KR20070069676A - Dna chip for detection of escherichia coli - Google Patents

Abstract

A DNA Chip for detection of Escherichia coli is provided to improve the rapidness and accuracy of detection by using a nucleic acid probe derived from 23S rRNA(ribosomal RNA) of Escherichia coli and eliminate influence of antibiotic administration used in a clinical test. The DNA Chip for detection of Escherichia coli comprises oligonucleotides which are nucleotide sequences specific to Escherichia coli from 23S rRNA of Escherichia coli and have the nucleotide sequences of SEQ ID NO:1(GTTAGCGGTAACGCG), SEQ ID NO:2(GTTAGTGGAAGCGTC), SEQ ID NO:3(GTGTTAGTGGAAGCGTCTGG), SEQ ID NO:4(ATGGGGTTAGCGGTAACGCGAAGCT), SEQ ID NO:5(ATGCACATATTGTGA), SEQ ID NO:6(GTGCTGAAGCAACAAATGCC) and SEQ ID NO:7(CGGTGCTGAAGCGACAAATGCCCTG).

Description

DNA chip for detecting Escherichia coli {DNA Chip for Detection of Escherichia coli}

FIG. 1A shows the design of a DNA chip for blind testing on specimens containing E. coli.

FIG. 1B shows the results of hybridization reactions detected using an ArrayWorks micro array scanner (ArrayWorks) in a blind test on specimens containing E. coli.

FIG. 2A shows the design of a DNA chip for blind testing on specimens containing E. coli.

FIG. 2B shows the results of hybridization reactions detected using the ArrayWorks microarray scanner in blind tests on specimens containing E. coli.

The present invention relates to an E. coli specific nucleic acid probe useful for detecting and identifying E. coli in a biological sample, and more particularly, to a DNA chip for detecting and identifying E. coli, wherein the nucleic acid probe derived from the 23S rRNA gene of E. coli is immobilized.

Infectious diseases are diseases that occur when pathogens start to live in the blood, body fluids, and tissues, and may be life-threatening if accurate identification and proper treatment of causative organisms are not performed. More recently, the misuse of antibiotics has resulted in a reduction in the incidence of pathogens. Diagnostic methods for diagnosing infectious diseases are increasingly difficult. Therefore, diagnosing it as soon as possible and identifying the type of causative agent occupies a very important position in the treatment.

Among these infectious pathogens, Escherichia coli produces vero toxin, causing hemorrhagic E. coli infections, and it is known that E. coli from more than 70 serogroups produces toxins. Intestinal hemorrhagic E. coli infection is a foodborne disease that occurs mainly in developed countries, including the United States and Japan, causing complications of hemolytic uremic syndrome and death in severe cases. In the United States, more than 20,000 deaths per year occur, causing 250 deaths (CDC 2001; James et al , 1998). In Japan, about 100 people die before 1996. Every year, after a sharp increase to 3,022 in 1996, More than 2,000 have been reported (NIID, 2003). In 2000, intestinal hemorrhagic E. coli infection was designated as a group 1 statutory epidemic, and sporadically occurred within 10 cases per year after 1 case in 1998. Until 2002, there was no mass epidemic of intestinal hemorrhagic E. coli infection, but the Korean dietary pattern is gradually westernizing, and the mass epidemic of hemorrhagic E. coli infection is sufficiently predicted.

 Due to this necessity, diagnostic methods for identifying Escherichia coli causing infectious diseases have been researched and developed for a long time. 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.

Except for viruses, all prokaryotic organisms contain rRNA genes that encode homologs of prokaryotic 5S, 16S and 23S rRNA molecules. For eukaryotic cells, these rRNA molecules are 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA, which are substantially similar to those of prokaryotic cells. Many probes have been published for the specific targeting of rRNA subsequences of specific organisms or groups of organisms in biological samples. By using these nucleic acid probes in combination with well-known polymerase chain reaction (PCR) many of these problems in conventional diagnostic methods can be solved. In the nucleic acid probe technology for diagnosis, the selection of target genes to be amplified is very important. In general, rRNA, in particular 23S rRNA gene, is widely used, and nucleic acid probe sequences thereof are advantageously cross-linked with nucleic acids derived from other organisms under moderate constriction conditions. It is known that the probability of reaction is low (P. Wattiau et. Al ., Appl. Microbiol. Biotechnol ., 56: 816, 2001; D, A. Stahlm et. Al ., J. Bacteriol ., 172: 116, 1990 Boddinghaus et al ., J. Clin, Microbiol ., 28: 1751, 1990; T. Rogall et al ., J. Gen. Microbiol ., 136: 1915, 1990; T. Rogall, et . , Int. J. System. Bacteriol ., 40: 323, 1990; K. Rantakokko-Jalava et. Al ., J. Clin., Mirobiol ., 38:32, 2000; Park et. Al ., J. Clin. , Mirobiol ., 38: 4080, 2000; A. Schmalenberger et. Al , Appl. Microbiol.Biotechnol ., 67: 3557, 2001; WO98 / 55646; US Pat. Nos. 6,025,132 and 6,277,577).

The present inventors have applied for a patent for a DNA chip using a nucleic acid sequence specific to the infectious organisms as a probe to detect and identify a non-viral infectious organism (Korean Patent Application No. 10-2005-7014626, etc.), Research continues to increase the sensitivity of DNA chips.

Therefore, the present inventors have made an effort to detect E. coli quickly and accurately in a sample of a patient suspected of E. coli infection. As a result, E. coli-specific sequences were found in the 23S rRNA gene of the E. coli genome, and the DNA chip was used as a probe for detecting E. coli. By the production, it was confirmed that E. coli can be detected quickly and accurately, and the present invention was completed.

An object of the present invention is to provide a DNA chip for E. coli detection and identification, characterized in that the oligonucleotide comprising a sequence specific for E. coli in the 23S rRNA gene of the E. coli genome is fixed.

Another object of the present invention to provide a probe for detecting and identifying E. coli consisting of the oligonucleotide.

In order to achieve the above object, the present invention is GTTAGCGGTAACGCG (SEQ ID NO: 1); GTTAGTGGAAGCGTC (SEQ ID NO: 2); GTGTTAGTGGAAGCGTCTGG (SEQ ID NO: 3); ATGGGGTTAGCGGTAACGCGAAGCT (SEQ ID NO: 4); ATGCACATATTGTGA (SEQ ID NO: 5); GTGCTGAAGCAACAAATGCC (SEQ ID NO: 6); And an oligonucleotide having a nucleotide sequence of CGGTGCTGAAGCGACAAATGCCCTG (SEQ ID NO: 7) is provided, which provides a DNA chip for E. coli detection and identification.

The invention also relates to GTTAGCGGTAACGCG (SEQ ID NO: 1); GTTAGTGGAAGCGTC (SEQ ID NO: 2); GTGTTAGTGGAAGCGTCTGG (SEQ ID NO: 3); ATGGGGTTAGCGGTAACGCGAAGCT (SEQ ID NO: 4); ATGCACATATTGTGA (SEQ ID NO: 5); GTGCTGAAGCAACAAATGCC (SEQ ID NO: 6); And one or more oligonucleotides selected from the group consisting of oligonucleotides having the nucleotide sequence of CGGTGCTGAAGCGACAAATGCCCTG (SEQ ID NO: 7).

Hereinafter, the present invention will be described in detail.

In the present invention, the 23S rRNA nucleotide sequence of E. coli is compared with the nucleotide sequence of the bacteria whose sequence has been identified so far, and the sequence specific to E. coli is identified, the probe candidate group is selected using the E. coli specific sequence, and E. coli specific. Probes were synthesized. The synthesized probe was fixed to a glass plate using an aldehyde-amine bond to prepare a DNA chip for detecting E. coli.

In addition, in order to verify the specificity of the DNA chip and the probe for detecting E. coli, the genome of the standard strain is isolated, and the PCR product obtained by PCR using the genome as a template is hybridized to the DNA chip so that the DNA chip is effective for detecting E. coli. Confirmed. In addition, although E. coli detection method through the conventional culture was affected by the addition of antibiotics, it was confirmed through experiments that the DNA chip of the present invention shows a high detection rate even when antibiotics.

Hereinafter, terms and expressions used in the present invention will be described.

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" or "nucleic acid probe" refers to an oligonucleotide specific for a single strand having a base sequence that is sufficiently complementary to the target base sequence to be detected and hybridizes.

"Composition" means that a probe complementary to a bacterial or fungal rRNA may be pure or combined with other probes. In addition, the probe may be combined with a salt or a buffer in a dried state, as a precipitate, in the form of an alcohol solution or an aqueous solution.

"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 single-stranded or double-stranded DNA (obtained after optional amplification) or RNA and contains a sequence having at least partial complementarity with at least one probe oligonucleotide.

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.

"Oligonucleotide" generally refers to a nucleotide polymer consisting of about 10 to about 100 nucleotides. However, the length of the nucleotides may be 100 or more or 10 or less.

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

"Homologous" is synonymous with identity, meaning that a polynucleic acid, for example 90% homology, exhibits 90% identical base pairs at the same position in the arrangement of the sequences.

"Hybridization" means the annealing of complementary sequences to a target nucleic acid (the sequence to be detected). The ability of two nucleic acid polymers, including complementary sequences, to find each other and hybridize through base pair interactions is a well known phenomenon.

"Primer" refers to a single stranded DNA oligonucleotide sequence that can serve as a starting point for the synthesis of extension products complementary to the nucleic acid strand to be replicated. The length and sequence of the primers may be such that they can initiate the synthesis of extension products, preferably consisting of about 5 to 50 nucleotides. The specific length and sequence will be determined by the complexity of the desired DNA or RNA target as well as the conditions of primer use such as temperature and ionic strength.

A "label" refers to any atom or molecule capable of providing a detectable (preferably quantifiable) signal and capable of binding to a nucleic acid. The label can provide a signal detectable by fluorescence, radioactivity, chromaticity, X-ray diffraction or absorption, magnetism, and the like.

A "hybrid" is a complex formed between two single stranded nucleic acid sequences by either Watson-Crick base pair or non-standard base pair between complementary bases.

"Probe specificity" refers to the characteristics of a probe to describe its ability to distinguish between target and nontarget sequences. In this regard, the specificity of the probe means that the nucleotide sequence hybridizes with a given target sequence and is substantially free of hybridization with the nontarget sequence or with minimal hybridization with the nontarget sequence. Probe specificity depends on sequence and assay conditions.

"Standard strain" refers to a strain that is commercially available or readily available.

Sympathy

In order to produce a nucleic acid probe for the detection of bacteria, it is necessary to have specificity only for each bacterium. For this purpose, a specific probe candidate group for each bacterium is selected. After the specific probe, the control group sorts and compares the sequences of all possible germs in a line through multiple alignments to separately classify the specific sequences present in each germ and prepare and select a probe candidate group present therein. . Probe candidates are determined within the 23S rRNA gene in each bacterium for bacteria and within the 18S rRNA gene region for fungi. 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. In addition, the nucleic acid probes selected as described above are adapted to clinical trials involving multiple biological samples to confirm their sensitivity.

The nucleic acid probes selected according to the invention are at least 15 oligonucleotides of oligonucleotide and preferably have at least 70%, 80%, 90% or 95% homology with the full complement sequence of the target to be detected. The length of the oligonucleotide of the nucleic acid probe for detecting and identifying each bacterium according to the present invention may be 50 or more. The nucleotides used in the present invention may also be modified nucleotides such as ribonucleotides, deoxyribonucleotides and nucleotides containing inosine or modified groups, but they must not affect hybridization characteristics.

Probe

Nucleic acid probes of the present invention can be used for all known hybridization techniques for testing the presence or absence of a target nucleic acid in a biological sample for diagnostic purposes, such as point deposition on a filter 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 Norson blot.

In addition, the nucleic acid probes of the present invention can be used in sandwich hybridization systems that enhance the specificity of nucleic acid probe-based assays. The principle and use of sandwich hybridization in nucleic acid probe-based assays is known (DUNN and HASSEL, Cell , 12:23, 1977; and RNAKI et al ., Gene , 21:77, 1983). The sandwich hybridization technique employs 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) has a region of the target specific for the desired bacterium. May hybridize. 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 a dissociation temperature that is 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 nucleic acid probes of the 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.

In addition, the nucleic acid probe of the present invention can be used for reversed hybridization ( Proc . Natl. Acad . Sci . USA , 86: 6230, 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 carried out without amplification. In such cases, the nucleic acid present in the biological sample must be labeled or modified specifically or nonspecifically by chemical or specific dye addition prior to hybridization.

The nucleic acid probe of the present invention can be included in a kit that can be used to quickly determine the presence of the pathogen of interest in the present invention. The kit includes all the components necessary to assay for the presence of the pathogen. In a conventional concept, a kit may contain a stable preparation of a labeled probe, a hybridization solution in anhydrous or liquid form to induce hybridization of a target and probe nucleic acid, a solution for washing and removing unwanted or unhybridized nucleic acid, and a labeled hybrid. Substrates for detection and optionally instruments for detecting labels.

One particular embodiment of the present invention includes a kit that utilizes the concept of a sandwich assay. The kit includes a first element for collecting a sample at a particular site of a patient, such as a sample scraping tool or patter point, a receiving vial, dispersion and lysis buffer. The second element includes an anhydrous or liquid medium for hybridization between the target and the probe nucleic acid and a medium for washing and removing unhybridized and unwanted forms. The third element comprises a solid support on which an unlabeled nucleic acid probe complementary to a portion of the target nucleic acid is immobilized or bound. In the case of analyzing multiple targets, one or more capture probes specific for their respective rRNAs are applied to different individual regions of the dipstick. The fourth element contains a labeled probe that is complementary to a second, different region of the same rRNA backbone where the fixed and unlabeled nucleic acid probe of the third element hybridizes. Here, the nucleic acid probe includes a combination of the probe either in anhydrous form such as lyophilized nucleic acid or in precipitate form such as alcohol precipitated nucleic acid or as a buffer. Any known label labeled may be possible. For example, the probe can be biotinylated by conventional means and the presence of the biotinylated probe is visually noticed when the avidin bound to an enzyme such as horseradish peroxidase is added and then reacted with the peroxidase. Detection can be made by contacting with a substrate that can be monitored or monitored using a colorimeter or spectrometer. Such labeling methods and labels of other enzyme forms have the advantage of being economical, highly sensitive and relatively safe compared to radiolabeling methods. The finished product of the assay kit includes various reagents for the detection of labeled probes and those included in other kits, such as instructions, positive and negative controls, and containers for mixing and reacting the various components.

DNA chip

In addition, the nucleic acid probe of the present invention is used as a gene chip. A preferred embodiment of the present invention provides a DNA chip in which the nucleic acid probe of the present invention is immobilized on a solid support. 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 silicon, or a polymer material such as acrylic, polyethylene terephtalate (PET), polystylene, polycarbonate, polypropylene, etc. The surface of the substrate may be flat or have a plurality of holes. 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 integrated using a microarray (Yoon et al , J. Microbiol . Biotechnol ., 10:21, 2000) prepared according to a known method in a state in which the probe is dissolved in a buffer solution do. 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 optimum conditions, suboptimal conditions or limit conditions, which conditions include the temperature, the reactant concentration, the presence of substances that lower the base pair optimal temperature of the nucleic acid (eg formamide, dimethylsulfoxide and urea) and the reaction volume. And / or the presence of a substance (eg, dextran sulfate, polyethyleneglycol or phenol) to reduce and / or accelerate hybrid formation.

Probe production

Nucleic acid probes can be cloned using conventional cloning methods (Maniatis, T., et al . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1982) or chemically using commercially available DNA synthesizers. It can be synthesized and obtained in large quantities.

The probe of the present invention can be produced in single strand or double strand according to a conventional method. As a method of obtaining a single chain probe, the most representative example is a synthesis by a DMT (dimethoxytrityl) off method in an automated synthesizer, subjected to a deprotection reaction, and then a probe composed of a desired number of bases. 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. Another method is to make a probe having a nucleotide sequence complementary to the DNA of a Japanese chain, and the primer is hybridized to the DNA of the template, and the Klenow enzyme and the fluorescent substance are attached from the primer. Base (dNTP) is 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. As a method of obtaining this heavy strand probe, a genomic DNA or plasmid DNA can be cut with a specific restriction enzyme to obtain a probe containing a gene or base portion of a desired portion. 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 also be synthesized by the fluorescent probe attached, DNA cleavage (DNase) I using a DNA cleavage of the double strands, 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. The double-stranded probe is made into a Japanese chain after denaturation and used for hybridization.

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 have residues (eg, antigens or haptens) with immune properties, residues (eg, ligands) with specific affinity for some reagents, residues (eg, enzymes) that provide a detectable enzymatic reaction. , Coenzymes, enzyme substrates or substrates participating in enzymatic reactions) or in combination with residues which provide the 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.

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 the desired strain, nucleic acid is sonicated from strain 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, a 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 DNA fragments by size. The second method is to amplify and use DNA of a desired portion using a PCR method. In this case, using the PCR method, the most common PCR that amplifies the forward and reverse primer pairs using the same amount and the asymmetric addition of the forward and reverse primer pairs of the double strand and the single strand Asymmetric PCR (Asymmetric PCR) to obtain bands at the same time, Multiple amplification method (Multiplex PCR) that can be amplified at the same time by putting several primer pairs, After amplification using four specific primers and Ligase (Ligase) Ligase Chain Reaction (LCR) method to determine the amount of fluorescence by enzyme immunoassay, 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 (SACE), Competitive PCR, Short tandem repeats; STR), 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 excreta) (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, including for example passing through a CHELEX 100 column (BioRad) and the like using the QIAAMP blood kit. 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 only necessary when incubating the saliva sample prior to analysis.

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 and reverse primers at 1: 5. In this case, the primers used are part of a nucleotide sequence existing in 16S rRNA to 23S rRNA in common in bacteria (Pirkko K. et al ., Clin. Micorbiol., 36: 2205, 1999).

Primer 1-S (Sense): P-TTGTACACACCGCCCGTC (1585Fw: SEQ ID NO: 8)

Primer 1-A (antisense): Cy3-TTTCGCCTTTCCCTCACGGTACT (23BR: SEQ ID NO: 9)

Primer 2-S (sense): P-AGTACCGTGAGGGAAAGGCGAA (23BFw: SEQ ID NO: 10)

Primer 2-A (antisense): Cy3-TGCTTCTAAGCCAACATCCT (MS37R: SEQ ID NO: 11)

Primer 3-S (sense): P-AGGATGTTGGCTTAGAAGCA (MS37F: SEQ ID NO: 12)

Primer 3-A (antisense): Cy3-CCCGACAAGGAATTTCGCTACCTT (MS38R: SEQ ID NO: 13)

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 was performed in 10X PCR buffer (100 mM Tris-HCl (pH8.3), 500 mM KCl, 15 mM MgCl ₂ ) 5ul, dNTP mixture (dATP, dGTP, dCTP, dTTP 2.5mM) 4ul, 10pmole before Total volume after adding 0.5ul aromatic primer, 2.5ul 10pmole reverse primer, 1ul template DNA (100ng) diluted with 1/10, 0.5ul Taq polymerase (5unit / ul, Takara Shuzo Co., Shiga, Japan) Add water to 50ul. 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. Thereafter, the third denaturation was performed at 94 ° C for 1 minute, hybridization at 54 ° C for 1 minute, extension at 72 ° C for 1 minute, and this was repeated 30 times. Thereafter, the last extension was performed once for 5 minutes at 72 ° C. The product produced by the PCR reaction is confirmed by agarose gel electrophoresis.

Hybridization  And washing

Hybridization techniques are not specific to the present invention. This technique is generally described in Nucleic Acid Hybridization: A Practical Approach, Ed. Hames, BD and Higgins, SJ, IRL Press, 1987; Gall and Pardue, Proc . Natl . Acad . Sci ., USA ., 63: 378, 1969, John, Burnsteil and Jones, Nature , 223: 582, 1969).

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. Contraction is especially a function of the base composition of the probe / target conjugate as well as 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. The helper oligonucleotide binds to a region of the target nucleic acid other than the region bound by the 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 probe and target is formed under conditions of excess target in lithium succinate buffer solution containing lithium laurylsulfate. 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 ° C. The 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 hybrids formed and the temperature upon washing. 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 hybridization 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.

In a particular preferred embodiment of the invention, hybridization buffer (6 × SSPE (0.15 M NaCl, 5 mM C ₆ H ₅ Na ₃ O ₇ , pH 7.0), 20% (v / v) formamide) is mixed with a PCR amplification gene After dispensing the probe on a fixed glass plate it is reacted for 6 hours at 30 ℃ to induce complementary binding. After the passage of time, wash for 3 minutes in the order of 3 × SPE, 2 × SSPE, 1 × SSPE.

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.

Escherichia coli usually does not show pathogenicity in the intestine, but when it enters the non-intestine area, it causes cystitis, pyelitis, peritonitis, sepsis, etc. It is called Escherichia coli especially because it can cause infectious diarrhea from adult to adult.

The E. coli detection and identification probe of the present invention is applied to a DNA chip to detect E. coli with high specificity, thereby accurately diagnosing an infectious disease caused by E. coli, and applying a specific probe to one or more other bacteria. By doing so, two or more causative agents of infectious disease can be diagnosed at the same time.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Selection of DNA Probe Candidates to Identify Escherichia Coli

In order to detect and identify E. coli, a probe specific for E. coli was prepared. For the production of specific probes, a specific probe candidate group having specificity for E. coli was selected. The specific probe candidate group was compared by comparing the 23S rRNA sequences of Escherichia coli known to GenBank with the nucleotide sequences of the known bacteria, and distinguishing the specific sequences present only in Escherichia coli to prepare probe candidate groups present therein. . Probe candidates were determined from the 23S rRNA gene of the E. coli genome. The specificity of the probe candidate group was confirmed by comparing the similarity between the fungus by BLAST search. As a result, the selected probe candidate group is shown in Table 1 below.

E. coli specific probe candidate group Probe number Sequence designation One GTTAGCGGTAACGCG Eco001 SEQ ID NO: 1 2 GTTAGTGGAAGCGTC Eco001m SEQ ID NO: 2 3 GTGTTAGTGGAAGCGTCTGG Eco001-20 SEQ ID NO: 3 4 ATGGGGTTAGCGGTAACGCGAAGCT Eco001-25 SEQ ID NO: 4 5 ATGCACATATTGTGA Eco002 SEQ ID NO: 5 6 GTGCTGAAGCAACAAATGCC Eco003-20 SEQ ID NO: 6 7 CGGTGCTGAAGCGACAAATGCCCTG Eco003-25 SEQ ID NO: 7

Example 2: Synthesis of Nucleic Acid Probes

Each probe candidate group selected in Example 1 was synthesized for application to a DNA chip. Mononucleotide (Proligo Biochemie GmbH Hamburg Co.) was put into an automated synthesizer (Expedite ^™ 8900, PE Biosystems Co.) and the nucleotide sequence of the desired probe and 0.05μmole synthesis scale were obtained to obtain a pure nucleic acid probe. The obtained probe was confirmed whether it was synthesized by electrophoresis.

Example 3: Preparation of DNA Chip

In order to fix the DNA probe synthesized in Example 2, the bond between the aldehyde group and the amine group 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) at the 3'-first position, The slide glass was coated with an aldehyde residue (CEL Associates, Inc. Huston, Texas, USA).

To fix the probe, use a microarray ( MicroGrid spotter, Biorobotics Inc, England) with the probe dissolved in 3 × SSC (0.45 M NaCl, 15 mM C ₆ H ₅ Na ₃ O ₇ , pH 7.0) buffer solution. After spotting, the DNA probe was immobilized by inducing a chemical reaction for at least 1 hour and leaving it for at least 6 hours under the condition of maintaining humidity of about 55%. The bacteria search probe was integrated at a concentration of 100 pmole / μl at intervals of 250 to 275 μm in order to prepare a DNA chip fixed with the probe.

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).

Example 4: Isolation and Amplification of Target Samples

Genomic DNA was separately extracted from the following 59 standard strains.

59 standard strains Strain name Gram Where to get Strain name Gram Where to get Neisseria Gonohea - ATCC10150 Acinetobacter Baumani - KCTC2771 Neisseria Meninzatidis - ATCC13100 Ichenella Corrodens - ATCC51724 Legionella Pneumophila - Clinical separation Actinomyces Israel - ATCC12102 Listeria monocytogenes + ATCC700603 Unaerobiospyroom Succinsi Prodersense - ATCC29305 Morgana Morgana - ATCC25830 Aeromonas Hydrophila - KCCM32586 Bacteroides vulgarus - KCCM11423 KCCM8482 Escherichia coli - ATCC25922 Bacteroides Obatos - ATCC8483 Enterobacter Aerogenes - KCCM11783 ATCC29751 Bacteroides Tetaiotao Micron - ACTC5015 Enterobacter cloake - KCCM40044 Bacteroides fragilis - ATCC25282 Enterococcus picalis + ATCC19433 Vulcoderia Sephacia - ATCC25416 Enterococos Pesium + ATCC19434 Branhamela Qatarihalis - KCCM4006 ATCC43617 Oklobactum Antropy - ATCC49188 Vibrio Bunnpickers - KCTC2962 Cardiobacterium Hominis - ATCC14900 Vibrio Cholera - KCTC2715 Corynebacterium Diphtheria + ATCC51696 Salmonella Enteritidis - KCCM12021 Comamonas Ashdoborance - ATCC9355 Salmonella typhimurium - ooooooooo Crevciela New Monie - AATCC700603 Serratia Marsense - KCTC1299 Crevciella Oxytoka - ATCC43863 Suterella Wausworthis - ATCC51579 Chrysbacterium meningocystum - ATCC13253 Shigella Sonei - KCCM11903 Clostridium Deep Sil + ATCC9689 Shigella Flexinary - ATCC11836 Clostridium Rammo Island + ATCC25582 Pseudomonas Aruginosa - KCTC1636 Kinzela Kingap - ATCC23330 Staphylococcus aureus + KCTC1621 Peptostreptococcus magnus + ATCC29328 Staphylococco Epidermis + KCTC1917 Peptostreptococcus free-air abyss + ATCC27337 Stomatococcus musilaginos + ATCC17931 Peptostreptococos Prevoti + KCTC3319 Stronttromonas Maltopila - ATCC13637 Popiromonas Ginzy Balis + ATCC33277 Streptococcus mutans + KCCM11823 ATCC25175 Fusobacterium Necroforum - ATCC25286 Streptococcus vulidans + ATCC35037 Proteus Mirabilis - KCCM11381 Streptococcus agalactia + 000000000 Proteus Bulgari - KCCM11539 Streptococcus piogen + KCCM11817 Haemophilus apropyls - ATCC13252 Streptococcus pneumoniae + KCCM40410 Haemophilus influenza - ATCC51907 Citrobacter Freundy - ATCC51579

First, the bacteria cultured in a titration medium was suspended in 200 μl sterile distilled water, centrifuged at 14,000 rpm for 10 minutes, and then the supernatant was discarded to obtain only cells.

In the case of Gram-negative bacteria, the cells were suspended in 180 µl of ATL solution (Tissue Lysis solution, DNeasy Tissue Kit, QIAGEN), 20 µl of Proteinase K was lysed, lysed, and reacted at 55 ° C for 1 hour, followed by AL solution (Lysis). 200 μl of a solution, DNeasy Tissue Kit, QIAGEN) was added and reacted at 70 ° C. for 10 minutes, and used as a lysate solution. 200 μl of ethanol (100%) was added to the lysate solution, and the whole solution was transferred to a 2mL tube containing a DNeasy mini column. The solution collected in the lower tube was removed by centrifugation at 8,000 rpm for 1 minute. Put 500 μl of AW1 solution (Wash solution 1, DNeasy Tissue Kit, Qiagen) into the upper tube and centrifuge at 8,000 rpm or more for 1 minute to remove the effluent, and then again, AW2 solution (Wash solution 2, DNeasy Tissue Kit, QIAGEN). 500 μl was added and centrifuged at 15,000 rpm for 3 minutes to dry the DNeasy membrane, and the effluent was removed. The elution solution was added to the DNeasy column and allowed to stand at room temperature for 15 minutes, followed by centrifugation at 8,000 rpm or more for 1 minute to elute genomic DNA.

In case of Gram-positive bacteria, the cells were suspended in 180 µl of lysozyme lysate solution (20mm Tris-Cl, pH8.0, 2mM EDTA, 1.2% TritonX-100, 20mg / ml lysozyme) and reacted at 37 ° C for 30 minutes or more. , 25 μl of Proteinase K and 200 μl of AL solution (solution solution, DNeasy Tissue Kit, QIAGEN) were mixed well and reacted at 70 ° C. for 30 minutes to use as a lysis solution, and then genome separation was carried out as described above.

DNA of the isolated standard strains as a template and PCR was performed using the following primers. In order to identify DNA bound to the probe during PCR, binding was confirmed by fluorescence by amplification using a reverse primer attached to Cy3 at the 5 'end. As the primers used for the PCR reaction, the following three pairs of primers were used simultaneously.

Primer 1-S (Sense): P-TTGTACACACCGCCCGTC (1585Fw: SEQ ID NO: 8)

Primer 1-A (antisense): Cy3-TTTCGCCTTTCCCTCACGGTACT (23BR: SEQ ID NO: 9)

Primer 2-S (sense): P-AGTACCGTGAGGGAAAGGCGAA (23BFw: SEQ ID NO: 10)

Primer 2-A (antisense): Cy3-TGCTTCTAAGCCAACATCCT (MS37R: SEQ ID NO: 11)

Primer 3-S (sense): P-AGGATGTTGGCTTAGAAGCA (MS37F: SEQ ID NO: 12)

Primer 3-A (antisense): Cy3-CCCGACAAGGAATTTCGCTACCTT (MS38R: SEQ ID NO: 13)

P at the 5 'end of the sequence represents a phosphate group, Cy3 represents a Cy3 fluorescent material.

PCR reaction was performed under the following conditions. 5 μl of 10 × PCR buffer (100 mM Tris-HCl, pH 8.3), 500 mM KCl, 15 mM MgCl ₂ , 1 μl of dNTP mixture (10 mM each of dATP, dGTP, dCTP, dTTP), 1 μl of 10 pmole forward primer, 1 μl of 10 pmole reverse primer , 1 μl of template DNA (100 ng) diluted to 1/10, 0.2 μl of Taq polymerase (5 unit / μl, Solgent Co., Korea) were added, and distilled water was added to a total volume of 50 μl.

After the first denaturation at 94 DEG C for 5 minutes, 10 cycles of 50 seconds at denaturation 94 DEG C, 50 seconds at 56 DEG C, and 1 minute 10 seconds at extension 72 DEG C, were repeated 10 times. After 20 cycles of 50 seconds at 94 DEG C, 50 seconds at 58 DEG C, and 1 minute 10 seconds at 72 DEG C extension, the final extension was performed once at 5 DEG C for 5 minutes. The product produced by the PCR reaction was confirmed by the agarose gel electrophoresis (agarose gel electrophoresis) DNA double strands were synthesized for each strain. DNA obtained through PCR was purified using a PCR purification kit (QIAGEN Co.), 1 μl of lambda exonuclease (New England Biolabs Inc., Netherlands) was added and reacted at 37 ° C. for 1 hour. Strand DNA was obtained. Lambda exonucleases are enzymes that selectively cleave and digest DNA strands with phosphate groups at the 5 'end. Therefore, when PCR is performed with an omnidirectional primer synthesized by attaching a phosphate group to the 5 'end, the phosphate group is attached to the DNA strand of the PCR product. In addition, when the lambda exonuclease is treated, the strand having the phosphate group attached to the 5 'end of the amplified DNA double strand is decomposed, so that only the single strand with the Cy3 fluorescent material attached to the 5' end remains. .

Example 5: Hybridization and Washing

In order to confirm the specificity and sensitivity of the probe candidate group, a hybridization reaction was performed by applying the PCR product amplified in Example 4 to the DNA chip to which the nucleic acid probe prepared in Example 3 was immobilized. When the DNA chip in which the nucleic acid probe was immobilized was hybridized with the PCR product of the genomic gene isolated from the E. coli standard strain, the signal was tested for cross-reaction with the 58 strains other than E. coli.

The DNA plate having the nucleic acid probe fixed on the glass plate was hydrolyzed by steam, and then immersed in 70% ethanol to remove the probe that was not fixed on the glass plate. Subsequently, during the hybridization process, the glass plate was blocked with a blocking solution (1.3 g NaBH ₄ , in order to prevent the fluorescent material from adhering to the aldehyde residues remaining on the surface of the glass plate, thereby increasing the overall signal and inhibiting the signal effect of the specific probe. 375ml PBS, 125ml 100% ethanol) and then reacted for 5 minutes in a stirrer. After washing for 5 minutes with 0.2% SDS, and then washed 5 times for 1 minute with sterile water, water was removed from the glass plate using a centrifuge (1,500rpm, 3 minutes).

To hybridization solution hybridization buffer solution (hybridization buffer: 6 × SSPE; 20X SSPE; 3M NaCl, 0.2M NaH 2 PO 4 · H 2 O, 0.02M EDTA, pH 7.4, 20% (v / v) formamide; Sigma 50 μl of the DNA fragment amplified in Example 4 was prepared in a total volume of 200 μl. The prepared solution was dispensed onto the glass plate on which the probe was immobilized and then covered with a probe-clip press-seal incubation chamber (Sigma Co., St. Louis, Mo.).

To prevent drying around the glass plate during hybridization, a wet tissue was placed in the hybridization chamber, followed by reaction for at least 8 hours in a 30 ° C. static incubator to induce complementary binding. After elapse of time, 3 × SSPE (0.45M NaCl, 15 mM C ₆ H ₅ Na ₃ O ₇ , pH 7.0), then 1 × SSPE (0.15M NaCl, 5mM C ₆ H ₅ Na ₃ O ₇ , pH 7.0) After washing for 5 minutes each, the water on the glass plate was removed using a centrifuge (1,500 rpm, 3 minutes).

Example 6 Detection of Hybrid

The results of the hybridization reactions were retrieved using an ArrayWorks micro array scanner (ArrayWorks, Applied Precision, Inc., USA) and the results are shown in Table 3 below.

Name Sequence Sequence number location Featured Castle Eco001 GTTAGCGGTAACGCG One 23S No cross reaction occurs with any target DNA derived from the strains in Table 2. Eco001m GTTAGTGGAAGCGTC 2 23S No cross-reaction occurred in 56 strains except S. enterica, S. sonnei and S. typhimurium . Eco001-20 GTGTTAGTGGAAGCGTCTGG 3 23S Among the strains of Table 2, B. cepacia , B. vulgatus, C. freundii , C. hominis, C. ramosum, E. aerogenes, E. cloacea, K. pneumoniae, M. morganii, O. anthropi , P. aeruginosa, P . Cross-reaction did not occur in 38 species except mirabilis , Rothia , S. agalactiae, S. enerica, S. flexneri, S. marcescens, S. sonnei, S. typhimurium, S. wadsworthi, and V. cholera . Eco001-25 ATGGGGTTAGCGGTAACGCGAAGCT 4 23S No cross-reaction occurred in 57 strains except P. aeruginosa and P. mirabilis . Eco002 ATGCACATATTGTGA 5 23S No cross reaction occurs with any target DNA derived from the strains in Table 2. Eco003-20 GTGCTGAAGCAACAAATGCC 6 23S No cross-reaction occurred in 55 strains except for K. pneumoniae, M. morganii, S. enterica, and S. typhimurium . Eco003-25 CGGTGCTGAAGCGACAAATGCCCTG 7 23S No cross-reaction occurred in 53 strains except C. freundii, E. cloacea, K. pneumoniae, M. morganii, S. enterica, and S. typhimurim .

Example 7: Blind Test

The blind test for Escherichia coli was carried out using the DNA chip prepared in Example 3. 1A and 2A illustrate the design of a DNA chip for blind testing. The names described in these figures indicate the positions where the probes listed in Table 1 above are fixed to the glass plates. Amine 3'-AAAAAAAAAAAAAAA-5'-FITC was used as the position marker, denoted M, and a buffer in which the probe was dissolved (3X SSC) was used as a negative control, and blanked.

The sample used in this example is a sample obtained after culturing for one day from a specific site of 63 pathogenic infection patients, and the bacterial infection was confirmed by the culture method.

Genomic DNA was extracted from the cultured samples as follows. If the sample was a bodily fluid, 10 ml of the bodily fluid was collected in an EDTA tube or a plain tube. If the amount of the sample is more than 10ml centrifuged for 15 minutes at 5,000 × g, if less than 10ml was centrifuged for 15 minutes at 14,000rpm and the precipitate was collected in a 1.5ml tube. The precipitate was suspended in 180 μl of lysozyme buffer (20 mM Tris-Cl, pH 8.0, 2 mM EDTA, 1.2% Triton X-100, 20 mg / ml lysozyme), incubated at 37 ° C. for 30 minutes, and then proteinase K 20 200 μl and 200 μL of AL solution (Lysis solution, QIAamp DNA Blood Mini Kit, QIAGEN) were gently mixed and reacted at 55 ° C. for 2 hours, and then at 95 ° C. for 10 minutes. After adding 200 μl of 100% ethanol and mixing, the whole solution was transferred to a 2 ml tube containing a QIAamp spin column, and centrifuged at 8,000 rpm for 1 minute to remove the liquid collected in the tube. 500 μl of AW1 solution (Wash solution 1, QIAamp DNA Blood Mini Kit, QIAGEN) was added to the spin column, and centrifuged at 8,000 rpm for 1 minute to discard the effluent. Then, AW2 solution (Wash solution 2, QIAamp DNA Blood Mini Kit, QIAGEN) was discarded. ) 500 μl was added and centrifuged at 14,000 rpm for 1 minute to discard the effluent. The QIAamp spin column was transferred to a new 1.5 ml tube, and 300 µl of AE solution (Elution solution, DNA Blood Mini Kit, QIAGEN) was placed at room temperature for 15 minutes, followed by centrifugation at 8,000 rpm for 3 minutes to elute genomic DNA. 750 μl of 100% ethanol was added to the eluate, and the mixture was allowed to stand at −20 ° C. for 1 hour, followed by centrifugation at 14,000 rpm for 20 minutes. The supernatant of ethanol was discarded and dried.

If the sample was blood, 10 ml blood was sampled into an EDTA tube. The plasma layer was divided into 1.5 ml tubes by centrifugation at 4 ° C. and 1,800 rpm for 10 minutes, centrifuged at 14,000 rpm for 10 minutes, and the precipitate was divided into 1.5 ml tubes, followed by concentrating genomic DNA. .

Amplification, hybridization reaction, washing and detection of hybrid were carried out in the same manner as described in Example 4 and Example 5 above. The results are shown in Table 4 below, the denominator indicates the number of times the sample is applied and the molecule indicates the number of times the signal by hybridization appeared.

blood Cerebrospinal fluid Juice Phlegm credit Pee Escherichia coli 9/9 3/3 3/3 19/19 1/1 28/28

FIG. 1B and FIG. 2B show hybridization reaction results in the DNA chips of FIGS. 1A and 2B searched using Scanarray 5000 in a blind test on a sample including E. coli.

Example 8 Experiment of Antibiotic Administration on DNA Chip Sensitivity

Antibiotic sensitivity of the DNA chip prepared in Example 3 was confirmed using a sample administered with antibiotics.

E. coli (ATCC 25922) was used as a strain and ceftriaxone (CJ, Seoul, Korea) that E. coli was not resistant to antibiotics.

First, with each strain activated through two passages, a minimal inhibitory concentration (MIC) test was conducted to determine how sensitive the strain was to antibiotics. MIC testing is based on the guidelines of The Clinical and Laboratory Standards Institute (CLIS), Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement, Clinical and Laboratory Standards Institute, M100-S15, Vol. 25, No. 1, 2005). The measured E. coli MIC was 0.120 μg / ml.

 Escherichia coli was diluted with BHI medium (Becton Dickinson, USA) and inoculated in two flasks containing 100 ml of BHI medium at 20-200 CFU / ml, respectively. Antibiotics were added to the prepared medium by diluting with Endotoxin-free sterile saline (Sino-Pharma, Korea) to 0.5 MIC, 1 MIC, and 2 MIC for each strain. For example, for E. coli, 2 MIC was added with 0.240 μg / ml antibiotic, 1 MIC was added with 2 dilutions of 2 MIC, and 0.5 MIC was added with 2 dilutions of 1 MIC.

After antibiotic administration, each flask was incubated at 37 ° C. and samples were taken six times over 0, 2, 4, 6, 12 and 24 hours of culture. Sampling was carried out from the flask by 15 ml pipette and placed in a 15 ml falcon tube. 5 ml of the sample was collected and placed in BACTEC / Alert medium (Biomerieux Inc., USA), and cultured in an Automated Blood Culture System (manufacturer, manufacturer) at 35 ° C. The colonies obtained through the culture were further biochemically tested to obtain the bacteria. I identified it. 5 ml of the remaining 10ml was collected and placed in 45ml BHI medium, and cultured by shaking at 250 rpm in a shaking incubator at 37 ° C. After 6 hours, all the bacteria were collected and genomic DNA was extracted. Finally, the remaining 5ml extracted genomic DNA immediately. Genomic DNA extraction was carried out in each 50ml of the culture medium extracted in each time, centrifuged at 12000rpm, 4 degrees, 10 minutes, followed by decanting the supernatant and pellets only, using the Wizard Genomic DNA Purification kit (Promega Co.) Obtained. On the other hand, in order to check the number of cells for each sampling time, take 100μl according to the plate count method, spread 50μl directly, spread 50μl by 10-fold dilution, 100-fold dilution, etc. The culture was carried out in the incubator.

After sampling, the obtained direct DNA extract and the DNA extract obtained after 6 hours of incubation are amplified DNA by the PCR method of Example 4, and then applied to the DNA chip, and the results are cultured method of Automated Blood Culture System and The results were compared with the results of bacterial identification obtained through biochemical tests.

Table 5 summarizes the results of three experiments. In the table, Y means identified, N means unidentified, and NT means untested. EC refers to E. coli, for example, EC-0.5-02 refers to a sample taken 2 hours after the 0.5 MIC antibiotic administered to the medium containing E. coli. The chip shown at the top of the table is the result of applying the sample to the DNA chip, Cxchip is the result of incubation for 6 hours after sampling and Cx is the result of microbial identification after incubation in the Automated Blood Culture System. Means.

Efficiency by detection method according to difference in antibiotic concentration and treatment time 1st experiment 2nd experiment 3rd experiment chip Cxchip Cx CFU chip Cxchip Cx CFU chip Cxchip Cx CFU start 1610 234 EC-0.5-00 Y Y Y 900 Y Y Y 130 Y Y Y 90 EC-1-00 Y Y Y 900 Y Y Y 90 N Y Y 230 EC-2-00 Y Y Y 1600 Y Y Y 170 N Y Y 250 EC-0.5-02 Y N Y 100 Y Y Y 100 Y Y Y 10 EC-1-02 Y Y Y 100 Y Y Y 0 Y Y Y 0 EC-2-02 Y Y Y 0 Y Y Y 0 Y Y Y 0 EC-0.5-04 Y Y Y 100 Y Y Y 0 Y N Y 0 EC-1-04 Y Y Y 0 Y Y N 0 Y N N 0 EC-2-04 Y Y N 0 Y N N 0 Y Y N 0 EC-0.5-06 Y NT Y 400 Y Y Y 0 Y Y Y 0 EC-1-06 N Y N 0 Y N N 0 Y Y N 0 EC-2-06 Y Y Y 0 Y Y N 0 Y Y N 0 EC-0.5-12 Y Y Y 10700 Y Y N 0 Y Y Y 110 EC-1-12 Y Y N 0 Y Y N 0 Y N N 0 EC-2-12 Y Y N 0 Y Y N 0 N N N 0 EC-0.5-24 N N Y 4.44E + 11 Y Y N 0 Y N Y 7.59E + 04 EC-1-24 Y Y N 0 Y Y N 0 Y Y N 0 EC-2-24 Y Y N 0 Y Y N 0 Y Y N 0

 Through the above experiments, the DNA chip consisting of the probe selected in the present invention was less sensitive to antibiotic administration than the conventional culture test results, and more accurate identification results could be obtained.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

The present invention provides an E. coli DNA chip for detecting and identifying E. coli and a probe for detecting and identifying E. coli consisting of the oligonucleotides. It works.

By using the DNA chip according to the present invention, not only can the bacterial infection be diagnosed more quickly and accurately than the method of diagnosing an infectious disease by sample culture, which is currently used, but also is not affected by the administration of antibiotics used in the clinic. Therefore, more accurate diagnosis can be made.

<110> MEDIGENES.Co., Ltd <120> DNA Chip for Detection of Escherichia coli <130> P05-B368 <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 1 gttagcggta acgcg 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 2 gttagtggaa gcgtc 15 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 3 gtgttagtgg aagcgtctgg 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 4 atggggttag cggtaacgcg aagct 25 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 5 atgcacatat tgtga 15 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 gtgctgaagc aacaaatgcc 20 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 7 cggtgctgaa gcgacaaatg ccctg 25 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttgtacacac cgcccgtc 18 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tttcgccttt ccctcacggt act 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 agtaccgtga gggaaaggcg aa 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgcttctaag ccaacatcct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aggatgttgg cttagaagca 20 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cccgacaagg aatttcgcta cctt 24

Claims (2)

E. coli detection and identification DNA chip characterized in that the oligonucleotide having the following nucleotide sequence is fixed: GTTAGCGGTAACGCG (SEQ ID NO: 1); GTTAGTGGAAGCGTC (SEQ ID NO: 2); GTGTTAGTGGAAGCGTCTGG (SEQ ID NO: 3); ATGGGGTTAGCGGTAACGCGAAGCT (SEQ ID NO: 4); ATGCACATATTGTGA (SEQ ID NO: 5); GTGCTGAAGCAACAAATGCC (SEQ ID NO: 6); And CGGTGCTGAAGCGACAAATGCCCTG (SEQ ID NO: 7). E. coli detection and identification probe comprising one or more oligonucleotides selected from the group consisting of oligonucleotides having the following nucleotide sequence: GTTAGCGGTAACGCG (SEQ ID NO: 1); GTTAGTGGAAGCGTC (SEQ ID NO: 2); GTGTTAGTGGAAGCGTCTGG (SEQ ID NO: 3); ATGGGGTTAGCGGTAACGCGAAGCT (SEQ ID NO: 4); ATGCACATATTGTGA (SEQ ID NO: 5); GTGCTGAAGCAACAAATGCC (SEQ ID NO: 6); And CGGTGCTGAAGCGACAAATGCCCTG (SEQ ID NO: 7).

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