KR20120038430A - Probe for detecting microbes causing sexually transmitted diseases and detection chip, detection kit, and detection method using the same - Google Patents

Abstract

PURPOSE: A probe for detecting pathogenic microorganism causing diseases infected by sexual contact is provided to ensure high specificity and sensitivity. CONSTITUTION: A probe composition for detecting pathogenic microorganism causing diseases infected by sexual contact contains at least one of probe selected oligonucleotides with 66-67th bases and oligonucleotides having base complementary to the oligonucleotide. A PCR kit for pathogenic microorganism gene contains a primer set for amplifying Ureaplasma parvum nucleic acid, DNA polymerase, dNTPs, PCR buffer solution, and single chain probe.

Description

Probe for detecting microbes causing sexually transmitted diseases and detection chip, detection kit, and detection method using the same }

The present invention It relates to a probe for testing pathogenic microorganisms causing diseases caused by sexual contact, and a test chip, test kit, and test method for pathogenic microorganisms using the same, and in more detail, a plurality of species of pathogenic microorganisms are quickly and conveniently and with high specificity and sensitivity. It relates to a probe for testing that can be tested with, and a test chip for pathogenic microorganisms, a test kit, and a test method using the same.

Specifically, the present invention relates to 11 kinds of pathogenic microorganisms (hereinafter abbreviated as "Sexually transmitted pathogens") causing diseases that are infected by sexual contact, that is, gonorrhea ( Nisseria gonorrhoeae ) , Chlamydia trachomatis , Trichomonas pants Trichomonas vaginalis , Treponema pallidum , Mycoplasma hominis , Mycoplasma genitalium , Ureaplasma parvum , Ureaplasma parvum ( Ureaplasma urealyticum ), Haemophilus ducreyi , Candida albicans and Gardnerella vaginalis (Ureaplasma urealyticum), a test probe that can quickly and easily test with high specificity and sensitivity, And it relates to a pathogenic microorganism test chip, test kit and test method using the same.

In general, sexually transmitted diseases are known as sexually transmitted diseases. Venereal disease refers to a disease in which a pathogen mediated by sexual contact invades only the genitals and causes initial symptoms. In recent years, based on the fact that a myriad of diseases other than consensual sexually transmitted diseases are caused by sexual contact, diseases that cause diseases by being infected by sexual contact are called sexually transmitted diseases (STDs) in a broad sense. In addition, sexually transmitted infections in the broadest sense include cases of STI (sexually transmitted infection), which is a symptomless state in the infected person, but is contagious.

Currently, more than 30 types of sexually transmitted diseases are known, and representative sexually transmitted diseases include gonorrhea, non-gonococcal urethritis, syphilis, trichomoniasis, and acquired immunodeficiency syndrome. As pathogens that cause such sexually transmitted diseases, bacteria, fungi, protozoa, viruses or external parasites are known. Therefore, sexually transmitted diseases can be classified according to the microorganisms that cause them.Bacterial sexually transmitted diseases include gonorrhea, clamedia infection, syphilis, and hypochondrium, and viral sexually transmitted diseases include acquired immunodeficiency syndrome, herpes simplex virus, and human papillomavirus infection. Etc. There are also infections caused by fungi (yeast) such as candida vaginitis and trichomoniasis, and protozoa.

Specifically, the representative microorganisms that cause sexually transmitted disease is gonorrhea (Neisseria gonorrhoeae), Cloud media trachomatis (Chlamydia trachomatis), trichomonas pants day lease (Trichomonas vaginalis), Trail Four nematic sold Doom (Treponema pallidum), Mycoplasma hoe varnish (Mycoplasma hominis ), Mycoplasma genitalium , Ureaplasma parvum , Ureaplasma urealyticum , Haemophilus ducreyi , Candida albicans, Candida albicans And Gardnerella vaginalis .

However, sexually transmitted diseases range from mild symptomatic diseases to life-threatening diseases, but they must be diagnosed early and treated. If treatment is neglected or the treatment period is delayed, it is not only life-threatening, but it can also cause recurrence of disease, infertility, and birth defects. As a method for testing such sexually transmitted diseases, culture tests and immunological tests are known. However, the conventional STD test method takes a long time to test, and it is often difficult to accurately detect the pathogen of an STD. In addition, the venereal disease test method must satisfy all of the sensitivity, specificity, and reproducibility, but the existing method has a limitation that does not satisfy these conditions. As an alternative to this, a genetic test that genetically analyzes a sexually transmitted pathogen, that is, a method of detecting the RNA and DNA of the pathogen, has been developed.

However, the sexually transmitted disease pathogen gene test method should be easy and inexpensive to test, obtain a result quickly, and be able to quickly and reliably test a large number of specimens and various types of sexually transmitted disease pathogens.

For example, Trichomonas vaginalis is the most frequently occurring non-viral sexually transmitted disease worldwide, with 18 million new infections reported per year. These infections caused by Trichomonas vaginalis are often asymptomatic and have a high correlation with HIV infection. Diagnosis of infection through conventional microscopy is low with a sensitivity of about 60%. Although the sensitivity can be increased by a diagnostic method through the culture method, the result of the experiment has a disadvantage that depends on the skill level of the experimenter and the quality of the sample. In order to solve this problem, a molecular genetic diagnostic method that can be identified by targeting the β-tubulin gene has been developed, but in certain strains, the deletion of the β-tubulin gene has a problem in its accuracy. It may occur.

In addition, microorganisms in the genus Neisseria, which cause gonorrhea (Neisseria gonorrhoeae ), are Gram-negative bacteria that reside on the surface of mucous membranes of many animal groups, belong to proteobacteria, and form dicocci similar to coffee beans. About 7 species are found in humans. Among them, Neisseria gonorrhoeae is a pathogenic bacterium that causes sexually infectious gonorrhea, and more than 60 million new infections are reported annually worldwide. In 2003 alone, 335,104 cases of gonorrhea infections were reported in the United States as the most frequently occurring sexually transmitted infection with Chlamydia trachomatis. Infection with gonorrhea (Nisseria gonorrhoeae) is primarily confined to the mucous membrane, and is considered a major cause of pelvic inflammatory disease in women, and a major cause of urethritis in men. Since most infections are asymptomatic, the propensity to not show these symptoms is considered an important factor in the transmission and management of gonorrhea. The cultivation method is widely used for the diagnosis of such gonorrhea ( Nisseria gonorrhoeae ), but it has a disadvantage that the sensitivity is relatively low compared to the molecular genetic diagnosis method. For this reason, molecular rheological diagnostic methods are widely used in recent years, but there are several problems with existing diagnostic methods. Neisseria spp , which belongs to non-pathogenic normal bacteria. This is the case with false-positives due to sequence similarity with and and false-negatives due to deletion of the cppB gene used as a target gene in a specific strain.

Next, Chlamydia trachomatis is a representative bacterium that causes non-gonococcal urethritis, and is a non-motile bacterium having a gram-negative polymorphism. Clamedia trachomatis is an intracellular pathogen with 15 serotypes. There are serotype AC, which causes trachoma, L1-L3, which causes sexually transmitted lymphogranulomas, and DK, which causes the most common sexually transmitted diseases. Methods for identifying such clamedia trachomatis include cell culture, antigen testing, identification using a probe, and nucleic acid amplification (NAATs). Among them, the cultivation method has high accuracy, but its sensitivity is relatively low compared to other molecular genetic methods, and molecular genetic methods are widely used in recent years. However, the conventional molecular genetic method using whether or not to amplify nucleic acids has high sensitivity and accuracy, but has a disadvantage in that it is difficult to identify a large number of pathogens in one experiment.

In this regard, Republic of Korea Patent Registration No. 10-0639783 (name of the invention: a kit for diagnosing multiple sexually transmitted diseases and a method for detecting sexually transmitted diseases using the same) discloses a method for identifying five types of sexually transmitted infection-related microorganisms using a PCR method. In the Republic of Korea Patent Publication No. 10-2007-0099208 (name of the invention: a sexually transmitted disease detection method and diagnostic kit using a polymerase chain reaction), a method for identifying 10 types of sexually transmitted infection-related microorganisms using a PCR method is disclosed. In addition, Republic of Korea Patent Registration No. 10-0860619 (name of the invention: oligonucleotide for detecting adult disease-causing pathogen nucleic acid) is a method for testing 12 STD pathogens through PCR amplification using primers designed in the form of bispecific oligonucleotides. Is being disclosed.

However, in the method of testing a sexually transmitted disease pathogen using PCR, careful attention and time are required when synthesizing primers, and there is a problem in that a false-positive product is generated by non-specific hybridization of the primers. In addition, when the number of STD pathogens to be tested increases, multiplex PCR (multiplex PCR) must be performed to diagnose multiple pathogens at the same time. In this case, many restrictions are imposed on designing the sequence of primers, and generation of false-positive products is difficult. It becomes even more problematic. In addition, the method of testing a sexually transmitted disease pathogen using PCR has a problem in that it is inconvenient to visually check a PCR product to determine whether it is positive or not, and is not suitable for making a database of test results and miniaturization of test equipment.

Accordingly, recently, as an alternative to the PCR method, a target gene specific to a sexually transmitted disease pathogen and useful for a sexually transmitted disease test has been discovered, and a microarray type test method using a probe for such a target gene and a DNA chip has been developed.

In this regard, Korean Patent Registration No. 10-0532665 discloses a method of testing Neisseria spp. bacteria using a DNA chip comprising a probe that complementarily binds to a nucleic acid of gonococcus or meningitis. No. 10-0532664 discloses a method for testing Chlamydia trachomatis using a probe and a DNA chip specific for the MOMP gene of Chlamydia trachomatis, and Korean Patent No. 10-0532666 discloses a trp gene of syphilis treponema. Disclosed is a method of detecting the infection of syphilis treponema and analyzing the treponema tpr subfamily using a probe and a DNA chip for.

In addition, Korean Patent Registration No. 10-0619189 A DNA chip capable of simultaneously diagnosing infections of the four bacteria with the highest incidence of sexually transmitted diseases, such as gonococcus, Chlamydia trachomatis, ureaplasma urealighticum, and Mycoplasma genitalium, is disclosed.

However, this microarray method of testing for STD pathogens has a problem to be overcome. That is, selection of a gene (hereinafter referred to as "target gene") useful for testing a sexually transmitted disease pathogen that is specific to a specific sexually transmitted disease pathogen and has no homology to other species, and a probe used in the DNA chip (a sequence complementary to the target gene) To minimize the occurrence of false-positive and/or false-negative results.

In particular, the above-described conventional microarray-type test method is for testing a limited number of sexually transmitted disease pathogens of around 5 types, and in the case of multiple analyzes of 10 or more types, the problem of false positives and/or false negatives is inevitably increased. Therefore, the prior art has a problem in terms of sensitivity and specificity, and it can be said that it is impossible to analyze a number of sexually transmitted pathogens from a practical point of view of a diagnosis site.

Therefore, in the technical field to which the present invention belongs, considering that there are currently more than 30 types of sexually transmitted diseases, a new target gene capable of providing sensitivity and specificity close to 100% while diagnosing multiple types of sexually transmitted pathogens quickly and conveniently. It can be said that selection and development of probes and DNA chips for this are required.

Republic of Korea Patent Publication No. 10-0639783 (2006.10.30) Republic of Korea Patent Publication No. 10-2007-0099208 (2007.10.09) Korean Registered Patent Publication No. 10-0860619 (2008.09.29) Korean Registered Patent Publication No. 10-0532665 (2005.12.02) Korean Registered Patent Publication No. 10-0532664 (2005.12.02) Korean Registered Patent Publication No. 10-0532666 (2005.12.02) Korean Registered Patent Publication No. 10-0619189 (2006.08.31)

The present invention overcomes the various problems of the conventional STD test method described above, and provides high reliability while quickly and simply diagnosing a plurality of STD pathogens, and a test chip, test kit, and test method using the same. Was conceived to develop.

An object of the present invention is to provide a primer that can accurately and highly amplify target genes of 11 sexually transmitted pathogens.

Another object of the present invention is to provide a target gene PCR amplification kit for 11 types of STD pathogens comprising the primers.

Another object of the present invention is to provide a probe capable of testing 11 types of STD pathogens quickly and conveniently and with high specificity and sensitivity.

Another object of the present invention is to provide an STD pathogen test chip comprising the probe.

Another object of the present invention is to provide a sexually transmitted disease pathogen test kit comprising the test chip.

Another object of the present invention is to provide a test method capable of testing 11 types of STD pathogens quickly and conveniently and with high specificity and sensitivity.

In addition, the present invention is to provide a method of effectively labeling a PCR amplified product amplified using a plurality of types of primers.

As a result of resolving the technical problem of the above invention and repeating intensive research to meet the purpose of the above invention, the present inventors selected each target gene specific to 11 types of STD pathogens, and the nucleotide sequence of the selected target gene Based on the synthesis of primers and probes that can amplify target genes specific to each STD pathogen, we have completed a test method that can quickly and conveniently test 11 STD pathogens with high specificity and sensitivity.

The probe for testing pathogenic microorganisms causing diseases infected by sexual contact of the present invention specifically binds to the gene nucleic acid of the pathogenic microorganism, and oligonucleotides having a nucleotide sequence of SEQ ID NO: 50 to SEQ ID NO: 54 And one or more first probe groups selected from the group consisting of oligonucleotides having a base sequence complementary to these oligonucleotides, an oligonucleotide having a base sequence of SEQ ID NO: 55, and a base sequence complementary to these oligonucleotides. Oligonucleotides having at least one second probe group selected from the group consisting of oligonucleotides having, and oligonucleotides having a nucleotide sequence of SEQ ID NO: 56 to SEQ ID NO: 57, and a nucleotide sequence complementary to these oligonucleotides At least one third probe group selected from the group consisting of, oligonucleotides having a nucleotide sequence of SEQ ID NO: 58 to SEQ ID NO: 60, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides Group consisting of at least one fourth probe group selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 61 to SEQ ID NO: 62, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides Selected from the group consisting of at least one fifth probe group selected from, oligonucleotides having a nucleotide sequence of SEQ ID NO: 63 to SEQ ID NO: 65, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides 1 selected from the group consisting of at least one sixth probe group, oligonucleotides having a nucleotide sequence of SEQ ID NO: 66 to SEQ ID NO: 67, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides One species selected from the group consisting of seven or more species of probe group, oligonucleotides having a nucleotide sequence of SEQ ID NO: 68 to SEQ ID NO: 71, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides At least one agent selected from the group consisting of the eighth probe group above, oligonucleotides having a nucleotide sequence of SEQ ID NO: 72 to SEQ ID NO: 73, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides At least one tenth probe selected from the group consisting of a group of 9 probes, oligonucleotides having a nucleotide sequence of SEQ ID NO: 74 to a nucleotide sequence of SEQ ID NO: 77, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides From the group, at least one eleventh probe selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 78 to SEQ ID NO: 79, and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides It includes at least one group of probes selected.

In the test probe of an embodiment of the present invention, the first probe group is a gene nucleic acid of Neisseria gonorrhoeae , and the second probe group is The gene nucleic acid of Chlamydia trachomatis , the third probe group is a gene nucleic acid of Trichomonas vaginalis , and the fourth probe group is Treponema pallidum (Treponema pallidum) gene nucleic acid, the fifth probe group is a gene nucleic acid of Haemophilus ducreyi (Haemophilus ducreyi), the sixth probe group is a gene nucleic acid of Candida albicans (Candida albicans) And, the seventh probe group is a gene nucleic acid of ureaplasma parvum (Ureaplasma parvum ), the eighth probe group is a gene nucleic acid of ureaplasma urealyticum (Ureaplasma urealyticum), the 9th probe group is mycoplasma homi Nice ( Mycoplasma hominis ) gene nucleic acid, the 10th probe group is Mycoplasma genitalium (Mycoplasma genitalium) gene nucleic acid, the 11th probe group is Gardnerella vaginalis (Gardnerella vaginalis) gene nucleic acid and It is characterized by specific binding.

The test chip for pathogenic microorganisms causing diseases that are infected by sexual contact includes oligonucleotides having a nucleotide sequence of SEQ ID NO: 50 to SEQ ID NO: 79 and an oligonucleotide having a nucleotide sequence complementary to these oligonucleotides. It includes at least one probe selected from the group consisting of.

In one embodiment of the inspection chip of the present invention, the probe is characterized in that the microarray on the substrate.

The gene PCR amplification kit for pathogenic microorganisms causing diseases infected by sexual contact of the present invention includes primers having one or more nucleotide sequences selected from SEQ ID NO: 1 to SEQ ID NO: 49, DNA polymerase, dNTPs, PCR buffer and PCR Contains single-chain labeling substances of amplification products. Preferably, the primer may be provided as a combination of at least one forward primer and a reverse primer selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 49.

In the PCR amplification kit of an embodiment of the present invention, there is no homology with the gene sequences specific to the pathogenic microorganism, and a non-homologous base belonging to a different animal group, another plant group, another microorganism group, or another virus group from the pathogenic microorganism. The sequence is tagged at the 5'end of the primer, and the single-chain labeling material of the PCR amplification product is a labeled probe having a sequence complementary to the non-homologous nucleotide sequence. For example, the non-homologous nucleotide sequence may be an M13 primer sequence, an SP6 sequence, or the like.

Alternatively, in the PCR amplification kit of an embodiment of the present invention, the single-chain labeling material of the PCR amplification product may be directly bound to the 5'end of the primer.

The test kit for pathogenic microorganisms causing diseases infected by sexual contact includes a test chip for pathogenic microorganisms defined as described above, a primer for amplifying a gene specific to the pathogenic microorganism, and hybridization with the test chip. It includes a labeling means for detecting the amplified gene that becomes.

In the test kit of an embodiment of the present invention, the primer is characterized in that it is a primer having one or more nucleotide sequences selected from SEQ ID NO: 1 to SEQ ID NO: 49. Preferably, the primer may be provided as a combination of at least one forward primer and a reverse primer selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 49.

In the test kit of an embodiment of the present invention, there is no homology with the gene sequences specific to the pathogenic microorganism, and a non-homologous nucleotide sequence belonging to a different animal group, another plant group, another microorganism group, or another virus group from the pathogenic microorganism. It is tagged at the 5'end of the primer, and the labeling means is a labeled probe having a sequence complementary to the non-homologous nucleotide sequence. For example, the non-homologous nucleotide sequence may be an M13 primer sequence, an SP6 sequence, or the like.

Alternatively, in the test kit of an embodiment of the present invention, the labeling means may be directly coupled to the 5'end of the primer.

In the test kit according to a preferred embodiment of the present invention, the non-homologous nucleotide sequence may be additionally included in the test chip as a position marker of the probe. At this time, the labeled probe having a sequence complementary to the non-homologous nucleotide sequence is hybridized with the non-homologous nucleotide sequence included in the test chip to confirm the position of the probe constituting the test chip.

The method for testing pathogenic microorganisms causing diseases caused by sexual contact of the present invention comprises the steps of PCR amplifying a gene of a sample using a primer for amplifying a gene specific to the pathogenic microorganism, and a test chip defined as described above. Hybridizing the amplified gene, and detecting a reactant hybridized with the probe of the test chip.

In the test method of an embodiment of the present invention, a primer having one or more nucleotide sequences selected from SEQ ID NO: 1 to SEQ ID NO: 49 is used for the PCR amplification. Preferably, the primer may be provided as a combination of at least one forward primer and a reverse primer selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 49.

In the test method of an embodiment of the present invention, there is no homology with the gene sequences specific to the pathogenic microorganism, and a non-homologous nucleotide sequence belonging to a different animal group, another plant group, another microorganism group, or another virus group from the pathogenic microorganism. Tagging the 5'end of the primer, and labeling the amplified gene with a labeled probe having a sequence complementary to the non-homologous nucleotide sequence. For example, the non-homologous nucleotide sequence may be an M13 primer sequence, an SP6 sequence, or the like.

In the test method of a preferred embodiment of the present invention, the non-homologous nucleotide sequence is included in the test chip as a position marker of the probe, and the position marker is a labeled probe having a sequence complementary to the non-homologous nucleotide sequence. It may further include the step of confirming the position of the probe by labeling.

In the present invention, the combination of at least one forward primer and reverse primer selected from SEQ ID NO: 1 to SEQ ID NO: 10 is to amplify the gene nucleic acid of Gonococcus (Nisseria gonorrhoeae ), at least one selected from SEQ ID NO: 11 to SEQ ID NO: 12 The combination of forward primer and reverse primer is In the amplification of the gene nucleic acid of Chlamydia trachomatis , the combination of at least one forward and reverse primer selected from SEQ ID NO: 13 to SEQ ID NO: 16 is used to amplify the gene nucleic acid of Trichomonas vaginalis, the sequence The combination of at least one forward primer and reverse primer selected from SEQ ID NO: 17 to SEQ ID NO: 24 is a gene nucleic acid amplification of Treponema pallidum , at least one forward primer selected from SEQ ID NO: 25 to SEQ ID NO: 28 And the combination of the reverse primer is to amplify the gene nucleic acid of Haemophilus ducreyi (Hemophilus ducreyi), the combination of at least one forward primer and reverse primer selected from SEQ ID NO: 29 to SEQ ID NO: 32 is Candida albicans ( Candida albicans ) In the amplification of the gene nucleic acid, a combination of at least one forward primer and a reverse primer selected from SEQ ID NO: 33 to SEQ ID NO: 35 is used to amplify the gene nucleic acid of ureaplasma parvum and ureaplasma urealyticum , The combination of at least one forward primer and reverse primer selected from SEQ ID NO: 36 to SEQ ID NO: 39 is used to amplify the gene nucleic acid of Mycoplasma hominis , and at least one forward primer selected from SEQ ID NO: 40 to SEQ ID NO: 45 And the combination of the reverse primer to amplify the gene nucleic acid of Mycoplasma genitalium (Mycoplasma genitalium), the combination of at least one forward primer and reverse primer selected from SEQ ID NO: 46 to SEQ ID NO: 49 Gardnerella genitalis (Gardnerella vaginalis ) characterized in that it is used for amplification of gene nucleic acids.

In the present invention, the labeling material is Cy5, Cy3, biotin-binding material, EDANS (5-(2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), x-rhodamine, or Texas Red, and the like, and a fluorescent substance such as Cy3 or Cy5 is preferable.

According to the present invention, 11 kinds of STD pathogens can be tested quickly and conveniently and with high specificity and sensitivity, and target genes of the 11 kinds of STD pathogens can be simultaneously amplified by using multiple kinds of primers, and amplified PCR The amplification product can be effectively labeled.

1 is a diagram illustrating a method of fluorescently labeling a PCR amplification product, a tag consisting of a sequence that is not homologous to an amplification product and a target gene sequence is bound to a primer, and a fluorescent material-labeled probe having a sequence complementary to the tag It is a diagram explaining a method of inexpensively and effectively labeling a single chain of a PCR amplification product by adding.
Figure 2 is a photograph showing the result of electrophoresis of the PCR amplification product of the target gene of gonorrhea (Nisseria gonorrhoeae ) amplified with a primer pair consisting of a combination of SEQ ID NO: 1 and SEQ ID NO: 2.
FIG. 3 is a hybridization reaction of a PCR amplification product of a target gene of Gonococcus (Nisseria gonorrhoeae ) amplified with a primer pair consisting of a combination of SEQ ID NO: 1 and SEQ ID NO: 2 on a DNA chip in which a probe specific to Gonorrhoeae (Nisseria gonorrhoeae) is arrayed. It is a graph showing the result of analysis by scanning fluorescence after being performed.
Figure 4 is a fluorescence image of the DNA chip confirmed after hybridizing the amplified PCR product to the DNA chip of the present invention.
5 is a PCR amplification product of a target gene of Treponema pallidum amplified with a primer pair consisting of a combination of SEQ ID NO: 17 and SEQ ID NO: 18, and a primer pair consisting of a combination of SEQ ID NO: 36 and SEQ ID NO: 37 This is a photograph showing the result of electrophoresis of the PCR amplification product of the target gene of Mycoplasma hominis amplified by. The first lane of FIG. 5 is a DNA marker, the second lane is an amplification product of Treponema pallidum, and the third to sixth lanes are an amplification product of Mycoplasma hominis.
6 is a PCR amplification product of a target gene of Mycoplasma hominis amplified with a primer pair consisting of a combination of SEQ ID NO: 36 and SEQ ID NO: 37, in which a probe specific to Mycoplasma hominis is arrayed. This is a graph showing the results of analysis by scanning fluorescence after hybridization reaction to the DNA chip.
7 is a PCR amplification product of a target gene of Mycoplasma genitalium amplified with a primer pair consisting of a combination of SEQ ID NO: 40 and SEQ ID NO: 41, a probe specific for Mycoplasma genitalium. This is a graph showing the results of analysis by scanning fluorescence after performing a hybridization reaction on the arrayed DNA chips.
Figure 8 is a PCR amplification product of the target gene of ureaplasma urealyticum amplified with a primer pair consisting of a combination of SEQ ID NO: 34 and SEQ ID NO: 35 is specific to ureaplasma urealyticum It is a graph showing the result of analysis by scanning fluorescence after performing a hybridization reaction on a DNA chip in which a probe (probe of SEQ ID NO: 69) is arrayed.
9 is a PCR amplification product of a target gene of ureaplasma parvum amplified with a primer pair consisting of a combination of SEQ ID NO: 33 and SEQ ID NO: 35, a probe specific for ureaplasma parvum (SEQ ID NO: 66). Probe) is a graph showing the result of analysis by scanning fluorescence after performing a hybridization reaction on the arrayed DNA chip. In FIG. 9, serotypes 1 and 6 of ureaplasma parvum can be identified.
10 is a PCR amplification product of the target gene of ureaplasma parvum amplified with a primer pair consisting of a combination of SEQ ID NO: 33 and SEQ ID NO: 35, a probe specific for ureaplasma parvum (SEQ ID NO: 67). Probe) is a graph showing the result of analysis by scanning fluorescence after performing a hybridization reaction on the arrayed DNA chip. In FIG. 10, serotypes 3 and 14 of ureaplasma parvum can be identified.

Hereinafter, the present invention will be described in more detail based on examples. It goes without saying that the following examples of the present invention are intended to embodi the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by experts in the technical field to which the present invention pertains from the detailed description and examples of the present invention is interpreted as belonging to the scope of the present invention. The references cited in the present invention are incorporated herein by reference.

Example 1: Selection of target genes specific to 11 STD pathogens

In order to provide primers and probes capable of analyzing 11 types of STD pathogens, a specific sequence is primarily selected from the 16S rRNA gene of each STD pathogen and the unique sequence of genes possessed by each STD pathogen. Then, a sequence capable of amplifying the selected specific sequence is secondly selected to determine a primer sequence, and thereafter, a process of synthesizing suitable primers and probes is performed.

In the conventional STD pathogen test method, in the case of Trichomonas vaginalis, a specific nucleotide sequence of the β-tubulin gene is used (JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2003, p. 5619-5622). , In the case of clamedia trachomatis ompA gene (JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2006, p. 4066-4071), and in the case of gonococcus cppB gene (JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1999, p. 386-390) Etc were being used.

On the other hand, in the present invention, in order to solve the problems of the prior art as described above , Neisseria gonorrhoeae , Chlamydia trachomatis , Trichomonas vaginalis , Treponema pallidum ), Mycoplasma hominis , Mycoplasma genitalium , Ureaplasma parvum , Ureaplasma urealyticum , Haemophilus ducreyi , Haemophilus ducreyi New target genes specific to each sexually transmitted disease pathogen such as Candida albicans and Gardnerella vaginalis were selected.

For example, in the case of gonococcus, porA pseudogene was used as a specific target gene, which was selected to improve the problem of false negative due to the loss of the cppB gene in a specific strain in the conventional test method using the cppB gene as a target gene ( Journal of Molecular Diagnostics, Vol. 8, No. 1, February 2006, 3-15). In addition, through homology analysis with other species belonging to the genus Neisseria, it was confirmed that the existing target sequence has a high homology and a possibility of false-positive. When synthesizing these features, the porA pseudogene was selected as the target gene. If so, the disadvantages of the prior art are further supplemented.

In addition, the 16S rRNA gene for Clamedia trachomatis and the 18S rRNA gene for Trichomonas vaginalis were used in the present invention to analyze pathogenic microorganisms that can be infected by sexual contact.

Example 2: Preparation of primers for amplifying target genes of pathogenic microorganisms capable of infection by sexual contact

Next, primers that can accurately and highly efficiently amplify target genes specific to 11 types of STD pathogens were prepared.

After determining the base sequence of a primer capable of amplifying each target gene of 11 sexually transmitted pathogens as shown in Table 1 below, the base sequence of Table 1 ( A primer for use in the present invention was prepared by synthesizing a primer having SEQ ID NO: 1 to SEQ ID NO: 49) or by requesting an organization specialized in oligonucleotide synthesis (eg, METABION, Germany).

[Table 1] Amplification of target genes of pathogenic microorganisms that can be infected by sexual contact Primer for

Example 3: Single-chain labeling of PCR amplification products

On the other hand, in the present invention, the conventional method of labeling a fluorescent substance in the DNA chip analysis method was improved, but only the amplified target gene product was selected by adding one fluorescent substance-labeled probe to the PCR amplification product for a plurality of target genes. Newly devised a method of labeling with.

In general, in a genotyping method using a DNA chip of a microarray method, a single chain complementary to a probe of a DNA chip among PCR amplification products of a specific gene amplified by a primer is labeled with a labeling material such as a fluorescent material.

That is, conventionally, when a probe is selected from the forward sequence information, a fluorescent material such as Cy3 is labeled at the 5'end of the reverse primer used in the PCR amplification process in order to label the fluorescent material only in the reverse single-chain amplification product. Then, the PCR amplification product is single-chained using heat denaturation or asymmetric amplification (amplified by adding the amount of the forward primer significantly lower than the amount of the reverse primer), and then hybridized with the probe of the DNA chip.

However, if the single chain of the PCR amplification product is labeled in this way, the number of primer pairs used for amplification increases as the type of gene to be amplified increases. Accordingly, the cost of labeling the fluorescent material on the reverse primer increases. A problem arises. That is, the conventional fluorescent substance labeling method has a problem in cost inefficient as the number of fluorescent substances to be labeled on the reverse primer increases as the type of target amplification product increases when the fluorescent substance is directly labeled on the reverse primer during the amplification process.

In view of this problem, in the present invention, there is no homology with the target gene sequences of 11 types of sexually transmitted pathogens, and "emergency" belonging to a different animal group, other plant group, other microbial group, or other virus group from the 11 types of sexually transmitted pathogens. A homologous nucleotide sequence" (eg, M13 primer sequence, SP6 sequence, etc.) is tagged to the primer sequence, and after PCR amplification, a fluorescent substance-labeled probe with a sequence complementary to this one kind of tag ("for primer labeling Probe") was added to label the single chain of the PCR amplification product (see FIG. 1). By labeling in this way, it is possible to label the fluorescent substance at a lower cost compared to the cost of directly labeling the fluorescent substance on the primer, and it is possible to label the fluorescent substance in the same way on the amplified product tagged with one kind of "non-homologous base sequence". . On the other hand, Figure 1 shows a case in which the M13 primer sequence is used as the tag sequence, but the present invention is not limited thereto, and a "non-homologous nucleotide sequence" that is not homologous to the target gene sequences of 11 types of STD pathogens. It will be readily understood by those skilled in the art to which the present invention pertains that any can be used.

In addition, for hybridization of the amplified product, the target DNA must be present in a single chain behavior. This process is generally accomplished through thermal denaturation of the amplification product or denaturation through chemical substances. However, this single-chain type amplification product can also be obtained through asymmetric PCR, and phosphate is conjugated to primers in other directions that are not tagged with “asymmetric nucleotide sequences”. After treatment with lambda exonuclease, a process of selectively decomposing a phosphate-labeled chain to form a single chain is also possible (see FIG. 1). In Figure 1, a process of making a single chain form using lambda exonuclease is described, but the conventional thermal denaturation, denaturation by chemical substances, or asymmetric polymerase chain reaction methods are replaced and used or in parallel. Can be used.

On the other hand, the present invention is not limited to the single-chain labeling method of the PCR amplified product as described above, and it will be readily understood by those skilled in the art that various labeling methods known in the art can be used. . For example, the conventional method of directly labeling a fluorescent substance on a primer sequence and a method of labeling using a fluorescent substance-labeled dNTP mix during the amplification process may be substituted or used in parallel.

Example 4: Preparation of a probe for analysis of pathogenic microorganisms capable of infection by sexual contact

A probe capable of complementary hybridization with a PCR amplification product of a target gene specific for 11 STD pathogens was constructed.

The probe was manufactured according to the method for synthesizing oligonucleotide probes generally known in the art, or was produced by requesting an oligonucleotide synthesis specialist (eg, METABION, Germany). To covalently bond oligonucleotides on an aldehyde-coated substrate. For this purpose, an amino link was attached to the 5'end of all probes, and 5-40 thymine groups after the 5'amine group were attached to reinforce the binding strength of the PCR amplification products and the specific probe for each of 11 STD pathogens. ) A probe was synthesized by attaching a base. In constructing a DNA chip, by modifying the amine functional group in the 5'direction of each probe and then adding oligo dTTP from pentagram to 40 mer, spatial interference can be minimized when the PCR amplification product and the probe are combined. On the other hand, the oligo dTTP may be replaced with another base or another chemical such as a carbon spacer.

A probe having a nucleotide sequence (SEQ ID NO: 50 to SEQ ID NO: 79) as shown in Table 2 below was synthesized in the same manner as described above.

[Table 2] Probes for analysis of pathogenic microorganisms that can be infected by sexual contact

Meanwhile, the sequence numbers of primer combinations capable of amplifying the target genes of 11 types of STD pathogens as described above, and PCR products amplified by these primer combinations can be detected and 11 types of STD pathogens can be tested. When the sequence numbers of the probes are matched to each other, they are shown in Table 3 below.

[Table 3] Primer combinations that amplify 11 STD pathogens and probes that can detect PCR products amplified by these primer combinations

Example 5: Fabrication of DNA chips for discriminating 11 types of sexually transmitted pathogens

According to the object of the present invention, an oligonucleotide microarray DNA chip for discriminating STD pathogens comprising a probe capable of analyzing the genotype of 11 kinds of STD pathogens with high specificity and sensitivity is prepared as follows. It was manufactured according to the manufacturing method widely known in.

That is, a DNA chip was fabricated by dipping the probes prepared in Example 4 on a microarray chip substrate made of glass or plastic with aldehyde attached to the substrate surface using a microarray device. The fabrication of the microarrayed DNA chip is carried out using a method well known to those skilled in the art.

For example, in an oligonucleotide probe imprinted on a glass substrate using a microarray device, an amine group at the 5'end causes an aldehyde group on the glass substrate to react with Schiff's base and is fixed on the glass substrate. In addition, it may include a step of reducing the aldehyde remaining unreacted. Although the conditions for reducing the aldehyde are not particularly limited thereto, it is preferable to use a reducing agent such as _{NaBH 4.}

The gene probe to be microarrayed is at least one probe selected from probes having nucleotide sequences of SEQ ID NO: 50 to SEQ ID NO: 79, and only one may be microarrayed or all 30 may be microarrayed.

In addition, in an embodiment of the present invention, a vector in which DNA of Arabidopsis thaliana was cloned (pGEM-T easy vector obtained from Promega) was used as a positive control. During the PCR amplification process of a target gene specific to a sexually transmitted disease pathogen, the clone vector and a pair of primers capable of amplifying it were added together to check for errors during the amplification process, and a probe capable of hybridizing with this amplification product was synthesized. It was included in the DNA chip construct for discriminating sexually transmitted pathogens of the invention. The primer sequence for amplification of the positive control DNA (SEQ ID NO: 80 and SEQ ID NO: 81) and the sequence of the probe included in the DNA chip (SEQ ID NO: 82) are as follows.

(SEQ ID NO: 80) ARPCF: 5'- GGA CAA CCA CTG CGT CTA T -3'

(SEQ ID NO: 81) ARPCR: 5'- CAA GGA AGG TCA CTG CAA -3'

(SEQ ID NO: 82) ARPCP: 5'- TCA CTT CTG GTG CGA ATC CCG -3'

In addition, in an embodiment of the present invention, an additional probe having no complementarity to a target gene sequence and a probe sequence specific for a sexually transmitted disease pathogen is prepared, integrated into the DNA chip structure for discriminating an sexually transmitted disease pathogen, and amplified complementary to these additional probes. By inducing a reaction with a product or a synthetic product, the position of each probe microarrayed on a DNA chip for a sexually transmitted disease pathogen can be identified. These additional probes can be used as a location marker to identify errors that may occur during the hybridization process between the PCR amplification product and the probe of the DNA chip, and to identify the location of the DNA chip component. The amplification product or synthetic product complementary to the additional probe may be labeled with a fluorescent material different from a fluorescent material that labels the PCR amplification product of a target gene specific to an STD pathogen, or may be labeled with the same fluorescent material.

On the other hand, the additional probe, which is a position marker integrated in the DNA chip for discriminating a sexually transmitted disease pathogen of the present invention, may use position markers well known in the art, but the so-called "non-homologous nucleotide sequence" described in Example 3 (for example, When the M13 primer sequence, SP6 sequence, etc.) are integrated into the DNA chip for discriminating sexually transmitted diseases of the present invention, there is an advantage that it is not necessary to prepare an additional probe for a position marker.

That is, as in Example 3, when amplifying the target gene sequences of 11 sexually transmitted pathogens, a so-called "non-homologous nucleotide sequence" (eg, M13 primer sequence, SP6 sequence, etc.) is tagged at the 5'end of the reverse primer. Then, PCR amplification was performed, and the single chain of the PCR amplified product was labeled with a fluorescent substance-labeled probe ("primer labeling probe") having a sequence complementary to the so-called "non-homologous nucleotide sequence", and thus labeled with a fluorescent substance. In the process of hybridizing the PCR amplified product to the DNA chip for discriminating STD pathogens of the present invention, a "non-homologous nucleotide sequence" such as the M13 sequence integrated as a position marker on the DNA chip for discriminating STD pathogens of the present invention and a fluorescent material complementary thereto By hybridization with the labeling sequence, the position of the probe constituting the DNA chip can be identified.

Example 6: gonococcal ( Neisseria gonorrhoeae ) Target gene amplification and hybridization experiment with DNA chip

Example 6-1: gonococcal ( Neisseria gonorrhoeae )-Specific target gene amplification confirmation experiment

The PCR amplification of the target gene of gonorrhea ( Nisseria gonorrhoeae ) was carried out by a combination of SEQ ID NO: 1 and SEQ ID NO: 2 of SEQ ID NO: 1 to SEQ ID NO: 10, which are primers for amplification of gonococcus in Table 1. The genomic genes (ATCC53420D, 53421D) of gonorrhea ( Nisseria gonorrhoeae ) were purchased from ATCC and used for PCR amplification.

Bionia's AccuPower HotStart PCR PreMix was used as the PCR construct (PCR premix) for amplifying the target gene of Neisseria gonorrhoeae , and the PCR amplification process was denatured at 95°C for 10 minutes, then denatured at 95°C for 15 seconds, 53°C. The process of 30 seconds and 30 seconds at 72°C was repeated 40 times and then reacted at 72°C for 10 minutes. Subsequently, the PCR amplified product was stored refrigerated at 4° C. until the experiment. In addition, the amplified product was confirmed by electrophoresis on a 2.5% agarose gel.

On the other hand, in order to prove the specificity and sensitivity of the primer for amplification of gonococcus of the present invention and the probe for gonococcus test, genes of STD pathogens other than gonococcus were used as templates.

For example, Trichomonas vaginalis (ATCC30001D), Trichomonas tenax (ATCC30207), Mycoplasma hominis (ATCC23114D), Mycoplasma genitalium (ATCC3530D) ) And Haemophilus ducreyi (ATTC700724D-5) were purchased from ATCC and used as a template, and Candida albicans (KCTC7270) was purchased from the Biological Resource Center. Was extracted and used as a template, and Chlamydia trachomatis was used as a template after cloning into a pGEM-T easy vector (a product amplified using DNA extracted from a clinical sample as a template). As shown in Figure 2, according to the result of electrophoresis of the amplified PCR product on a 3% agarose gel, it can be seen that the target gene of gonococcus (Nisseria gonorrhoeae ) was selectively amplified, and the size of the amplified product was confirmed to be 124bp. Became.

Example 6-2: gonococcal ( Neisseria gonorrhoeae ) Target gene and DNA chip hybridization experiment

The amplification of the target gene for gonorrhea ( Nisseria gonorrhoeae ) was performed in the same manner as in Example 6-1, and the primers for amplification were selected from SEQ ID NO: 1 and SEQ ID NO: 2.

And, hybridization experiment of the amplified PCR product and a DNA chip composed of a probe of SEQ ID NO: 50, a probe of SEQ ID NO: 51, a probe of SEQ ID NO: 52, and a probe of SEQ ID NO: 54, which are probes specific to Neisseria gonorrhoeae Proceeded.

In the hybridization experiment, 10 µl of the PCR amplification product after amplification was taken, diluted with 90 µl of oligonucleotide microarray hybridization buffer (manufactured by Genochek Co., Ltd.) on the prepared DNA chip and applied. The DNA chip was reacted in an incubation oven at 50° C. for 1 hour, washed with a wash solution kit, and scanned with an axon chip scanner to check the hybridization result. It was confirmed that the performance (see Fig. 3), the signals are strongly analysis of the results gonorrhea (Neisseria gonorrhoeae) was analyzed by scanning the fluorescence of the DNA chip-specific probe to Neisseria gonorrhoeae (Neisseria gonorrhoeae) as shown in Figure 3 Was determined to be effective for gonococcal examination (fluorescence intensity in FIG. 3 is an arbitrary unit for comparison as the median signal intensity minus the background value).

Example 7: Treponema Palidum ( Treponema pallidum ) And mycoplasma hominis ( Mycoplasma hominis ) Amplification of the target gene

TRE to Po nematic PCR amplification of the target gene Farley Doom (Treponema pallidum) are shown in Table 1 of the tray Four nematic sold combination of Doom (Treponema pallidum) amplification primers of SEQ ID NO: 17 to SEQ ID NO: 24 of SEQ ID NO: 17 and SEQ ID NO: 18 Proceeded. And, as Mycoplasma hoe varnish combination of PCR amplification of target genes are shown in Table 1 of Mycoplasma hoe varnish (Mycoplasma hominis) amplification primers of SEQ ID NO: 36 to SEQ ID NO: 39 SEQ ID NO: 36 and SEQ ID NO: 37 of for the (Mycoplasma hominis) Proceeded.

Meanwhile, the amplification experiment of the target gene was carried out in the same manner as in Example 6-1. As shown in FIG. 5, according to the result of electrophoresis of the amplified PCR product on 3% agarose gel, target genes of Treponema pallidum and Mycoplasma hominis were selectively amplified. You can see that it is.

Example 8: Mycoplasma Hominis ( Mycoplasma hominis ) Target gene and DNA chip hybridization experiment

A hybridization experiment was conducted using a specific probe capable of identifying Mycoplasma hominis. The amplification product obtained through the process of Example 7 was converted to a single base chain form by treatment with lambda exonuclease, and then a hybridization experiment with a DNA chip composed of SEQ ID NO: 73 was conducted.

Meanwhile, the hybridization experiment between the amplification product and the DNA chip was carried out in the same manner as in Example 6-2.

The, the signal for analysis by scanning the fluorescence of the DNA chip Mycoplasma hoe varnish (Mycoplasma hominis) is strongly analysis-specific probe to Mycoplasma hoe varnish (Mycoplasma hominis), as shown in Figure 6, Mycoplasma hoe It was determined to be valid for the Nice Test.

Example 9: Mycoplasma genitalium ( Mycoplasma genitalium ) Target gene and DNA chip hybridization experiment

A hybridization experiment was conducted using a specific probe capable of identifying Mycoplasma genitalium. The target gene sequence was PCR-amplified using the primer pairs of SEQ ID NO: 40 and SEQ ID NO: 41, and a hybridization experiment was performed with the amplified product with a DNA chip composed of SEQ ID NO: 74.

On the other hand, the amplification experiment of the target gene was carried out in the same process as in Example 6-1, and the hybridization experiment between the amplification product and the DNA chip was carried out in the same process as in Example 6-2.

The, the signal for the result of Mycoplasma Jenny de Solarium (Mycoplasma genitalium) was analyzed by scanning the fluorescence of the DNA chip is strongly analysis-specific probe to Mycoplasma Jenny de Solarium (Mycoplasma genitalium), as shown in Fig. 7 M. It was determined to be effective for the plasma genitalium test.

Example 10: Urea Plasma Urea Lighticum ( Ureaplasma urealyticum ) And Urea Plasma Paboom ( Ureaplasma parvum ) Target gene and DNA chip hybridization experiment

A hybridization experiment was conducted using a specific probe (SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69) that can identify ureaplasma parvum and ureaplasma urealyticum.

Using a primer pair from the primer of SEQ ID NO: 33, the primer of SEQ ID NO: 34, and the primer of SEQ ID NO: 35, the target gene sequence of ureaplasma parvum , ureaplasma urealyticum (Ureaplasma urealyticum) was PCR amplified and The amplification product was subjected to a hybridization experiment with a DNA chip composed of a probe of SEQ ID NO: 66, a probe of SEQ ID NO: 67, and a probe of SEQ ID NO: 69.

On the other hand, the amplification experiment of the target gene was carried out in the same process as in Example 6-1, and the hybridization experiment between the amplification product and the DNA chip was carried out in the same process as in Example 6-2.

A were analyzed by scanning the fluorescence of the DNA chip, as shown in FIG urea plasma Wu reahra ET Com (Ureaplasma urealyticum) the signal is strong and analyzed for urea plasma Wu reahra ET Com-specific probe in (Ureaplasma urealyticum) (Probe of SEQ ID NO: 69) was determined to be effective for ureaplasma urea lighticum test.

In addition, Figure 9 and that the signal for analysis by scanning the fluorescence of the DNA chip urea plasma pabum (Ureaplasma parvum) is strongly analysis urea plasma pabum specific probe in (Ureaplasma parvum), as shown in Figure 10 ( The probe of SEQ ID NO: 66 and the probe of SEQ ID NO: 67) were determined to be effective in the ureaplasma phagocytosis test. In particular, the probe of SEQ ID NO: 66 was able to identify serotypes 1 and 6 of ureaplasma parvum (see Fig. 9), and the probe of SEQ ID NO: 67 is serotypes 3 and 14 of ureaplasma parvum. Could be confirmed (see Fig. 10).

On the other hand, the primers and probes used in the examples are only one example used for the actual experiment, and all primers and probes for amplification of the target gene as shown in Tables 1 to 3 can be used. Of course.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. Those skilled in the art will be able to make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

<110> Genocheck Co., Ltd. <120> Probe for detecting microbes causing sexually transmitted diseases and detection chip, detection kit, and detection method using the same <130> P12-0284KR <160> 82 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NGporAP1F Primer <400> 1 cgaagtcaaa gctggtgt 18 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NGporAP1R Primer <400> 2 gcttttggcc ttagtaactg ta 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGporAP2F Primer <400> 3 aaggctgttt ggcagctcga 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NGporAP2R Primer <400> 4 tggcatcgcc aaacggat 18 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NGporAP3F Primer <400> 5 tcaaacgcca cgacggtat 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NGporAP3R Primer <400> 6 gctgaaaccg gaaaatccg 19 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NGporAP6F Primer <400> 7 aaaatggcgg ctttttcgga aatta 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGporAP6R Primer <400> 8 ggatcggtat cactcgctct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGporAP7F Primer <400> 9 ttcggtaata cagtcccgcg 20 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NGporAP7R Primer <400> 10 gcttggaaaa atcgtaatcg acacc 25 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CT16SF81 Primer <400> 11 gaacggagca attgtttcga cg 22 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CT16SR261 Primer <400> 12 gctgatatca catagactct cccttaac 28 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TV18SP1F Primer <400> 13 gccaattatt tactttgccg aag 23 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TV18SP1R Primer <400> 14 ctcttccacc tgctaaaatc g 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TV18SP2F Primer <400> 15 gcgtgctaca atgttagga 19 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TV18SP2R Primer <400> 16 gattataaga gtagcgcacc c 21 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TP16SF164 Primer <400> 17 tttgagatgg ggatagcctc tag 23 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TP16SR253 Primer <400> 18 gcccctttcc tctcaaagac 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TPpolAF1210 Primer <400> 19 gacggctgca catcttctc 19 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TPpolAF1648 Primer <400> 20 tggcgccata cctatagaaa ata 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TPpolAR1770 Primer <400> 21 gcacacgcgc aatcaataat acg 23 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TPpolAF2407 Primer <400> 22 aatgccgctt ttgcctatcc ta 22 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TPpolAF2766 Primer <400> 23 cagggcgtgt acatactagc tttg 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TPpolAR2889 Primer <400> 24 aacgcctgcc ttatttttct tcct 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HD16S1135F Primer <400> 25 cgcgaagaac cttacctact c 21 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HD16S1135R Primer <400> 26 accttcctcc agtttatcac tg 22 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HDPSSDBPF190 Primer <400> 27 atagccacca aagccatc 18 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HDPSSDBPR289 Primer <400> 28 ccaaaaatca actagtttta ctcatcg 27 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CAITS1F406 Primer <400> 29 gacggtagtg gtaaggc 17 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CAITS1R496 Primer <400> 30 aagatatacg tggtggacg 19 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CANRG1F331 Primer <400> 31 gcagctactc cattgtca 18 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CANRG1R467 Primer <400> 32 ggaggtactt gggaaggat 19 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> UPMBAF361-1 Primer <400> 33 tacgaaattg aaaactttaa agatctaagt 30 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> UUMBAF362-2 Primer <400> 34 acgacattga aaatttcgat gatttaa 27 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> UsppMBAR528 Primer <400> 35 ttttggttca cgwggtaatt taac 24 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MH16S549F Primer <400> 36 gcacgatgaa ggtcttcgg 19 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MH16S549R Primer <400> 37 aggtaccgtc agtctgcaat 20 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MHhitBLF22 Primer <400> 38 aaagaagtta caaaacaagt tttaacattt ga 32 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> MHhitBLR243 Primer <400> 39 tcatgcatat tatactagac cttcaacttt 30 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> MG4.F Primer <400> 40 caaacagtag ccaacttgta aaagtg 26 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MG4.R Primer <400> 41 gattactagt gattccagct tcatgag 27 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MGtufF60 Primer <400> 42 agaacttact acctttttca caagc 25 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> MGtufR278 Primer <400> 43 aaatttaaag ctgagatcta tgctttaaag 30 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MGprP65F881 Primer <400> 44 aactttcaaa gcacaactct tatcc 25 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MGprP65R1103 Primer <400> 45 tcacggtaac cccttcttct aaa 23 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GVVLYF339 Primer <400> 46 tcgcgtatac ccaggtgctc tttt 24 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GVVLYR839 Primer <400> 47 actggtgggc gcttgttgtc a 21 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GVVLYF455 Primer <400> 48 acggcggcga aagtgctgta 20 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GVVLYR972 Primer <400> 49 atggaattcg gtgtttggct tgat 24 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGporAP1 Probe <400> 50 tcagaaggtc aaacgggcaa 20 <210> 51 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NGporAP2 Probe <400> 51 gacccgttgg ggtaacag 18 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NGporAP3 Probe <400> 52 cgtgcgttac gattcccc 18 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> NGporAP6 Probe <400> 53 atgaggggca tgatgctttc tttttg 26 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NGporAP7 Probe <400> 54 cgactttgtc gaacgcagtc agaaa 25 <210> 55 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CT16SP220 Probe <400> 55 gatatttggg catccgagta acgtt 25 <210> 56 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> TV18SP1-2 Probe <400> 56 gccgaagtcy ttcggttaaa gttctaatt 29 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TV18SP2-2 Probe <400> 57 tcaataggac tgcgagcctg ag 22 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TP16SP228 Probe <400> 58 ccgaatacgc tcttttggac gta 23 <210> 59 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TPpolAF1210 Probe <400> 59 tggtgttact gaccctgtag aact 24 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TPpolAP2831 Probe <400> 60 ccaaatttac aaaacattcc cattaaaagc 30 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HD16S1135P Probe <400> 61 agcatgtagt gatgggaact caa 23 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HDPSSDBPP232 Probe <400> 62 agtatgcttg tatttaacct cacctgt 27 <210> 63 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CAITS1P423 Probe <400> 63 gggatcgctt tgacaatg 18 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CANRG1P380 Probe <400> 64 cactacaaca acctcagcca tacc 24 <210> 65 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CANRG1P412 Probe <400> 65 tactacaact atcagtatgc tgctcc 26 <210> 66 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> UPMBAP452-1 Probe <400> 66 acaaaacaga aaatctagtt acaaaaggtc at 32 <210> 67 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> UPMBAP454-2 Probe <400> 67 aaaacagaaa gtacacttga aaaaggtca 29 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> UUMBAP465-3 Probe <400> 68 cgcaacaaca aaaggtcact tac 23 <210> 69 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> UUMBAP462-4 Probe <400> 69 aaacgcaact ataaaaggtc acttactt 28 <210> 70 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> UUMBAP467-5 Probe <400> 70 caacaacaaa aggtcactta cttaacaaaa aa 32 <210> 71 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> UUMBAP467-6 Probe <400> 71 caactataaa aggtcactta cttaacaaga aa 32 <210> 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MH16S549P Probe <400> 72 aagggaagaa catttgcaat agg 23 <210> 73 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> MHhitBLP178 Probe <400> 73 gtttcagaaa caaayaaatc tgtttatgat atat 34 <210> 74 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MGP4 Probe <400> 74 gcaaatctga aaagttggtc tcag 24 <210> 75 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MGtufP190 Probe <400> 75 atagaattga ggacggtaac cgttt 25 <210> 76 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MGprP65P1018 Probe <400> 76 caatactacc ctcaacccta tccatac 27 <210> 77 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> MGprP65P1047 Probe <400> 77 aagaccttac tatgatgaac ctatttacgc 30 <210> 78 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> GVVLYP787 Probe <400> 78 agatttcttt gctccttgca ctacgc 26 <210> 79 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GVVLYP891 Probe <400> 79 caccagcaag agcactgatt tcca 24 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ARPCF Primer <400> 80 ggacaaccac tgcgtctat 19 <210> 81 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ARPCR Primer <400> 81 caaggaaggt cactgcaa 18 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ARPCP Probe <400> 82 tcacttctgg tgcgaatccc g 21

Claims (14)

An oligonucleotide having a nucleotide sequence of SEQ ID NO: 66 to a base sequence of SEQ ID NO: 67 and at least one probe selected from the group consisting of oligonucleotides having a nucleotide sequence complementary to these oligonucleotides; In the probe composition for the examination of pathogenic microorganisms that cause diseases infected by sexual contact, specifically binding to the gene nucleic acid of Ureaplasma parvum ),
Oligonucleotides having a nucleotide sequence of SEQ ID NO: 66 to a base sequence of SEQ ID NO: 67 and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides specifically bind to a gene nucleic acid of Ureaplasma parvum . Characterized in that, the probe composition for the examination of pathogenic microorganisms causing diseases infected by sexual contact. An oligonucleotide having a nucleotide sequence of SEQ ID NO: 66 to a base sequence of SEQ ID NO: 67, and at least one probe selected from the group consisting of oligonucleotides having a nucleotide sequence complementary to these oligonucleotides; Oligonucleotides having a nucleotide sequence of SEQ ID NO: 67 to the base sequence of and oligonucleotides having a base sequence complementary to these oligonucleotides are characterized in that the specific binding to the gene nucleic acid of ureaplasma parvum ( Ureaplasma parvum ) Pathogenic microbial test chip, causing disease infected by sexual contact. The method of claim 2,
And the probe is microarrayed on a substrate, wherein the pathogenic microbial test chip causes a disease infected by sexual contact. Sexual contact, comprising a set of primers for amplifying the ureaplasma paboom gene nucleic acid having the nucleotide sequences of SEQ ID NO: 33 and SEQ ID NO: 35, DNA polymerase, dNTPs, PCR buffer and PCR amplification products, PCR amplification kit for pathogenic microbial genes causing disease by infection. The method of claim 4, wherein
There is no homology with the gene sequences specific to the pathogenic microorganisms and non-homologous sequences belonging to different animal groups, different plant groups, different microbial groups or different viral groups than the pathogenic microorganisms are tagged at the 5 'end of the primer. , The single-chain label of the PCR amplification product is a labeled probe having a sequence complementary to the heterologous nucleotide sequence, pathogenic microorganism gene PCR amplification kit causing a disease infected by sexual contact. The method of claim 5,
The non-homologous nucleotide sequence is M13 primer sequence or SP6 sequence, pathogenic microbial gene PCR amplification kit causing a disease infected by sexual contact. A pathogenic microbial test chip for causing a disease infected by sexual contact as defined in claim 2,
As a primer set for amplifying a gene specific for the pathogenic microorganism, a primer set for amplifying a gene nucleic acid of ureaplasma paboom having a nucleotide sequence of SEQ ID NO: 33 and SEQ ID NO: 35,
And a labeling means for detecting the amplified gene hybridized with the test chip, the pathogenic microorganism test kit causing a disease infected by sexual contact. The method of claim 7, wherein
There is no homology with the gene sequences specific to the pathogenic microorganisms and non-homologous sequences belonging to different animal groups, different plant groups, different microbial groups or different viral groups than the pathogenic microorganisms are tagged at the 5 'end of the primer. And said labeling means is a labeled probe having a sequence complementary to said non-homologous sequencing, said pathogenic microorganism test kit causing a disease infected by sexual contact. The method of claim 8,
The non-homologous base sequence is characterized in that the M13 primer sequence or SP6 sequence, pathogenic microorganism test kit causing a disease infected by sexual contact. The method of claim 8,
The non-homologous sequence is further included in the test chip as a position marker of the probe, pathogenic microorganism test kit causing a disease infected by sexual contact. In the test for pathogenic microorganisms causing diseases infected by sexual contact,
PCR amplifying a gene of a sample using a primer set for amplifying a ureaplasma paboom gene nucleic acid having a nucleotide sequence of SEQ ID NO: 33 and SEQ ID NO: 35 as a primer set for gene amplification specific to the pathogenic microorganism;
Hybridizing the amplified gene to a test chip as defined in claim 2,
Detecting a reactant hybridized with a probe of the test chip, wherein the pathogenic microorganism test method causes a disease infected by sexual contact. The method of claim 11,
Tagging the non-homologous sequences belonging to different animal groups, different plant groups, different microbial groups or different viral groups than the genetic sequences specific for the pathogenic microorganisms at the 5 'end of the primer Wow,
Labeling the amplified gene with a labeled probe having a sequence complementary to the non-homologous sequence, pathogenic microorganism testing method for causing a disease infected by sexual contact. The method of claim 12,
The non-homologous base sequence is characterized in that the M13 primer sequence or SP6 sequence, pathogenic microbial test method for causing diseases infected by sexual contact. The method of claim 12,
Incorporating the heterologous nucleotide sequence into the test chip as a position marker of the probe, and identifying the position of the probe by labeling the position marker with a labeled probe having a sequence complementary to the heterologous nucleotide sequence. A pathogenic microorganism testing method comprising a disease caused by sexual contact.

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