KR100532664B1 - Probe for detecting chlamydia trachomatis, detection kit and detection method for chlamydia trachomatis using the same - Google Patents

Abstract

The present invention relates to a kit for detecting Chlamydia trachomatis, a detection method, and probes, primers, and DNA chips used therein. Kits and methods for detecting Chlamydia trachomatis using these probes or DNA chips are faster, easier and more accurate than the culture assays or antigen-antibody assays.

Description

 Chlamydia trachomatis detection probe, Chlamydia trachomatis detection kit including the same, and Chlamydia trachomatis detection method using the same

[Technical Field]

The present invention relates to a detection kit and detection method for detecting Chlamydia trachomatis infection, and probes and DNA chips used therein. Using such DNA chips, kits and methods for diagnosing infection of Chlamydia trachomatis are provided.

[Private Technology]

Chlamydia trachomatis (Chlamydia trachomatis) is a spherical bacterium having a size of 0.2 ~ 1.5 m. Chlamydia trachomatis is not infected by insects, but by human contact or respiratory infections. In particular, it is one of the most serious bacteria causing sexually transmitted diseases along with Neisseria gonorrhoeae .

 Chlamydia infection causes the EB to form a Reticulate Body (RB) after the Elementary Body (EB) invades the host cell. Thereafter, the RB splits several times and in the process reproduces the EB. This regenerated EB can migrate to other cells in the process of breaking the host cell, and the intercellular infection spreads.

Chlamydia genus includes Chlamydia psittaci , Chlamydia pneumoniae , Chlamydia pecorum in addition to Chlamydia trachomatis. Among them, bacteria that affect humans are Chlamydia trachomatis and Chlamydia pneumoniae. Among them, Chlamydia trachomatis is a bacterium that is related to human sexually transmitted diseases.

Chlamydia trachomatis is divided into at least 15 serotypes. The criteria is to distinguish Chlamydia trachomatis major Outer Membrane Protein (MOMP) by antigen-antibody response ( Infection and Immunity, 66 (8), 1998). , 3618-3625 ). In addition, the diseases caused by the serotypes are different, serotypes A, B, and C cause ocular trachoma, and serotypes D, E, F, G, H, I, J, and K. It causes urethritis, epididymitis, cervicitis, salpingitis, and so on. In addition, serotypes L1, L2, and L3 cause STIs. In large division, serotypes A to K are infected with epidermal cells of the mucosa, and serotypes L1, L2, and L3 proliferate in lymphoid tissues or cause systemic infection (Journal of Clinical Microbiology, 129). 6), 1991, 1132-1136).

 Chlamydia trachomatis infection is one of the most common sexually transmitted infections in the world, and more than 50% of non-acute infections without subjective symptoms must be tested. If found early, the treatment is easy, but if the treatment is delayed, complications such as pelvic inflammatory disease and infertility can be caused.

Currently, the most accurate method for the detection of Chlamydia trachomatis is the method of isolation and culture by cell culture. However, this method is not suitable for testing many Chlamydia trachomatis samples because of the difficult testing methods and excessive time and cost.

As a method of detecting Chlamydia trachomatis without cell culture, Kimja staining method is suitable for eye infection only, and it is not suitable when the sample is urethra or cervix.

In addition, direct fluorescent antibodies against major Outer Membrane Protein (MOMP) antigens have the advantage of being rapid in detecting Chlamydia trachomatis, but require much experience and skill in observation (STI Guidelines 2002, National Institutes of Health).

An object of the present invention does not require a separate specific antibody to Chlamydia trachomatis and can more accurately detect Chlamydia trachomatis infection with high specificity and sensitivity quickly and easily without sequencing To provide a probe for detecting teeth.

Another object of the present invention is to provide a primer for amplifying chlamydia trachomatis to more accurately detect whether Chlamydia trachomatis is infected.

Another object of the present invention to provide a Chlamydia trachomatis detection DNA chip comprising the Chlamydia trachomatis detection probe.

Another object of the present invention to provide a Chlamydia trachomatis detection kit comprising the Chlamydia trachomatis detection probe.

Another object of the present invention is to provide a Chlamydia trachomatis detection method using the Chlamydia trachomatis detection probe.

The present invention is SEQ ID. NO: 1 oligonucleotide having a nucleotide sequence of SEQ ID NO: 11, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides, and selected from the group consisting of DNA of Chlamydia trachomatis ( Chlamydia trachomatis ) It relates to a probe that binds complementarily.

The invention also relates to SEQ ID. Chlamydia trachomatis, comprising one or more probes selected from the group consisting of an oligonucleotide having a nucleotide sequence of NO: 1 to a nucleotide sequence of SEQ ID NO: 11, and an oligonucleotide having a nucleotide sequence complementary to these oligonucleotides. It relates to a DNA chip for detection.

The present invention also provides (a) a DNA chip for detecting Chlamydia trachomatis, (b) a primer for amplifying the sample DNA of Chlamydia trachomatis by PCR, and (c) detecting amplified DNA hybridized with the DNA chip. It relates to a kit for detecting Chlamydia trachomatis comprising labeling means for.

The present invention also comprises the steps of (a) amplifying Chlamydia trachomatis sample DNA using a primer for DNA amplification of Chlamydia trachomatis;

(b) the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO. Hybridizing the amplified DNA to a DNA chip comprising a single probe or two or more probe sets selected from the group consisting of 12 nucleotide sequences;

(c) a method for detecting Chlamydia trachomatis, comprising detecting a hybridized reactant with the probe.

The present invention also comprises the steps of (a) amplifying the sample DNA of Chlamydia trachomatis using a primer for DNA amplification of Chlamydia trachomatis;

(b) hybridizing the amplified DNA with a single probe or a set of two or more probes selected from the group consisting of a nucleotide sequence of SEQ ID NO: 1 to a nucleotide sequence of SEQ ID NO: 12; And

(c) a method for detecting Chlamydia trachomatis, comprising detecting a hybridized reactant with the probe.

The present invention also relates to a primer for amplifying Chlamydia trachomatis having a nucleotide sequence of SEQ ID NO: 14 to a nucleotide sequence of SEQ ID NO: 16.

The present inventors have tried to establish a method for analyzing chlamydia trachomatis by analyzing a patient's sample by a simple method, and have developed a probe capable of complementarily binding to the DNA of chlamydia trachomatis, and a DNA chip comprising the same. It was. In addition, a DNA chip comprising a probe capable of complementarily binding to the DNA of Chlamydia trachomatis, a primer for amplifying the DNA taken from the sample by PCR method and detecting the amplified DNA hybridized with the DNA chip Chlamydia trachomatis analysis kit comprising a labeling means for producing, and by analyzing the DNA collected from the patient's sample using the analysis kit, it was confirmed that the infection of Chlamydia trachomatis can be analyzed quickly and accurately, The present invention has been completed.

Chlamydia trachomatis detection method of the present invention has the advantage that can be derived faster, easier and more accurate than the culture test or antigen-antibody test.

 Hereinafter, the present invention will be described in detail.

The present invention relates to a probe that complementarily binds to DNA of Chlamydia trachomatis , preferably SEQ ID. NO: 1 to an oligonucleotide having a nucleotide sequence of SEQ ID NO: 11, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides. The probe is hybridizable with DNA or RNA of Chlamydia trachomatis.

Chlamydia trachomatis DNA, a target of PCR, is an omp1 gene that expresses Major Outer Membrane Protein (MOMP). After PCR amplification of this gene, the presence or absence of chlamydia trachomatis can be observed depending on the DNA amplification. In particular, since Major Outer Membrane Protein is a protein that forms a direct correlation between Chlamydia trachomatis cells and host cells, it is a protein that can be decisive evidence in determining whether Chlamydia trachomatis is detected. Therefore, testing the omp1 gene expressing MOMP is a valid method for the detection of Chlamydia trachomatis.

The present invention provides a nucleotide sequence of Chlamydia trachomatis DNA, preferably a nucleotide sequence complementarily binding to the omp1 DNA of Chlamydia trachomatis as a probe. Probes of the invention comprise the nucleotide sequences set forth in SEQ ID NOS: 1-11. In Table 1 below, the underlined portions of the sequences in SEQ ID NOs: 5, 6, 10, 11 are sequences added to provide a suitable form for probe function.

Seq ID No. designation Sequence Length One CT II 1-1 5'-CCT CTT GAT CTT ACA GCA GGA ACA GA-3 ' 26 mer 2 CT II 1-2 5'-CTT GAT CTT ACA GCA GGA ACA GAT G-3 ' 25 mer 3 CT II 1-3 5'-CCT CTT GAT CTT ACA GCA GGA ACA GAT G-3 ' 28 mer 4 CT II 2-1 5'-CTT GAT ATT ACC GCA GGA ACA GAA G-3 ' 25 mer 5 CT II 2-2 5'- CTT CT T TGA TAT TAC CGC AGG AAC AGA AG-3 ' 29 mer 6 CT II 2-3 5'- CTT CTT GAT ATT ACC GCA GGA ACA GAA G-3 ' 28 mer 7 CT II 3-1 5'-CCT CTT GCA CTC AYA GCA GGA ACT GAT G-3 ' 28 mer 8 CT II 3-2 5'-CTT GCA CTC AYA GCA GGA ACT GAT G-3 ' 25 mer 9 CT III 1 5'-GAC RGG VAC TAA RGA TGC CTC TAT TGA-3 ' 27 mer 10 CT III 2 5'-GAC RGG VAC TAA RGA TGC CTC TAT TGA TGT C -3 ' 31 mer 11 CT III 3 5'-GAC RGG VAC TAA RGA TGC CTC TAT TGA GTC -3 ' 30 mer 12 BG probe 5'-TGC ACC TGA CTC CTG AGG AGA AGT CTG CCG-3 ' 30 mer 13 Negative 5'-GGT AAA AAT CGA AGG ATA WNR TCA ACC-3 ' 27 mer

R (A, G): Y (C, T): M (A, C): K (G, T): S (G, C): W (A, T), V (A, C) , G): H (A, T, C): B (G, T, C): D (G, A, T): N (A, G, C, T), which means It is widely known to experts.

The probe of the present invention can be used to analyze the presence of chlamydia trachomatis with high specificity and sensitivity, and to quickly test for chlamydia trachomatis.

 The Chlamydia trachomatis analysis kit invented so far has a long experiment time, is expensive to experiment, or difficult to test, but in this experiment, the DNA chip is used based on PCR. Quick and accurate experimental results can be derived.

In another aspect, the present invention is a microarray, preferably a DNA chip containing the oligonucleotide probe of Chlamydia trachomas. In one embodiment of the invention, the DNA chip also includes a DNA chip comprising all probe sequence sets that can be readily combined by one of ordinary skill in the art. DNA chip according to the present invention is SEQ ID. At least one probe selected from the group consisting of an oligonucleotide having a nucleotide sequence of NO: 1 to a nucleotide sequence of SEQ ID NO: 11, and an oligonucleotide having a nucleotide sequence complementary to these oligonucleotides.

In addition, the DNA chip of the present invention, in addition to the probe for Chlamydia trachomas, Neisseria gonorrhoeae , Syphilis ( Treponema pallidum ), Trichomonas vaginalis , Mycobacterium tuberculosis , Human Papillomavirus ( Human Papillomavirus ) DNA probes capable of detecting Herpes Simplex virus can be fixed to one slide at the same time to produce one DNA chip. An example of such various DNA chip formats is shown in FIG.

When immobilized DNA probes of various bacteria or viruses on a slide, it is easy to detect which bacteria or viruses are present in a sample in a single experiment. In addition, when the DNA probes of various bacteria and viruses, in addition to the above-mentioned bacteria or viruses, are placed on a single slide, there is an advantage in that the sample can be accurately analyzed in a single experiment.

The DNA chip may further comprise a negative control probe, a preferred example of which is shown in SEQ ID NO: 13 in the Sequence Listing.

In addition, the DNA chip may further include a reference marker as a control for the global position marker and the hybridization reaction. Examples of reference markers may further include β-globin, actin, or glyceraldehyde-3-phosphate dehydrogenase genes as reference markers. When the reference marker is β-globin, preferably has the nucleotide sequence shown in SEQ ID NO: 12, and a primer for amplification thereof may include the nucleotide sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18. It may be a primer for amplifying β-globin DNA having.

 On the other hand, the method of manufacturing a DNA chip included in the detection kit for Chlamydia trachomatis comprises the steps of preparing a DNA probe in which the amine is bound to the 5 'end of the base sequence that can complementarily bind to the DNA of Chlamydia trachomatis; Binding the prepared DNA probe to the surface of the solid to which the aldehyde is bound; And reducing aldehydes not bound to the amine present on the solid surface to which the DNA probe is bound.

The binding of the probe DNA to the aldehyde on the surface of the solid is carried out by a method known in the art, that is, through the Schiffs base reaction of amines and aldehydes at temperatures of 30-40 ° C. and humidity conditions of 70-100%. Can be. Reduction of the aldehyde may be carried out using a reducing agent such as NaBH ₄ . The concentration of probe DNA reacting with aldehyde present on the surface of the solid is preferably 100-300 pmole / l. When used in other ranges, the probe DNA may not bind to the aldehyde on a solid surface.

The solid may be selected from glass, silicon dioxide, plastic or ceramic.

Hereinafter, the DNA chip of the present invention and its manufacturing method will be described in more detail by dividing the process.

 First Step: Probe Preparation

A DNA probe having an amine bound to five ends of a nucleotide sequence capable of complementarily binding to DNA of Chlamydia trachomatis is prepared. In this case, the probe is designed and synthesized to have a nucleotide sequence complementary to the DNA of Chlamydia trachomatis, preferably the omp1 DNA of Chlamydia trachomatis, so that the synthesized base sequence can be bound to the surface of the solid. A probe is prepared by binding an amine group to the 5 'end of the nucleotide sequence.

Step 2: Immobilize the Probe

The prepared DNA probe is bound to the surface of the aldehyde-bound solid: the solid is not particularly limited, but is preferably glass, and the aldehyde of the solid surface is a method known in the art, that is, the 5 'end of the probe. The probe is bound to a solid surface by carrying out a Schiffs base reaction at 30-40 ° C. and 70-100% humidity with an amine bound to the amine. At this time, the concentration of the probe is preferably 100 to 300 pmole / l, most preferably 200 pmole / l. It also attaches β-globin to the surface of the solid, which serves as a control for global position markers and hybridization reactions.

Third Step: Manufacture of DNA Chip

A DNA chip is prepared by reducing aldehydes not bound to the amine present on the solid surface to which the probe is bound. At this time, the reducing conditions of the aldehyde is not particularly limited, it is preferable to use a reducing agent such as NaBH ₄ .

The present invention relates to a kit for detecting Chlamydia trachomatis, preferably one or more probes selected from the group consisting of oligonucleotide probes having the nucleotide sequence of SEQ ID NO: 1 to 11 or complementary thereto It contains a DNA chip containing. In a specific example of the invention,

(a) a DNA chip for detecting Chlamydia trachomatis, (b) a primer for amplifying the sample DNA of Chlamydia trachomatis by PCR, and (c) a labeling means for detecting amplified DNA hybridized with the DNA chip. It provides a kit for the detection of Chlamydia trachomatis comprising a.

The label means may be used in the present invention a variety of labels known to those skilled in the art, for example Cy5, Cy3, biotin-binding material, EDANS (5- (2'-aminoethyl) amino- 1-naphthalene sulfuric acid), tetramethyltamine (TMR), tetramethyltamine isocyanate (TMRITC), x-rhodamine or Texas red can be used. The labeling means may add a label gene for Chlamydia trachomatis to label the gene during amplification and label amplification using a PCR reaction. In addition, it can be labeled using a biotintin-binding material which is a general labeling means. The biotin-binding material includes, for example, streptavidin-R-phycoerythrin. In case of labeling using Cy5, the fluorescent signal can be analyzed directly using an analyzer such as a confocal laser scanner when detecting the labeled reactant, which is more efficient and compared to labeling using biotin-binding material. More sensitive results can be obtained.

The primer may be a primer for Chlamydia trachomatis amplification having a nucleotide sequence of SEQ ID NO: 14 to a nucleotide sequence of SEQ ID NO: 16. The sequences of the primers are shown in Table 2 below.

PCR primers Seq ID No.  designation Sequence Length 14 CT 1 5'-TTA GGA GCT TCT TTC CAA TA-3 ' 20 mer 15 CT 2-1 5'-TAA ACT TGC TTG CCA YTC AT-3 ' 20 mer 16 CT 2-2 5'-GGA GTG AAC ATA TTY ART CT-3 ' 20 mer 17 BG I 5'-ATA CAA GTC AGG GCA GAG-3 ' 18 mer 18 BG II 5'-CTT AAA CCT GTC TTG TAA CC-3 ' 20 mer

In the table, R (A, g): Y (C, T): M (A, C): K (g, T): S (g, C): W (A, T), V (A, C , g): H (A, T, C): B (g, T, C): D (g, A, T): N (A, g, C, T), which means It is widely known to experts.

The present invention also relates to a method for detecting Chlamydia trachomatis using the probe and amplification primers of Chlamydia trachomatis.

In one example of the present invention, a) amplifying the sample DNA of Chlamydia trachomatis using a primer for DNA amplification of Chlamydia trachomatis;

(b) hybridizing the amplified DNA with a single probe or a set of two or more probes selected from the group consisting of a nucleotide sequence of SEQ ID NO: 1 to a nucleotide sequence of SEQ ID NO: 12; And

(c) providing a method for detecting Chlamydia trachomatis, comprising detecting a hybridized reaction with the probe.

In another example of the invention,

(a) amplifying chlamydia trachomatis sample DNA using a primer for amplifying the DNA of chlamydia trachomatis;

(b) the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO. Hybridizing the amplified DNA to a DNA chip comprising a single probe or two or more probe sets selected from the group consisting of 12 nucleotide sequences; And

(c) providing a method for detecting Chlamydia trachomatis, comprising detecting a hybridized reaction with the probe.

The primer is preferably selected from the group consisting of Chlamydia trachomatis amplification primers having base sequences of SEQ ID NO: 14 to SEQ ID NO: 16, examples of which are shown in Table 2 above.

In addition, the present invention may use a microarray, preferably a DNA chip containing the oligonucleotide probe of Chlamydia trachomas, wherein the DNA chip is β-globin, actin, or glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) gene may be further included as a position marker, and the position marker may be used in the DNA chip.

The detection can be performed using a labeling means, and the labeling means listed above can be used.

In one example of the invention, amplification of the sample DNA can be carried out by a conventional PCR reaction. In other words, the sample DNA can be amplified by a PCR reaction consisting of total denaturation, denaturation, primer binding, and chain extension.

In a preferred embodiment of the present invention, the Chlamydia trachomatis primer and β-globin primer, dATP, dCTP, dGTP, dTTP, labeling agent, and Taq polymerase, and optionally, by using BSA and DMSO as an additive Place in a thermocycler, denature for 5 minutes at 94 ° C, denature for 1 minute at 94 ° C, primer annealing at 45-60 ° C for 1 minute, and extend 20-45 seconds at 72 ° C. extension) was repeated 30 to 35 times, followed by further extension reaction at 72 ° C. for 2 minutes to obtain an amplified DNA sample bound with a labeled molecule.

Hereinafter, the Chlamydia trachomatis detection method using the analysis kit of Chlamydia trachomatis of the present invention will be described in more detail by dividing step by step.

Step 1: Obtain and Amplify the Sample

Amplify the sample to be tested to obtain an amplified sample containing CY5 (Cyanine 5): At this time, an amplified sample is obtained by PCR using CY5-dUTP so that the amplified sample contains CY5.

Second Step: Hybridization Reaction

The amplification sample is applied to a DNA chip to perform a hybridization reaction with a probe.

Third Process: Detection

The probe and the hybridized DNA are labeled with a labeling means and detected to determine whether Chlamydia trachomatis is infected. The detection method is performed according to the detection method of each display means, for example, can be read by scanning with a confocal laser scanner as the detection means.

Therefore, using the analysis kit of Chlamydia trachomatis using the DNA chip of the present invention, it is possible to quickly and simply detect the presence of Chlamydia trachomatis in the sample, such as urethritis, cervicitis, etc. The spread of a sexually transmitted infection can be suppressed.

Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments. 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 by these examples in accordance with the gist of the present invention. . In particular, since the type and number of probes are not particularly limited, DNA chips using probes having nucleotide sequences derived from Chlamydia trachomatis and various analysis kits using the DNA chips are also within the scope of the present invention.

Example 1 Preparation of Probes

 Each probe used for detecting Chlamydia trachomatis and a probe specific for β-globin were prepared to include an amine group at the 5 'end. The base sequence of each probe is shown in SEQ ID NO: 1 to 11 in Sequence Listing.

Example 2: Preparation of DNA Chips

Each probe prepared in Example 1 was dissolved in 3X SSC (0.05 M sodium citrate, 0.45 M NaCl, pH 7.0) at 200 pmole / l, and an aldehyde-linked slide (silylated slide, CSS-100, CEL, Houston, TX, USA) Using the arrayer (GMS 417 Arrayer, TaKaRa, Japan) on the surface, spotting at 150m size and 300m spacing, and then sieving for 4 hours at 37 ℃ and 70% humidity. Schiff base reaction was performed. The slides were then washed with 0.2% (w / v) sodium dodecyl sulfate (SDS) solution, washed with tertiary distilled water, and then washed with NaBH ₄ solution (0.1 g NaBH ₄ , 30 ml phosphate buffered saline). PBS), immersed in 10 ml ethanol) for 5 minutes to reduce the amine (Amine) bound aldehyde, washed with tertiary distilled water, and dried to prepare a DNA chip.

Also attached to the surface of the solid was β-globin, which serves as a control for global position markers and hybridization reactions.

Example 3: Optimal PCR Amplification Reaction (Chlamydia Trachomatis Amplification)

Method 1)

After denaturation at 94 ℃ for 5 minutes, denaturation at 94 ℃ for 1 minute, primer annealing at 45 ~ 60 ℃ for 1 minute, extension at 72 ℃ for 30 seconds 30 ~ 35 times After repeating, the reaction was further extended at 72 ° C. for 2 minutes to obtain an amplified DNA sample having Cy5 bound thereto. The result is shown in detail in FIG. 1.

Method 2)

Except for performing primer binding (primer annealing) for 1 minute at 45 ~ 60 ℃ was carried out in the same manner as in Method 1 to obtain an amplified DNA sample with Cy5 binding.

Example 4 Optimal PCR Amplification Reaction ( β -globin Amplification)

Method 1)

After denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 1 minute, primer annealing at 50 ° C for 2 minutes, extension at 72 ° C for 30 seconds, and then repeated at 5 ° C at 94 ° C. 1 minute denaturation, primer annealing for 2 minutes at 50 ° C, extension for 15 seconds at 72 ° C for 35 minutes, followed by extension for 2 minutes at 72 ° C Amplified DNA samples were obtained. The result is shown in detail in FIG.

Method 2)

Except for performing primer binding (primer annealing) for 30 seconds at 50 ℃ was carried out in the same manner as in Method 1 to obtain an amplified DNA sample conjugated with Cy5.

Example 5 Obtaining a Sample

To determine whether human cervical swabs were infected with Chlamydia trachomatis, DNA was first extracted from the tissue and purified. Using the purified DNA as a template, PCR is performed using a β-globin primer having a nucleotide sequence shown in SEQ ID NOs: 17 and 18, and the DNA amplified by the β-globin gene is selected and searched. Used as a sample. Amplified β-globin DNA is shown in FIG. 2. Chlamydia trachomatis positive standard strain samples and clinical samples were received from the National Institutes of Health, and the DNA samples obtained from them were used for PCR and DNA chip experiments. PCR amplification experiments were performed using the extracted Chlamydia trachomatis DNA using the primers shown in SEQ ID NOS: 14-16.

Amplified Chlamydia trachomatis DNA is shown in FIG. 1. 1 is a PCR amplified picture of Chlamydia trachomatis DNA using primers CT1 and CT2-1, and primers CT1 and CT2-2. Each lane is as follows. 1: Chlamydia trachomatis clinical sample DNA amplification photograph (CT1 ~ CT2-1), 2: Chlamydia trachomatis clinical sample DNA amplification photograph (CT1 ~ CT2-2), 3: Chlamydia trachomatis clinical sample DNA amplification photograph (CT1 ~ CT2) -1), 4: Chlamydia trachomatis clinical sample DNA amplification photograph (CT1-CT2-2), 5: Chlamydia trachomatis positive standard strain DNA amplification photograph (CT1-CT2-1), 6: Chlamydia trachomatis positive standard strain DNA Amplified photograph (CT1 ~ CT2-2).

5-1: Obtaining Chlamydia trachomatis positive standard strain sample

Using the purified Chlamydia trachomatis DNA as a template, PCR was performed using primers of SEQ ID NO: 14 to SEQ ID NO: 16 as follows: 1 x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 0.1 μg DNA, 3 mM MgCl ₂ , each 10 pmol primer, 50 ° C. each dATP, dCTP, dGTP, dTTP (Pharmacia), 0.05 nM Cy5-dUTP (NEN, USA) and 2 unit Taq polymerase ( 50 μl of the reaction mixture containing TaKaRa, Japan) was placed in a PCR thermocycler (Perkin-Elmer Cetus, Calif., USA), denatured at 94 ° C. for 5 minutes, and denatured at 94 ° C. for 1 minute. denaturation, primer annealing at 50 ° C. for 1 minute, extension for 30 seconds at 72 ° C. for 30 minutes, and then extension reaction at 72 ° C. for 2 minutes to obtain amplified DNA sample with Cy5 binding. Obtained.

5-2: Obtaining Chlamydia Trachomatis Clinical Sample

Using the clinical sample DNA as a template, using the nucleotide sequence shown in SEQ ID NO: 14 to 16 as a primer, an amplified DNA sample conjugated with Cy5 was obtained in the same manner as in Example 5-1.

5-3: Obtaining β -globin Sample

Using the clinical sample DNA as a template and using a primer having the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, an amplified DNA sample conjugated with Cy5 was obtained in the same manner as in Example 5-1.

Example 6: Analysis of Standard Strains Using DNA Chips

By applying the amplified sample obtained in Example 5-1 to the DNA chip prepared in Example 2, the hybridization reaction was carried out as follows. In this case, a 100 μl cover slip (GRACE Bio-Labs, USA, PC4L-1.0) was used as a hybridization reaction chamber.

In the case of Chlamydia trachomatis DNA, a mixture of 10 μl of amplified product and 5 μl of β-amplified product was used as a reaction sample. 10% (v / v) of 3N sodium hydroxide (NaOH) aqueous solution was added to the reaction sample, and denatured at room temperature for 5 minutes, and neutralized by adding 5% (v / v) of 1M Tris-HCl (pH 7.2). Thereafter, 10% (v / v) of 3N hydrochloric acid was added to the resultant, and the resultant was allowed to stand on ice for 5 minutes. Then, silylation at 40 ° C using hybridization solution (6X Saline-Sodium Phosphate-EDTA buffer (SSPE, Sigma Chemical Co., St. Louis, MO, USA) and 0.2% sodium dodecyl sulfate (SDS)) The reaction was fixed with a probe fixed on a silylated slide for 2 hours, the DNA chip was washed for 2 minutes with 3X SSPE and 2 minutes with 1X SSPE, and then air dried at room temperature or dried using a spin dryer.

The fluorescent signal was analyzed using a confocal laser scanner (GSI Lumonics, Germany) (excitation 650 nm, emission 668 nm). The results are shown in FIGS. 4 to 5.

Figure 3 is a schematic diagram showing the type and location of the probes located on the DNA chip, the number denotes each probe, (○) represents the site to which the probe can bind to the sample, contained in (○) The letters indicate the names of the probes. The analysis results are shown well in FIGS. 4 and 5. Figure 4 shows the result of amplifying the positive standard strain DNA using primers CT1 ~ CT2-1, DNA chip test, in the case of Figure 5 after amplifying the positive standard strain DNA using primers CT1 and CT2-1 DNA chip test results. In each case, we compared the probe's ability to detect Chlamydia trachomatis on a DNA chip. Six probes, CTIII-1, CTIII-2, CTIII-3, CTIII1, CTIII2, and CTIII3, showed excellent detection effects.

Example 7: Analysis of Clinical Samples Using DNA Chips

The experiment was conducted in substantially the same manner as in Example 6, except that the amplified DNA of the clinical sample obtained in Example 5-2 was applied to the DNA chip prepared in Example 2, and the results are shown in FIG. 7 and FIG. 8.

Figure 7 shows the results of amplifying the clinical sample DNA using primers CT1 ~ CT2-1, DNA chip test, in the case of Figure 8 after amplifying the clinical sample DNA using primers CT1 and CT2-1, DNA This is the result of a chip test. In each case, we compared the probe's ability to detect Chlamydia trachomatis on a DNA chip.

The present invention provides a detection kit and detection method for detecting Chlamydia trachomatis infection, and a probe and a DNA chip used therein, which are faster, easier and more accurate than the culture test or the antigen-antibody test. It has the advantage of providing a detection method for Chlamydia trachomatis.

1 is a PCR amplified photograph of Chlamydia trachomatis DNA using primers CT1 and CT2-1, and primers CT1 and CT2-2.

2 shows the results of PCR amplification of β-globin using primers BGI and BGII.

Figure 3 is a schematic diagram showing the type and position of the probe located in the DNA chip according to an example of the present invention.

4 is a result of PCR amplifying DNA of Chlamydia trachomatis positive standard strain using primers CT1 and CT2-1, and then hybridizing the DNA chip to DNA chips.

5 is a result of PCR amplifying DNA of Chlamydia trachomatis positive standard strain using primers CT1 and CT2-2, and then hybridizing the DNA chip to the DNA chip.

FIG. 6 shows Chlamydia trachomatis, Neisseria gonorrhoeae , Treponema pallidum , Trichomonas vaginalis , Mycobacterium tuberculosis , Human Papillomavirus, Herpes Simplex virus An example of the DNA chip format including all of the DNA probes capable of detecting the back is shown.

Figure 7 shows the result of PCR amplification of the DNA of Chlamydia trachomatis clinical sample using primers CT1 and CT2-1, and then hybridization reaction to the DNA chip.

FIG. 8 shows the results of PCR amplification of the DAN of Chlamydia trachomatis clinical sample using primers CT1 and CT2-2, followed by hybridization reaction to DNA chips.

<110> BIOMEDLAB <120> PROBE FOR DETECTING CHLAMYDIA TRACHOMATIS, DETECTION KIT AND DETECTION METHOD FOR CHLAMYDIA TRACHOMATIS USING THE SAME <130> DPP2002-1790 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CT II 1-1 probe <400> 1 cctcttgatc ttacagcagg aacaga 26 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CT II 1-2 Probe <400> 2 cttgatctta cagcaggaac agatg 25 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CT II 1-3 Probe <400> 3 cctcttgatc ttacagcagg aacagatg 28 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CT II 2-1 Probe <400> 4 cttgatatta ccgcaggaac agaag 25 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CT II 2-2 Probe <400> 5 cttctttgat attaccgcag gaacagaag 29 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CT II 2-3 Probe <400> 6 cttcttgata ttaccgcagg aacagaag 28 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CT II 3-1 Probe <400> 7 cctcttgcac tcayagcagg aactgatg 28 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CT II 3-2 Probe <400> 8 cttgcactca yagcaggaac tgatg 25 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CT III 1 Probe <400> 9 gacrggvact aargatgcct ctattga 27 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CT III 2 Probe <400> 10 gacrggvact aargatgcct ctattgatgt c 31 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CT III 3 <400> 11 gacrggvact aargatgcct ctattgagtc 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Beta-globin Probe <400> 12 tgcacctgac tcctgaggag aagtctgccg 30 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe for negative control <400> 13 ggtaaaaatc gaaggatawn rtcaacc 27 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CT I Primer <400> 14 ttaggagctt ctttccaata 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CT 2-1 Primer <400> 15 taaacttgct tgccaytcat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CT 2-2 Primer <400> 16 ggagtgaaca tattyartct 20 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BG I Primer <400> 17 atacaagtca gggcagag 18 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BG II Primer <400> 18 cttaaacctg tcttgtaacc 20

Claims (14)

SEQ ID. NO: 1 oligonucleotide having a nucleotide sequence of SEQ ID NO: 11, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides, and selected from the group consisting of a nucleic acid of Chlamydia trachomatis ( Chlamydia trachomatis ) Complementary binding probes. SEQ ID. Chlamydia trachomatis, comprising one or more probes selected from the group consisting of an oligonucleotide having a nucleotide sequence of NO: 1 to a nucleotide sequence of SEQ ID NO: 11, and an oligonucleotide having a nucleotide sequence complementary to these oligonucleotides. DNA chip for detection. The DNA chip of claim 2, wherein the DNA chip further comprises a reference marker probe selected from the group consisting of β-globin, actin, and glyceraldehyde-3-phosphate dehydrogenase genes. DNA chip for detecting Chlamydia trachomatis. 4. The DNA chip of claim 3, wherein the β-globin probe is a β-globin probe having a nucleotide sequence shown in SEQ ID NO: 12. The DNA chip according to claim 2, wherein the 5 'terminal amine group of the probe DNA is immobilized by a cip group reaction with an aldehyde group on the solid surface of the chip. (a) a DNA chip for detecting Chlamydia trachomatis according to any one of claims 2 to 5, (b) a primer for amplifying the sample DNA of Chlamydia trachomatis by PCR, and (c) hybridized with the DNA chip Chlamydia trachomatis detection kit comprising a label means for detecting amplified DNA. The method of claim 6, wherein the label means Cy5, Cy3, biotin-binding material, EDANS (5- (2'-aminoethyl) amino-1- naphthalene sulfate), tetramethyltamine (TMR), tetramethyltamine Kit for detection of Chlamydia trachomatis is selected from the group consisting of isocyanate (TMRITC), x- rhodamine and Texas red. The kit for detecting Chlamydia trachomatis according to claim 6, wherein the primer is a Chlamydia trachomatis amplification primer having a nucleotide sequence of SEQ ID NO: 14 to a nucleotide sequence of SEQ ID NO: 16. The kit for detecting Chlamydia trachomatis according to claim 6, wherein the primer comprises a primer for amplifying β-globin DNA having a nucleotide sequence of SEQ ID NO: 17 to a nucleotide sequence of SEQ ID NO: 18. (a) amplifying chlamydia trachomatis sample DNA using a primer for amplifying the DNA of chlamydia trachomatis; (b) hybridizing the amplified DNA to a DNA chip comprising a single probe or a set of two or more probes selected from the group consisting of a base sequence of SEQ ID NO: 1 to a base sequence of SEQ ID NO: 11; And (c) detecting Chlamydia trachomatis comprising detecting the hybridized reactant with the probe. The method of claim 10, wherein the primer is selected from the group consisting of Chlamydia trachomatis amplification primers having a nucleotide sequence of SEQ ID NO: 14 to SEQ ID NO: 16. 11. The method of claim 10, wherein the detection is Chlamydia trachomatis detection method characterized in that for analyzing the fluorescent signal using a confocal laser scanner when the gene is labeled with Cy5. (A) amplifying the sample DNA of Chlamydia trachomatis using a primer for DNA amplification of Chlamydia trachomatis; And (b) hybridizing the amplified DNA with a single probe or a set of two or more probes selected from the group consisting of a nucleotide sequence of SEQ ID NO: 1 to a nucleotide sequence of SEQ ID NO: 12; (c) detecting Chlamydia trachomatis comprising detecting the hybridized reactant with the probe. delete