47-54). Samples containing relatively few copies of the target nucleic acid to be amplified, such as cerebrospinal fluid, can be added directly to the PCR. Blood samples should be specially handled during PCR due to the inhibitory properties of red blood cells, and red blood cells should be removed before using blood for PCR. Many methods are used for this purpose, for example passing through a CHELEX 100 column (BioRad) and the like and using QIAAMP blood kits. Extracting nucleic acids from saliva requires a preliminary purification process that kills or inhibits growth of other bacterial species other than the desired organism. This purification process is accomplished by treating the sample with N-acetyl-L-cysteine and NaOH. This purification process is necessary only when incubating before saliva samples are analyzed.

In a preferred embodiment of the present invention, a fragment gene is prepared by using DNA of an isolated sample as a template and performing asymmetric amplification (Asymmetric PCR). The fragment gene is obtained by one polymerase chain reaction by differentiating the ratio of forward primer and reverse primer 1: 5. The primers used here were part of the nucleotide sequence present in 16S rRNA to 23S rRNA in common to bacteria (Pirkko K. et al., Clin. Micorbiol ., 36 (8), 2205-2209, 1999):

Primer 1 (sense): TTGTACACACCGCCCGTC (SEQ ID NOs: 278, 1585F)

Primer 2 (antisense): F-TTTCGCCTTTCCCTCACGGTACT (SEQ ID NOs: 279, 23BR);

Primer 3 (sense): AGTACCGTGAGGGAAAGG (SEQ ID NOs: 280, 23BF)

Primer 4 (antisense): F-TGCTTCTAAGCCAACATCCT (SEQ ID NOs: 281, 37R); And

Primer 5 (Sense): AGGATGTTGGCTTAGAAGCA (SEQ ID NOs: 282, 37F)

Primer 6 (antisense): F-CCCGACAAGGAATTTCGCTACCTTA (SEQ ID NOs: 283, 38R).

Approximate location maps for these primers are shown in FIG. 1 and F indicates the FITC fluorescent material attached to the 5 'end. In order to identify DNA bound to the probe during PCR, amplification using a 5-FITC attached primer is performed to confirm the binding through fluorescence so that the infection can be identified and the type of infection. In addition, in order to obtain a portion that cannot be amplified by these primers, primers are designed by performing multi-alignment and BLAST by a bioinformatics method.

As a preferred embodiment of the present invention, the PCR reaction is carried out under the following conditions. The first denaturation is carried out at 94 ° C. for 7 minutes and the second denaturation at 94 ° C. for 1 minute. Annealing is performed at 52 ° C. for 1 minute and extension at 72 ° C. for 1 minute, and this is repeated 10 times. Then, the third denaturation was performed at 94 ° C. for 1 minute, annealing at 54 ° C. for 1 minute, and extension at 72 ° C. for 1 minute, and this was repeated 30 times. The final extension is then performed once at 5 ° C. for 5 minutes. The resulting product is confirmed by agarose gel electrophoresis.

Hybridization and Washing

Hybridization conditions are determined by stringency, i.e. the stringency of the operating conditions. The higher the tightening that hybridization takes place, the more specific the hybridization becomes. Constriction is a function of not only the base composition of the probe / target conjugate but also the degree of mismatch between the two nucleic acids. Constriction may also be a function of hybridization reaction variables such as the concentration and type of ions present in the hybridization solution, the nature and concentration of the denaturant and / or the hybridization temperature. The basis of the conditions under which the hybridization reaction should be carried out depends in particular on the probe used. All these data are well known and the appropriate conditions can be determined by routine experimentation in the individual case. Generally, depending on the length of the probe used, the temperature of the hybridization reaction is about 20 ° C. to 65 ° C., in particular 35 ° C. to 65 ° C., in a saline solution at a concentration of about 0.8 to 1 M.

Hybridization between the labeled oligonucleotide probe and the target nucleic acid can be increased using unlabeled helper oligonucleotides following the procedure described in US Pat. No. 5,030,557 to Hogan et al. Helper oligonucleotides bind to a region of the target nucleic acid other than the region bound by the assay probe. This binding takes on new secondary and tertiary structures in the target region of single-stranded nucleic acids, accelerating the binding speed of the probe.

Those skilled in the art will appreciate that factors affecting the thermal stability of the hybrid formed between the probe and the target may also affect probe specificity. Therefore, the melting point profile, including the melting point temperature of the probe and target hybrid, must be determined empirically for each probe and hybrid of the target. This determination can be made according to the method described in US Pat. No. 5,283,174 to Arnold et al. One method of determining the melting point temperature of the probe and target hybrids involves performing hybridization protection assays. According to this assay method a hybrid of the probe and the target is formed under conditions of excess target in a lithium succinate buffer solution containing lithium lauryl sulfate. A portion of the preformed hybrid is diluted in hybridization buffer and incubated for 5 minutes at various temperatures starting at or below the expected melting point temperature (typically 55 ° C.) and increasing 2 to 5 degrees. This solution is then diluted with weakly alkaline borate buffer and incubated at lower temperature (eg 50 ° C.) for 10 minutes. The acridinium esters linked to single chain probes are hydrolyzed under these conditions while the acridinium esters linked to hybridized probes are relatively protected. This procedure is called hybridization protection assay. The amount of chemiluminescence remaining is proportional to the amount of hybrid and is measured with a luminometer by adding hydrogen peroxide followed by alkali. This data is dipped as a percentage of the maximum signal (usually the lowest temperature) to the temperature. The melting point temperature is defined as the point at which 50% of the maximum signal remains. Alternatively, the melting point temperature for the hybrid of the probe and the target can be determined using a probe labeled with a radioisotope. In all cases, the melting point temperature for a given hybrid may vary by the concentration of salts, detergents and other solutes contained in the hybridization solution. All these factors affect the relative hybrid stability during heat denaturation (Molecular Cloning: A Laboratory Manual Sambrook et al., Eds. Cold Spring Harbor Lab Publ., 9.51 (1989)).

Hybridization conditions can be monitored taking into account several variables, for example, the hybridization temperature, the nature and concentration of the media components, the temperature at the time of washing of the formed hybrid, and the like. Hybridization and wash temperatures are limited to higher values depending on the base sequence, type and length of the probe and the maximum hybridization or wash temperature is about 30 ° C. to 60 ° C. At higher temperatures it competes with duplexing and dissociation or denaturation of the hybrid formed between the probe and the target. Hybridization media are for example about 3xSSC (1xSSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), about 25 mM phosphate buffer pH 7.1 and 20% deion formamide, 0.02% picol, 0.02% bovine serum albumin, 0.02 % Polyvinylpyrrolidone and about 0.1 mg / ml cut and denatured salmon sperm DNA. The wash medium contains, for example, about 3 × SSC, 25 mM phosphate buffer pH 7.1 and 20% deion formamide. Depending on the modification of the probe and medium, the hybridization or washing temperature should be changed according to known associations to achieve the desired specificity (BD HAMES and SJ HIGGINS, (eds.). Nucleic acid hybridzation.A practical approach, IRL Press, Oxford , UK, 1985). In this regard, since DNA: DNA hybrids are generally less stable than RNA: DNA or RNA: RNA hybrids, hybridization conditions must be appropriately adjusted to achieve specific detection, depending on the nature of the hybrid to be detected.

As an embodiment of the present invention, hybridization buffer (hybridization buffer: 6 × SSPE (0.15M NaCl, 5mM C6H5Na3O7, pH 7.0), 20% (v / v) formamide) is mixed with the PCR amplification gene and the probe is fixed on a glass plate After dispensing, the reaction was conducted at 30 ° C. for 6 hours to induce complementary binding. After the passage of time, wash for 3 minutes each in 3 x SSPE, 2 x SSPE, and 1 x SSPE.

Kit

The present invention provides a kit for detecting each of the above bacteria. The kit of the present invention comprises: (i) a probe specific for each of the above bacteria, that is, a nucleic acid molecule comprising any one of the base sequences 1 to 49; (ii) a forward and reverse primer pair, optionally used to amplify 23S rDNA genes and / or ITS or fragments thereof in a biological sample; (iii) a buffer or component that provides a buffering capacity to induce a hybridization reaction between these probes and only the DNA and / or RNA of each of the above bacteria; (iv) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (v) means for detecting the hybrid formed optionally as a result of the hybridization reaction that occurred previously.

Quantification of the hybrid can be accomplished by labeling the target with fluorescent or radioisotopes, as in the probes described above, according to conventional methods. Labeling may be on the primer itself or by using base residues labeled with fluorescent and radioisotopes for amplification and transcription.

Diagnostic purpose

The probe of the present invention can be diagnosed by infecting a disease caused by each of the bacteria by applying the kit, in particular, a DNA chip to specifically detect the bacteria, or by applying two or more probes to cause two or more infection diseases. By detecting the disease, several infectious diseases can be diagnosed simultaneously. Those skilled in the art will understand that the infectious disease of the bacterium according to the invention can be diagnosed alone or simultaneously using the probe of the invention.

In order to confirm the probe specificity of the present invention, the names and sequences of the probes integrated and used in the DNA chip in the following examples and other experiments not presented herein are as follows. Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention and the scope of the present invention is not limited by these examples.

Acti002: ATAGTGTTGCAAGGC derived from ITS of Acinetobacter baumanii (SEQ ID NO: 104)

Acti004: TGATGGAACTTGCTT (SEQ ID NO: 105) derived from 23S rRNA of Acinetobacter baumanii

Act3 (Acti I): ATACACAGTACTTCG derived from ITS of Acinetobacter baumanii (SEQ ID NO: 106)

Acti23S01: AGGGCACACATAATG derived from 23S rRNA of Acinetobacter baumanii (SEQ ID NO: 107)

Acti23S02: ACGCTGTTGTTGGTG derived from 23S rRNA of Acinetobacter baumanii (SEQ ID NO: 108)

Acii1: AACCTGGCTGGTGGC derived from 23S rRNA of Actinomyces israelii (SEQ ID NO: 1)

Acii2: GACACTTTTGTGTCA derived from 23S rRNA of Actinomyces israelii (SEQ ID NO: 50)

Acii3: GTTGGGTGGTTGCCT derived from ITS of Actinomyces israelii (SEQ ID NO: 109)

Acti1: GGGCACACATAATGA (SEQ ID NO: 110) derived from 23S rRNA of Acinetobacter baumanii

Acti2: CGGGGTACTCTATAC derived from 23S rRNA of Acinetobacter baumanii (SEQ ID NO: 111)

Acti3: ATACACAGTACTTCG derived from ITS of Acinetobacter baumanii (SEQ ID NO: 112)

Ah: GGCGCCTCGGTAGGG (SEQ ID NO: 30) derived from 23S rRNA of Aeromonas hydrophila

Anas23S03: CAGTTGGAAGCAGAG derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 275)

Anas1: AAAGTGCAGGGCACA derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 276)

Anas2: TGGATTGTGGTGAAA derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 277)

Anas3: TAGCGTTCTGCGAGG derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 113)

Anas4: TTAAAAGACTGGTAT derived from 23S rRNA of Anaerosirillum succiniciproducens

(SEQ ID NO: 114)

Anas001: TGACTCGTGCCCATG derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 115)

Anas002: TACCGGGGTTAAAAG derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 116)

Anas003: ATCAGTGATCTGAGA derived from 23S rRNA of Anaerospirillum succiniciproducens

(SEQ ID NO: 117)

Anas005: GTTCTTGATTCATTGC derived from ITS of Anaerospirillum succiniciproducens

(SEQ ID NO: 118)

Anas008: CAGCCCAAAAGTTGA from ITS of Anaerospirillum succiniciproducens (SEQ ID NO: 119)

Anas009: AAACTGCAGGGCACA (SEQ ID NO: 120) derived from ITS of Anaerospirillum succiniciproducens

Anas010: ATACTACCTGACGAC derived from ITS of Anaerospirillum succiniciproducens (SEQ ID NO: 121)

Bacf1 (Bf23): GGTAACCGAAGCGTA (SEQ ID NO: 122) derived from 23S rRNA of Bacteroides fragilis

Bacf2 (Bf): CTCGGAAAACGGTAA (SEQ ID NO: 123) derived from 23S rRNA of Bacteroides fragilis

Bacf3: GGTTCAGATCCTTTT from ITS of Bacteroides fragilis (SEQ ID NO: 124)

CACGGTGGATGCCCT derived from BaP1 01: Bacteria positive control (SEQ ID NO: 125)

GACCGATAGTGAACC (SEQ ID NO: 126) derived from BaP1 06: Bacteria positive control

AGAACCTGAAACCGT from Bap2 01: Bacteria positive control (SEQ ID NO: 127)

GTACCTTTTGTATAA derived from BaP2 03: Bacteria positive control (SEQ ID NO: 128)

B.ovatus01:Bacteroides ovatusof TAGAAGGAAGCATTC derived from 23S rRNA (SEQ ID NO: 129)

B.ovatus02:Bacteroides ovatusof CCAATGTTGTTACGG (SEQ ID NO: 130) derived from 23S rRNA

b.theta01: Bacteroides thetaiotaomicronof TTATTGTACTACTGG from ITS (SEQ ID NO: 131)

b.theta03: Bacteroides thetaiotaomicronof TTGTCGTTGCCAATA derived from ITS (SEQ ID NO: 132)

b.theta04: Bacteroides thetaiotaomicronof CAGTGTTGGAATGTT from ITS (SEQ ID NO: 133)

b.theta05: Bacteroides thetaiotaomicronof ACTATACTATAGTCA derived from 23S rRNA

(SEQ ID NO: 134)

b.thetaio01: Bacteroides thetaiotaomicronof TTATTGTACTACTGG from ITS (SEQ ID NO: 135)

b.thetaio02: Bacteroides thetaiotaomicronof ATCAGGTAGACAAGG from ITS (SEQ ID NO: 136)

b.thetaio03: Bacteroides thetaiotaomicronof TTGTCGTTGCCAATA derived from ITS (SEQ ID NO: 137)

b.thetaio04: Bacteroides thetaiotaomicronof CAGTGTTGGAATGTT from ITS (SEQ ID NO: 138)

b.thetaio05: Bacteroides thetaiotaomicronof ACTATACTATAGTCA from ITS (SEQ ID NO: 139)

B.theta01:Bacteroides thetaiotaomicronof TTATTGTACTACTGG from ITS (SEQ ID NO: 140)

B.theta02:Bacteroides thetaiotaomicronof ATCAGGTAGACAAGG from ITS (SEQ ID NO: 141)

B.theta03:Bacteroides thetaiotaomicronof TTGTCGTTGCCAATA derived from ITS (SEQ ID NO: 142)

B.theta04:Bacteroides thetaiotaomicronof CAGTGTTGGAATGTT from ITS (SEQ ID NO: 143)

B.theta05:Bacteroides thetaiotaomicronof ACTATACTATAGTCA derived from 23S rRNA

(SEQ ID NO: 144)

B.theta006:Bacteroides thetaiotaomicronof GCTAACGCAGGGAAC derived from 23S rRNA

(SEQ ID NO: 145)

Bur (Bur23): TTGTTAGCCGAACGC (SEQ ID NO: 4) derived from 23S rRNA of Burkholderia cepacia

Bur01: GGGTGTGGCGCGAGC (SEQ ID NO: 6) derived from 23S rRNA of Burkholderia cepacia

b.vulga01: CATCTTGAGATGTGC derived from 23S of Bacteroides vulgatus (SEQ ID NO: 146)

b.vulga02: AGTCGGGCGTGGATA (SEQ ID NO: 147) derived from 23S of Bacteroides vulgatus

b.vulga03: AGTCAGCGTCGAAGG (SEQ ID NO: 148) derived from 23S rRNA of Bacteroides vulgatus

b.vulga04: ACGCTAATCGGATCA derived from 23S of Bacteroides vulgatus (SEQ ID NO: 149)

b.vulga05:Bacteroides vulgatusof GACCGATAGAGCATG derived from 23S (SEQ ID NO: 150)

b.vulga06:Bacteroides vulgatusof TGACACACTGTAACT derived from 23S (SEQ ID NO: 151)

b.vulga07:Bacteroides vulgatusof CGAATGCGCATCAGT (SEQ ID NO: 152) derived from 23S rRNA

b.vulga08:Bacteroides vulgatusof ATTGTCATGAGCCAC (SEQ ID NO: 153) from 23S

Car3 (CarI): AAAGAGAGAACACAGCA (SEQ ID NO: 154) derived from 23S rRNA of Cardiobacterium hominis

Car2 (CarM): CCAGCACACTGTTGG (SEQ ID NO: 155) derived from 23S rRNA of Cardiobacterium hominis

Car001: TTGGCGACAACAGGC derived from ITS of Cardiobacterium hominis (SEQ ID NO: 156)

Car002: GCCCCGGGAAGCTGA (SEQ ID NO: 157) derived from ITS of Cardiobacterium hominis

Car003: TAGACTGCGGAAGCG (SEQ ID NO: 158) derived from ITS of Cardiobacterium hominis

Car004: AATTAAGTTGCGTAT from ITS of Cardiobacterium hominis (SEQ ID NO: 159)

Car006: AACCCTGGTGAAGGG (SEQ ID NO: 160) derived from 23S rRNA of Cardiobacterium hominis

Car007: ATATGAAGATATGTG (SEQ ID NO: 161) derived from 23S rRNA of Cardiobacterium hominis

Car008: TAGATTGACTTACGG (SEQ ID NO: 162) derived from 23S rRNA of Cardiobacterium hominis

Car009: GTAAAGTTTTACTAC (SEQ ID NO: 163) derived from 23S rRNA of Cardiobacterium hominis

C.diffc001:Clostridium diffcileof Derived from 23S rRNA GCATATATATTTAGT (SEQ ID NO: 164)

C.diph001:Corynebacterium diphtheriaDerived from ITS of ACCACGCAGCAGTTT (SEQ ID NO: 165)

C.diph002: CGAGTCGGTAGGGTA derived from ITS of Corynebacterium diphtheria (SEQ ID NO: 166)

C.diph003: ACCATCTTCCCAAGG (SEQ ID NO: 167) derived from 23S rRNA of Corynebacterium diphtheria

C.dipht01: TAACTAGATAAGAAA derived from ITS of Corynebacterium diphtheria (SEQ ID NO: 168)

Chr003: TAACCCCTTAGATTA derived from ITS of Chryseobacterium meningosepticum (SEQ ID NO: 169)

Chr004: TCAAACCTCAAACTA (SEQ ID NO: 170) derived from ITS of Chryseobacterium meningosepticum

Chr005: AAGAAATCGAAGAGA (SEQ ID NO: 171) derived from the ITS of Chryseobacterium meningosepticum

Chr23S04: GGCATATTTAGATGA derived from 23S rRNA of Chryseobacterium meningosepticum

(SEQ ID NO: 172)

Chr23S05: ATCGTGAGGTTACGA derived from 23S rRNA of Chryseobacterium meningosepticum

(SEQ ID NO: 173)

ATTTAGATGATAAAT derived from 23S rRNA of Chry1: Chryseobacterium meningosepticum

(SEQ ID NO: 174)

Chry2: TAATCTTACTAGCGA derived from 23S rRNA of Chryseobacterium meningosepticum

(SEQ ID NO: 175)

Chry3: TCCTTGAGTGCAGAG derived from ITS of Chryseobacterium meningosepticum (SEQ ID NO: 176)

Coma1: AAAACCGACTGGTGG derived from 23S rRNA of Comamonas acidovorans (SEQ ID NO: 177)

Coma2 (Com7): TGAGCTAGAGAAAAG derived from 23S rRNA of Comamonas acidovorans (SEQ ID NO: 178)

Coma3 (ComM): ATCCGCCGGGCTTAG (SEQ ID NO: 179) derived from 23S rRNA of Comamonas acidovorans

Coma4: ACGCGCGAGGTGAGA derived from ITS of Comamonas acidovorans (SEQ ID NO: 180)

C.ramo 001:Clostidium ramosumof Derived from ITS TAGTTGATGATAGTA (SEQ ID NO: 181)

C.ramo 002:Clostidium ramosumof GCTTATCTGTGGATG from ITS (SEQ ID NO: 182)

C.ramo 003:Clostidium ramosumof GGAATCCCTCCTTGT (SEQ ID NO: 183) derived from 23S rRNA

c.ramo 004: CCCGGGAAGGGGAGT derived from 23S rRNA of Clostidium ramosum (SEQ ID NO: 184)

c.ramo 05: GTATTGGAGTTGCTA derived from 23S rRNA of Clostidium ramosum (SEQ ID NO: 185)

Citf: CCGGTACCTTTTTAA derived from 23S rRNA of Citrobacter freundii (SEQ ID NO: 89)

Citf02: GGAGGTTCCAGGTAA derived from 23S rRNA of Citrobacter freundii (SEQ ID NO: 92)

Citf03: GGTACCTTTTTAACG derived from 23S rRNA of Citrobacter freundii (SEQ ID NO: 93)

Com004: TAGGGCGTCCAGTCG derived from 23S rRNA of Comamonas acidovorans (SEQ ID NO: 186)

Com005: CGCAGAGTACAGCTT (SEQ ID NO: 187) derived from 23S rRNA of Comamonas acidovorans

Coma3ComM: ATCCGCCGGGCTTAG (SEQ ID NO: 188) derived from 23S rRNA of Comamonas acidovorans

e.aero01: TTCCGACGGTACAGG derived from 23S rRNA of Enterobacter aerogenes (SEQ ID NO: 189)

e.aero02: GAGCGGGGTAGTTGA (SEQ ID NO: 190) derived from 23S rRNA of Enterobacter aerogenes

e.aero03: GTATCAGTAAGTGCG derived from 23S rRNA of Enterobacter aerogenes (SEQ ID NO: 191)

e.aero04: TTATCCAGGCAAATC derived from 23S rRNA of Enterobacter aerogenes (SEQ ID NO: 192)

Eco1 (Eco23): GAGCCTGAATCAGTG (SEQ ID NO: 8) derived from 23S rRNA of Escherichia coli

Eco2 (Ecoli): GTTAGCGGTAACGCG derived from 23S rRNA of Escherichia coli (SEQ ID NO: 53)

E coli 003: CTGAAGCGACAAATG derived from 23S rRNA of Escherichia coli (SEQ ID NO: 10)

E.faecium002: TTACGATTGTGTGAA derived from 23S rRNA of Enterococcus faecium (SEQ ID NO: 34)

E.faecium003: ATAGCACATTCGAGG (SEQ ID NO: 35) derived from 23S rRNA of Enterococcus faecium

Efalis: GCTTCTTTTCTTAAG derived from 23S rRNA of Enterococcus faecalis (SEQ ID NO: 193)

Efclum1 (Enfaeci23): GTTCTTTCAGATAGT derived from 23S rRNA of Enterococcus faecium

(SEQ ID NO 32)

Efclum2 (EnfaeciM): CTGAAGAGGAGTCAA derived from 23S rRNA of Enterococcus faecium

(SEQ ID NO: 33)

Ente1: Enterobacter spp. GTACACGAAAATGCA derived from 23S rRNA of (SEQ ID NO: 194)

Ente2 (Efacalis23): TCTGTTAGTATAGTT derived from 23S rRNA of Enterococcus faecalis

(SEQ ID NO: 195)

Ente3: GCTTCTTTTCTTAAG derived from 23S rRNA of Enterococcus faecalis (SEQ ID NO: 196)

fnecro 01: TTTCGCAGACGTAAG derived from 23S rRNA of Fusobacterium necrophorum (SEQ ID NO: 197)

fnecro 02: GTTTTCTTGCGCTGT derived from 23S rRNA of Fusobacterium necrophorum (SEQ ID NO: 198)

fnecro 03: CCGTATTCATGTCAA (SEQ ID NO: 199) derived from 23S rRNA of Fusobacterium necrophorum

fnecro 04: GGGGTAGAGCCTAAA (SEQ ID NO: 200) derived from 23S rRNA of Fusobacterium necrophorum

fnecro 06: CAGACGTAAGCAAAG (SEQ ID NO: 201) derived from 23S rRNA of Fusobacterium necrophorum

fnecro 07: CCTGTATTGGTAGTT (SEQ ID NO: 202) derived from 23S rRNA of Fusobacterium necrophorum

h.actino01:Haemophilus actinomycetmcomiof AAGTCTGGGTCGTGG derived from 23S rRNA

(SEQ ID NO: 203)

h.actino02:Haemophilus actinomycetmcomiof AATCCTAGGGTAATG derived from 23S rRNA

(SEQ ID NO: 204)

h.actino03:Haemophilus actinomycetmcomiof AACCGGGTAGAACTG derived from 23S rRNA

(SEQ ID NO: 205)

Hin (Hin23): GGAGAATGTGTTGGG (SEQ ID NO: 206) derived from 23S rRNA of Haemophilus influenza

k.king01: TGATTCAATGCGATG derived from 23S rRNA of Kingella kingae (SEQ ID NO: 207)

k.king02: GGTTAGCAAACTGTT derived from 23S rRNA of Kingella kingae (SEQ ID NO: 208)

k.king03: CCAGTAGGTGGAAAG derived from 23S rRNA of Kingella kingae (SEQ ID NO: 209)

k.king04: AACACCGAGACGTGA (SEQ ID NO: 210) derived from 23S rRNA of Kingella kingae

k.king05: TATAATTAAACGCAT derived from ITS of Kingella kingae (SEQ ID NO: 211)

k.king06: AATGTTGTCGATTTG from ITS of Kingella kingae (SEQ ID NO: 212)

k.king07: AGGCAACAAATCGAA from ITS of Kingella kingae (SEQ ID NO: 213)

k.king08: TATCAACTAATCTTG from ITS of Kingella kingae (SEQ ID NO: 214)

k.king09: TATTCAATGCGATGG (SEQ ID NO: 215) derived from 23S rRNA of Kingella kingae

K.pneu002: GCTGAGACCAGTCGA (SEQ ID NO: 13) derived from 23S rRNA of Klebsiella pneumoniae

Ko001: GAACGTTACTAACGC derived from 23S rRNA of Klebsiella oxytoca (SEQ ID NO: 216)

Koxy1: CCGGAACGTTACTAA derived from 23S rRNA of Klebswiella oxytoca (SEQ ID NO: 217)

Koxy2: CGCGACACGACGATG (SEQ ID NO: 218) derived from ITS of Klebswiella oxytoca

Koxy3: AAGAGCGCCAGCTCA derived from 23S rRNA of Klebswiella oxytoca (SEQ ID NO: 219)

Kpneu (Kpneu23): GTACACCAAAATGCA derived from 23S rRNA of Klebsiella pneumoniae (SEQ ID NO: 11)

Kpneu01: ACCTTCGGGTGTGAC derived from 23S rRNA of klebsiella pneumoniae (SEQ ID NO: 14)

LM: GGGTGCAAGCCCGAG derived from 23S rRNA of Listeria monocytogenes (SEQ ID NO: 31)

Nm1 (Nm): CCCTGGAGGGTCGCA derived from 23S rRNA of Neisseria meningitidis (SEQ ID NO: 87)

Nm2 (Nm-1): TTTGAATTGAACCGT derived from 23S rRNA of Neisseria meningitidis (SEQ ID NO: 88)

Nm002: AGATGTGAGAGCATC derived from 23S rRNA of Neisseria meningitidis (SEQ ID NO: 42)

Mor1: TAGGGTGCAAGCCCA derived from 23S rRNA of Morganella morganii (SEQ ID NO: 220)

AGAACACTCACAGA (SEQ ID NO: 221) derived from ITS of Mor2: Morganella morganii

Ochr1: CGGCGCGTGAGCGAG (SEQ ID NO: 222) derived from 23S rRNA of Ochrobacterium anthropi

Ochr2: GAACACCTGTTTCC (SEQ ID NO: 223) derived from 23S rRNA of Ochrobacterium anthropi

Ochr3: GATCCGACGATTTCC derived from ITS of Ochrobacterium anthropi (SEQ ID NO: 224)

Ochr04: GGACCAGGCCAGTGG derived from 23S rRNA of Ochrobacterium anthropi (SEQ ID NO: 225)

Ochr05: GACCAGGCCAGTGGC derived from 23S rRNA of Ochrobacterium anthropi (SEQ ID NO: 226)

Ochr004: GTTGATTGACACTTG (SEQ ID NO: 227) derived from ITS of Ochrobacterium anthropi

Ochr005: TACCGCTCACGAGCC derived from ITS of Ochrobacterium anthropi (SEQ ID NO: 228)

Ochr007: GGGTCCGGAGGTTCA derived from ITS of Ochrobacterium anthropi (SEQ ID NO: 229)

Pa: GTTTCCCGTGAAGGC derived from 23S rRNA of Pseudomonas aeruginosa (SEQ ID NO: 67)

Pa03: GGATCTTTGAAGTGA derived from 23S rRNA of Pseudomonas aeruginosa (SEQ ID NO: 29)

P.anae001:Peptostreptococcus anaerobiusof TGCATTACTAAGTGA from 23S rRNA

(SEQ ID NO: 230)

P.anae002:Peptostreptococcus anaerobiusof GTAAGGTCGATACCC derived from 23S rRNA

(SEQ ID NO: 231)

P.anae003:Peptostreptococcus anaerobiusof AGGAGGAAGAGAAAG derived from 23S rRNA

(SEQ ID NO: 232)

Pep1: ACTAAATAAACCAGG derived from 23S rRNA of Peptostreptococcus prevotii

(SEQ ID NO: 233)

Pep2: ATAATCAACATCTAC derived from 23S rRNA of Peptostreptococcus prevotii

(SEQ ID NO: 234)

Pep3: TCTGTATAATAGTTC from ITS of Peptostreptococcus prevotii (SEQ ID NO: 235)

Pep002: ACTAGGGAGAGCTCA Derived from 23S rRNA of Peptostreptococcus prevotii (SEQ ID NO: 236)

Pep003: GCTTAGTAAAGCAAG (SEQ ID NO: 237) derived from 23S rRNA of Peptostreptococcus prevotii

Pep23S02: GTCGAATCATCTGGG (SEQ ID NO: 238) derived from 23S rRNA of Peptostreptococcus prevotii

Pep23S03: TAAAACGTATCGGAT (SEQ ID NO: 239) derived from 23S rRNA of Peptostreptococcus prevotii

Pm: GTTACCAACAATCGT derived from 23S rRNA of Proteus mirabilis (SEQ ID NO: 15)

Pm01: AGGCAGAGTGATTAG (SEQ ID NO: 21) derived from 23S rRNA of Proteus mirabilis

Pm002: GGCGACGGTCGTCCC derived from 23S rRNA of Proteus mirabilis (SEQ ID NO: 17)

Pm003: GATGACGAACCACCA derived from 23S rRNA of Proteus mirabilis (SEQ ID NO: 18)

Pm004: TGAAGCAATTGATGC derived from 23S rRNA of Proteus mirabilis (SEQ ID NO: 19)

Por1: GTTGGATGTTATCAT derived from 23S rRNA of Porphylomonas gingivalis (SEQ ID NO: 240)

Por2: CGGGCAGCTAAAACC (SEQ ID NO: 241) derived from 23S rRNA of Porphylomonas gingivalis

Por3: TGTTTGTGCGACGTG derived from ITS of Porphylomonas gingivalis (SEQ ID NO: 242)

Por23S08: CTGAGCTGTCGTGCA (SEQ ID NO: 243) derived from 23S rRNA of Porphylomonas gingivalis

P.vulga01: ATACGTGTTATGTGC derived from ITS of Proteus vulgaris (SEQ ID NO: 244)

P.vulga02: CTCACACAGACTTGT derived from ITS of Proteus vulgaris (SEQ ID NO: 245)

P.vulga03: ATATCCAATGGATAT (SEQ ID NO: 246) derived from 23S rRNA of Proteus vulgaris

P.vulga04: AGAGGAGGCTTAGTG (SEQ ID NO: 247) derived from 23S rRNA of Proteus vulgaris

Rot1 (Rot23): TGGTAGGCGAACTGG derived from 23S rRNA of Stomatococcus mucilaginosus (Rothia)

(SEQ ID NO: 248)

Rot2 (RotM): GCCGGTTGTGTGTCT derived from 23S rRNA of Stomatococcus mucilaginosus (Rothia)

(SEQ ID NO: 249)

Rot3 (RotI): TATATAGTGGACGCG derived from ITS of Stomatococcus mucilaginosus (Rothia)

(SEQ ID NO: 250)

Rot4 (Rot7): TAAGAGCGTGTGGAG derived from 23S rRNA of Stomatococcus mucilaginosus (Rothia)

(SEQ ID NO: 251)

Saur: GTTAACGCCCAGAAG derived from 23S rRNA of Staphylococcus aureus (SEQ ID NO: 84)

S.aureus004: GATTGCACGTCTAAG (SEQ ID NO: 38) derived from 23S rRNA of Staphylococcus aureus

S.aureus 005: AATCCGGTACTCGTT derived from 23S rRNA of Staphylococcus aureus (SEQ ID NO: 39)

Saure03 (Saureus03): TCTTCGAGTCGTTGA derived from 23S rRNA of Staphylococcus aureus

(SEQ ID NO 40)

S.dysen01: CAGCCTGTTAAGTCT derived from ITS of Shigella dysenteriae (SEQ ID NO: 252)

S.dysen02: CCTCTTTAATGGGGT (SEQ ID NO: 253) derived from 23S rRNA of Shigella dysenteriae

Se1: TTCTCTCTTGAGTGG derived from 23S rRNA of Staphylococcus epidermidis (SEQ ID NO: 51)

Se2: CGTGCTGTTGGAGTG (SEQ ID NO: 52) derived from 23S rRNA of Staphylococcus epidermidis

Se3: GCTATTTATTTTGAA derived from ITS of Staphylococcus epidermidis (SEQ ID NO: 254)

SeM01: GATAGATAACAGGTG (SEQ ID NO: 2) derived from 23S rRNA of Staphylococcus epidermidis

SeM02: AGGGTTCACGCCCAG (SEQ ID NO: 3) derived from 23S rRNA of Staphylococcus epidermidis

Sflex: GCTGATACGTAGGTG derived from 23S rRNA of Shigella flexneri (SEQ ID NO: 61)

Sflexneri01: AATCAAGGCCGAGGC (SEQ ID NO: 25) derived from 23S rRNA of Shigella flexineri

Sflexneri04: GCCGAGGCGTGATGA (SEQ ID NO: 26) derived from 23S rRNA of Shigella flexineri

Sm: TTTGGATCTTTGGCA derived from 23S rRNA of Strentrophomonas maltophila (SEQ ID NO: 255)

Sm02: AAGCTGGATTGGTTC (SEQ ID NO: 256) derived from 23S rRNA of Strentrophomonas malthophilia

S.mutans01:Streptococcus mutansof GAAAAACGAAGGGTA (SEQ ID NO: 257) derived from 23S rRNA

S.mutans02:Streptococcus mutansof ATGACTACGTGGTCG derived from 23S rRNA (SEQ ID NO: 258)

S.mutans03:Streptococcus mutansof GTAATGCAAGATATC derived from 23S rRNA (SEQ ID NO: 259)

S.mutan001: TAGGTATTCTCTCCT derived from 23S rRNA of Streptococcus mutans (SEQ ID NO: 260)

S.mutan002: TAGCACAGGAATACT (SEQ ID NO: 261) derived from 23S rRNA of Streptococcus mutans

S.mutan003: AAGGGGTACAGATCC derived from 23S rRNA of Streptococcus mutans (SEQ ID NO: 262)

Spy1: TTGAGCCCTAGCAGT (SEQ ID NO: 263) derived from 23S rRNA of Streptococcus pyogenes

Spy2: TTCTATGTGTGAAGA (SEQ ID NO: 264) derived from 23S rRNA of Streptococcus pyogenes

Spy3: ACGCTGTGATTATTT (SEQ ID NO: 265) derived from 23S rRNA of Streptococcus pyogenes

Ss1: TGAAACACTGAACAA derived from 23S rRNA of Shigella sonnei (SEQ ID NO: 266)

Styp (Stpy23): GCCTGAATCAGCATG derived from 23S rRNA of Salmonella spp. (Enteritidis)

(SEQ ID NO: 7)

Strepp (StreppM): TAGGACTGCAATGTGCAGCATG derived from 23S rRNA of Streptococcus pneumoniae

(SEQ ID NO: 22)

s.wad 01: CTCAGTAAGACGTTT from ITS of Sutterella wadsworthii (SEQ ID NO: 267)

S.wad 02: GCTCCGACAAGAACT derived from ITS of Sutterella wadsworthii (SEQ ID NO: 268)

S.wad 03: CGAGTTGTTGAATTC (SEQ ID NO: 269) from ITS of Sutterella wadsworthii

S.wad 04: GTCGTCTTGTGCTTT from ITS of Sutterella wadsworthii (SEQ ID NO: 270)

S.wad 05: TCTTCAAAGAACCGA (SEQ ID NO: 271) from ITS of Sutterella wadsworthii

Svi1 (SviM): ACAGGTGCTAATACC derived from 23S rRNA of Streptococcus viridans (SEQ ID NO: 272)

Svi2 (Svi2M): GAGTGAAGAAGGTTT (SEQ ID NO: 273) derived from 23S rRNA of Streptococcus viridans

Vcho: CCCAACTGCATAAGC derived from 23S rRNA of Vibrio cholerae (SEQ ID NO: 274)

Vvul02: GTTGACGATGCATGT (SEQ ID NO: 24) derived from 23S rRNA of Vibrio vulnificus

VvulM (Vvul): AGTAACAGCCACTTG derived from 23S rRNA of Vibrio vulnificus (SEQ ID NO: 23)

Example 1

Actinomyces Israel bacillus (G +)

A. Screening of DNA Probes for Actinomyces Israel Identification

To identify the presence and identification of bacteria, Actinomyces Israel produced probes specific to the bacteria. In order for Actinomyces Israel to produce a specific probe, Actinomyces Israel should have specificity only to bacteria. The specific probe candidate group is known that the active Actinomyces Israel sequenced all the possible sequences along with the 23S rRNA and ITS sequences of the bacteria in a line through multiple alignments. The base sequence was separately divided to prepare a candidate candidate group present therein. Probe candidates were determined within the 23S rRNA gene site in all bacteria. The specificity of the probe candidate group was first confirmed by comparing the similarities between the different strains through BLAST search, and actually, the strains were applied to the hybridization reaction to select the probe in the candidate group for actinomyces Israel to respond only to the strain.

The locations and base sequences of the probes identified are shown in Table 20 below.

Sympathetic strain Probe name location Sequence SEQ ID NO: Actinomyces israelii ATCC 12102 Acii1 23S rRNA AACCTGGCTGGTGGC One

B. Immobilization of the Bacterial Identification Probe

In order to immobilize the DNA probe, the binding of the aldehyde group-amine period was used. When synthesizing all DNA fragment probes to immobilize the DNA probe, a base having an amino residue was inserted using an aminolinker column (Aminolinker column, Cruachem, Glasgrow, Scotland) in the 3-first position, and a glass plate Slide glass was coated with an aldehyde residue (CEL Associates, Inc. Huston, Texas, USA).

When fixing the probe, the probe was dissolved in 3X SSC (0.45M NaCl, 15 mM C6H5Na3O7, pH 7.0) buffer solution using a microarrayer prepared according to the known technique (Yoon. SH, et al, J.). Microbiol.Biotechnol.10 (1), 21-26, 2000), and then immobilized the DNA probe by inducing a chemical reaction for at least 1 hour and leaving it for at least 6 hours under conditions of 55% humidity. . The bacteria search probe was integrated with the total probes at a concentration of 100 μM at intervals of 280 μm to prepare a DNA chip composed of a bacteria identification probe.

Figure 2 shows the DNA chip produced. The designation indicated in FIG. 2 indicates a specific probe candidate group for the above-mentioned strain.

In order to confirm that the reaction between the amine group of the probe and the aldehyde on the glass plate was smooth and well immobilized, it was confirmed by staining with SYBRO green II (Molecular Probe, Inc., Leiden, Netherlands).

C. Isolation and Amplification of Specimen Samples

Genomic DNA extraction from bacteria present in the specimen was performed as follows. The bacteria grown in the titration medium were suspended in 200 µl sterile distilled water, centrifuged at 14,000 rpm for 10 minutes, and the supernatant was discarded, leaving only pellets. Actinomyces Israel is a Gram-positive bacterium, so it is suspended in 180 µl of lysozyme lysate solution (20mm Tris-Cl, pH8.0, 2mM EDTA, 1.2% TritonX-100, 20mg / ml lysozyme) for 30 minutes at 37 ℃. After culturing, 25 μl of Proteinase K and 200 μl of AL solution (solution solution, DNeasy Tissue Kit, QIAGEN) were mixed well and incubated at 70 ° C. for 30 minutes. After 200 μl of ethanol (100%) was added and mixed, the solution was transferred to a 2-ml tube containing a DNeasy mini column and centrifuged at 8,000 rpm for 1 minute to discard the liquid collected in the tube. Add 500 µl of AW1 solution (Wash solution 1, DNeasy Tissue Kit, Qiagen), centrifuge at 8,000 rpm or more for 1 minute, discard the effluent, and add 500 µl of AW2 solution (Wash solution 2, DNeasy Tissue Kit, QIAGEN). The DNeasy membranes were dried by centrifugation at full speed for 3 minutes and the effluent was discarded. After inserting the DNeasy mini-column for 15 minutes at room temperature, the genomic DNA was eluted by centrifugation for 1 minute at 8,000 rpm or more.

A fragment gene was prepared by using the DNA of the sample isolated as described above as a template and performing asymmetric amplification (Asymmetric PCR). Fragment gene was obtained by one polymerase chain reaction by differentiating the ratio of forward primer and reverse primer 1: 5. In order to identify DNA bound to the probe during PCR, amplification was carried out using a primer with 5-FITC attached to confirm the binding through fluorescence to determine whether the infection and the type of infection. Primers used for PCR reactions are as follows:

Primer 1 (sense): TTGTACACACCGCCCGTC (SEQ ID NOs: 278, 1585F)

Primer 2 (antisense): F-TTTCGCCTTTCCCTCACGGTACT (SEQ ID NOs: 279, 23BR)

Primer 3 (sense): AGTACCGTGAGGGAAAGG (SEQ ID NOs: 280, 23BF)

Primer 4 (antisense): F- TGCTTCTAAGCCAACATCCT (SEQ ID NOs: 281, 37R)

Primer 5 (Sense): AGGATGTTGGCTTAGAAGCA (SEQ ID NOs: 282, 37F)

Primer 6 (antisense): F-CCCGACAAGGAATTTCGCTACCTTA (SEQ ID NOs: 283, 38R)

(F: FITC)

PCR reaction was performed under the following conditions. The first denaturation was performed at 94 ° C. for 7 minutes and the second denaturation at 94 ° C. for 1 minute. Annealing was performed at 52 ° C. for 1 minute and extension at 72 ° C. for 1 minute, which was repeated 10 times. Then, the third denaturation was performed at 94 ° C. for 1 minute, annealing at 54 ° C. for 1 minute, and extension at 72 ° C. for 1 minute, which was repeated 30 times. Then, the last extension was performed once at 72 ° C. for 5 minutes. The product produced as a result of the PCR reaction was confirmed by agarose gel electrophoresis. As a result, it was confirmed that DNA double strand and fragment strand were synthesized at the same time for each bacteria, and 23S rRNA and ITS of all bacterial species using the primers used in the present invention when using 23S rRNA and ITS for the identification of bacteria It was found that can be amplified.

D. Hybridization and Washing

In order to confirm the specificity and sensitivity of the selected probe, the PCR product (step C) amplified from the sample was applied to the DNA chip (step B) to which the probe was immobilized to perform hybridization reaction. 30 μl of amplified gene (Asymmetric PCR) was added to the hybridization buffer (hybridization buffer: 6 × SSPE, 20% (v / v) formamide) to prepare a total volume of 200 μl. After dispensing the prepared solution on the glass plate to which the probe is immobilized, and then covered with a probe-clip press-seal incubation chamber (Sigma Co., St. Louis, MO.), 30 ℃ constant temperature shaking The reaction was induced for 6 hours in a shaking incubator to induce complementary binding. After elapse of time 3 × SSPE (0.45M NaCl, 15mM C6H5Na3O7, pH 7.0), 2 × SSPE (0.3M NaCl, 10mM C6H5Na3O7, pH 7.0), 1 × SSPE (0.15M NaCl, 5mM C6H5Na3O7, pH 7.0) 5 minutes each.

F. Detection and Interpretation of Hybrids

The results of the hybridization reaction were searched using Scanarray 5000 (GSI Lumonics Inc., Bedford, MA.) And are shown in FIG. 3. In FIG. 3, a yellow rectangle means a positive control (probe) and a red rectangle means fungal specific probe. Therefore, the selected probe of the present invention is a specific probe with no cross-reaction within 37 species of the standard strain. When the probe is used, the presence of the bacterium and the infection of Actinomyces Israel in the clinical specimens were detected. You can check.

Example 2

Staphylococcus epidermidis strain (G +)

The same procedure as in Example 1 was performed. The location and base sequence of the identified probe of Staphylococcus epidermidis strain are shown in Table 21 below.

Identification strain Probe name location Sequence SEQ ID NO: Staphylococcus epidrmidis KCTC 1917 (ATCC 1228) SeM01 23S rRNA GATAGATAACAGGTG 2 SeM02 23S rRNA AGGGTTCACGCCCAG 3

The produced DNA chip is shown in FIG. The designation indicated in FIG. 4 indicates a specific probe candidate group for the above-mentioned strain. The results of the hybridization reaction are shown in FIG. 5. In FIG. 5, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 15 kinds of the standard strain group. When the probe is used, the presence of Staphylococcus epidermidis strain in clinical specimens and whether it is infected with the bacterium You can check.

Example 3

Vulcoderia Sephacia strain (G-)

The same procedure as in Example 1 was performed except for the extraction of genomic DNA from the specimen sample during the isolation and amplification of the specimen sample. The locations and base sequences of the identified probes of the Vulcoderia cephacia strain are shown in Table 22 below.

Identification strain Probe name location Sequence SEQ ID NO: Burkholderia cepacia ATCC 25416 Bur23 23S rRNA TTGTTAGCCGAACGC 4 Bur01 23S rRNA GGGTGTGGCGCGAGC 6

Since the Vulcoderia Sephacia strain is a gram-negative bacterium, the method of eluting genomic DNA from bacteria present in the sample is suspended in 180 µl of ATL solution (Tissue Lysis solution, DNeasy Tissue Kit, QIAGEN), and 20 µl of Proteinase K is added. After lysis, the cells were incubated at 55 ° C. for 1 hour. After vortexing for 15 seconds, 200 μl of AL solution (Lysis solution, DNeasy Tissue Kit, QIAGEN) was mixed and incubated at 70 ° C. for 10 minutes. After 200 μl of ethanol (100%) was added and mixed, the solution was transferred to a 2-ml tube containing a DNeasy mini column and centrifuged at 8,000 rpm for 1 minute to discard the liquid collected in the tube. Add 500 µl of AW1 solution (Wash solution 1, DNeasy Tissue Kit, Qiagen), centrifuge at 8,000 rpm or more for 1 minute, discard the effluent and add 500 µl of AW2 solution (Wash solution 2, DNeasy TissueKit, QIAGEN) centrifuged for 3 min at speed to dry the DNeasy membrane and discard the effluent. After inserting the DNeasy mini-column for 15 minutes at room temperature, the genomic DNA was eluted by centrifugation for 1 minute at 8,000 rpm or more.

The produced DNA chip is shown in FIG. The designation indicated in FIG. 4 indicates a specific probe candidate group for the above-mentioned strain. The results of the hybridization reaction are shown in FIG. 6. In Figure 6, the yellow ellipse means a positive control (probe) and the red ellipse means a bacteria specific probe. Therefore, the selected probe of the present invention is a specific probe with no cross-reaction within the 15 kinds of the standard strain group. When the probe is used, it is determined whether the presence of Vulcoderia Sephacia strain in the clinical specimen and whether it is infected with the bacterium. You can check it.

Example 4

Salmonella enteritidis strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of Salmonella enteritidis strain are shown in Table 23 below.

Identification strain Probe name location Sequence SEQ ID NO: Salmonella spp. (enteritidis ) KCCM 12021 Styp23 23S rRNA GCCTGAATCAGCATG 7

The produced DNA chip is shown in FIG. The results of the hybridization reaction are shown in FIG. In FIG. 7, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 15 kinds of the standard strain group. When the probe is used, the presence of Salmonella enteritidis bacteria in clinical specimens and whether they are infected Can be.

Example 5

Escherichia coli strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Escherichia coli strain are shown in Table 24 below.

Identification strain Probe name location Sequence SEQ ID NO: Escherichia coli ATCC 25922 Eco23 23S rRNA GAGCCTGAATCAGTG 8

The produced DNA chip is shown in FIG. The results of the hybridization reaction are shown in FIG. 8. In FIG. 8, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 15 kinds of the standard strain group. When the probe is used, the presence of Escherichia coli bacteria in clinical specimens and whether they are infected Can be.

Example 6

Crevciella pneumoniae strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Crevciella pneumoniae strain are shown in Table 25 below.

Identification strain Probe name location Sequence SEQ ID NO: Klebsiella pneumoniae ATCC 700603 Kpneu 23 23S rRNA GTACACCAAAATGCA 11 Kpneu01 23S rRNA ACCTTCGGGTGTGAC 14

The produced DNA chip is shown in FIG. The designation indicated in FIG. 9 indicates a specific probe candidate group for the above-mentioned strain. The result of the hybridization reaction is shown in FIG. 10. In FIG. 10, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe having no cross-reaction within the 17 kinds of the standard strain group, and when the probe is used, the presence or absence of the presence of the Crevciella pneumoniae strain in the clinical sample and infection with the bacterium You can check whether

Example 7

Proteus mirabilis strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Proteus mirabilis strain are shown in Table 26 below.

Identification strain Probe name location Sequence SEQ ID NO: Proteus mirabilis KCCM 11381 (ATCC 21100) Pm 23S rRNA GTTACCAACAATCGT 15 Pm01 23S rRNA AGGCAGAGTGATTAG 21

The produced DNA chip is as shown in FIG. The results of the hybridization reaction are shown in FIG. In FIG. 11, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within 17 species of the standard strain, and when the probe is used, it is confirmed whether the Proteus mirabilis bacterium is present in the clinical specimen and whether it is infected with the bacterium. Can be.

Example 8

Streptococcus pneumoniae strain (G +)

The same procedure as in Example 1 was performed. The location and base sequence of the identified probe of the Streptococcus pneumoniae strain are shown in Table 27 below.

Identification strain Probe name location Sequence SEQ ID NO: Streptococcus pneumoniae KCCM 40410, 41568, 41569,41570 StreppM 23S rRNA TAGGACTGCAATGTG 22

The produced DNA chip is shown in FIG. The result of the hybridization reaction is shown in FIG. 12. In FIG. 12, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe with no cross-reaction in 17 kinds of the standard strain group. When the probe is used, it is determined whether or not the presence of the Streptococcus pneumoniae bacteria in clinical specimens is infected with the bacteria. You can check it.

Example 9

Vibrio Bernoullican strain (G-)

The same procedure as in Example 3 was performed. The locations and base sequences of the identified probes of the Vibrio Bernique Picus strain are shown in Table 28 below.

Identification strain Probe name location Sequence SEQ ID NO: Vibrio vulnificus KCTC 2962 (ATCC 3385) VvulM 23S rRNA AGTAACAGCCACTTG 23 Vvul02 23S rRNA GTTGACGATGCATGT 24

The produced DNA chip is shown in FIG. The result of the hybridization reaction is shown in FIG. 13. In Figure 13, the yellow ellipse means a positive control (probe) and the red ellipse means a bacteria specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within 17 kinds of the standard strain group, and the use of this probe confirms the presence of Vibrio Bernickus bacteria in clinical specimens and whether they are infected with the bacteria. Can be.

Example 10

Shigella flexneri strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Shigella flexneri strain are shown in Table 29 below.

Identification strain Probe designation location Sequence Sequence Listing Shigella flexneri ATCC 11836 Sflexneri01 23S rRNA AATCAAGGCCGAGGC 25 Sflexneri04 23S rRNA GCCGAGGCGTGATGA 26

The produced DNA chip is as shown in FIG. The results of the hybridization reaction are shown in FIG. 14. In FIG. 14, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 17 kinds of the standard strain group. When the probe is used, the presence of Shigella flexneri bacteria in clinical specimens and whether they are infected Can be.

Example 11

Pseudomonas aeruginosa strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of Pseudomonas aeruginosa strain are shown in Table 30 below.

Identification strain Probe name location Sequence SEQ ID NO: Pseudomonas aeruginosa KCTC 1636 Pa03 23S rRNA GGATCTTTGAAGTGA 29

The produced DNA chip is as shown in FIG. The results of the hybridization reaction are shown in FIG. 15. In FIG. 15, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 17 kinds of the standard strain group. When the probe is used, the presence of Pseudomonas aeruginosa bacteria in clinical specimens and whether the infection is confirmed Can be.

Example 12

Eromonas hydrophila strain (G-)

The same procedure as in Example 3 was performed. Aeromonas Hydrophila The location and base sequence of the identified probe of the strain are shown in Table 31 below.

Identification strain Probe designation location Sequence SEQ ID NO: Aeromonas hydrohila KCCM 32586 (ATCC 11163) Ah 23S rRNA GGCGCCTCGGTAGGG 30

The produced DNA chip is as shown in FIG. The result of the hybridization reaction is shown in FIG. 16. In FIG. 16, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 17 kinds of the standard strain group. When the probe is used, the presence of the E. monas hydrophila bacterium in the clinical sample and the presence of the bacterium are confirmed. Can be.

Example 13

Listeria monocytogenes strain (G +)

The same procedure as in Example 1 was performed. The location and base sequence of the identified probe of the Listeria monocytogenes strain are shown in Table 32 below.

Identification strain Probe name location Sequence SEQ ID NO: Listeria monocytogenes ATCC 700603 Lm 23S rRNA GGGTGCAAGCCCGAG 31

The produced DNA chip is shown in FIG. The result of the hybridization reaction is shown in FIG. 17. In FIG. 17, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 17 kinds of the standard strain group. When the probe is used, the presence of Listeria monocytogenes bacteria in clinical specimens and whether the infection is confirmed Can be.

Example 14

Enterococcus pesium strain (G +)

The same procedure as in Example 1 was performed. The location and base sequence of the identified probe of Enterococcus pesium strain are shown in Table 33 below.

Identification strain Probe name location Sequence SEQ ID NO: Enterococcus faecium ATCC 19434 Enfaeci23 23S rRNA GTTCTTTCAGATAGT 32 EnfaeciM 23S rRNA CTGAAGAGGAGTCAA 33

The produced DNA chip is shown in FIG. The results of the hybridization reaction are shown in FIG. 18. In FIG. 18, the yellow ellipse means a positive control (probe) and the red ellipse means a bacterial specific probe. Therefore, the selected probe of the present invention is a specific probe without cross-reaction within the 17 kinds of the standard strain group, and when the probe is used, it is possible to confirm the presence of Enterococcus psium bacteria in clinical specimens and whether they are infected with the bacteria. have.

Example 15

Staphylococcus aureus strain (G +)

The same procedure as in Example 1 was performed. The location and base sequence of the identified probe of Staphylococcus aureus strain are shown in Table 34 below.

Identification strain Probe name location Sequence SEQ ID NO: Staphylococcus aureus KCTC 1621C S.aureus004 23S rRNA GATTGCACGTCTAAG 38 S.aureus005 23S rRNA AATCCGGTACTCGTT 39 Saure03 23S rRNA TCTTCGAGTCGTTGA 40

The selected probe of the present invention was a specific probe without cross-reaction within 59 species of the standard strain group, and when the probe was used, the presence of Listeria monocytogenes bacteria in clinical specimens and whether it was infected with the bacterium were confirmed. .

Example 16

Naaceria meningitidis strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Neisseria meningitidis strain are shown in Table 35 below.

Identification strain Probe name location Sequence SEQ ID NO: Neisseria meningitidis ATCC 13100 Nm001 23S rRNA GTTTACTGGCATGGT 41 Nm002 23S rRNA AGATGTGAGAGCATC 42 Nm02 23S rRNA CCGGGTCTTCTTAAC 43

The selected probe of the present invention is a specific probe without cross-reaction within 59 species of the standard strain group, and when the probe is used, the presence of Neisseria meningitidis bacteria in clinical specimens and whether they are infected with the bacteria are identified. Could.

Example 17

Serasia Malcesense Strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of Seracia malcesense strain are shown in Table 36 below.

Identification strain Probe name location Sequence SEQ ID NO: Serratia marcescens KCTC 1299, 2197 Seme01 23S rRNA ATCGAACTCACGACC 44 Seme02 23S rRNA CTGTGTATTTTAGTG 45

The selected probe of the present invention is a specific probe without cross-reaction within 59 species of the standard strain group, and when the probe is used, it is possible to confirm the presence of Ceracia Malcesense bacteria in clinical specimens and whether they are infected with the bacterium. have.

Example 18

Citrobacter Prundai strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Citrobacter prundi strain are shown in Table 37 below.

Identification strain Probe name location Sequence SEQ ID NO: Citrobacter freundii KCTC 2006, 2195 Citf001 23S rRNA CGGAGGTTCCAGGTA 46

The selected probe of the present invention is a specific probe without cross-reaction within 59 species of the standard strain group, and when the probe is used, it is possible to confirm the presence and presence of Citrobacter prundai bacteria in clinical specimens. have.

Example 19

Legionella pneumophila strain (G-)

The same procedure as in Example 3 was performed. The location and base sequence of the identified probe of the Legionella pneumophila strain are shown in Table 38 below.

Identification strain Probe name location Sequence SEQ ID NO: Legionella pneumophila L.pneu011 23S rRNA TGGAGAGCATTTTAT 47 L.pneu012 23S rRNA GTGATTTTGAGGTGA 48 L.pneu013 23S rRNA AGATGGTAAAGAAGA 49

The selected probe of the present invention is a specific probe without cross-reaction within 59 species of the standard strain group, and when the probe is used, the presence of Legionella pneumophila in clinical specimens and the presence of the bacterium can be confirmed.

The probes of the present invention exhibit a high degree of specificity.

Claims (117)

Nucleic acid molecule comprising the following base sequence derived from the 23S rRNA of the bacterium Actinomyces: AACCTGGCTGGTGGC (Acii1, SEQ ID NO: 1). A nucleic acid probe for detecting the actinomyces Israeli bacterium comprising a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1. A composition comprising a nucleic acid probe for the detection of an actinomyces Israeli bacterium comprising a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1. (a) a composition comprising a nucleic acid probe for the detection of an actinomyces Israeli bacterium comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or a component necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed. 18. A kit for detecting and identifying fungi of actinomyces israel in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization and washing conditions of the polynucleic acid of step (a) and (b) with a composition comprising a nucleic acid probe for the detection of the actinomyces Israeli bacterium comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1. Contacting under; (d) detecting the hybrid formed in step (c); (e) identifying a strain present in the sample from the different hybridization signals obtained in step (d), wherein the actinomyces israel is detected and identified in the biological sample. An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from Staphylococcus epidermidis 23S rRNA: GATAGATAACAGGTG (SeM01, SEQ ID NO: 2); And AGGGTTCACGCCCAG (SeM02, SEQ ID NO: 3). A nucleic acid probe for detecting Staphylococcus epidermidis bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 2 and 3. A composition comprising a nucleic acid probe for detecting Staphylococcus epidermidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 2 and 3. (a) a composition comprising a nucleic acid probe for detecting Staphylococcus epidermidis bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 2 and 3; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein said kit is for detecting and identifying Staphylococcus epidermidis bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 2 and 3. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) a composition comprising a nucleic acid probe for detecting Staphylococcus epidermidis bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 2 and 3 in the polynucleic acid of steps (a) and (b); Contacting under appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Staphylococcus epidermidis bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from 23 S rRNAs of Vulcoderia cephacia: TTGTTAGCCGAACGC (Bur23, SEQ ID NO: 4); GCCAGGAGGGTGAAG (Bur001, SEQ ID NO: 5); And GGGTGTGGCGCGAGC (Bur01, SEQ ID NO: 6). A nucleic acid probe for the detection of Bulcoderia cephacia bacteria, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 4-6. A composition comprising a nucleic acid probe for the detection of Bulcoderia cephacia bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 4-6. (a) a composition comprising a nucleic acid probe for the detection of Bulcoderia sefacia bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 4 to 6; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed. 17. A kit for detecting and identifying Bulcoderia cephacia bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 4-6. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) the polynucleic acid of step (a) and (b) is suitable for a composition comprising a nucleic acid probe for the detection of the baculovirus Sephacia bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOS: 4-6 Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strains present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying Vulcoderia cephacia bacteria in the biological sample. Nucleic acid molecule comprising the following nucleotide sequence derived from Salmonella enteritidis bacteria 23S rRNA: GCCTGAATCAGCATG (Styp23, SEQ ID NO: 7). A nucleic acid probe for detecting Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7. A composition comprising a nucleic acid probe for detecting Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7. In another aspect, the present invention (a) composition comprising a nucleic acid probe for the detection of Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Salmonella enteritidis bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) contacting the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7 under appropriate hybridization and washing conditions To; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Salmonella enteritidis bacteria in the biological sample, characterized in that the strains present in the sample are identified from the different hybridization signals obtained in step (d). An isolated nucleic acid molecule selected from the group consisting of isolated nucleic acid molecules comprising the following base sequence derived from 23S rRNA of Escherichia coli: GAGCCTGAATCAGTG (Eco23, SEQ ID NO: 8); GTTAGCGGTAACGCG (E coli 001, SEQ ID NO: 9); And CTGAAGCGACAAATG (E coli 003, SEQ ID NO: 10). A nucleic acid probe for detecting Escherichia coli, characterized in that it comprises a nucleic acid molecule comprising any one of SEQ ID NOs: 8 to 10. A composition comprising a nucleic acid probe for detecting Escherichia coli, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 8-10. (a) a composition comprising a nucleic acid probe for detecting Escherichia coli, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 8 to 10; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Escherichia coli bacteria in a biological sample. A DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 8-10. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of step (a) and (b) with a composition comprising a nucleic acid probe for detecting Escherichia coli comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 8 to 10 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Escherichia coli bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from the 23S rRNA of Crevciella pneumoniae: GTACACCAAAATGCA (Kpneu 23, SEQ ID NO: 11); ACGCTGGTGTGTAGG (K.pneu001, SEQ ID NO: 12); GCTGAGACCAGTCGA (K.pneu002, SEQ ID NO: 13); And ACCTTCGGGTGTGAC (Kpneu01, SEQ ID NO: 14). A nucleic acid probe for detecting Crebesciella pneumoniae, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 11-14. A composition comprising a nucleic acid probe for detecting Crebesciella pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14. (a) a composition comprising a nucleic acid probe for detecting Crebesciella pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying CrebSiela pneumoniae in a biological sample. In another aspect, the present invention is a DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) a composition comprising a nucleic acid probe for detecting Crebsiella pneumoniae comprising the nucleic acid molecule of any one of SEQ ID NOs: 11 to 14 in the polynucleic acid of steps (a) and (b) Contacting with appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strains present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying Crebesciella pneumoniae in the biological sample. Provided are isolated nucleic acid molecules selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from the 23S rRNA of Proteus mirabilis: GTTACCAACAATCGT (Pm, SEQ ID NO: 15); AAGGCTAGGTTGTCC (Pm001, SEQ ID NO: 16); GGCGACGGTCGTCCC (Pm002, SEQ ID NO: 17); GATGACGAACCACCA (Pm003, SEQ ID NO: 18); TGAAGCAATTGATGC (Pm004, SEQ ID NO: 19); TAAAGTCCCTCGCGG (Pm005, SEQ ID NO: 20); And AGGCAGAGTGATTAG (Pm01, SEQ ID NO: 21). A nucleic acid probe for detecting Proteus mirabilis bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 15 to 21. A composition comprising a nucleic acid probe for detecting Proteus mirabilis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 15-21. (a) a composition comprising a nucleic acid probe for detecting Proteus mirabilis, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 15 to 21; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a previously formed hybrid. 2. A kit for detecting and identifying Proteus mirabilis bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 15-21. a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of step (a) and (b) with a composition comprising a nucleic acid probe for detecting the proteus mirabilis comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 15-21 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Proteus mirabilis bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). In another aspect, the present invention provides a nucleic acid molecule comprising the following base sequence derived from the Streptococcus pneumoniae 23S rRNA: TAGGACTGCAATGTG (StreppM, SEQ ID NO: 22). A nucleic acid probe for detecting Streptococcus pneumoniae bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22. In another aspect, the present invention is a composition comprising a nucleic acid probe for the detection of Streptococcus pneumoniae bacteria comprising a nucleic acid molecule comprising the base sequence of SEQ ID NO: 22. (a) a composition comprising a nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Streptococcus pneumoniae in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) the polynucleic acid of steps (a) and (b) under appropriate hybridization and washing conditions with a composition comprising a nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 22. Contact; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Streptococcus pneumoniae bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). Provided is an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from the Vibrio Bernespickus 23S rRNA: AGTAACAGCCACTTG (VvulM, SEQ ID NO: 23); And GTTGACGATGCATGT (Vvul02, SEQ ID NO: 24). A nucleic acid probe for the detection of Vibrio Bernickus bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 23 to 24. A composition comprising a nucleic acid probe for the detection of Vibrio Bernique Picus bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 23 and 24. (a) a composition comprising a nucleic acid probe for the detection of Vibrio Bernique Picus bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 23 and 24; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed. 17. A kit for detecting and identifying Vibrio Bernoullican bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 23 and 24. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of Vibrio Bernoulcus bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 23 and 24 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strains present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying Vibrio Bernoullican bacteria in the biological sample. An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from Shigella flexneri strain 23S rRNA: AATCAAGGCCGAGGC (Sflexneri01, SEQ ID NO: 25); And GCCGAGGCGTGATGA (Sflexneri04, SEQ ID NO: 26). In another aspect, the present invention is a nucleic acid probe for the detection of Shigella flexneri bacteria, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NO: 25 and 26. A composition comprising a nucleic acid probe for detecting Shigella flexneri bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 25 and 26. (a) a composition comprising a nucleic acid probe for the detection of Shigella flexneri bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 25 and 26; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein said kit is for detecting and identifying Shigella flexneri bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 25 and 26. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of Shigella flexneri bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 25 and 26 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strain present in the sample from the different hybridization signals obtained in step (d), and detecting and identifying Shigella flexneri bacteria in the biological sample. In another aspect, the present invention is an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following base sequence derived from Pseudomonas aeruginosa bacteria 23S rRNA. GAAGTGCCGAGCATG (P.aeru001, SEQ ID NO: 27); GTGTCACGTAAGTGA (P.aeru002, SEQ ID NO: 28); And GGATCTTTGAAGTGA (Pa03, SEQ ID NO: 29). A nucleic acid probe for detecting Pseudomonas aeruginosa, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 27-29. A composition comprising a nucleic acid probe for detecting Pseudomonas aeruginosa, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 27 to 29. (a) a composition comprising a nucleic acid probe for detecting Pseudomonas aeruginosa, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 27 to 29; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed. 16. A kit for detecting and identifying Pseudomonas aeruginosa bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 27-29. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of step (a) and (b) with a composition comprising a nucleic acid probe for detecting Pseudomonas aeruginosa, comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 27-29 And under washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Pseudomonas aeruginosa bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). Nucleic acid molecule comprising the following nucleotide sequence derived from 23S rRNA of Eromonas hydrophila: GGCGCCTCGGTAGGG (Ah, SEQ ID NO: 30). A nucleic acid probe for the detection of E. hydronas bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30. A nucleic acid probe for the detection of Eromonas hydrophile bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30. (a) a composition comprising a nucleic acid probe for the detection of E. hydronas bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) means for detecting a hybrid previously formed, wherein said kit is for detecting and identifying said E. monas hydrophila bacterium in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) contacting the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of Eromonas hydrophila comprising the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30 under appropriate hybridization and washing conditions To; (d) detecting the hybrid formed in step (c); (e) identifying the strain present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying the E. hydronas bacteria in said biological sample. Nucleic acid molecule comprising the following nucleotide sequence derived from 23S rRNA of Listeria monocytogenes: GGGTGCAAGCCCGAG (LM, SEQ ID NO: 31). A nucleic acid probe for detecting Listeria monocytogenes bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31. A composition comprising a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31. (a) a composition comprising a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Listeria monocytogenes bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) contacting the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for detecting Listeria monocytogenes bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31 under appropriate hybridization and washing conditions To; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Listeria monocytogenes bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). An isolated nucleic acid molecule comprising the following nucleotide sequence derived from 23S rRNA of Enterococcus pesium: GTTCTTTCAGATAGT (Enfcium1 (Enfaeci23), SEQ ID NO: 32); CTGAAGAGGAGTCAA (Enfcium2 (Enfaeci M), SEQ ID NO: 33); TTACGATTGTGTGAA (E.faecium002, SEQ ID NO: 34); And ATAGCACATTCGAGG (E.faecium003, SEQ ID NO: 35). A nucleic acid probe for detecting Enterococcus pesium bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35. A composition comprising a nucleic acid probe for the detection of Enterococcus pesium bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35. (a) a composition comprising a nucleic acid probe for detecting enterococcus pesium bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed. 16. A kit for detecting and identifying Enterococcus pesium bacteria in a biological sample. A DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 32 to 35. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of steps (a) and (b) with a composition comprising a nucleic acid probe for the detection of Enterococcus pesium bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 32-35; Contacting under washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strains present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying enterococcus pesium bacteria in the biological sample. An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from 23S rRNA of Staphylococcus aureus: AGGACGACATTAGAC (S.aureus 001, SEQ ID NO: 36); CGAAGCGTGCGATTG (S. aureus 003, SEQ ID NO: 37); GATTGCACGTCTAAG (S.aureus 004, SEQ ID NO: 38); AATCCGGTACTCGTT (S.aureus 005, SEQ ID NO: 39); And TCTTCGAGTCGTTGA (Saure03 (Saureus03), SEQ ID NO: 40). A nucleic acid probe for detecting Staphylococcus aureus bacteria, comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 36 to 40. In another aspect, the present invention is a composition comprising a nucleic acid probe for the detection of Staphylococcus aureus bacteria comprising a nucleic acid molecule comprising a nucleotide sequence of any one of SEQ ID NOs: 36 to 40. (a) a composition comprising a nucleic acid probe for detecting Staphylococcus aureus bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 36 to 40; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Staphylococcus aureus bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 36 to 40. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) a composition comprising a nucleic acid probe for detecting Staphylococcus aureus comprising the polynucleic acid of step (a) and (b) comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 36 to 40; Contacting under appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Staphylococcus aureus bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from 23S rRNA of Neisseria meningitidis bacteria: GTTTACTGGCATGGT (Nm001, SEQ ID NO: 41); AGATGTGAGAGCATC (Nm002, SEQ ID NO: 42); And CCGGGTCTTCTTAAC (Nm02, SEQ ID NO: 43). A nucleic acid probe for detection of Neisseria meningitidis bacteria, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 41 to 43. A composition comprising a nucleic acid probe for the detection of Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 41 to 43. (a) a composition comprising a nucleic acid probe for the detection of Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 41 to 43; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Neisseria meningitidis bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 41-43. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) a composition comprising a nucleic acid probe for detection of Neisseria meningitidis bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 41 to 43 for the polynucleic acid of steps (a) and (b); Contacting under appropriate hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) detecting and identifying strains of Neisseria meningitidis in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising the following nucleotide sequences derived from 23S rRNA of Seracia malcesense: ATCGAACTCACGACC (Seme01, SEQ ID NO: 44); And CTGTGTATTTTAGTG (Seme02, SEQ ID NO: 45). A nucleic acid probe for detection of Seracia malcesense bacteria, characterized in that it comprises a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 44 and 45. A composition comprising a nucleic acid probe for detecting Seracia malcesense bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 44 and 45. (a) a composition comprising a nucleic acid probe for detecting a Seracia malcesense bacterium comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 44 and 45; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Seracia malcesense bacteria in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 44 and 45. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) the polynucleic acid of step (a) and (b) is suitable for a composition comprising a nucleic acid probe for the detection of Seracia malcesense bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 44 and 45. Contacting under hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) identifying the strain present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying Ceracia Malcesense bacteria in the biological sample. An isolated nucleic acid molecule comprising the following nucleotide sequence derived from Citrobacter prundi 23S rRNA: CGGAGGTTCCAGGTA (Citf001, SEQ ID NO: 46). A nucleic acid probe for detecting Citrobacter prundi, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46. A composition comprising a nucleic acid probe for detecting Citrobacter prune, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46. (a) a composition comprising a nucleic acid probe for detecting Citrobacter prune, comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying Citrobacter prundi bacteria in a biological sample. A DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) the polynucleic acid of steps (a) and (b) under appropriate hybridization and washing conditions with a composition comprising a nucleic acid probe for detecting Citrobacter prundi comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 46; Contact; (d) detecting the hybrid formed in step (c); (e) identifying the strains present in the sample from the different hybridization signals obtained in step (d), thereby detecting and identifying Citrobacter prundi in the biological sample. An isolated nucleic acid molecule selected from the group consisting of isolated nucleic acid molecules comprising the following base sequence derived from the Legionella pneumophila 23S rRNA: TGGAGAGCATTTTAT (L.pneu011, SEQ ID NO: 47); GTGATTTTGAGGTGA (L.pneu012, SEQ ID NO: 48); And AGATGGTAAAGAAGA (L.pneu013, SEQ ID NO: 49). A nucleic acid probe for detecting Legionella pneumophila, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 47 to 49. A nucleic acid probe for detecting Legionella pneumophila, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 47 to 49. (a) a composition comprising a nucleic acid probe for detecting Legionella pneumophila, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 47 to 49; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein said kit is for detecting and identifying Legionella pneumophila in a biological sample. A DNA chip comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 47 to 49. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) appropriate hybridization of the polynucleic acid of step (a) and (b) with a composition comprising a nucleic acid probe for detecting Legionella pneumophila comprising the nucleic acid molecule comprising any one of SEQ ID NOs: 47 to 49; Contacting under washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying Legionella pneumophila bacteria in the biological sample, characterized by identifying the strains present in the sample from the different hybridization signals obtained in step (d). In still another aspect, the present invention provides a nucleic acid probe for detecting a bacterium of Actinomyces Israel comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, comprising the nucleotide sequence of any one of SEQ ID NOs: 2 and 3. Nucleic acid probe for the detection of the bacteriophage bacterium bacterium comprising a nucleic acid molecule comprising any one of the nucleotide sequence of SEQ ID NO: 4 to 6, at least one of the nucleic acid probe for the detection of Staphylococcus epidermidis bacteria containing a nucleic acid molecule A nucleic acid probe for detecting Salmonella enteritidis bacteria comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7, an Escherichia coli comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 8 to 10 Creb comprising a nucleic acid molecule comprising the base sequence of any one or more of the nucleic acid probe for detecting the bacteria, SEQ ID NO: 11-14 A nucleic acid probe for detecting Proteus mirabilis, comprising one or more of the nucleic acid probes for detecting Ella pneumoniae, a nucleic acid molecule comprising any one of SEQ ID NOs: 15 to 21, and a nucleotide sequence of SEQ ID NO: 22. At least one of a nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid molecule, and a nucleic acid probe for detecting Vibrio Bunnpickus bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 23 and 24, One or more of the nucleic acid probes for detecting Shigella flexneri comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 25 and 26, and a nucleic acid molecule comprising any one of SEQ ID NOs: 27 to 29. At least one nucleic acid probe for detecting Pseudomonas aeruginosa, and includes a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30 Nucleic acid probe for detecting the E. monophyllosis, nucleic acid probe for detecting Listeria monocytogenes comprising the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31, nucleic acid molecule comprising any one of SEQ ID NOs: 32 to 35 At least one of the nucleic acid probes for detecting enterococcus fungi comprising: at least one of the nucleic acid probes for detecting Staphylococcus aureus comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 36 to 40, A nucleic acid molecule comprising the base sequence of any one of SEQ ID NOs: 44 and 45, at least one of the nucleic acid probes for detecting the bacterium Meningitidis, including the nucleic acid molecule comprising any one of SEQ ID NOs: 41 to 43; One or more of the nucleic acid probes for detecting the strain of Ceracia malcesense, comprising the nucleotide sequence of SEQ ID NO: 46 Among the group consisting of one or more of the nucleic acid probe for detecting the strain of Citrobacter Prundi comprising a nucleic acid molecule comprising a nucleic acid probe for detecting Legionella pneumophila comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS: 47-49 A composition comprising one or more probes selected; (b) a forward and reverse primer pair, optionally used to amplify polynucleic acid in a biological sample; (c) a buffer or component necessary to produce such a buffer to induce a hybridization reaction between the probe contained in the composition and the polynucleic acid or amplified product thereof present in the biological sample; (d) a solution for washing the hybrid formed under suitable washing conditions or the components necessary to produce such a solution; And (e) optionally means for detecting a hybrid previously formed, wherein the kit is for detecting and identifying one or more species of said organism in a biological sample. A DNA chip comprising at least one nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 1-49. (a) optionally isolating and / or concentrating polynucleic acid present in the biological sample, and (b) optionally amplifying the polynucleic acid with the appropriate forward and reverse primer pairs; (c) any one of the nucleic acid probes for detecting the actinomyces Israeli bacterium, wherein the polynucleic acid of steps (a) and (b) comprises a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NOs: 2 and 3 Bulcoderia cephacia bacterium comprising a nucleic acid molecule comprising any one of the base sequences of SEQ ID NOs: 4 to 6, at least one of the nucleic acid probes for detecting Staphylococcus epidermidis bacterium comprising a nucleic acid molecule comprising a nucleotide sequence of At least one of the nucleic acid probes for detection of the nucleic acid probes for detecting Salmonella enteritidis comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7, and a nucleic acid molecule comprising any one of SEQ ID NOs: 8 to 10 One or more of the nucleic acid probe for detecting the Escherichia coli strain, comprising a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID NOs: 11-14 Nucleic acid probe for detecting Proteus mirabilis, comprising the nucleic acid molecule comprising any one of SEQ ID NOS: 15-21 At least one of a nucleic acid probe for detecting Streptococcus pneumoniae comprising a nucleic acid molecule comprising a nucleic acid probe for detecting Vibrio Bernoulcus bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 23 and 24 , At least one of the nucleic acid probes for the detection of Shigella flexneri bacteria comprising the nucleic acid molecule of any one of SEQ ID NOs: 25 and 26, and a nucleic acid molecule comprising any one of SEQ ID NOs: 27 to 29. At least one nucleic acid probe for detecting Pseudomonas aeruginosa bacteria, the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 30 Nucleic acid probe for detection of E. coli bacteria, nucleic acid probe for detecting Listeria monocytogenes comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 31, nucleic acid comprising any one of SEQ ID NOs: 32 to 35 One or more nucleic acid probes for detecting enterococcus fungus containing molecules, or one or more nucleic acid probes for detecting Staphylococcus aureus comprising nucleic acid molecules comprising any one of SEQ ID NOs: 36-40 , A nucleic acid molecule comprising any one of SEQ ID NOs: 44 and 45, or at least one of nucleic acid probes for detecting Neisseria meningitidis bacteria comprising a nucleic acid molecule comprising any one of SEQ ID NOs: 41 to 43. One or more of the nucleic acid probe for the detection of Ceracia malcesense bacteria comprising, the base of SEQ ID NO: 46 A group consisting of at least one of a nucleic acid probe for detecting Citrobacter prundi comprising a nucleic acid molecule comprising a row and a nucleic acid probe for detecting Legionella pneumophila comprising a nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS: 47-49 Contacting the composition comprising at least one probe selected from among suitable hybridization and washing conditions; (d) detecting the hybrid formed in step (c); (e) A method for detecting and identifying one or more species of said bacteria in said biological sample, characterized by identifying strains present in the sample from different hybridization signals obtained in step (d).