WO2025003787A1 - Amorces pour la détection et la quantification de pathogènes sexuellement transmissibles et kit de test pcr en temps réel multiplex prévu aux mêmes fins - Google Patents

Amorces pour la détection et la quantification de pathogènes sexuellement transmissibles et kit de test pcr en temps réel multiplex prévu aux mêmes fins Download PDF

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WO2025003787A1
WO2025003787A1 PCT/IB2024/054896 IB2024054896W WO2025003787A1 WO 2025003787 A1 WO2025003787 A1 WO 2025003787A1 IB 2024054896 W IB2024054896 W IB 2024054896W WO 2025003787 A1 WO2025003787 A1 WO 2025003787A1
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seqid
nucleotide
sequence
sequence identity
encodes
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Acácio Agostinho GONÇALVES RODRIGUES
Carmen Maria LISBOA SILVA
Micael FERREIRA MOTA GONÇALVES
Ângela Rita VIEIRA FERNANDES
Isabel MARCOS MIRANDA
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Universidade do Porto
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

  • STIs sexually transmitted infections
  • PCR methods sometimes gives false-positive results associated with non-specific annealing of primers, in particular the conventional PCR that produce much more false-positive results. In this way, multiplex real-time PCR amplification is conducted for detecting simultaneously pathogens and is more accurate.
  • WO2018158618A1 discloses a kit for detecting silent sexually transmitted diseases (sstds) in a urine sample.
  • This invention claims the simultaneous detection of 7 STD-causing pathogens comprising 8 probes, and 14 primers based on multiplex real time PCR to detect herpes virus type 1, herpes virus type 2, Chlamydia trachomatis, Ureaplasma urealitycum, Mycoplasma hominis, Mycoplasma genitalium, and Human papillomavirus (HPV).
  • the Real-Q STIs Kit assay can simultaneously detect six STD pathogens. It showed 100% sensitivity and specificity in the detection of Mycoplasma hominis, Mycoplasma genitalium, Chlamydia trachomatis, Trichomonas vaginalis, and Neisseria gonorrhoeae , and 94.1% sensitivity and 100% specificity in the detection of Ureaplasma urealyticum .
  • the FTD STD9 kit had high sensitivity and specificity for in detecting seven pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum/Ureaplasma parvum, Trichomonas vaginalis, and HSV-1/2 from urine samples and genital or rectal swabs of human origin.
  • pathogens including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum/Ureaplasma parvum, Trichomonas vaginalis, and HSV-1/2 from urine samples and genital or rectal swabs of human origin.
  • the present application relates to a primer for the detection and quantification of sexually transmitted pathogens in a biological sample selected from at least one of the following:
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:4 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:4, wherein said nucleotide of SEQID NO:4 represents reverse primer to amplify Neisseria gonorrhoeae ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:5 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:5, wherein said nucleotide of SEQID NO:5 represents forward primer to amplify Trichomonas vaginalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:6 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:6, wherein said nucleotide of SEQID NO:6 represents reverse primer to amplify Trichomonas vaginalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:7 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:7, wherein said nucleotide of SEQID NO:7 represents forward primer to amplify Chlamydia trachomatis, A – K strains;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:8 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:8, wherein said nucleotide of SEQID NO:8 represents reverse primer to amplify Chlamydia trachomatis, A – K strains;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:9 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:9, wherein said nucleotide of SEQID NO:9 represents forward primer to amplify Chlamydia trachomatis, serovar L;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:10 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:10, wherein said nucleotide of SEQID NO:10 represents reverse primer to amplify Chlamydia trachomatis, serovar L;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:11 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:11, wherein said nucleotide of SEQID NO:11 represents forward primer to amplify Treponema pallidum ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:12 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:12, wherein said nucleotide of SEQID NO:12 represents reverse primer to amplify Treponema pallidum ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:13 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:13, wherein said nucleotide of SEQID NO:13 represents forward primer to amplify Haemophilus ducreyi ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:14 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:14, wherein said nucleotide of SEQID NO:14 represents reverse primer to amplify Haemophilus ducreyi ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:15 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:15, wherein said nucleotide of SEQID NO:15 represents forward primer to amplify Herpes simplex type 1 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:16 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:16, wherein said nucleotide of SEQID NO:16 represents reverse primer to amplify Herpes simplex type 1 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:17 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:17, wherein said nucleotide of SEQID NO:17 represents forward primer to amplify Herpes simplex type 2 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:18 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:18, wherein said nucleotide of SEQID NO:18 represents reverse primer to amplify Herpes simplex type 2 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:19 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:19, wherein said nucleotide of SEQID NO:19 represents forward primer to amplify Candida albicans ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:20 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:20, wherein said nucleotide of SEQID NO:20 represents reverse primer to amplify Candida albicans ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:21 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:21, wherein said nucleotide of SEQID NO:21 represents forward primer to amplify Candida glabrata ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:22 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:22, wherein said nucleotide of SEQID NO:22 represents reverse primer to amplify Candida glabrata ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:23 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:23, wherein said nucleotide of SEQID NO:23 represents forward primer to amplify Candida krusei ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:24 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:24, wherein said nucleotide of SEQID NO:24 represents reverse primer to amplify Candida krusei ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:25 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:25, wherein said nucleotide of SEQID NO:25 represents forward primer to amplify Candida tropicalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:26 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:26, wherein said nucleotide of SEQID NO:26 represents reverse primer to amplify Candida tropicalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:27 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:27, wherein said nucleotide of SEQID NO:27 represents forward primer to amplify Candida parapsilosis ;
  • the invention also relates to a primer pair for the detection and quantification of sexually transmitted pathogens in a biological sample wherein each pair comprises:
  • the invention also relates to a reaction mixture for multiplex real-time PCR for the detection and quantification of sexually transmitted pathogens in a biological sample comprising:
  • At least one primer sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described in claim 1 or a part of it or a nucleotide having 90% sequence identity with said selected sequence; or
  • At least one primer pair sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described in claim 3 or a part of it or a nucleotide having 90% sequence identity with said selected sequence;
  • the internal control gene is at least one sequence selected from the list comprising sequences SEQID NO: 29 to SEQID NO:32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the internal control gene is a sequence pair selected from:
  • sequences SEQID NO: 31 and SEQID NO: 32 selected from sequences SEQID NO: 31 and SEQID NO: 32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the invention also relates to a multiplex real-time PCR test kit for the detection and quantification of sexually transmitted pathogens in a biological sample, comprising:
  • At least one primer sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described in claim 1 or a part of it or a nucleotide having 90% sequence identity with said selected sequence; or
  • At least one primer pair sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described in claim 3 or a part of it or a nucleotide having 90% sequence identity with said selected sequence;
  • the internal control gene is at least one sequence selected from the list comprising sequences SEQID NO: 29 to SEQID NO:32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the internal control gene is a sequence pair selected from:
  • sequences SEQID NO: 31 and SEQID NO: 32 selected from sequences SEQID NO: 31 and SEQID NO: 32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the invention also relates to a method for the detection and quantification of sexually transmitted pathogens in a biological sample comprising the steps of:
  • At least one primer sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 described in claim 1 or a part of it or a nucleotide having 90% sequence identity with said selected sequence; or
  • At least one primer pair sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described in claim 3 or a part of it or a nucleotide having 90% sequence identity with said selected sequence;
  • the internal control gene is selected from:
  • sequences SEQID NO: 31 and SEQID NO: 32 selected from sequences SEQID NO: 31 and SEQID NO: 32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the pathogens are selected from Mycoplasma genitalium, Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis: A – K strains, Chlamydia trachomatis: serovar L, Treponema pallidum, Haemophilus ducreyi, Herpes simplex type 1, Herpes simplex type 2, Monkeypox virus, Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis or Candida parapsilosis.
  • the biological sample is a non-invasive test sample selected from urine, genital or oropharynx exudates.
  • the biological sample is genitourinary biopsy.
  • the present invention describes oligonucleotides hybridizable with nucleic acids, i.e., primers, a PCR reaction mixture comprising at least one primer selected from a list, and a testing kit comprising at least one primer selected from a list for the detection and quantification of sexually transmitted pathogens based on multiplex real-time PCR, resulting in a novel test kit for clinical testing for sexually transmitted pathogens in a biological sample. It is also disclosed a method for detection and quantification of sexually transmitted pathogens in a biological sample.
  • the difference of the presently disclosed invention and the prior art is mainly in the target microorganisms which is much more comprehensive and clinically relevant, supporting its use as a syndromic diagnostic panel in a broader scope or targeting 2 or 3 specific organisms.
  • trachomatis A – K strains, and serovar L, and detect and quantify the Monkeypox virus and other Candida species, such as C. glabrata, C. krusei, C. tropicalis and C. parapsilosis .
  • the present invention is intended for the fast, automated and convenient diagnostic approach for prevention, and management of STD by screening microbiological risk factors.
  • this invention detects and quantifies sexually transmitted pathogens present in multiple types of biological samples, such as in non-invasive (e.g., urine, genital and oropharynx exudates), and invasive test samples (e.g., genitourinary biopsies). These types of samples usually have a low load of microbial DNA, making the present invention a very sensitive and quantitative approach on sexually transmitted pathogens diagnostics.
  • non-invasive e.g., urine, genital and oropharynx exudates
  • invasive test samples e.g., genitourinary biopsies
  • this invention relates to primers for the detection and quantification of sexually transmitted pathogens and a multiplex real-time PCR testing kit suitable to be used as a syndromic panel for the detection and quantification of sexually transmitted pathogens.
  • This invention further relates to a reaction mixture, a testing kit and a method for the multiplex PCR which enables the detection and quantification of sexually transmitted pathogens.
  • the testing kit comprises the primers for the detection and quantification of sexually transmitted pathogens with a reaction mixture containing DNA polymerase and an internal control gene for checking the validity of the reaction.
  • the kit advantageously helps in the detection and quantification of sexually transmitted pathogens, given the efficiency of the primers to detect samples with low copy number.
  • This invention allows for the single or simultaneous detection and quantification of sexually transmitted pathogens.
  • the present invention relates to a primer for the detection and quantification of sexually transmitted pathogens in a biological sample, to a reaction mixture comprising at least one primer selected from a list, a multiplex real-time PCR test kit for the detection and quantification of sexually transmitted pathogens comprising at least one primer, and a method for the detection and quantification of sexually transmitted pathogens in a biological sample.
  • the sequences representing all sexually transmitted pathogens were downloaded from the GenBank nucleotide database and aligned using the online program Clustal Omega. A highly conserved region of each chosen genes was selected for the design of real-time PCR primers.
  • the primer sequences for use in the present invention are mentioned in Table 1.
  • the present invention also aims to include at least one primer for the detection and quantification of Monkeypox defined as SEQID NO: 33 or SEQID NO: 34, or both.
  • a variety of internal control genes can be used in the context of the present invention as well.
  • Human ⁇ -actin gene is expressed at relatively constant levels and highly conserved that are involved in cell motility, structure, and integrity.
  • Housekeeping gene is typically a constitutive gene that is required for the maintenance of basic cellular functions and expressed in all cells of an organism.
  • the primers sequences for non-limitative examples of internal control genes are mentioned in Table 2.
  • the present invention relates to a primer for the detection and quantification of sexually transmitted pathogens in a biological sample selected from at least one of the following:
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:1 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:1, wherein said nucleotide of SEQID NO:1 represents forward primer to amplify Mycoplasma genitalium ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:2 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:2, wherein said nucleotide of SEQID NO:2 represents reverse primer to amplify Mycoplasma genitalium ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:3 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:3, wherein said nucleotide of SEQID NO:3 represents forward primer to amplify Neisseria gonorrhoeae ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:4 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:4, wherein said nucleotide of SEQID NO:4 represents reverse primer to amplify Neisseria gonorrhoeae ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:5 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:5, wherein said nucleotide of SEQID NO:5 represents forward primer to amplify Trichomonas vaginalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:6 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:6, wherein said nucleotide of SEQID NO:6 represents reverse primer to amplify Trichomonas vaginalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:7 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:7, wherein said nucleotide of SEQID NO:7 represents forward primer to amplify Chlamydia trachomatis (A – K strains);
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:8 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:8, wherein said nucleotide of SEQID NO:8 represents reverse primer to amplify Chlamydia trachomatis (A – K strains);
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:9 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:9, wherein said nucleotide of SEQID NO:9 represents forward primer to amplify Chlamydia trachomatis (serovar L);
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:10 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:10, wherein said nucleotide of SEQID NO:10 represents reverse primer to amplify Chlamydia trachomatis (serovar L);
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:11 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:11, wherein said nucleotide of SEQID NO:11 represents forward primer to amplify Treponema pallidum ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:12 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:12, wherein said nucleotide of SEQID NO:12 represents reverse primer to amplify Treponema pallidum ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:13 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:13, wherein said nucleotide of SEQID NO:13 represents forward primer to amplify Haemophilus ducreyi ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:14 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:14, wherein said nucleotide of SEQID NO:14 represents reverse primer to amplify Haemophilus ducreyi ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:15 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:15, wherein said nucleotide of SEQID NO:15 represents forward primer to amplify Herpes simplex type 1 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:16 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:16, wherein said nucleotide of SEQID NO:16 represents reverse primer to amplify Herpes simplex type 1 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:17 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:17, wherein said nucleotide of SEQID NO:17 represents forward primer to amplify Herpes simplex type 2 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:18 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:18, wherein said nucleotide of SEQID NO:18 represents reverse primer to amplify Herpes simplex type 2 ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:19 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:19, wherein said nucleotide of SEQID NO:19 represents forward primer to amplify Candida albicans ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:20 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:20, wherein said nucleotide of SEQID NO:20 represents reverse primer to amplify Candida albicans ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:21 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:21, wherein said nucleotide of SEQID NO:21 represents forward primer to amplify Candida glabrata ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:22 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:22, wherein said nucleotide of SEQID NO:22 represents reverse primer to amplify Candida glabrata ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:23 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:23, wherein said nucleotide of SEQID NO:23 represents forward primer to amplify Candida krusei ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:24 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:24, wherein said nucleotide of SEQID NO:24 represents reverse primer to amplify Candida krusei ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:25 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:25, wherein said nucleotide of SEQID NO:25 represents forward primer to amplify Candida tropicalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:26 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:26, wherein said nucleotide of SEQID NO:26 represents reverse primer to amplify Candida tropicalis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:27 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:27, wherein said nucleotide of SEQID NO:27 represents forward primer to amplify Candida parapsilosis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:28 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:28, wherein said nucleotide of SEQID NO:28 represents reverse primer to amplify Candida parapsilosis ;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:33 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:33, wherein said nucleotide of SEQID NO:33 represents forward primer to amplify Monkeypox virus;
  • nucleic acid molecule that encodes a nucleotide sequence of SEQID NO:34 or a part of it or a nucleotide having 90% sequence identity with said SEQID NO:34, wherein said nucleotide of SEQID NO:34 represents reverse primer to amplify Monkeypox virus.
  • each pair comprises:
  • the present invention also relates to reaction mixture for multiplex real-time PCR for the detection and quantification of sexually transmitted pathogens in a biological sample comprising:
  • At least one primer sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described above, or a part of it or a nucleotide having 90% sequence identity with said selected sequence; or
  • At least one primer pair sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described above, or a part of it or a nucleotide having 90% sequence identity with said selected sequence;
  • the reaction mixture comprises all sequences SEQID NO: 1 to SEQID NO:28, or a part of them or a nucleotide having 90% sequence identity with said sequences.
  • the internal control gene is at least one sequence selected from the list comprising sequences SEQID NO: 29 to SEQID NO:32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the internal control gene is a sequence pair selected from:
  • sequences SEQID NO: 31 and SEQID NO: 32 selected from sequences SEQID NO: 31 and SEQID NO: 32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the present invention also relates to a multiplex real-time PCR test kit for the detection and quantification of sexually transmitted pathogens in a biological sample, comprising:
  • At least one primer sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described above, or a part of it or a nucleotide having 90% sequence identity with said selected sequence; or
  • At least one primer pair sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described above, or a part of it or a nucleotide having 90% sequence identity with said selected sequence;
  • the kit comprises all the primer sequences SEQID NO: 1 to SEQID NO:28, or a part of them or a nucleotide having 90% sequence identity with said sequences.
  • the internal control gene is at least one sequence selected from the list comprising sequences SEQID NO: 29 to SEQID NO:32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the internal control gene is a sequence pair selected from:
  • sequences SEQID NO: 31 and SEQID NO: 32 selected from sequences SEQID NO: 31 and SEQID NO: 32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • At least one primer sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 described above, or a part of it or a nucleotide having 90% sequence identity with said selected sequence; or
  • At least one primer pair sequence selected from the list comprising sequences SEQID NO: 1 to SEQID NO:28 as described above, or a part of it or a nucleotide having 90% sequence identity with said selected sequence;
  • the internal control gene is at least one sequence selected from the list comprising sequences SEQID NO: 29 to SEQID NO:32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the internal control gene is a sequence pair selected from:
  • sequences SEQID NO: 31 and SEQID NO: 32 selected from sequences SEQID NO: 31 and SEQID NO: 32, or a part of it or a nucleotide having 90% sequence identity with said selected sequence.
  • the listed primer sequences, the method, and testing kit are suitable to detect and quantify in a biological sample the presence of microbial DNA from Mycoplasma genitalium, Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis (A – K strains), Chlamydia trachomatis ( serovar L ), Treponema pallidum, Haemophilus ducreyi, Herpes simplex type 1, Herpes simplex type 2, Monkeypox virus, Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis or Candida parapsilosis .
  • the biological sample is a non-invasive test samples selected from, but not limited to, urine, genital or oropharynx exudates.
  • the biological sample is an invasive test sample selected from, but not limited to, genitourinary biopsy.
  • the present invention for amplifying nucleic acids may be applied to the amplification of nucleic acids of any sexually transmitted pathogens.
  • the nucleic acid as starting material is the DNA or RNA molecule.
  • General methods for extracting DNA from a biological sample are available on the market and known in the art. Where a mRNA is employed as starting material sample, a reverse transcription step is necessary prior to performing annealing step.
  • DNA polymerases can be used in the amplification step of the present method.
  • the polymerase is a thermostable DNA polymerase which may be isolated from a variety of bacterial species or obtained commercially.
  • the amplification step of the present method can be done in a single reaction.
  • Annealing or hybridization in the present method is performed under strict conditions that allow for specific binding between the selected primer sequence and the template nucleic acid.
  • the annealing temperature ranges from 60°C to 64° C.
  • the advantages of this invention are the disclosed primer sequences ensure to completely overcome problems of false-negative and false-positive products because of their higher specificity; and the present primers work in multiplex PCR, enabling to detect and quantify various sexually transmitted pathogens in a single PCR reaction.
  • the designed primers were tested (individually) with DNA strains from local isolates and reference type strains from American Type Culture Collection (ATCC).

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Abstract

La présente invention concerne un ensemble d'amorces pour la détection et la quantification de pathogènes sexuellement transmissibles, qui sont un ensemble de 28 séquences sens et antisens spécifiques pour plusieurs pathogènes associés à des infections sexuellement transmissibles. L'invention concerne également un kit de test PCR en temps réel multiplex approprié pour être utilisé en tant que panel syndromique pour la détection et la quantification d'agents pathogènes sexuellement transmissibles.
PCT/IB2024/054896 2023-06-28 2024-05-20 Amorces pour la détection et la quantification de pathogènes sexuellement transmissibles et kit de test pcr en temps réel multiplex prévu aux mêmes fins Ceased WO2025003787A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070092871A1 (en) * 2005-10-20 2007-04-26 Combimatrix Corporation Microarray for pathogen identification
US20090280486A1 (en) 2006-02-23 2009-11-12 Jong Yoon Chun Oligonucleotides for Detecting Nucleic Acids of Pathogen Causing Sexually Transmitted Diseases
US20100055755A1 (en) * 2006-12-21 2010-03-04 Novozymes, Inc. Methods of Eliminating or Reducing Expression of Genes in Filamentous Fungal Strains by Transitive RNA Interference
US20160017442A1 (en) 2013-03-05 2016-01-21 Indian Council Of Medical Research Multiplex Real Time PCR Testing Kit for the Simultaneous Detection of Hepatitis Virus
WO2017039144A1 (fr) * 2015-09-04 2017-03-09 주식회사 바이오세움 Trousse et procédé de détection de bactéries entraînant des maladies sexuellement transmises
WO2018158618A1 (fr) 2017-03-03 2018-09-07 Universidad De La Frontera Trousse de détection d'infections sexuellement transmissibles asymptomatiques dans un échantillon d'urine
EP3737776A1 (fr) * 2018-01-09 2020-11-18 Abbott Molecular Inc. Dosage pour détecter chlamydia trachomatis, neisseria gonorrhoeae, trichomonas vaginalis, et mycoplasma genitalium
CN115074456A (zh) * 2022-08-09 2022-09-20 江苏格诺生物科技有限公司 一种基于多重荧光pcr检测性传播病原体的引物探针组和试剂盒及其使用方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070092871A1 (en) * 2005-10-20 2007-04-26 Combimatrix Corporation Microarray for pathogen identification
US20090280486A1 (en) 2006-02-23 2009-11-12 Jong Yoon Chun Oligonucleotides for Detecting Nucleic Acids of Pathogen Causing Sexually Transmitted Diseases
US20100055755A1 (en) * 2006-12-21 2010-03-04 Novozymes, Inc. Methods of Eliminating or Reducing Expression of Genes in Filamentous Fungal Strains by Transitive RNA Interference
US20160017442A1 (en) 2013-03-05 2016-01-21 Indian Council Of Medical Research Multiplex Real Time PCR Testing Kit for the Simultaneous Detection of Hepatitis Virus
WO2017039144A1 (fr) * 2015-09-04 2017-03-09 주식회사 바이오세움 Trousse et procédé de détection de bactéries entraînant des maladies sexuellement transmises
WO2018158618A1 (fr) 2017-03-03 2018-09-07 Universidad De La Frontera Trousse de détection d'infections sexuellement transmissibles asymptomatiques dans un échantillon d'urine
EP3737776A1 (fr) * 2018-01-09 2020-11-18 Abbott Molecular Inc. Dosage pour détecter chlamydia trachomatis, neisseria gonorrhoeae, trichomonas vaginalis, et mycoplasma genitalium
CN115074456A (zh) * 2022-08-09 2022-09-20 江苏格诺生物科技有限公司 一种基于多重荧光pcr检测性传播病原体的引物探针组和试剂盒及其使用方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BARRIENTOS-DURÁN ANTONIO ET AL: "Detection of sexually transmitted disease-causing pathogens from direct clinical specimens with the multiplex PCR-based STD Direct Flow Chip Kit", EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES, SPRINGER, WIESBADEN, DE, vol. 39, no. 2, 4 January 2020 (2020-01-04), pages 235 - 241, XP037016990, ISSN: 0934-9723, [retrieved on 20200104], DOI: 10.1007/S10096-019-03686-W *
CHOE HYUN-SOP ET AL: "Performance of Anyplex(TM) II multiplex real-time PCR for the diagnosis of seven sexually transmitted infections: comparison with currently available methods", INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, vol. 17, no. 12, 1 December 2013 (2013-12-01), CA, pages e1134 - e1140, XP093048961, ISSN: 1201-9712, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S1201971213002464?via%3Dihub> DOI: 10.1016/j.ijid.2013.07.011 *
KIM YOONJUNG ET AL: "Analytical Performance of Multiplex Real-Time PCR for Six Sexually Transmitted Pathogens", CLINICAL LABORATORY, vol. 61, no. 11/2015, 1 January 2015 (2015-01-01), DE, XP093196281, ISSN: 1433-6510, Retrieved from the Internet <URL:https://dx.doi.org/10.7754/Clin.Lab.2015.150413> DOI: 10.7754/Clin.Lab.2015.150413 *

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