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WO2006038752A1 - Sonde de bacteries provoquant des maladies sexuellement transmissibles, puce a adn et trousse de genotypage - Google Patents

Sonde de bacteries provoquant des maladies sexuellement transmissibles, puce a adn et trousse de genotypage Download PDF

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Publication number
WO2006038752A1
WO2006038752A1 PCT/KR2005/000773 KR2005000773W WO2006038752A1 WO 2006038752 A1 WO2006038752 A1 WO 2006038752A1 KR 2005000773 W KR2005000773 W KR 2005000773W WO 2006038752 A1 WO2006038752 A1 WO 2006038752A1
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seq
dna
std
amplifying
pathogens
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PCT/KR2005/000773
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English (en)
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Woo-Chul Moon
Myung-Ryurl Oh
Jung-Eun Ko
Min-Koo Park
Jin-Sun Lee
Eun-Ju Jang
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Goodgene Inc.
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Priority to JP2007535593A priority Critical patent/JP2008515423A/ja
Publication of WO2006038752A1 publication Critical patent/WO2006038752A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a DNA chip for diagnosis with quickness and accuracy of infection with Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticum and Mycoplasma genitaliiun, which are four bacteria having highest attack rate among pathogens for sexually transmitted diseases (STD), a genotyping kit and assay, and an oligonucleotide probe used therefor.
  • STD sexually transmitted diseases
  • STD Sexually transmitted diseases
  • NGU nongonococcal urethritis
  • the nongonococcal urethritis herein refers the case where Neisseria gonorrhoeae is not found in a male attacked by urethritis.
  • the most common pathogen for nongonococcal urethritis is Chlamydiae trachomatis, and besides, Ureaplasma urealyticum and Mycoplasma genitalium are also pointed out as a major pathogen.
  • the four kinds of bacteria i.e., Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium are in absolute majority of STD causes.
  • These bacteria are all major causes in common for anterior urethritis in a male, and may be complicated with epididymitis and prostatitis, and are also crucial causes for sterility. Also, they are major causes for cervicitis and pelvic inflammatory diseases in a female, and may cause complications such as sterility and ectopic pregnancy by causing fallopian tube occlusion.
  • Chlamydiae infection is most common in Western countries, and also in Korea, Chlamydiae infection and nongonococcal urethritis are on an increasing trend recently while gonorrhea has decreased.
  • STD lies latent with no symptom differently from other diseases, which leads to severe symptoms in many cases, and it is easy for reinfection. Therefore, it is important to develop a method of detecting the STD pathogens in its early stages, and also diagnosing effectively a carrier with no symptoms.
  • urethritis When urethritis is found in some patient, first, it should be discriminated if it is gonococcal or non gonococcal because the treatment and the progress of the disease are different from each other. However, due to recent increase of bacterial variation and mixed infection according to abuse and misuse of antibiotics, discrimination between them is likely to be unclear, so accurate diagnosis needs molecular genetic assay as in the present invention.
  • Neisseria gonorrhoeae which causes gonococcal urethritis, belongs to the genus Neisseria.
  • Neisseria gonorrhoeae To make up for Gram staining, culture of Neisseria gonorrhoeae has been tried.
  • the culture of Neisseria gonorrhoeae is an assay which is useful in many views and accurate, but has a shortcoming that it consumes much time and cost until the assay results are found.
  • Nongonococcal urethritis is on the increase in the attack rate, while gonococcal urethritis has decreased recently. Nongonococcal urethritis is a kind of syndrome which brings about from various microorganisms, and not from single disease. Most important microorganism causing nongonococcal urethritis is Chlamydiae trachomatis.
  • Chlamydiae trachomatis is a microorganism which has a life cycle of intracellular parasitism, which has both of DNA and RNA on the genome, and which has 15 serum types in total. D-K types cause nongonococcal urethritis among them. Human diseases which can be developed by them include STD trachoma, respiratory infection and STD comprising urethritis, cervicitis, epididymitis and pelvic inflammatory diseases. It has been reported recently that Chlamydiae causes myocarditis.
  • a method for identifying Chlamydiae trachomatis some methods have been used such as (1) a method for identifying the bacteria by culturing and isolating a material gathered from an infected region, (2) a method for identifying the bacteria by direct smear staining of the epithelium in an infected region, and (3) a method for identifying the bacteria by isolating antibody from the serum of a patient, and recently, a method using molecular genetic technique is preferred such as (4) a method using polymerase chain reaction (PCR) and hybridization.
  • PCR polymerase chain reaction
  • the method of culturing and isolating the sample is not suitable for the purpose of clinical diagnosis due to taking long time, low diagnosis sensitivity and the like.
  • the method for identifying the bacteria by direct smear staining of epithelium in an infected region is advantageously simple, but has low diagnosis sensitivity.
  • the fluorescent antibody method using a reagent wherein FITC (fluorescein isothiocynate), a fluorescent substance, is adhered to monoclonal antibody has relatively high sensitivity, but it has a shortcoming that it requires high cost and dedicated apparatus, resulting in its limited use.
  • Ureaplasma urealyticum and Mycoplasma genitaliiim which belong to the genuses Mycoplasma and Ureaplasma which are widely present in human and various mammalian animals, and which may cause infection in urogenital organs, are very difficult to culture technically, and also difficult to find by staining or immunological method. Accordingly, recent tendency for diagnosis is molecular genetic assay based on PCR. In this case, use is mainly made of a method of identifying the bacteria by PCR-ampl ifying DNA base sequence of gene specific for each bacterial species, and identifying it by sequencing reaction, hybridization and the like.
  • an assay should have nearly 100% sensitivity, specificity and reproductivity.
  • the conventional bacterial phenotype assay such as the culture assay or the immune assay has trouble to satisfy such conditions.
  • genetic assay analyzing genotype of bacterial genome i.e., base sequence of DNA and RNA, can fulfill all the three conditions in theory (Olive DM et al. Journal of Clinical Microbiology. 1999:37:1661-9), and a diagnosis kit for assaying gonococcal infection and Chlamydiae infection using PCR technique, has been already marketed and used (Shafer M. et al . , J. Clin. Microbiol. 2003; 41: 4395 ⁇ 9).
  • the genetic assay for bacterial finding has many conditions to satisfy in addition to the mentioned conditions so as to be lively used for the clinical examination and treatment or in other laboratories, and give substantial help to clinical examination and treatment.
  • the genetic assay should be easy, easily interpreted and simple, give quick results and has no need of special expensive apparatus.
  • the assay should be automated to allow quick assay of many samples, and also not should be expensive in the assay cost. Therefore, PCR needs to be multiplex type PCR if possible, with which the subject bacteria are assayed all in one tube in one PCR reaction.
  • the gene and base sequence region to be assayed should be suitably selected so as to discriminate each subject bacteria accurately to strain level in PCR, and in accordance with this, optimal PCR condition should be established.
  • multiplex type PCR which searches for four major pathogens for urethritis and cervicitis, i.e., Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticim or Mycoplasma genitalium at a time, and such PCR kit has not been marketed yet .
  • an assay is needed which identifies accurately if amplified products after PCR is accurately DNA of desired bacteria.
  • a genetic assay is needed which reads accurately bacterial genotype, and thereby allows to predict accurately antibiotic resistance, biological invasion rate and the like. Therefore, it is urgent to develop a DNA chip (or DNA microarray) which assays various STD pathogens at a time, and furthermore gives information such as antibiotic resistance.
  • a multiplex type PCR method which is capable of amplifying genes of Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma iirealyticum and Mycoplasma genitaliiim, which are in absolute majority by 90% or more of STD causes, being four major pathogens for urethritis, cervicitis and pelvic inflammatory diseases, and control DNA of human gene in one tube in one PCR reaction at a time, and assay them.
  • a primer which is effective for PCR amplification of the four major pathogens and human b-globulin.
  • an oligonucleotide probe which allows more accurate detection for a gene of the base sequence that is unique for each of the four bacteria, control gene and antibiotic-resistant gene.
  • a DNA chip for detection and genotyping of the four major pathogens which comprises the probe collected.
  • a kit for detection and genotyping of the four major pathogens which comprises the probe.
  • the gene and its region of the four bacteria to be assayed and human b-globulin to be assayed have been determined, and PCR primers necessary for amplification of them have been designed (Example 1), DNA clones for the control strain and a target gene of each strain have been secured (Example 2), a method of gathering and storing a sample has been established (Example 3), a method of isolating DNA from a sample has been established (Example 4), single PCR conditions for the target gene of bacteria have been established (Example 5), single PCR and sequencing has been performed in a clinical sample, and the results have been made as database (Examples 6 and 7), multiplex PCR conditions for genes of the four bacteria and human b-globulin have been established (Example 8), and the multiplex assay has been performed in a human sample and thereby quality control of the assay has been checked (Example 9).
  • a probe for assaying hybridization of the four bacteria and human b-globulin gene has been designed and a DNA chip with use of this has been constructed (Examples 10 and 11), an artificial sample has been assayed with the DNA chip to thereby establish assay conditions (Example 12), a clinical sample has been assayed with use of the DNA chip (Example 13), and thereby it has been found that infection with the four bacteria has been diagnosed, and genotype of the infection bacteria has been discriminated as well. Thereby, the present invention has been completed.
  • the primers of the present invention comprise a primer for amplifying nucleic acid of Neisseria gonorrhoeae having the base sequence of SEQ ID NO: l or SEQ ID NO: 2, a primer for amplifying nucleic acid of Chlamydiae trachomatis having the base sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a primer for amplifying nucleic acid of Ureaplasma urealyticum having the base sequence of SEQ ID NO: 5 or SEQ ID NO: 6, a primer for amplifying nucleic acid of Mycoplasma genitaliim having the base sequence of SEQ ID NO: 7 or SEQ ID NO: 8, a primer for amplifying nucleic acid of human b-globulin having the base sequence of SEQ ID NO: 9 or SEQ ID NO: 10.
  • the PCR amplification method of the present invention is a single or multiplex PCR amplification method with use of at least one of primers.
  • the probe for detection of a STD pathogen of the present invention comprises oligonucleotide comprising 16S rRNAs of Neisseria gonorrhoeae and Mycoplasma genitalium and 16S-23S intergenic region of Ureaplasma urealyticum, or a part of cryptic plasmid gene of Chlamydiae trachomatis. More preferably, the probe is selected from a group consisting of oligonucleotides having the base sequences of SEQ ID NO: 11 to SEQ ID NO: 33, and oligonucleotides having base sequences complementary to these oligonucleotides.
  • the DNA chip of the present invention comprises one or more kinds of probe which is selected from a group consisting of oligonucleotides having the base sequences of SEQ ID NO: H to SEQ ID NO: 33 and oligonucleotides having base sequences complementary to these oligonucleotides.
  • SEQ ID NOS: Ii to 16 are probes for detection of Ureaplasma urealyticum
  • SEQ ID NOS: 17 to 21 are probes for detection of Neisseria gonorrhoeae
  • SEQ ID NOS: 11 to 28 are probes for detection of Chlamydiae trachomatis
  • SEQ ID NOS: 29 to 33 are probes for detection of Mycoplasma genitaliiw.
  • the DNA chip for detection of a STD pathogen in another embodiment of the present invention uses 5 to 7 probes per gene of the target bacteria, thereby to avoid false negative and false positive which can be caused when only one probe is used per one target gene, and to maximize diagnosis sensitivity and specificity.
  • the DNA chip for detection of a STD pathogen in another embodiment of the present invention may further comprise b-globulin, actin or glyceraldehyde-3-phosphate dehydrogenase gene as a reference marker, and when the reference marker is b-globin, it has preferably the base sequence represented by sequence No. 40 and a primer for its amplification is a primer for amplifying b-globulin DNA having the base sequences of SEQ ID NO: 9 and SEQ ID NO: 10.
  • Use of the reference marker makes it possible to evaluate the hybridization in the DNA chip, and quality control for DNA isolation procedure and PCR amplification procedure, which are prior procedures, and find false negative.
  • the DNA chip for detection of a STD pathogen in another embodiment of the present invention may further comprise one or more kinds of probes for detection of tetracycline resistance gene (TetC). Assaying tetracycline antibiotic-resistant gene as well can accurately find in advance resistance for tetracycline antibiotics, which is most widely used antibiotics in STD treatment, helping with determination of treatment plan, and treatment.
  • the probe of the tetracycline antibiotics gene preferably has the base sequence of SEQ ID NOS: 34 to 39.
  • a production method for the DNA chip of the present invention comprises a process of producing a DNA probe which is a base sequence which can bind complementarily to the nucleic acid of a STD pathogen, and has 5' end amine bound; a process of having the produced DNA probe bound to the surface of the solid to which aldehyde is bound; and a process of reducing aldehyde which is not bound to the amine present on the solid surface to which the DNA probe is bound.
  • Binding of the probe DNA and aldehyde on the solid surface can be performed through Schiff base reaction of amine and aldehyde, and the solid is selected from glass, silicon dioxide, plastics or ceramics.
  • the kit for detection of a sexually transmitted diseases (STD) pathogen of the present invention comprises a primer set for PCR amplification of the DNA sample of a STD pathogen, a DNA chip for detection of the STD pathogen, and a marker for detection of the DNA chip and amplified DNA to be hybridized.
  • STD sexually transmitted diseases
  • a method of detecting a sexually transmitted diseases (STD) pathogen of the present invention comprises (a) a step of amplifying the DNA sample of a STD pathogen with use of a primer for amplifying STD pathogen DNA; (b) a step of hybridizing the amplified DNA on a DNA chip comprising one or more kinds of probe which is selected from a group consisting of oligonucleotides having the base sequences of SEQ ID NO: 11 to SEQ ID NO: 33 and oligonucleotides having base sequences complementary to these oligonucleotides; and (c) a step of detecting the probe and the hybridized reactants.
  • STD sexually transmitted diseases
  • the assay kit and a detection method for detection of a STD pathogen may comprise the primers and the marker.
  • the primer may be a primer for amplifying a STD pathogen which is selected from a group consisting of the base sequences of SEQ ID NO: 1 to SEQ ID NO: 8, and may comprise b-globulin primer consisting of the base sequences of SEQ ID NOS: 9 and 10.
  • ⁇ 4i> As the marker, use can be made of publicly known various labels, for example, Cy5, Cy3, a biotin-binding substance, EDANS (5-(2' ⁇ aminoethyl)amino-l-naphthalene sulfuric acid), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), ⁇ -rhodamine or Texas red.
  • TMR tetramethylrhodamine
  • TRITC tetramethylrhodamine isocyanate
  • ⁇ -rhodamine or Texas red When Cy5 is used for labeling, fluorescent signal can be assayed directly with use of an analyzer such as confocal laser scanner without additional reaction in detection of the labeled reactants, which leads to effective and sensitive results.
  • Fig. 1 is a photograph for electrophoresis on 2% agarose gel of products which is obtained by PCR amplification for 16S ribosome RNA (16s rRNA) gene of Neisseria gonorrhoeae with use of a primer set of certain base sequences (Lanes 1 and 9: the 100 bp DNA size marker, Lane 2- ' the negative control group, Lane 3: the positive control group, an artificial sample which is made by mixing 10 copies of the plasmid clone of 16s rRNA of Neisseria gonorrhoeae, with the urine of a normal person, Lane 4: the positive control group, an artificial sample which is made by mixing 100 copies of the plasmid clone of 16s rRNA of Neisseria gonorrhoeae, with the urine of a normal person, Lane 5: the urethral discharge of a male who is infected with Neisseria gonorrhoeae, Lane 6- the
  • Fig. 2 is a photograph for electrophoresis on 2% agarose gel of products which is obtained by PCR amplification for cryptic plasmid DNA of Chlamydiae trachomatis with use of a primer set of certain base sequences (Lanes 1 and 9: the 100 bp DNA size marker, Lane 2- the negative control group, Lane 3: the positive control group, an artificial sample which is made by mixing 10 copies of the plasmid into which is cryptic plasmid clone of Chlamydiae trachomatis inserted, with the urine of a normal person, Lane 4: the positive control group, an artificial sample which is made by mixing 100 copies of the plasmid into which is cryptic plasmid clone of Chlamydiae trachomatis inserted, with the urine of a normal person, Lane 5: the urethral discharge of a male who is infected with Chlamydiae trachomatis, Lane 6: the
  • Fig. 3 is a photograph for electrophoresis on 1.5% agarose gel after PCR amplification for the intergenic space region of 16S ribosome RNA and 23S ribosome RNA of Ureaplasma urealyticiun (16S-23S RNA intergenic space region) with use of a primer set of certain base sequences (Lanes 1 and 9: 100 bp & 1 kb DNA size markers, Lane 2: the negative control group, Lane 3: the positive control group, an artificial sample which is made by mixing 10 copies of the plasmid of 16S-23S ribosome RNA intergenic space region of Ureaplasma urealyticuw, with the urine of a normal person, Lane 4- the positive control group, an artificial sample which is made by mixing 100 copies of the plasmid of 16S-23S ribosome RNA intergenic space region of Ureaplasma urealyticum, with the urine of a normal person, Lane 5: the urethral discharge of a male who is in
  • Fig. 4 is a photograph for electrophoresis on 2% agarose gel after PCR amplification for 16S ribosome RNA (16s rRNA) gene of Mycoplasma genitalium with use of a primer set of certain base sequences
  • Lane 1 and 9 the 100 bp DNA size marker
  • Lane 2 the negative control group
  • Lane 3 the positive control group
  • an artificial sample which is made by mixing 10 copies of 16s rRNA plasmid clone of Mycoplasma genitalium, with the urine of a normal person
  • Lane 4 the positive control group, an artificial sample which is made by mixing 100 copies of 16s rRNA plasmid clone of Mycoplasma genitalium, with the urine of a normal person
  • Lane 5 the urethral discharge of a male who is infected with Mycoplasma genitalium
  • Lane 6 the sample which is obtained by scraping the urethra of a male who is infected with Mycoplasma gen
  • Fig. 5 is a photograph for electrophoresis of products which are assayed on 1.8% agarose gel, which is obtained by isolating DNA from the urine samples of a normal person who has no STD infection and a patient of STD infection, and PCR amplification of human b-globulin (HBB) gene which is a housekeepeeping gene, at 58 ° C, to check quality control (QC) for DNA isolation procedure and PCR amplification procedure in the human sample (Lane l: the 100 bp DNA size marker, Lane 2: the urine sample of the normal person, Lanes 3 & 4' the urine sample of the STD ⁇ infected patient).
  • HBB human b-globulin
  • Fig. 6 is a graph of sequencing for 16S rRNA gene of Neisseria gonorrhoeae among the results found with an automated sequencer after amplifying genes of Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium in a clinical sample with use of the multiplex PCR method of the present invention.
  • Fig. 7 is a graph of sequencing for the cryptic piasmid of Chlamydiae among the results found with an automated sequencer after amplifying genes of Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium in a clinical sample with use of the multiplex PCR method of the present invention.
  • Fig. 8 is a graph of sequencing for the 16S-23S rRNA intergenic space region of Ureaplasma urealyticum among the results found with an automated sequencer after amplifying genes of Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium in a clinical sample with use of the multiplex PCR method of the present invention.
  • Fig. 9 is a graph of sequencing for the 16S rRNA of Mycoplasma genitalium among the results found with an automated sequencer after amplifying genes of Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium in a clinical sample with use of the multiplex PCR method of the present invention.
  • Fig. 10 is a photograph for electrophoresis on 1.8% agarose gel, which is obtained by mixing a sample storage reagent with 10 copies and 100 copies, respectively of each plasmid clone, into which inserted are DNA of each target gene of Neisseria gonorrhoeae, Mycoplasma genitaliiim, Ureaplasma urealyticum and Chlamydiae trachomatis, and DNA of human b- globulin gene, respectively, adding the mixture with a primer set which is bound to the DNAs specifically to one tube, and then performing multiplex PCR at 56V (Lanes 1 & 6: the 100 bp & 1 Kb DNA ladder, Lane 2: the negative control group (b-globulin), Lane 3: the case where the concentration of the primer set is the same for all by 10 pmo ⁇ e/ ⁇ l ⁇ , Lane 4: the case where the concentration of the primer set is 10 pmole/ ⁇ ?.
  • Fig. 11 is a photograph for electrophoresis on 1.8% agarose gel after performing STD multiplex PCR of the present invention at 58 " C for 4 ⁇ Jt DNA sample which is extracted from a clinical sample, which has been identified already for STD infection and the pathogen (Lanes 1 & 8: the 100 bp & 1 kb DNA ladder, Lane 2: the negative control group (human b- globulin), Lane 3: the positive control group (an artificial sample obtained by mixing a sample storage reagent with 10 copies and 100 copies, respectively of each plasmid clone, into which inserted are DNA of each target gene of the four pathogens and DNA of human b-globulin gene, respectively), Lane 4- the urine sample of a male who was found as infected with Mycoplasma genitalium, Lane 5: the urine sample of a male who was found as infected with Chlamydiae trachomatis, Lane 6: the urine sample of a male who was found as infected
  • Fig. 12 is a photograph for electrophoresis on 1.8% agarose gel for products which is obtained by performing STD multiplex PCR of the present invention for 4 ⁇ i DNA sample of a patient who has been already found as hyperinfected by two or more kinds of bacteria
  • Lane 1 and 10 the 100 bp DNA ladder, Lane 9- 1 kb DNA ladder, Lane 2' the negative control group (HBB), Lane 3: the positive control group (an artificial sample obtained by mixing a sample storage reagent with 100 copies of each of plasmid clones, into which inserted are DNA of each target gene of the four pathogens and DNA of human b-globulin gene, respectively)
  • Lane 4 a swab sample for the cervix of a female who was found as complex-infected with Chlamydiae trachomatis and Ureaplasma urealyticum
  • Lane 5- the urine sample of a male who was found as complex-infected with Ureaplasma urealyticum and of
  • Lane T the urine sample of a male who was found as complex-infected with Mycoplasma genitalium and Chlamydiae trachomatis
  • Lane 8 the urine sample of a male who was found as complex-infected with Neisseria gonorrhoeae, Chlamydiae trachomatis and Ureaplasma urealyticum).
  • Fig. 13 is a schematic view of a DNA chip (STD genotyping oligonucleotide microarray) on which the products after multiplex PCR are placed, and hybridization reaction is performed for identification.
  • Fig. 14 is a photograph of a DNA chip which has eight grids, which is one kind of the STD DNA chips of the present invention.
  • Fig. 15 is a fluorescent scanner image of a clinical sample which is infected with Neisseria gonorrhoeae, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 16 is a fluorescent scanner image of a clinical sample which is infected with Chlamydiae trachomatis, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 17 is a fluorescent scanner image of a clinical sample which is infected with Ureaplasma urealyticum, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 18 is a fluorescent scanner image of a clinical sample which is infected with Mycoplasma genitalium, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 16 is a fluorescent scanner image of a clinical sample which is infected with Chlamydiae trachomatis, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 17 is a fluorescent scanner image of a clinical sample which is infected with Ureaplasma urealyticum, which is
  • Fig. 19 is a fluorescent scanner image of a clinical sample which is complex-infected with Neisseria gonorrhoeae and Chlamydiae trachomatis, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 20 is a fluorescent scanner image of a clinical sample which is complex-infected with Neisseria gonorrhoeae and Ureaplasma urealyticum, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 20 is a fluorescent scanner image of a clinical sample which is complex-infected with Neisseria gonorrhoeae and Ureaplasma urealyticum, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 20 is a fluorescent scanner image of a clinical sample which is complex-infected with Neisseria gonorrhoeae and Ureaplasma
  • Fig. 21 is a fluorescent scanner image of a clinical sample which is complex-infected with Neisseria gonorrhoeae and Mycoplasma genitalium, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 22 is a fluorescent scanner image of a clinical sample which is complex-infected with Chlamydiae trachomatis and Ureaplasma urealyticum, which is assayed with the DNA chip for STD genotype diagnosis of the present invention.
  • Fig. 23 is a fluorescent scanner image of a negative control group which is not infected with STD, which is assayed with the DNA chip for STD genotype diagnosis of the present invention, wherein the signal of human b-globulin gene is only seen.
  • the gene region to be assayed was selected primarily from 16S rRNA or 23S rRNA, or the middle of them, and the space intergenic region, which are most widely used for finding phylogeny of bacteria (Olsen G.J. et al., Ribosomal RNA: a key to phylogeny. FASEB 1993; 7: 113-123; Lane DJB et al., Proc. Natl. Acad. Sci. USA. 1985; 92: 6955-9).
  • cytosine methyltransferase gene may be also assayed instead of 16S rRNA
  • ompl gene of major outer membrane protein (MOMP) may be also assayed.
  • MOMP major outer membrane protein
  • NG Neisseria gonorrhoeae
  • CT Chlamydia trachomatis
  • UU Ureaplasma ureaJyticum
  • MG Mycoplasma genitaliiw
  • HBB human b- globulin
  • F forward direction
  • R Reverse direction
  • ⁇ iO3> The strains of standard positive control group for each of the four bacteria to be assayed, were purchased from ATCC, USA (Manassas, VA20108, USA.), and also, a sample which had been identified to be already infected with each bacterium, was obtained. DNA was isolated from these, and then the target gene region to be assayed was PCR-amplified for each bacterium and identified through cloning and sequencing reaction, thereby plasmid clone was secured for each of them.
  • ⁇ I 2O> 8 About 100 ⁇ t of the culture solution was applied to the LB/ampici1lin/IPTG/X-Gal plate, and then the plate was cultured as flipped in a 37 ° C-adjusted incubator for 16 to 24 hours. Then, colony number was counted (colony counting), and only white colony was selected, and cultured in 3 ml LB/ampici11in culture solution, and then plasmid DNA was isolated and purified with Mini-Prep procedure. Then it was identified if the insertion DNA was inserted properly with use of PCR and cleavage reaction by restriction enzyme, and the base sequences of thus obtained clone was assayed with use of an automated sequencer.
  • Example 3 Gathering of Clinical Sample ⁇ I 25> A suitable method was established for gathering from a human body various samples such as discharge from the urethra, cervix or mouth, a swab sample which is obtained by gathering the cells with a cotton swab or brush, the urine, urethral washing solution and the prostate solution, and carrying and storing them before an assay.
  • gathering of the male urine was performed in division of VB (voided bladder) 1, VB2, VB3 and expressed prostatic secretion (EPS) according to the traditional 3-glass test.
  • VBl refers to the 10 to 20 ml urine which comes first in urination, i.e., early stream urine.
  • VB2 refers to mid-stream urine which comes in the middle of urination.
  • the prostate secretion refers to secretion which comes to the urethra following the prostate massage.
  • VB3 refers to the 10 to 20 ml urine which comes first in urination following the prostate massage.
  • VBl represents a urethral sample
  • VB2 represents a bladder sample
  • VB3 represents a prostate sample.
  • the standard samples for a male were a swab sample of the urethra and a sample of the early stream urine (VBl), and if necessary, the urethral discharge or the urine (VB3) after the prostate massage was added thereto.
  • the standard samples for a female were the swab or scrape sample of the cervix, and if necessary, a swab sample or discharge of the vagina or the urine was added thereto.
  • samples may be further obtained from the rectum or the anus.
  • the cotton swab from COPAN COPAN innovation, USA
  • Pap-brush from Sang-A Medical Seoul, ROK
  • the cotton part or brush part of the cotton swab was put into a tube, which has sterilized sample storage solution inside, and has a screw cap, moved and stored.
  • the sample gathered such as the urine or the swab sample should be carried within 48 hours to the laboratory if possible, and should be kept at refrigerating temperature during transportation. This helps to isolation of bacteria which is stringent for gene amplification, and inhibits overgrowth of rapid-growing bacteria.
  • a sterilized sample storage solution was filled in advance in the sample gathering tube The composition was made by mixing 100 ml of 37 to 40% formaldehyde with 15 ml methanol, 6.5 g of Na2HP04 and 4.0 g of NaH2P04, adding the sterilized triple-distilled water thereto to a final volume of 1 1iter.
  • DNA was isolated and purified by a method of using a commercialized kit and a method of using a manually prepared reagent for isolating DNA from various human samples of Example 3
  • QIA amp DNA blood mini kit (Cat No. 51106, Qiagen, Valencia, CA, USA) was used as follows
  • ⁇ i3s> A urine or swab sample was centrifuged using a swing rotor centrifuge for 30 minutes at 3000 rpm The supernatant was poured out the pellet was resuspended with 500 ⁇ l PBS, and then the suspension was transferred to a 15 ml-microcentrifuge tube The suspension was centrifuged for 2 minutes, and again the supernatant was poured out.
  • the concentration of the isolated DNA was measured in a spectrometer or an agarose gel. In the case where the DNA concentration of about 20 ng/ ⁇ C was obtained, 3 ⁇ t of the isolated DNA was used in the PCR, and in the case where the DNA band was not shown on the 1.5% agarose gel, 6 ⁇ i thereof was used.
  • ⁇ I36> The method of using a manually prepared reagent by the present inventors is as follows. About 1 ml of a urine sample or swab sample was centrifuged for 30 minutes at 15,000 rpm, the supernatant was discarded, and then the residue was washed with PBS (pH 7.4). Then, a 100 ⁇ i of cell lysis buffer composed of 10 mM Tris HCl (pH 8.0), 1 mM EDTA, 0.5% Tween- 50, 0.5% Nonidet P-40 and 200 #g/ml Protenase K was added thereto and reacted for 30 minutes at 55 ° C.
  • PBS pH 7.4
  • a 100 ⁇ i of cell lysis buffer composed of 10 mM Tris HCl (pH 8.0), 1 mM EDTA, 0.5% Tween- 50, 0.5% Nonidet P-40 and 200 #g/ml Protenase K was added thereto and reacted for 30 minutes at 55
  • the lysis buffer may be composed of 10 mM Tris HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl 2 , 0.5% sodium dodecylsulfate (SDS), 0.5% Tween-50, 0.5% Nonidet P-40 and 700 ⁇ g/ml Protenase K, and 500 ⁇ i thereof can be used for the treatment.
  • the reaction resultant is heated for 15 minutes at 94 ° C for inactivation of Protenase K, and then centrifuged again to obtain a concentrated precipitates.
  • the precipitates were treated with phenol-chloroform to obtain DNA, which was precipitated with ethanol, and then centrifuged.
  • the precipitates were dissolved in 50 ⁇ i of the sterilized triple-distilled water or a TE buffer (10 mM Tris-Hcl [pH 8.0] and 1 mM EDTA), and 3 to 6 ⁇ i thereof was used in PCR.
  • Example 5 PCR for establishing conditions of single PCR ⁇ 138> Artificial samples were made by adding plasmid clones of target genes, which were obtained in Example 2 for testing on each bacterium in multiple copies of every 10, 100, 1,000 and 10,000, to the sterilized triple-distilled water, a sample storing solution of Example 3 and a fresh urine (VBl) of a normal male without symptoms of infection. Then, a single PCR on target genes for each bacterium was performed repeatedly, thereby established appropriate conditions for the single PCR. At the time of performing PCR, PCR of human b-globulin, which was an internal reference gene, was surely performed together. Moreover, in consideration of a multiplex PCR afterwards, it is designed such that the size of each PCR product was distinctively different from each other, and that the annealing temperature had no big difference.
  • Example 6 Single PCR on clinical sample ⁇ I71> From the adult male and female patients of 541 persons who had visited urology department and obstetrics and gynecology department of domestic hospitals in the suspicion of contraction of STD and the adult male and female of 103 persons without symptoms of sexual infection, various samples such as fresh urine (VBl) or swab of urethritis, cervicitis, and the like are collected via the method of Example 3. Then, DNA was isolated therefrom via the method of Example 4, and single PCR was performed according to Example 5.
  • VBl fresh urine
  • swab of urethritis cervicitis
  • Example 7 Sequencing of PCR products on clinical sample ⁇ 173>
  • the PCR products of Example 6 were subjected to sequencing reaction using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer Biosystems, USA), and then subjected to assay of base sequence using an ABI Prism 377 Automated Base Sequencer (Perkin Elmer, USA). These processes were carried out in the following order.
  • Example 8 Experiment for establishing conditions for multiplex PCR ⁇ 186> Artificial samples were made by adding plasmid clones of specific genes comprising one to four types, which were obtained in Example 2 for testing on each bacterium in multiple copies of every 10, 100, 1,000 and 10,000, to the sterilized triple-distilled water, a sample storing solution of Example 3 and a fresh urine (VBl) of a normal adult male without symptoms of STD. Then, a multiplex PCR, which performs PCR simultaneously by adding 4 types of primers of target genes for every bacterium with the artificial samples in one tube, was performed.
  • a human b-globulin gene which is a housekeeping gene, was also amplified as a control gene.
  • Table 10 The compositions and conditions in the multiplex PCR are listed in Table 10. After the multiplex PCR, its product was confirmed by subjecting to electrophoresis on a 1.5 to 2.0% agarose gel (Figs. 6 to 9).
  • the product of Ureaplasma urealyticiim is 812 bp
  • the product of Neisseria gonorrhoeae is 386 bp
  • the product of Chlamydiae trachomatis is 314 bp
  • the product of Mycoplasma genitaliim is 207 bp
  • the amplified product of human b-globulin gene is 110 bp, respectively.
  • the DNAs may be searched accurately using a multiplex PCR at once. At this time, it was always possible to distinguish each DNA as long as 10 to 100 copies of plasmid clones of bacteria genes different from each other were contained in 1 ml of the sample (Fig. 10).
  • Example 9 Multiplex PCR on clinical sample ⁇ I 92>
  • DNA samples included as a test subject are 50 examples of Neisseria gonorrhoeae infected samples, 50 examples of Chlamydiae trachomatis infected samples, 50 examples of Ureaplasma urealyticum infected samples, 50 examples of Mycoplasma genitaliwi infected samples and 50 examples of non-infected samples.
  • 50 examples of infected samples 40 examples of complicated infection were also included.
  • the sensitivity and specificity of the detection of bacteria for causing STD by comparative assay between the results of the multiplex PCR and sequencing thereof were studied. Thereby, the assay was carried out on whether the multiplex PCR may be used in selecting and searching the four major pathogens for causing STD, clinically.
  • Figs. 6 to 9 are the results of sequencing confirmed by an automated sequencer after amplifying genes of Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma urealyticiun and Mycoplasma genitaliiim using the multiplex PCR method of the invention. It was known that the PCR products having very high selectivity can be produced by using 4 bacteria designed in the invention, primers of a human b-globulin gene and PCR methods.
  • Example 10 Probe designing for assay of hybridization ⁇ 200> In order to prepare a DNA chip for assaying genes of four major pathogens for causing STD and the PCR products of the control gene at the same time by hybridization reaction on one chip, first, a combination oligonucleotide probe having an appropriate base sequence was designed.
  • the database of the genetic bank of National Center for Biotechnology Information in the USA, and the database of four pathogens for causing STD and a human b-globulin gene found on Koreans obtained in Example 7 were assayed, and each genotype base sequence was obtained.
  • the obtained DNA sequence was subjected to the pairwise alignment and the multiple sequence alignment by means of ClustalW using the computer program of DNASTAR
  • the salt concentration of an oligo probe is standardized at 50 mil.
  • TetC Tetracycline resistance type C ⁇ 206> * Location of a probe sequence in the base sequence of the PCR product, excluding the primer sequence in the forward and reverse directions.
  • Example 11 Preparation of DNA chip ⁇ 208>
  • the oligonucleotide probes designed in Example 10 were mixed with appropriate reagents, and then by integrating on a glass slide for microscopes using an arrayer, an oligonucleotide microarray, or an oligo DNA chip for detection of genotypes of pathogens for causing STD was prepared by the following order and methods. Furthermore, a modified chip having 8 grids on one chip wherein 8 different samples may be integrated thereon for simultaneous assay was also prepared (Figs. 13 and 14).
  • ⁇ 2io> In the invention, grids are written in groups such that after hybridization reaction on a chip, specific bacteria can be easily found by the fluorescence signal shown according to the STD genotypes.
  • the mimetic diagram of the grid arrangement is illustrated in Fig. 13. On the most left side, 6 probes for 16S-23S ribosome boundary spacer of Ureaplasma urealyticiim were integrated, and to its right side, 6 probes for 16S rRNA of Neisseria gonorrhoeae were integrated. In the center, 7 probes for cryptic plasmid of Chlamydiae trachomatis were integrated, and to its right side, 5 probes for 16S rRNA of Mycoplasma genitalium were integrated.
  • oligonucleotide probes specific to the human b-globulin gene which is a control gene, as a corner marker, were integrated such that two sets thereof were located on the upper left and right sides and one each was located on the lower left and right sides.
  • oligonucleotide probe was spotted or integrated using an arrayer. At this time, the same probes were integrated in duplicate so that each genotype of bacteria was designed to show at least twice and at most four times.
  • oligonucleotide probes designed according to Example 10 synthesized by attaching an amine onto C6 position was purified using a high performance liquid chromatography, and then dissolved in the sterilized triple-distilled water to the final concentration of 200 pM.
  • prepared oligo probes were mixed with micro spotting solution Plus (Telechem, TC-MSP, USA), which is a spotting solution, in a proportion of 4.3 times so as to the final concentration of 38 pM.
  • micro spotting solution Plus Telechem, TC-MSP, USA
  • 32.4 ⁇ JL of spotting solution was mixed so as to the total of 40 ⁇ l.
  • prepared mixture was divided into a 96-well master plate in the order, respectively.
  • a glass slide to become a base material for a chip it is preferably a BMT aldehyde glass slide (Biometrix technology, Korea) or a product corresponding thereto having the size of 7.5 D 2.5 cm, and having coated with super aldehyde.
  • an arrayer it is preferably a GMS 417 arrayer (Affymetrix, USA), MGII (Biorobotics Inc. MA01801, USA) or equipments corresponding thereto.
  • the fixated slide was baked in a dry oven for 1 hour and 30 minutes at 120 ° C. Then, the slide was washed in a 0.2% sodium dodecylsulfate (SDS) twice for 2 minutes, and then transferred to a triple distilled water and washed twice for 2 minutes. Thereafter, the slide was dipped into heated triple distilled water at 95 ° C for 3 minutes, whereby the oligonucleotide probes attached on the slide were denatured, and then transferred to triple distilled water and washed for 1 minute.
  • SDS sodium dodecylsulfate
  • the slide after washing was reduced for 15 minutes in a blocking solution (1 g of NaBH 4 , 300 ml of PBS and 100 ml of ethanol), washed in a 0.2% SDS solution twice for 2 minutes, and then transferred to triple distilled water and washed twice for 2 minutes. Water on the slide was removed using a centrifuge for 1 minute 30 seconds at 800 rpm, and then stored in a slide box at room temperature in a desiccator.
  • Hybridization reaction On a slide chip spotted with 30 types of oligonucleotide probes, the products amplified with PCR of 16S rRNA of Neisseria gonorrhoeae and Mycoplasma genitalium, 16S-23S intergenic region of Ureaplasma urealyticum, cryptic plasmid of Chlamydiae trachomatis and control gene of a human trglobulin were integrated by having the DNA of the samples as a main substances, and the hybridization reaction was performed. At this time, a perfusion 8-well chamber (Schleicher & Schuell BioScience, Germany) having 100 ⁇ i capacity was used as a hybridization reaction chamber.
  • hybridization reaction solution was composed by mixing 2 ml of 2OX SSC, 1.7 ml of 90% glycerol and 6.3 ml of a 50 mM phosphate buffer solution to the final volume of 10 ml.
  • the slide was further washed with a IX SSPE (NaCl (8.765 g), NaH 2 PO 4 -IH 2 O (1.38 g), Na 2 EDTA (0.37 g) dissolved in 1 liter of distilled water, and pH being adjusted to 7.4 with 10 N NaOH) solution for 2 minutes at room temperature, and dried by centrifugation for 1 minute and 30 seconds at 800 rpm. ⁇ 230> 4. Scanning assay
  • ⁇ 23i> The slide, which was hybridized, washed to remove nonspecific signal and then dried, was assayed of its fluorescent signals and images using a confocal laser fluorescence scanner.
  • a scanner it is preferably Affymetrix 428 Array Scanner (Affrymetrix, USA), Scan Array Lite (Packard Bioscience, USA) or equipments corresponding thereto.
  • DNA samples included as a test subject are 50 examples of Neisseria gonorrhoeae infected samples, 50 examples of Chlamydiae trachomatis infected samples, 50 examples of Ureaplasma urealyticum infected samples, 50 examples of Mycoplasma genitalium infected samples and 50 examples of non-infected samples.
  • 50 examples of Neisseria gonorrhoeae infected samples 50 examples of Chlamydiae trachomatis infected samples, 50 examples of Ureaplasma urealyticum infected samples, 50 examples of Mycoplasma genitalium infected samples and 50 examples of non-infected samples.
  • 40 examples of complicated infection were also included.
  • ⁇ 234> The sensitivity, specificity and reproducibility of the DNA chip assay were studied by repeating the same test twice having a time interval in between by the different observers. Thereby, the DNA chip of the invention was assayed whether the DNA chip is capable of detecting the four major pathogens of STD accurately in the clinic, and in particular, whether the DNA chip is capable of detecting the complicated infection as well.
  • the multiplex type PCR method and DNA chip may detect Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma iirealyticuw and Mycoplasma genitalium, which are in absolute majority by 90% or more of STD causes, being four major pathogens for urethritis, cervicitis and pelvic inflammatory diseases, with accuracy and quickness at the same time.
  • the DNA chip of the invention is prominent as to show nearly 100% of sensitivity, specificity and reproducibility at STD diagnosis. The DNA chip may detect the complicated infection accurately and the test method is convenient, and the assay of the results is easy.
  • the DNA chip is more prominent and economical compared with the conventional methods. Therefore, it may be assumed that the invention may be used widely for primary detection and diagnosis of STD and for determining therapeutic policies. Moreover, the invention is very effective from the viewpoint of medicinal industry, which will replace the conventional DNA test kits such as culturing or staining, and the single PCR.
  • SEQ ID NOS: 1 to 10 composed of the base sequence of a primer for nucleic acid amplification
  • SEQ ID NOS: 11 to 40 composed of the base sequence of a probe connecting complementariIy with nucleic acids, and these are appended hereto.

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Abstract

La présente invention se rapporte à une puce à ADN et à une trousse de génotypage permettant de diagnostiquer rapidement et précisément une infection par l'un des agents pathogènes à l'origine des quatre plus fréquentes flambées de maladies sexuellement transmissibles (MST), du type Neisseria gonorrhoeae, Chlamydiae trachomatis, Ureaplasma uréalyticum, Mycoplasma génitalium, ainsi qu'à une sonde oligonucléotidique utilisée dans cette trousse de génotypage. La puce à ADN et la trousse de génotypage présentent d'excellentes caractéristiques de sensibilité, spécificité et reproductibilité lors du diagnostic d'une MST, et elles permettent efficacement et rapidement une analyse automatisée des quatre types de bactéries dans des échantillons variés.
PCT/KR2005/000773 2004-10-08 2005-03-17 Sonde de bacteries provoquant des maladies sexuellement transmissibles, puce a adn et trousse de genotypage WO2006038752A1 (fr)

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WO2007137650A1 (fr) * 2006-05-29 2007-12-06 Bio-Rad Pasteur Détection multiplex en temps réel de trois espèces bactériennes responsables de maladies sexuellement transmissibles
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WO2011073675A3 (fr) * 2009-12-17 2011-09-15 Atlas Genetics Limited Dosage microbien
EA027074B1 (ru) * 2009-12-17 2017-06-30 Этлас Дженетикс Лимитид СПОСОБ ОБНАРУЖЕНИЯ ГЕНЕТИЧЕСКОГО МАТЕРИАЛА Chlamydia trachomatis, ЗОНД НУКЛЕИНОВОЙ КИСЛОТЫ (ВАРИАНТЫ), ПРЯМОЙ (ВАРИАНТЫ) И ОБРАТНЫЙ (ВАРИАНТЫ) ПРАЙМЕРЫ ПЦР, ИХ СОДЕРЖАЩИЙ КОМПОНЕНТ ПЦР, СОДЕРЖАЩИЙ ВЫШЕУКАЗАННЫЙ КОМПОНЕНТ НАБОР И СПОСОБ ОБНАРУЖЕНИЯ ГЕНЕТИЧЕСКОГО МАТЕРИАЛА Pectobacterium atrosepticum
US9982312B2 (en) 2009-12-17 2018-05-29 Atlas Genetics Limited Microbial assay
CN111304295A (zh) * 2019-12-19 2020-06-19 武汉中帜生物科技股份有限公司 同时检测淋病奈瑟菌、沙眼衣原体及解脲脲原体核酸的试剂盒及其应用
CN111304295B (zh) * 2019-12-19 2022-08-05 武汉中帜生物科技股份有限公司 同时检测淋病奈瑟菌、沙眼衣原体及解脲脲原体核酸的试剂盒及其应用
CN112458195A (zh) * 2020-12-28 2021-03-09 广州迈景基因医学科技有限公司 基于高通量测序检测性传播病原体的多重pcr引物组、试剂盒及其方法

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