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WO1998011259A2 - Composition, procede et necessaire pour la detection et l'identification de micro-organismes porteurs de maladies sexuellement transmissibles - Google Patents

Composition, procede et necessaire pour la detection et l'identification de micro-organismes porteurs de maladies sexuellement transmissibles Download PDF

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Publication number
WO1998011259A2
WO1998011259A2 PCT/CA1997/000660 CA9700660W WO9811259A2 WO 1998011259 A2 WO1998011259 A2 WO 1998011259A2 CA 9700660 W CA9700660 W CA 9700660W WO 9811259 A2 WO9811259 A2 WO 9811259A2
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WIPO (PCT)
Prior art keywords
seq
sexually transmitted
ttg
transmitted disease
primer pairs
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PCT/CA1997/000660
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English (en)
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WO1998011259A3 (fr
Inventor
Max Chernesky
Kathleen Luinstra
Dan Jang
Sylvia Chong
James B. Mahony
James M. Dunn
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Visible Genetics Inc.
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Publication date
Application filed by Visible Genetics Inc. filed Critical Visible Genetics Inc.
Priority to AU41962/97A priority Critical patent/AU4196297A/en
Publication of WO1998011259A2 publication Critical patent/WO1998011259A2/fr
Publication of WO1998011259A3 publication Critical patent/WO1998011259A3/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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • This invention relates to combinations of oligonucleotides useful in detecting infectious genitourinary micro-organisms and sexually transmitted disease pathogens such as bacteria, fungi and viruses, by multiplex poiymerase chain reaction.
  • the invention also relates to kits containing such combinations and to methods of using said oligonucleotide combination for such detection.
  • Genitourinary tract infections due to infectious microorganisms such as Chlamydia trachomatis and Neisseria gonorrhoeae are a major cause of morbidity in sexually active individuals.
  • De Schryver, et al. "Epidemiology of sexually transmitted diseases: the global picture" Bull. W.H.O. 68:639-6.54 (1990).
  • N. gonorrhoeae rates are on the increase and C. trachomatis is now the most common bacterial sexually transmitted disease (STD) with asymptomatic carriage rates in the general population of 2 to 5%
  • STD bacterial sexually transmitted disease
  • STDs found in genitourinary samples such as Mycoplasma genitalium and Ureaplasma urealyticum, are also etiologic agents of serious genitourinary infection that compromise the health of individuals around the world.
  • PCR is the well known process described in US Patents 4,683,202, 4,683,195, and 4,965,188, and Current Protocols in Molecular Biology, (Eds. Ausubel, F.M. et al, (John Wiley & Sons; 1995)), all of which are incorporated herein by reference, whereby a generally short (50-1000 nt) fragment of a complex DNA sample is reproduced billions of times to generate a substantially pure sample of the fragment DNA. Labeled pathogen specific probes are added to the amplified mixture, and detected if hybridization to the target DNA occurs.
  • AmplicorTM a test which detects C. trachomatis or N. gonorrohoeae by the hybridization of a pathogen specific probe to PCR amplified products, detectable by a color change/optical density technique.
  • the test format permits testing a single patient sample for both pathogens using a multiplex PCR amplification, but still requires that two separate detection reactions be performed.
  • UriProbe also a test for C. trachomatis and N. gonorrohoeae, which relies on the ligase chain reaction (LCR).
  • LCR ligase chain reaction
  • the LCR method described in Patent Applications WO 9320227, WO 9300447, WO 9408047, WO 9403636, EP 477 912 uses thermostable ligase enzyme to ligate two DNA probes which hybridize in ligatable juxtaposition on a template DNA strand, thus generating a detectable ligated DNA fragment only if the template DNA is present.
  • Direct detection of PCR amplified fragments is not utilized in any of the above noted diagnostic techniques. It would be advantageous to provide a method and compositions that allow the direct detection of PCR amplified fragments, for example, on a high speed automated detection electrophoresis apparatus such as the MicroGene Blaster (Visible Genetics Inc., Toronto).
  • SUBSTTTUTE SHEET (RULE 26) would simplify the complex determination of the etiological basis of infection, and would therefore be desirable.
  • M-PCR Multiplex PCR
  • M-PCR is a technique which permits the amplification of multiple distinct genes or portions of genes in a single reaction mixture.
  • M-PCR is described by Chamberlain et al., "Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification” Nucleic Acids. Res. 16: 11141-11156 (1988) and by Edwards et al. "Multiplex PCR” in PCR primer: A Laboratory Manual eds. Dieffenbach et al., CSHL Press; Plainview, NY, 1995.
  • M-PCR has been used to distinguish species of Legionella (Bej et al. "Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water” Mol. Cell.
  • the present invention provides a method for co-amplification and detection of at least three sexually transmitted disease pathogens in DNA isolated from a patient sample suspected of containing at least one sexually transmitted disease pathogen.
  • the isolated DNA is combined with at least first, second and third oligonucleotide primer pairs for amplification of a gene fragments from first, second and third different sexually transmitted disease pathogens.
  • One of the primers in each primer pair is labeled with a detectable label.
  • the first, second and third oligonucleotide primer pairs are reacted with the isolated DNA in an amplification reaction to generate an amplification product mixture that contains amplified gene fragments from either or both of the first and second sexually transmitted disease pathogens, depending on whether or not the first and second sexually transmitted disease pathogens are present in the patient sample.
  • Amplified gene fragments in the amplification product mixture are then evaluated, for example by gel electrophoresis, to determine whether amplified gene fragments from the first, second or third sexually transmitted disease pathogen are present.
  • the instant invention teaches a single test that can simultaneously diagnose the presence of three or more pathogens associated with urethritis and/or sexually transmitted disease (STD) in a patient genitourinary sample.
  • the test detects nucleic acids specific for these pathogens by a modified poiymerase chain reaction technique called Multiplex PCR (M-PCR).
  • M-PCR Multiplex PCR
  • PCR technique is quite well known. The following is a brief summary of the diverse information on PCR, and is not meant to detail the information which is readily available to one skilled in the art, in texts such as Current Protocols in Molecular Biology, (Eds. Ausubel, F.M. et al, (John Wiley & Sons; 1995)).
  • a “primer pair” comprises of a pair of oligonucleotides ("primers”) which hybridize to DNA of the target organism.
  • the oligonucleotide is usually synthetically produced. It acts as a point of initiation of DNA synthesis.
  • the exact size and sequence of each primer will vary depending upon the use contemplated, the complexity of the targeted sequence, reaction temperature and the source of the primer. Generally, the primers used in this invention will have from 12 to 60 nucleotides (“nt”), and preferably, they have from 15 to 30 nt.
  • each primer hybridizes to a different template strand, in an orientation such that the 3' or "extendable" end of each primer points towards each other.
  • the amplification reaction is induced by a polymerization agent (such as a DNA poiymerase) and deoxyribonucleotide triphosphates under the appropriate conditions.
  • a polymerization agent such as a DNA poiymerase
  • deoxyribonucleotide triphosphates under the appropriate conditions.
  • the result is the formation of primer extension products along the strands, the products having added thereto nucleotides which are complementary to the templates. Once the primer extension products are denatured, one copy of each of the templates has been prepared.
  • thermostable enzymes and reagents By using thermostable enzymes and reagents, cycles of priming, extending and denaturation can be carried out by controlling the temperature of the reaction mixture for as many times as desired to provide an exponential increase in the amount of nucleic acid which has the same sequence as the target nucleic acid. This is the essence of the Poiymerase Chain Reaction (PCR). In effect, the target nucleic acid is duplicated (or "amplified”) many times so that it is more easily detected.
  • PCR Poiymerase Chain Reaction
  • primers are idiosyncratic and may be influenced by slight changes in salt concentration or temperature.
  • Preferred primers are without internal homology or primer-primer homology.
  • Primers used in the present invention are preferably exactly complementary to the target site of the template DNA.
  • primers that form more stable duplexes with the target DNA at the 5' -end of the primer than at the 3'-end thereof can be useful, and can result in less false priming.
  • Mismatched primers tend not to hybridize at higher temperatures. For that reason, it is generally desirable to carry out hybridizations for PCR at the highest possible temperature where both members of the primer set will form stable hybrids with the target site.
  • Expected stability (or melting temperature) of a hybridized primer can be approximated by GC content, since GC base pairs are more stable than AT pairs, or by nearest neighbor thermodynamic parameters. Breslauer et al., "Predictinq DNA duplex stability from base sequence", Proc. Nat'IAcad. Sci. USA 83: 3746-3750 (1986).
  • the melting temperature, Tm can be calculated using either of the following equations:
  • Tm (°C) 2 X (A + T) + 4 X (G+C)
  • A, T, G, and C represent the number of adenosine, thymidine, guanosine and cytosine residues in the primer.
  • primers for coaamplification should be selected to have predicted melting temperatures differing by less than 4°C. Notwithstanding the proposed equations for calculating Tm, it is sometimes found that only trial and error can help the investigator determine the actual Tm Of the primer sets selected.
  • Hybridization sites are the sites of hybridization and initiation of synthesis on the target DNA.
  • the sites are selected to be specific to some part of the target DNA which is unique to the target.
  • the resulting amplified fragment can be any gene fragment, including part of a gene, an entire gene, or contiguous parts of several genes from the target DNA, as long as the hybridization sites of the primer set is specific for that microorganism. It is desirable that the primer sets do not amplify any human DNA which is likely to be contaminating patient samples (mostly patient sample DNA, but also DNA from technicians who handle the sample).
  • the present invention provides primers sets which permit co- amplification and detection of a plurality of sexually transmitted disease pathogens that may be found in human genitourinary samples in a single test.
  • sexually transmitted disease pathogens include but are not limited to C. trachoma, N. gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum.
  • FVU First Void Urine
  • urethral swab urethral swab
  • vaginal swab vaginal introitus swab
  • tissue samples blood or organ, or tumor
  • nucleic acid amplification technologies such as PCR and LCR have indicated that FVU specimens are the preferred specimen for diagnosing C. trachomatis infections in both men and women. FVU is also usually the easiest sample to obtain from patients.
  • DNA can be isolated from virtually all pathogens found in a FVU sample, by using the technique described in Mahony et al. "Confirmatory poiymerase chain reaction testing for Chlamydia trachomatis in first void urine from asymptomatic and symptomatic men" J. Clin. Microbiol. 30:2241-2245 (1992) which is incorporated herein by reference.
  • Mahony et al. "Confirmatory poiymerase chain reaction testing for Chlamydia trachomatis in first void urine from asymptomatic and symptomatic men” J. Clin. Microbiol. 30:2241-2245 (1992) which is incorporated herein by reference.
  • Mahony et al. "Confirmatory poiymerase chain reaction testing for Chlamydia trachomatis in first void urine from asymptomatic and symptomatic men” J. Clin. Microbiol. 30:2241-2245 (1992) which is incorporated herein by reference.
  • the isolated DNA from a sample is treated with at least two sets of primers to produce amplified gene fragments from different species of sexually transmitted disease pathogens in a multiplex PCR process.
  • Primer sets used in M-PCR are subject to constraints beyond those of PCR alone.
  • primer sets must successfully co-amplify under the same temperature cycling regime. Primers must not hybridize to other co-amplification products, i.e. they must be specific for single pathogen species only, and should not amplify related or unrelated pathogens. Additional factors applying to the selection of primers for M-PCR are discussed in Rylchik, W., Selection of Primers for Poiymerase Chain Reaction", in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current, Methods and Applications, White B.A. ed., Humana press, Totowa, N.J., 1993.
  • Optimization of reactions can be achieved by modifying reaction variables such as primer concentrations, dNTP amounts, number of thermal cycles, reagent concentrations, sample preparation etc. An optimized result produces approximately the same quantity of reaction product for each fragment when all target organisms are present in the same amounts.
  • the length of respective amplification products must be sufficiently different so that respective amplification products can be detected by electrophoretic (size based) separation.
  • the amplification product from a first pathogen is designed to be lOOnt; a second pathogen will be 150nt, a third will be 200 ⁇ t and a fourth will be 250nt.
  • This size difference ensures that when a certain pathogen is not present or is below detectable level, no amplification product is present.
  • the expected fragment length of the amplification product is selected to be specific for each pathogen, and is known from the distance lying between the hybridization sites of the primers in the pathogen target sequence. Differences of only one base in length are sufficient, provided a high resolution gel capable of resolving one base differences is used in analyzing the amplification products. Greater differences in length are preferred.
  • the primer set selected must also amplify DNA from all strains of the same species, if a primer is situated on a region of variability between strains, some strains will not be detected. A region of DNA that is highly conserved among strains is the preferred primer target sequence. On the other hand, strain specific primer sets could provide useful information if it became clear that certain strains responded to different treatments.
  • Another primer set representing a control amplification can be included in the reaction mix to monitor the reliability of reaction reagents and sample preparation.
  • a primer set known to amplify DNA from a commensal microorganism normally found in the type of patient sample being tested i.e. lactobaciilus from vaginal fluid
  • Human DNA Primer sets may also be used, since some patient DNA is expected in body fluid samples and tissue samples.
  • An alternative control can be the addition to the patient sample of a known non-human DNA composition, such as a purified plasmid DNA, prior to amplification reactions.
  • primer sets are added to the patient sample DNA in conditions that induce primer extension by PCR (generally requiring the addition of a thermostable poiymerase, dNTPs and other reagents). After sufficient thermal cycles, the PCR is halted, and the reaction products, if any, are detected.
  • a detectable label is a fluorescent dye, although other labels including radio-labels, chemical couplers such as biotin which can be detected with streptavidin-iinked enzymes, and epitope tags such as digoxigenin detected using antibodies available from Boehringer-Mannheim (Mannheim DE) can be used.
  • Fluorescently-labeled, amplified fragments can be detected using automated fluorescence detection electrophoresis apparatuses such as instruments provided by Visible Genetics Inc. (Toronto, ON), Applied Biosystems Inc. (Foster City, CA), Pharmacia Biotech, Inc. (Piscataway, NJ), Li-Cor, Inc. (Lincoln, NE), and Molecular Dynamics, inc. (Sunnyvale, CA).
  • automated fluorescence detection electrophoresis apparatuses such as instruments provided by Visible Genetics Inc. (Toronto, ON), Applied Biosystems Inc. (Foster City, CA), Pharmacia Biotech, Inc. (Piscataway, NJ), Li-Cor, Inc. (Lincoln, NE), and Molecular Dynamics, inc. (Sunnyvale, CA).
  • fluorescent label is fluorescein, which is a standard label used in nucleic acid sequencing systems using laser light as a detection system.
  • Further fluorescent labels may include: Rhodamine dyes, cyanine dyes such as CY5 and Cy5.5 (Amersham Life Sciences), dyes with infra-red excitation spectra and many others. These fluorescent labels may be conjugated to the 5' end of the primer using well known methods such as the phosphoramidite (see US Patent No. 4415732, which is incorporated herein by reference) or amino-linker methods.
  • the use of a different fluorophore attached to the 5' and 3' primers, respectively, can prove advantageous when used in an automated fluorescence detection apparatus with a two-dye detector.
  • the results from one primer can be used as a control to check the results of the other primer.
  • two or more different patient samples could be run in the same channel if one patient's fragments were labeled with one dye, while the other patients' fragments had the different label. This technique would increase the throughput capacity of the apparatus two-fold or more, depending on the number of dyes employed.
  • Detection of unlabeled amplification products may be made by ethidium bromide staining of such amplification products after electrophoretic separation in an agarose gel.
  • Agarose gel techniques are generally not as precise in their separation as the polyacrylamide gels used in the above noted instruments. However, where amplification products are significantly different in size, such as greater than 40-50 nt, they can often readily be discerned on an agarose gel.
  • Agarose gel electrophoresis and detection techniques are detailed in Protocols in Molec, Biol. at chapter 4, incorporated herein by reference.
  • probes for micro-organismal STDs are known in the art.
  • primer sets have been found to successfully co-amplify DNA fragments from different sexually transmitted disease pathogens present in a patient sample, in the reaction conditions described in the examples below.
  • the primers of set I amplify an open reading frame (orf) of the cryptic plasmid of C. trachomatis, generating a 241 nt DNA fragment.
  • the primers of set 2 amplify the cppB gene on the 4.2 kb cryptic plasmid of N. gonorrhoeae, generating a 390 nt fragment.
  • the primers of set 3 amplify part of the MgPa adhesin gene of M. genitalium, generating a DNA fragment Of 281 nt.
  • the primers Of set 4 amplify the urease gene of U. urealyticum generating a 167 nt DNA fragment.
  • the primers used in the present invention are selected to be "substantially complementary" to the specific target nucleic acid priming sites. This means that they must be sufficiently complementary to hybridize with the respective nucleic acid sequences to form the desired hybridization products and then be extendable by a DNA poiymerase. In the preferred and most practical situation, the primers have exact complementarity to the nucleic acid sequences of interest.
  • These primers may be conveniently provided in a kit format. Such a kit may include enough reagents for a single assay or for multiple assays, for example for up to 200 or more assays.
  • the primer sets may be conveniently mixed in a cocktail of primers, with the concentration of each primer optimized for the reaction. Additionally the kit may contain reaction buffer mix, thermostable poiymerase such as Taq Poiymerase (Perkin-Elmer) or Thermo Sequenase (Amersham Life Sciences), internal standards and/or loading buffer.
  • FVU specimens (20 ml) were collected from men presenting with non-gonoccocal urethritis to the Kenya City Commission Special Treatment Clinic. Two urethral swabs and a FVU specimen were collected for culture, EIA and PCR as described previously. The FVU specimen was collected first. The study was approved by the University of Kenya's Scientific and Ethical Review Committee.
  • the optimal conditions for M-PCR were established by varying the concentrations of MgCI 2 , dNTPs, and primers at different annealing temperatures. The optimal conditions determined were an annealing temperature of 55°C, primer concentrations of 0.5 uM, a MgCI 2 concentration of 2.5 uM and dNTP concentrations of 200 uM. After the addition of Taq poiymerase (Amersham Life Sciences), the reaction mixtures were thermal cycled as follows: 94°C for two minutes then 40 cycles of 60 sec at 55°C; 120 sec at 72°C; and 60 sec at 94°C. The final extension phase was allowed to proceed for 300 sec before being placed on ice at 4°C.
  • PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization using fluorescein-11-dUTP labeled oligonucleotide probes prepared with terminal deoxynucieotidyl transf erase using an ECL 3'-oligolabeling and detection kit (Amersham Life Sciences, Arlington Heights, III).
  • the hybridization probes used were the KL3 probe specific for C. trachomatis 241 bp PCR product and the JMGC3 probe derived from the published plasmid sequence, Korch, et al., "Cryptic plasmid of Neisseria gonorrhoeae: Complete nucleotide sequence and genetic organization", J. Bacteriol. 163: 430-438 (1985), and specific for the 390 bp NG-PCR product.
  • M-PCR total analytical sensitivity of M-PCR was 10 fg of total DNA equivalent to 1-2 genome copies for both C. trachomatis and N. gonorrhoeae and equal to that of the individual CT-PCR and NG-PCR assays.
  • the bands observed in the gel demonstrated the specificity of the primers used in M-PCR; KL1-KL2 primers amplified only C. trachomatis DNA while HOI-HO3 primers amplified only N. gonorrhoeae DNA in the presence of both sets of primers and DNA from both organisms.
  • This protocol detects four bacteria associated with urethretis and/or sexually transmitted diseases (STDs): Chlamydia trachomatis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Ureaplasma urealyticum in a single M-PCR reaction.
  • STDs sexually transmitted diseases
  • FVU samples are prepared for PCR amplification as follows. Deposit 100 ul Of F O in a sterile microcentrifuge tube. Centrifuge at 12,000 x g for 20 min and remove the supernatant. Add 100 ul of Lysis Solution (Proteinase K @ 100 ug/ml; 1% Tween 20) to the bacteria pellet. Incubate sample for 1 h at 55°C, or 18 h at room temperature. Incubate further at 95°C for 10 min. Add 200 ul of Geneclean II glass milk. Elute the DNA in 10 ul of ddH 2 0.
  • a lysis solution control can be made as follows: add the lysis solution to a sterile tube (a tube without any urine pellet), and treat this tube like the others.
  • PCR amplification is obtained for 10 reactions (including 10% extra for the loss during pipetting) as follows: Take a sterile 1.5 or 2.0 ml tube. Add 88 ul of the PCR Master mix (per 880 ul: add 275 ul of 10x PCR buffer without MgCI 2 (Perkin- Elmer); 275 ul of MgCI 2 @ 25 mM (Perkin-Elmer); 330 ul of each dNTPs @ 2.5 mM). Add 6.9 ul of the Primer Mix in which each of the following primers are present at 2 urn: The following primers are present:
  • the label for use with a Visible Genetics Inc. MicroGene Blaster is a Cy5.5 fluorophore (Amersham Life Sciences) amino-linked to the 5' end nucleotide.
  • the Internal Standards are provided in a solution containing 4 fragments, labeled with CY5-5, that is added to the PCR tube after amplification.
  • 4 fragments of appropriate length can be made from the plasmid template pGEM-7Zf(-) using the following primers:
  • the following annealing temperature can be used:
  • T7-GEM99 (generates a 138-bp fragment): 52.5 ⁇ C
  • T7-GEM161 (generates a 200-bp fragment): 52.2°C
  • T7-GEM312 (generates a 351-bp fragment): 53.3°C
  • T7-GEM387 (generates a 426-bp fragment): 54.3°C
  • Each fragment can be generated separately, purified (by gel or otherwise), and calibrated to give good peaks on the MicroGene Blaster. These peaks can be used to calibrate both length and quantity of fragments observed.
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL no
  • ANTI-SENSE yes
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (A) ORGANISM: C. trachomatis (D) OTHER INFORMATION: primer for C. trachomatis (xi) SEQUENCE DESCRIPTION.
  • SEQ ID NO:l TCCGGAGCGA GTTACGAAGA 20
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL no
  • ANTI-SENSE yes
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (A) ORGANISM: Neisseria gonorrohoeae (D) OTHER INFORMATION: primer for Neisseria gonorrhoeae (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCTACGCATA CCCGCGTTGC 20
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL no
  • ANTI-SENSE yes
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (A) ORGANISM: Mycoplasma genitalium (D) OTHER INFORMATION: primer for Mycoplasma genitalium (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AGTTGATGAA ACCTTAACCC CTTGG 25
  • ORGANISM Mycoplasma genitalium
  • D OTHER INFORMATION: primer for Mycoplasma genitalium
  • ORGANISM Ureaplasma urealyticum
  • ORGANISM Ureaplasma urealyticum
  • D OTHER INFORMATION: primer for Ureaplasma urealyticum
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL No
  • ANTI-SENSE No
  • FRAGMENT TYPE No
  • ORIGINAL SOURCE (A) ORGANISM:

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Abstract

On procède à l'amplification conjuguée et à la détection d'au moins trois agents pathogènes de maladies sexuellement transmissibles dans l'ADN isolé dans un échantillon prélevé sur un patient, en combinant l'ADN isolé avec au moins une première, deuxième et troisième paire d'amorces oligonucléotidiques pour l'amplification de fragments de gènes à partir d'un premier, deuxième et troisième agent pathogène différent de maladies sexuellement transmissibles. L'une des amorces de chaque paire d'amorces est marquée au moyen d'un marqueur détectable. On met les première, deuxième et troisième paire d'amorces oligonucléotidiques en réaction avec l'ADN isolé dans une réaction d'amplification pour générer un mélange de produit d'amplification contenant des fragments de gène amplifiés de l'un du premier ou deuxième (ou des deux) agent(s) pathogène(s) de maladie sexuellement transmissible, selon que le premier et le second agent pathogène de maladie sexuellement transmissible sont présents dans l'échantillon ou non. On évalue ensuite les fragments de gène amplifiés dans le mélange de produit d'amplification, par exemple, par électrophorèse sur gel, pour déterminer si des fragments de gène amplifiés du premier, deuxième ou troisième agent pathogène de maladies sexuellement transmissibles sont présents.
PCT/CA1997/000660 1996-09-09 1997-09-09 Composition, procede et necessaire pour la detection et l'identification de micro-organismes porteurs de maladies sexuellement transmissibles WO1998011259A2 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000017391A1 (fr) * 1998-09-24 2000-03-30 Innogenetics N.V. Identification des micro-organismes responsables d'infections respiratoires
WO2006038752A1 (fr) * 2004-10-08 2006-04-13 Goodgene Inc. Sonde de bacteries provoquant des maladies sexuellement transmissibles, puce a adn et trousse de genotypage
WO2007020656A1 (fr) * 2005-08-17 2007-02-22 University Of Delhi Procédé de détection basé sur pcr du chlamydia trachomatis
GB2431469A (en) * 2005-10-19 2007-04-25 Ivan Petyaev Pathogen diagnosis
WO2007097582A1 (fr) * 2006-02-23 2007-08-30 Seegene, Inc. Oligonucléotides utilisés pour détecter des acides nucléiques de pathogène responsable de maladies sexuellement transmissibles
EP1862557A1 (fr) * 2006-05-29 2007-12-05 Bio-Rad Pasteur Détection multiplex en temps réel de trois espèces bactériennes responsables de maladies à transmission sexuelle
WO2010062001A1 (fr) * 2008-11-25 2010-06-03 Goodgene Inc. Puce à adn, kit pour la détection ou le génotypage de bactéries causant des maladies sexuellement transmissibles, le génotypage de résistance aux médicaments antibactériens et procédé de détection ou de génotypage utilisant ceux-ci
WO2011115465A2 (fr) * 2010-03-19 2011-09-22 주식회사 진진바이오 Méthode et kit pour la détection d'agent pathogène d'organes génitaux féminins faisant intervenir un système à réseau liquide multiplex
WO2011114146A1 (fr) 2010-03-15 2011-09-22 Knight Scientific Limited Méthode de détection d'une inflammation dans les voies urinaires ou l'urètre
US8778611B2 (en) 2006-05-26 2014-07-15 The Secretary Of State For Health Mycoplasma genitalium detection assay based on the Mg219 gene
US9139883B2 (en) 2005-08-17 2015-09-22 University Of Delhi PCR-based detection method for Chlamydia trachomatis
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
CN113186316A (zh) * 2021-05-26 2021-07-30 北京华诺奥美基因生物科技有限公司 一种生殖支原体核酸实时荧光pcr检测引物、探针及试剂盒
CN113215322A (zh) * 2021-05-26 2021-08-06 北京毅新博创生物科技有限公司 Maldi-tof ms检测生殖道病原体的引物组合物及用途

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EP0648845B1 (fr) * 1993-07-08 2002-10-09 Johnson & Johnson Clinical Diagnostics, Inc. Procédé pour la coamplification de deux acides nucléiques différents utilisant la réaction en chaíne par polymérase
US5508168A (en) * 1994-05-16 1996-04-16 Hoffmann-La Roche Inc. Methods and reagents for the detection of herpes simplex virus, treponema pallidum, and haemophilus ducreyi

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WO2000017391A1 (fr) * 1998-09-24 2000-03-30 Innogenetics N.V. Identification des micro-organismes responsables d'infections respiratoires
WO2006038752A1 (fr) * 2004-10-08 2006-04-13 Goodgene Inc. Sonde de bacteries provoquant des maladies sexuellement transmissibles, puce a adn et trousse de genotypage
KR100619189B1 (ko) * 2004-10-08 2006-08-31 굿젠 주식회사 성교전파성질환 원인균 탐지용 프로브 및 이를 이용한성교전파성질환 원인균 유전자형 분석용 ｄｎａ 칩,분석키트 및 유전형 분석방법
JP2008515423A (ja) * 2004-10-08 2008-05-15 グッジェン インク 性交渉感染症を引き起こす細菌のプローブ、ｄｎａチップ及び遺伝子型解析キット
WO2007020656A1 (fr) * 2005-08-17 2007-02-22 University Of Delhi Procédé de détection basé sur pcr du chlamydia trachomatis
US9139883B2 (en) 2005-08-17 2015-09-22 University Of Delhi PCR-based detection method for Chlamydia trachomatis
GB2431469A (en) * 2005-10-19 2007-04-25 Ivan Petyaev Pathogen diagnosis
KR100860619B1 (ko) * 2006-02-23 2008-09-29 주식회사 씨젠 성인성 질환 유발 병원체 핵산 검출용올리고뉴클레오타이드
WO2007097582A1 (fr) * 2006-02-23 2007-08-30 Seegene, Inc. Oligonucléotides utilisés pour détecter des acides nucléiques de pathogène responsable de maladies sexuellement transmissibles
US8778611B2 (en) 2006-05-26 2014-07-15 The Secretary Of State For Health Mycoplasma genitalium detection assay based on the Mg219 gene
AU2007268172B2 (en) * 2006-05-29 2011-12-01 Bio-Rad Innovations Real-time multiplex detection of three bacterial species responsible for sexually-transmitted diseases
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
EP1862557A1 (fr) * 2006-05-29 2007-12-05 Bio-Rad Pasteur Détection multiplex en temps réel de trois espèces bactériennes responsables de maladies à transmission sexuelle
WO2010062001A1 (fr) * 2008-11-25 2010-06-03 Goodgene Inc. Puce à adn, kit pour la détection ou le génotypage de bactéries causant des maladies sexuellement transmissibles, le génotypage de résistance aux médicaments antibactériens et procédé de détection ou de génotypage utilisant ceux-ci
WO2011114146A1 (fr) 2010-03-15 2011-09-22 Knight Scientific Limited Méthode de détection d'une inflammation dans les voies urinaires ou l'urètre
WO2011115465A2 (fr) * 2010-03-19 2011-09-22 주식회사 진진바이오 Méthode et kit pour la détection d'agent pathogène d'organes génitaux féminins faisant intervenir un système à réseau liquide multiplex
WO2011115465A3 (fr) * 2010-03-19 2012-02-23 주식회사 진진바이오 Méthode et kit pour la détection d'agent pathogène d'organes génitaux féminins faisant intervenir un système à réseau liquide multiplex
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
CN113186316A (zh) * 2021-05-26 2021-07-30 北京华诺奥美基因生物科技有限公司 一种生殖支原体核酸实时荧光pcr检测引物、探针及试剂盒
CN113215322A (zh) * 2021-05-26 2021-08-06 北京毅新博创生物科技有限公司 Maldi-tof ms检测生殖道病原体的引物组合物及用途

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