WO2024045683A1 - 通用探针、引物探针组、应用和试剂盒 - Google Patents

通用探针、引物探针组、应用和试剂盒 Download PDF

Info

Publication number
WO2024045683A1
WO2024045683A1 PCT/CN2023/092551 CN2023092551W WO2024045683A1 WO 2024045683 A1 WO2024045683 A1 WO 2024045683A1 CN 2023092551 W CN2023092551 W CN 2023092551W WO 2024045683 A1 WO2024045683 A1 WO 2024045683A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nucleotide sequence
fragment
gene
primer pair
Prior art date
Application number
PCT/CN2023/092551
Other languages
English (en)
French (fr)
Inventor
夏江
余皓
朱海涛
Original Assignee
领航基因科技（杭州）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 领航基因科技（杭州）有限公司 filed Critical 领航基因科技（杭州）有限公司
Priority to US18/326,300 priority Critical patent/US11987850B2/en
Publication of WO2024045683A1 publication Critical patent/WO2024045683A1/zh

Links

Classifications

    • 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
    • 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
    • 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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/22—Klebsiella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/385—Pseudomonas aeruginosa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/725—Candida albicans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/74—Candida tropicalis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biological detection technology, and in particular to universal probes, primer probe sets, applications and kits.
  • Pathogenic microorganisms refer to some microorganisms that can cause infectious diseases or even infectious diseases in the human body after invading the human body.
  • Common pathogenic microorganisms include prions, bacteria, mycoplasma, chlamydia, rickettsiae, fungi, spirochetes and viruses.
  • chemotherapeutic drugs After certain pathogenic microorganisms are repeatedly exposed to certain chemotherapeutic drugs, their reactivity continues to weaken, so that eventually the pathogenic microorganisms can resist the drugs without being killed or inhibited. This causes the pathogenic microorganisms to develop tolerance to the drugs, which is called resistance. drug resistance or resistance.
  • Resistance genes are a nucleotide sequence encoding drug-resistant traits, located on the bacterial chromosome or extrachromosomal plasmid. When pathogenic microorganisms develop drug resistance, the therapeutic effect is often reduced or completely lost, which directly affects the therapeutic effect of the disease. Therefore, it is necessary to establish a systematic, efficient and fast detection method for the identification of nucleic acids and drug-resistant genes of various pathogenic microorganisms. It can effectively promote the development of medical industry.
  • qPCR real-time fluorescence quantitative PCR
  • SYBR GreenI method fluorescent dye methods
  • TaqMan method fluorescent probe methods
  • the SYBR GreenI dye method is widely used because of its advantages such as ease of use and low price.
  • its biggest disadvantage is its lack of specificity, that is, the dye can bind to any dsDNA, affecting the accuracy of quantitative results.
  • TaqMan detection adds a fluorescent probe component.
  • the probe acts through fluorescence resonance energy transfer (FRET), which has the characteristics of high specificity and is suitable for multiplex qPCR.
  • FRET fluorescence resonance energy transfer
  • TaqMan probe technology usually requires one probe for each target; when detecting multiple targets in one fluorescence channel, too many probes labeled with the same fluorescence will cause the background signal to be too high, resulting in a decrease in contrast, making it difficult to detect signals. interpretation, thus limiting the number of detection targets.
  • the technical problem to be solved by the present invention is to provide primer probe sets and kits for digital PCR detection of multiple pathogenic microorganisms and drug-resistant genes.
  • the present invention provides a digital PCR platform based on 7-color fluorescence channels and combines multiple Digital PCR technology achieves coverage of multiple bacterial strains or drug-resistant sites under each species.
  • the present invention provides a structure of a universal probe, whose 5' end to 3' end includes: quenching group-fragment A-fluorophore-fragment B-C3.
  • nucleotide sequence of fragment A is as shown in any one of SEQ ID Nos: 1 to 7.
  • the nucleotide sequence of the fragment B is shown in any one of SEQ ID NO: 8 to 14.
  • the 5' end to the 3' end of the universal probe include in sequence:
  • the fluorescent group of the universal probe in the present invention is selected from FAM, VIC, ROX, CY5, A425, CY5.5 or CY7; the quenching group is selected from BHQ1, BHQ2 or BHQ3.
  • the fluorescent group A425 is the fluorescent group ATTO425.
  • the 5’ end to the 3’ end of the universal probe are in sequence:
  • the universal probe cooperates with the single-modified non-fluorescent probe, wherein the single-modified non-fluorescent probe specifically binds to the amplification product, and after hybridization between the universal probe and the hydrolyzed fragment of the single-modified non-fluorescent probe, Generate fluorescent signals to achieve detection purposes.
  • the present invention also provides a primer probe set for detecting pathogenic microorganisms and drug-resistant genes, including the universal probe of the present invention.
  • the primer probe set provided by the invention also includes a specific primer pair, which targets pathogenic microorganisms and drug-resistant genes.
  • the pathogenic microorganisms include Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Enterobacter cloacae, Candida albicans, Candida glabrata bacteria, Candida parapsilosis, Candida tropicalis;
  • the drug resistance genes include carbapenem resistance-KPC gene, carbapenem resistance-NDM gene, carbapenem resistance-OXA48 gene, carbapenem resistance-IMP gene, Vancomycin resistance-vanA gene, vancomycin resistance-vanB gene, vancomycin resistance-vanM gene, methicillin resistance-mecA gene or methicillin resistance- mecC gene.
  • the primer pair targeting Pseudomonas aeruginosa has the nucleotide sequence shown in SEQ ID NO: 37 ⁇ 38;
  • the primer pair targeting E. coli has the nucleotide sequence shown in SEQ ID NO: 39-40;
  • the primer pair targeting Klebsiella pneumoniae has the nucleotide sequence shown in SEQ ID NO: 41 ⁇ 42;
  • the primer pair targeting Acinetobacter baumannii has the nucleotide sequence shown in SEQ ID NO: 43 ⁇ 44;
  • the primer pair targeting Staphylococcus aureus has the nucleotide sequence shown in SEQ ID NO: 45-46;
  • the primer pair targeting Enterococcus faecium has the nucleotide sequence shown in SEQ ID NO: 47 ⁇ 48;
  • the primer pair targeting Enterococcus faecalis has the nucleotide sequence shown in SEQ ID NO: 49-50;
  • the primer pair targeting Enterobacter cloacae has the nucleotide sequence shown in SEQ ID NO: 51 ⁇ 52;
  • the primer pair targeting Candida albicans has the nucleotide sequence shown in SEQ ID NO: 53 ⁇ 54;
  • the primer pair targeting Candida glabrata has the nucleotide sequence shown in SEQ ID NO: 55 ⁇ 56;
  • the primer pair targeting Candida parapsilosis has the nucleotide sequence shown in SEQ ID NO: 57 ⁇ 58;
  • the primer pair targeting Candida tropicalis has the nucleotide sequence shown in SEQ ID NO: 59 ⁇ 60;
  • the primer pair targeting the carbapenem resistance-KPC gene has the nucleotide sequence shown in SEQ ID NO: 61-62;
  • the primer pair targeting the carbapenem resistance-NDM gene has the nucleotide sequence shown in SEQ ID NO: 63 ⁇ 64;
  • the primer pair targeting carbapenem resistance-OXA48 has the nucleotide sequence shown in SEQ ID NO: 65 ⁇ 66;
  • the primer pair targeting the carbapenem resistance-IMP gene has the nucleotide sequence shown in SEQ ID NO: 67-68;
  • the primer pair targeting the vancomycin resistance-vanA gene has the nucleotide sequence shown in SEQ ID NO: 69-70;
  • the primer pair targeting the vancomycin resistance-vanB gene has the nucleotide sequence shown in SEQ ID NO: 71-72;
  • the primer pair targeting the vancomycin resistance-vanM gene has the nucleotide sequence shown in SEQ ID NO: 73 ⁇ 74;
  • the primer pair targeting the methicillin-resistant-mecA gene has the nucleotide sequence shown in SEQ ID NO: 75-76;
  • the primer pair targeting the methicillin-resistant-mecC gene has the nucleotide sequence shown in SEQ ID NO: 77-78.
  • the primer probe set provided by the invention also includes a single-modified non-fluorescent probe.
  • the nucleic acid sequence of the single-modified non-fluorescent probe is reverse complementary to fragment B in the universal probe, and the 3' end is labeled with a quencher.
  • the single-modified non-fluorescent probe targeting Pseudomonas aeruginosa has the nucleotide sequence shown in SEQ ID NO: 15, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting E. coli has the nucleotide sequence shown in SEQ ID NO:16, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Klebsiella pneumoniae has the nucleotide sequence shown in SEQ ID NO:17, with a 3' end label The MGB quenching group is recorded;
  • the single-modified non-fluorescent probe targeting Acinetobacter baumannii has the nucleotide sequence shown in SEQ ID NO:18, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Staphylococcus aureus has the nucleotide sequence shown in SEQ ID NO:19, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Enterococcus faecium has the nucleotide sequence shown in SEQ ID NO:20, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Enterococcus faecalis has the nucleotide sequence shown in SEQ ID NO:21, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Enterobacter cloacae has the nucleotide sequence shown in SEQ ID NO:22, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Candida albicans has the nucleotide sequence shown in SEQ ID NO:23, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Candida glabrata has the nucleotide sequence shown in SEQ ID NO:24, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Candida parapsilosis has the nucleotide sequence shown in SEQ ID NO:25, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting Candida tropicalis has the nucleotide sequence shown in SEQ ID NO:26, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting the carbapenem resistance-KPC gene has the nucleotide sequence shown in SEQ ID NO:27, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting the carbapenem resistance-NDM gene has the nucleotide sequence shown in SEQ ID NO:28, and the 3' end is labeled with a BHQ2 quenching group;
  • the single-modified non-fluorescent probe targeting carbapenem resistance-OXA48 has the nucleotide sequence shown in SEQ ID NO:29, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting the carbapenem resistance-IMP gene has the nucleotide sequence shown in SEQ ID NO:30, and the 3' end is labeled with an MGB quenching group;
  • the single-modified non-fluorescent probe targeting the vancomycin resistance-vanA gene has the nucleotide sequence shown in SEQ ID NO:31, and the 3' end is labeled with a BHQ1 quenching group;
  • the single-modified non-fluorescent probe targeting the vancomycin resistance-vanB gene has the nucleotide sequence shown in SEQ ID NO:32 Column, 3' end labeled with BHQ1 quenching group;
  • the single-modified non-fluorescent probe targeting the vancomycin resistance-vanM gene has the nucleotide sequence shown in SEQ ID NO:33, and the 3' end is labeled with a BHQ1 quenching group;
  • the single-modified non-fluorescent probe targeting the methicillin-resistant mecA gene has the nucleotide sequence shown in SEQ ID NO:34, and the 3' end is labeled with a BHQ2 quenching group;
  • the single-modified non-fluorescent probe targeting the methicillin-resistant-mecC gene has the nucleotide sequence shown in SEQ ID NO:35, and the 3' end is labeled with an MGB quenching group.
  • the present invention also provides the application of the universal probe or the primer probe set in preparing digital PCR detection reagents for various pathogenic microorganisms and drug-resistant genes.
  • the invention also provides a kit for detecting multiple pathogenic microorganisms and drug-resistant genes, including the universal probe or primer probe set of the invention.
  • the kit provided by the invention also includes a primer and probe combination of the internal standard gene.
  • the specific sequence is: IC-UP7: ctgcacgaagctctttttcccgcgacggatctacgtcacagcg (SEQ ID NO: 36); IC-P: cgcgacggatctacgtcacagcg (SEQ ID NO: 102); IC-F: gcttcttgtggagctcgacaa (SEQ ID NO: 79); IC-R: ccgtcagcaacttcgttttca (SEQ ID NO: 80).
  • the present invention also provides a method for multiplex digital PCR detection of multiple pathogenic microorganisms and drug-resistant genes, using the primer probe set of the present invention or the kit of the present invention for detection.
  • the detection method based on the multiplex digital PCR platform of the present invention includes the following steps:
  • the primer probe of the present invention can be used to detect 12 kinds of pathogenic microorganisms, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Enterobacter cloacae, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and 9 resistance genes including carbapenem resistance-KPC gene, carbapenem resistance-NDM gene, carbapenem resistance Class resistance-OXA48 gene, carbapenem resistance-IMP gene, vancomycin resistance-vanA gene, vancomycin resistance-vanB gene, vancomycin resistance-vanM gene, methoxygen Joint detection of cillin resistance-mecA gene and methicillin resistance-mecC gene.
  • pathogenic microorganisms including Pseudomonas a
  • This invention specifically designed a total of 42 primers, 7 universal probes containing different fluorescent groups and 21 specific single-modified non-fluorescent group probes for the conserved regions of 12 pathogenic microorganisms and 9 drug-resistant genes.
  • needle, universal probe Combined with a single modified probe to generate a fluorescent signal, it can detect whether the sample contains Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Cloacae Enterobacteriaceae, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and whether they contain carbapenem resistance-KPC gene, carbapenem resistance-NDM gene, carbapenem resistance Resistance-OXA48 gene, carbapenem resistance-IMP gene, vancomycin resistance-vanA gene, vancomycin resistance
  • the present invention adopts a single-plex amplification system of conventional double-modified probes and a multiplex amplification system of universal probes and single-modified probes.
  • the detection results of the two systems are consistent, ensuring that the target genes are designed to contain different fluorescent groups.
  • the present invention provides a primer probe set and a kit for digital PCR detection of various pathogenic microorganisms and drug-resistant genes.
  • Persons skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the present invention. Invent technology.
  • test materials used in this invention are all common commercial products and can be purchased in the market.
  • UP1-UP7 represent 7 universal probes of different colors.
  • F represents the upstream primer
  • R represents the downstream primer
  • P represents the probe.
  • TL-F represents the upstream primer of Pseudomonas aeruginosa
  • TL-R represents the downstream primer of Pseudomonas aeruginosa
  • TL-P represents the conventional double-modified probe of Pseudomonas aeruginosa
  • TL-UP1 represents Pseudomonas aeruginosa.
  • Cytozoa-specific single-modified non-fluorophore probe
  • Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, and Enterobacter cloacae only have detection values in VIC and CY7, and there are no detection values in other channels.
  • Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis only have detection values in ROX and CY7, others There is no detection value in any channel.
  • Carbapenem resistance genes only have detection values in CY5 and CY7, and there are no detection values in other channels.
  • Vancomycin resistance genes only have detection values in A425 and CY7, and there are no detection values in other channels.
  • Methicillin-resistant genes only have detection values in CY5.5 and CY7, and there are no detection values in other channels.
  • the primers and probes provided by the present invention can accurately detect simulated samples, and the detection results are completely consistent with expectations, indicating that the accuracy of the detection system is normal; the system for detecting multiple pathogenic microorganisms and drug-resistant genes in the present invention has Specificity; the detection results of the two detection systems are basically consistent, indicating that the multiple detection system of the present invention is stable.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

涉及生物检测技术领域，尤其涉及通用探针、引物探针组、应用和试剂盒。提供了一种通用探针，其5'端至3'端依次包括：淬灭基团-片段A -荧光基团-片段B-C3；片段A 的核苷酸序列如SEQ ID NO:1~7任意一种所示；片段B的核苷酸序列如SEQ ID NO:8~14任意一种所示。还提供了引物探针组、应用及试剂盒。

Description

通用探针、引物探针组、应用和试剂盒 技术领域
本发明涉及生物检测技术领域，尤其涉及通用探针、引物探针组、应用和试剂盒。
背景技术
病原微生物，是指一些侵入人体后，可以导致人体发生感染性疾病，甚至是传染病的微生物。常见的病原微生物包括朊毒体，细菌，支原体，衣原体，立克次体，真菌，螺旋体以及病毒。某些病原微生物反复接触某些化学治疗药物后，其反应性不断减弱，以致最后病原微生物可抵抗该药而不被杀灭或抑制，这就使病原微生物对药物产生耐受性，称为耐药性或抗药性。耐药基因是编码耐药性状的一段核苷酸序列，位于细菌的染色体或者染色体外的质粒上。病原微生物产生耐药性后常使疗效减小或完全失效，直接影响了疾病的治疗效果，因此，针对多种病原微生物核酸和耐药基因鉴定，建立一套系统、高效、快捷的检测方法，可以有效地促进医疗事业的发展。
目前，常用的针对病原微生物核酸和耐药基因的检测方法主要是实时荧光定量PCR(简称qPCR)，qPCR可分为荧光染料法(SYBR GreenⅠ法为代表)和荧光探针法(TaqMan法为代表)。SYBR GreenⅠ染料法因具有使用简便，价格便宜等优点而被广泛使用，但其最大的缺点是缺乏特异性，即染料可以与任何dsDNA结合，影响定量结果的准确性。与染料法qPCR不同，TaqMan检测中会增加荧光探针成分，探针通过荧光共振能量转移(FRET)发生作用，具有特异性高、适用多重qPCR的特点。但传统的TaqMan探针技术，通常每个靶标需要一条探针；在一个荧光通道检测多个靶标时，同一荧光标记的探针过多，会导致背景信号过高，造成对比度下降，难以进行信号判读，因而限制了检测靶标数量。
发明内容
有鉴于此，本发明要解决的技术问题在于提供多种病原微生物和耐药基因数字PCR检测的引物探针组及其试剂盒，本发明提供了基于7色荧光通道数字PCR平台，并结合多重数字PCR技术实现了每个种类下多个菌种或耐药位点的覆盖。
本发明提供了通用探针的结构，其5’端至3’端依次包括：淬灭基团-片段A-荧光基团-片段B-C3。
在一些实施方案中，所述片段A的核苷酸序列如SEQ ID NO:1～7任意一种所示。
所述片段B的核苷酸序列如SEQ ID NO:8～14任意一种所示。
在一些实施方案中，所述通用探针的5’端至3’端依次包括：
淬灭基团-SEQ ID NO:1所示的片段A-荧光基团-SEQ ID NO:8所示的片段B-C3；
淬灭基团-SEQ ID NO:2所示的片段A-荧光基团-SEQ ID NO:9所示的片段B-C3；
淬灭基团-SEQ ID NO:3所示的片段A-荧光基团-SEQ ID NO:10所示的片段B-C3；
淬灭基团-SEQ ID NO:4所示的片段A-荧光基团-SEQ ID NO:11所示的片段B-C3；
淬灭基团-SEQ ID NO:5所示的片段A-荧光基团-SEQ ID NO:12所示的片段B-C3；
淬灭基团-SEQ ID NO:6所示的片段A-荧光基团-SEQ ID NO:13所示的片段B-C3；和/或
淬灭基团-SEQ ID NO:7所示的片段A-荧光基团-SEQ ID NO:14所示的片段B-C3。
本发明中所述通用探针的荧光基团选自FAM、VIC、ROX、CY5、A425、CY5.5或CY7；淬灭基团选自BHQ1、BHQ2或BHQ3。
一些实施例中，荧光基团A425为荧光基团ATTO425。
一些实施例中，所述通用探针的5’端至3’端依次为：
BHQ1-SEQ ID NO:1所示的片段A-dT-FAM-SEQ ID NO:8所示的片段B-C3；
BHQ1-SEQ ID NO:2所示的片段A-dT-VIC-SEQ ID NO:9所示的片段B-C3；
BHQ2-SEQ ID NO:3所示的片段A-dT-ROX-SEQ ID NO:10所示的片段B-C3；
BHQ2-SEQ ID NO:4所示的片段A-dT-CY5-SEQ ID NO:11所示的片段B-C3；
BHQ1-SEQ ID NO:5所示的片段A-dT-A425-SEQ ID NO:12所示的片段B-C3；
BHQ3-SEQ ID NO:6所示的片段A-dT-CY5.5-SEQ ID NO:13所示的片段B-C3；
BHQ3-SEQ ID NO:7所示的片段A-dT-CY7-SEQ ID NO:14所示的片段B-C3。
本发明中，所述通用探针与所述单修饰无荧光探针配合，其中单修饰无荧光探针与扩增产物特异性结合，通用探针与单修饰无荧光探针水解片段杂交后，产生荧光信号达到检测目的。
本发明还提供了检测病原微生物和耐药基因的引物探针组，包括本发明所述的通用探针。
本发明提供的引物探针组，还包括特异性引物对，所述特异性引物对靶向病原微生物和耐药基因。
其中，所述病原微生物包括铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌；
所述耐药基因包括碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-vanA基因、万古霉素类耐药-vanB基因、万古霉素类耐药-vanM基因、甲氧西林类耐药-mecA基因或甲氧西林类耐药- mecC基因。
具体地，靶向铜绿假单胞菌的引物对具有如SEQ ID NO:37～38所示的核苷酸序列；
靶向大肠杆菌的引物对具有如SEQ ID NO:39～40所示的核苷酸序列；
靶向肺炎克雷伯菌的引物对具有如SEQ ID NO:41～42所示的核苷酸序列；
靶向鲍曼不动杆菌的引物对具有如SEQ ID NO:43～44所示的核苷酸序列；
靶向金黄葡萄球菌的引物对具有如SEQ ID NO:45～46所示的核苷酸序列；
靶向屎肠球菌的引物对具有如SEQ ID NO:47～48所示的核苷酸序列；
靶向粪肠球菌的引物对具有如SEQ ID NO:49～50所示的核苷酸序列；
靶向阴沟肠杆菌的引物对具有如SEQ ID NO:51～52所示的核苷酸序列；
靶向白色念珠菌的引物对具有如SEQ ID NO:53～54所示的核苷酸序列；
靶向光滑念珠菌的引物对具有如SEQ ID NO:55～56所示的核苷酸序列；
靶向近平滑念珠菌的引物对具有如SEQ ID NO:57～58所示的核苷酸序列；
靶向热带念珠菌的引物对具有如SEQ ID NO:59～60所示的核苷酸序列；
靶向碳青霉烯类耐药-KPC基因的引物对具有如SEQ ID NO:61～62所示的核苷酸序列；
靶向碳青霉烯类耐药-NDM基因的引物对具有如SEQ ID NO:63～64所示的核苷酸序列；
靶向碳青霉烯类耐药-OXA48的引物对具有如SEQ ID NO:65～66所示的核苷酸序列；
靶向碳青霉烯类耐药-IMP基因的引物对具有如SEQ ID NO:67～68所示的核苷酸序列；
靶向万古霉素类耐药-vanA基因的引物对具有如SEQ ID NO:69～70所示的核苷酸序列；
靶向万古霉素类耐药-vanB基因的引物对具有如SEQ ID NO:71～72所示的核苷酸序列；
靶向万古霉素类耐药-vanM基因的引物对具有如SEQ ID NO:73～74所示的核苷酸序列；
靶向甲氧西林类耐药-mecA基因的引物对具有如SEQ ID NO:75～76所示的核苷酸序列；
靶向甲氧西林类耐药-mecC基因的引物对具有如SEQ ID NO:77～78所示的核苷酸序列。
本发明提供的引物探针组，还包括单修饰无荧光探针，所述单修饰无荧光探针的核酸序列与所述通用探针中的片段B反向互补，且3’端标记有淬灭基团，其中，所述淬灭基团选自MGB、BHQ1、BHQ2和BHQ3。
具体地，靶向铜绿假单胞菌的单修饰无荧光探针具有如SEQ ID NO:15所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向大肠杆菌的单修饰无荧光探针具有如SEQ ID NO:16所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向肺炎克雷伯菌的单修饰无荧光探针具有如SEQ ID NO:17所示的核苷酸序列，3’端标 记有MGB淬灭基团；
靶向鲍曼不动杆菌的单修饰无荧光探针具有如SEQ ID NO:18所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向金黄葡萄球菌的单修饰无荧光探针具有如SEQ ID NO:19所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向屎肠球菌的单修饰无荧光探针具有如SEQ ID NO:20所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向粪肠球菌的单修饰无荧光探针具有如SEQ ID NO:21所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向阴沟肠杆菌的单修饰无荧光探针具有如SEQ ID NO:22所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向白色念珠菌的单修饰无荧光探针具有如SEQ ID NO:23所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向光滑念珠菌的单修饰无荧光探针具有如SEQ ID NO:24所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向近平滑念珠菌的单修饰无荧光探针具有如SEQ ID NO:25所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向热带念珠菌的单修饰无荧光探针具有如SEQ ID NO:26所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向碳青霉烯类耐药-KPC基因的单修饰无荧光探针具有如SEQ ID NO:27所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向碳青霉烯类耐药-NDM基因的单修饰无荧光探针具有如SEQ ID NO:28所示的核苷酸序列，3’端标记有BHQ2淬灭基团；
靶向碳青霉烯类耐药-OXA48的单修饰无荧光探针具有如SEQ ID NO:29所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向碳青霉烯类耐药-IMP基因的单修饰无荧光探针具有如SEQ ID NO:30所示的核苷酸序列，3’端标记有MGB淬灭基团；
靶向万古霉素类耐药-vanA基因的单修饰无荧光探针具有如SEQ ID NO:31所示的核苷酸序列，3’端标记有BHQ1淬灭基团；
靶向万古霉素类耐药-vanB基因的单修饰无荧光探针具有如SEQ ID NO:32所示的核苷酸序 列，3’端标记有BHQ1淬灭基团；
靶向万古霉素类耐药-vanM基因的单修饰无荧光探针具有如SEQ ID NO:33所示的核苷酸序列，3’端标记有BHQ1淬灭基团；
靶向甲氧西林类耐药-mecA基因的单修饰无荧光探针具有如SEQ ID NO:34所示的核苷酸序列，3’端标记有BHQ2淬灭基团；
靶向甲氧西林类耐药-mecC基因的单修饰无荧光探针具有如SEQ ID NO:35所示的核苷酸序列，3’端标记有MGB淬灭基团。
本发明还提供了所述通用探针或所述引物探针组在在制备多种病原微生物和耐药基因的数字PCR检测试剂中的应用。
本发明还提供了一种检测多种病原微生物和耐药基因的试剂盒，包括本发明所述的通用探针或引物探针组。
本发明提供的试剂盒中，还包括内标基因的引物和探针组合，具体序列为：IC-UP7：ctgcacgaagctctttttcccgcgacggatctacgtcacagcg(SEQ ID NO：36)；IC-P：cgcgacggatctacgtcacagcg(SEQ ID NO：102)；IC-F：gcttcttgtggagctcgacaa(SEQ ID NO：79)；IC-R：ccgtcagcaacttcgttttca(SEQ ID NO：80)。
本发明还提供了多种病原微生物和耐药基因多重数字PCR检测的方法，利用本发明引物探针组或本发明试剂盒进行检测。
本发明基于多重数字PCR平台的检测方法包括以下步骤：
(1)提取待测样本核酸；
(2)配置多重数字PCR扩增体系；
(3)运行扩增程序:95℃5min,【95℃15s，60℃1min】×40；
(4)输出结果。
利用本发明引物探针可实现12种病原微生物包括铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌，和9种耐药基因包括碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-vanA基因、万古霉素类耐药-vanB基因、万古霉素类耐药-vanM基因、甲氧西林类耐药-mecA基因、甲氧西林类耐药-mecC基因的联合检测。
本发明针对12种病原微生物、9种耐药基因的保守区域，共特异性设计了42条引物、7条含有不同荧光基团的通用探针和21条特异性的单修饰无荧光基团探针，通用探针 和单修饰探针配合产生荧光信号，可以检测样品中是否含有铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌，以及是否含有碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-vanA基因、万古霉素类耐药-vanB基因、万古霉素类耐药-vanM基因、甲氧西林类耐药-mecA基因、甲氧西林类耐药-mecC基因位点。
本发明采用常规双修饰探针的单重扩增体系以及通用探针和单修饰探针配合的多重扩增体系，两种体系的检测结果一致，保证了针对靶标基因设计含有不同荧光基团的通用探针，并结合特异性单修饰无荧光基团探针进行多重数字PCR的可行性。
具体实施方式
本发明提供了多种病原微生物和耐药基因数字PCR检测的引物探针组及其试剂盒，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1本发明的引物探针组设计
本实施例涉及的引物探针组序列如表1所示。
其中：UP1-UP7表示7条不同颜色的通用探针。F表示上游引物，R表示下游引物，P表示探针。例如TL-F表示铜绿假单胞菌的上游引物，TL-R表示铜绿假单胞菌的下游引物，TL-P表示铜绿假单胞菌常规的双修饰探针，TL-UP1表示铜绿假单胞菌特异性的单修饰无荧光基团的探针；
表1





实施例2数字PCR扩增体系
一、单重扩增体系如表2所示
表2
二、多重扩增体系如表3所示
表3


三、扩增程序
95℃5min,【95℃15s，60℃1min】×40
四、结果判断
1、单重扩增体系检测结果如表4所示
表4
2、多重扩增体系检测结果如表5所示
表5
结果显示，铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌仅在FAM和CY7有检测值，其他通道均无检测值。
金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌仅在VIC和CY7有检测值，其他通道均无检测值。
白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌仅在ROX和CY7有检测值，其他 通道均无检测值。
碳青霉烯类耐药基因仅在CY5和CY7有检测值，其他通道均无检测值。
万古霉素类耐药基因仅在A425和CY7有检测值，其他通道均无检测值。
甲氧西林类耐药基因仅在CY5.5和CY7有检测值，其他通道均无检测值。
综合上述，本发明提供的引物和探针能够准确对模拟样本进行检测，检测结果与预期完全相符，说明本检测体系的准确性正常；本发明中检测多种病原微生物以及耐药基因的体系具有特异性；两种检测体系的检测结果基本一致，说明本发明的多重检测体系具有稳定性。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围

Claims (12)

  1. 通用探针，其特征在于，其5’端至3’端依次包括：淬灭基团-片段A-荧光基团-片段B-C3；
    所述片段A的核苷酸序列如SEQ ID NO:1～7任意一种所示；
    所述片段B的核苷酸序列如SEQ ID NO:8～14任意一种所示。
  2. 根据权利要求1所述的通用探针，其特征在于，其5’至3’端依次包括：
    淬灭基团-SEQ ID NO:1所示的片段A-荧光基团-SEQ ID NO:8所示的片段B-C3；或
    淬灭基团-SEQ ID NO:2所示的片段A-荧光基团-SEQ ID NO:9所示的片段B-C3；或
    淬灭基团-SEQ ID NO:3所示的片段A-荧光基团-SEQ ID NO:10所示的片段B-C3；或
    淬灭基团-SEQ ID NO:4所示的片段A-荧光基团-SEQ ID NO:11所示的片段B-C3；或
    淬灭基团-SEQ ID NO:5所示的片段A-荧光基团-SEQ ID NO:12所示的片段B-C3；或
    淬灭基团-SEQ ID NO:6所示的片段A-荧光基团-SEQ ID NO:13所示的片段B-C3；或
    淬灭基团-SEQ ID NO:7所示的片段A-荧光基团-SEQ ID NO:14所示的片段B-C3。
  3. 根据权利要求1或2所述的通用探针，其特征在于，所述荧光基团选自FAM、VIC、ROX、CY5、A425、CY5.5和CY7；所述淬灭基团选自BHQ1、BHQ2和BHQ3。
  4. 检测病原微生物和耐药基因的引物探针组，其特征在于，包括权利要求1～3任一项所述的通用探针、特异性引物对和单修饰无荧光探针；
    所述特异性引物对靶向病原微生物和耐药基因。
  5. 根据权利要求4所述的引物探针组，其特征在于，
    所述单修饰无荧光探针的核酸序列与所述通用探针中的片段B反向互补。
  6. 根据权利要求5所述的引物探针组，其特征在于，所述单修饰无荧光探针的3’端标记有淬灭基团。
  7. 根据权利要求6所述的引物探针组，其特征在于，所述单修饰无荧光探针3’端标记的淬灭基团选自MGB、BHQ1、BHQ2和BHQ3。
  8. 根据权利要求4所述的引物探针组，其特征在于，所述单修饰无荧光探针具有如SEQ ID NO:15～36任一项所示的核苷酸序列。
  9. 根据权利要求4-8中任一项所述的引物探针组，其特征在于，
    所述病原微生物包括铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌；
    所述耐药基因包括碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐 药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-vanA基因、万古霉素类耐药-vanB基因、万古霉素类耐药-vanM基因、甲氧西林类耐药-mecA基因或甲氧西林类耐药-mecC基因。
  10. 根据权利要求9所述的引物探针组，其特征在于，
    靶向铜绿假单胞菌的引物对具有如SEQ ID NO:37～38所示的核苷酸序列；
    靶向大肠杆菌的引物对具有如SEQ ID NO:39～40所示的核苷酸序列；
    靶向肺炎克雷伯菌的引物对具有如SEQ ID NO:41～42所示的核苷酸序列；
    靶向鲍曼不动杆菌的引物对具有如SEQ ID NO:43～44所示的核苷酸序列；
    靶向金黄葡萄球菌的引物对具有如SEQ ID NO:45～46所示的核苷酸序列；
    靶向屎肠球菌的引物对具有如SEQ ID NO:47～48所示的核苷酸序列；
    靶向粪肠球菌的引物对具有如SEQ ID NO:49～50所示的核苷酸序列；
    靶向阴沟肠杆菌的引物对具有如SEQ ID NO:51～52所示的核苷酸序列；
    靶向白色念珠菌的引物对具有如SEQ ID NO:53～54所示的核苷酸序列；
    靶向光滑念珠菌的引物对具有如SEQ ID NO:55～56所示的核苷酸序列；
    靶向近平滑念珠菌的引物对具有如SEQ ID NO:57～58所示的核苷酸序列；
    靶向热带念珠菌的引物对具有如SEQ ID NO:59～60所示的核苷酸序列；
    靶向碳青霉烯类耐药-KPC基因的引物对具有如SEQ ID NO:61～62所示的核苷酸序列；
    靶向碳青霉烯类耐药-NDM基因的引物对具有如SEQ ID NO:63～64所示的核苷酸序列；
    靶向碳青霉烯类耐药-OXA48的引物对具有如SEQ ID NO:65～66所示的核苷酸序列；
    靶向碳青霉烯类耐药-IMP基因的引物对具有如SEQ ID NO:67～68所示的核苷酸序列；
    靶向万古霉素类耐药-vanA基因的引物对具有如SEQ ID NO:69～70所示的核苷酸序列；
    靶向万古霉素类耐药-vanB基因的引物对具有如SEQ ID NO:71～72所示的核苷酸序列；
    靶向万古霉素类耐药-vanM基因的引物对具有如SEQ ID NO:73～74所示的核苷酸序列；
    靶向甲氧西林类耐药-mecA基因的引物对具有如SEQ ID NO:75～76所示的核苷酸序列；
    靶向甲氧西林类耐药-mecC基因的引物对具有如SEQ ID NO:77～78所示的核苷酸序列。
  11. 权利要求1～3任一项所述通用探针，或权利要求4～8任一项所述的引物探针组在制备多种病原微生物和耐药基因的数字PCR检测试剂中的应用。
  12. 试剂盒，其特征在于，其包括权利要求1～3任一项所述的通用探针，或权利要求4～8任一项所述的引物探针组以及助剂或载体。
PCT/CN2023/092551 2022-08-31 2023-05-06 通用探针、引物探针组、应用和试剂盒 WO2024045683A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/326,300 US11987850B2 (en) 2022-08-31 2023-05-31 Universal probe, primer-probe set and kit

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211052186.6 2022-08-31
CN202211052186.6A CN116064859B (zh) 2022-08-31 2022-08-31 多种病原微生物和耐药基因数字pcr检测的引物探针组及其试剂盒

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/326,300 Continuation US11987850B2 (en) 2022-08-31 2023-05-31 Universal probe, primer-probe set and kit

Publications (1)

Publication Number Publication Date
WO2024045683A1 true WO2024045683A1 (zh) 2024-03-07

Family

ID=86173803

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/092551 WO2024045683A1 (zh) 2022-08-31 2023-05-06 通用探针、引物探针组、应用和试剂盒

Country Status (3)

Country Link
US (1) US11987850B2 (zh)
CN (1) CN116064859B (zh)
WO (1) WO2024045683A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064859B (zh) * 2022-08-31 2024-08-23 领航基因科技（杭州）有限公司 多种病原微生物和耐药基因数字pcr检测的引物探针组及其试剂盒
CN117448471B (zh) * 2023-12-26 2024-03-08 苏州锐讯生物科技有限公司 一种用于检测目标基因的组合及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080233572A1 (en) * 2006-09-28 2008-09-25 The University Of North Carolina At Chapel Hill Methods and compositions for the detection and quantification of e.coli and enterococcus
CN103781908A (zh) * 2011-08-24 2014-05-07 盖立复治疗公司 用于核酸杂交的组合物、方法和试剂盒
CN105734119A (zh) * 2014-12-09 2016-07-06 常州金麦格生物技术有限公司 利用通用探针的核酸目标待测位点检测方法
CN110564824A (zh) * 2019-08-22 2019-12-13 领航基因科技（杭州）有限公司 一种检测人类致病菌的引物和探针组合、试剂盒
CN113584191A (zh) * 2021-06-24 2021-11-02 领航基因科技（杭州）有限公司 7种耐药基因多重pcr检测的引物、探针及其试剂盒
CN113621727A (zh) * 2021-06-24 2021-11-09 领航基因科技（杭州）有限公司 5种病原菌多重pcr检测的引物、探针及其试剂盒
CN116064859A (zh) * 2022-08-31 2023-05-05 领航基因科技（杭州）有限公司 多种病原微生物和耐药基因数字pcr检测的引物探针组及其试剂盒

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866336A (en) * 1996-07-16 1999-02-02 Oncor, Inc. Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon
GB9812768D0 (en) * 1998-06-13 1998-08-12 Zeneca Ltd Methods
CN110628940A (zh) * 2019-11-08 2019-12-31 领航基因科技（杭州）有限公司 一种基于液滴数字pcr检测四种念珠菌的试剂盒
CN111088378B (zh) * 2020-01-09 2023-12-19 中国科学院大学宁波华美医院 检测重症肺炎常见病原菌的引物探针系统、试剂盒及方法
CN111593131A (zh) * 2020-04-26 2020-08-28 领航基因科技（杭州）有限公司 一种可同时检测多种耐药基因的引物探针系统及试剂盒

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080233572A1 (en) * 2006-09-28 2008-09-25 The University Of North Carolina At Chapel Hill Methods and compositions for the detection and quantification of e.coli and enterococcus
CN103781908A (zh) * 2011-08-24 2014-05-07 盖立复治疗公司 用于核酸杂交的组合物、方法和试剂盒
CN105734119A (zh) * 2014-12-09 2016-07-06 常州金麦格生物技术有限公司 利用通用探针的核酸目标待测位点检测方法
CN110564824A (zh) * 2019-08-22 2019-12-13 领航基因科技（杭州）有限公司 一种检测人类致病菌的引物和探针组合、试剂盒
CN113584191A (zh) * 2021-06-24 2021-11-02 领航基因科技（杭州）有限公司 7种耐药基因多重pcr检测的引物、探针及其试剂盒
CN113621727A (zh) * 2021-06-24 2021-11-09 领航基因科技（杭州）有限公司 5种病原菌多重pcr检测的引物、探针及其试剂盒
CN116064859A (zh) * 2022-08-31 2023-05-05 领航基因科技（杭州）有限公司 多种病原微生物和耐药基因数字pcr检测的引物探针组及其试剂盒

Also Published As

Publication number Publication date
CN116064859A (zh) 2023-05-05
US20240084405A1 (en) 2024-03-14
CN116064859B (zh) 2024-08-23
US11987850B2 (en) 2024-05-21

Similar Documents

Publication Publication Date Title
WO2024045683A1 (zh) 通用探针、引物探针组、应用和试剂盒
AU2018299445B2 (en) Application of Cas protein, method for detecting target nucleic acid molecule and kit
CN108754000B (zh) 耐药基因mcr-4/5/8荧光定量PCR检测方法
CN110982945A (zh) 一种检测2019新型冠状病毒的核酸组合物、试剂盒以及方法
CN102822352B (zh) 利用核酸色谱法的肺炎病原菌的检出方法
WO2018014614A1 (zh) 一种同时鉴别8种牛病原体的引物组合及GeXP检测方法
CN107937578A (zh) 15种呼吸道感染病原体联检的引物探针组合及试剂盒
WO2023109032A1 (zh) 一种多重核酸检测系统及其制备方法与应用
CN112458212B (zh) 同时检测甲型流感病毒、乙型流感病毒及呼吸道合胞病毒的试剂盒
Jeong et al. Evaluation of peptide nucleic acid-mediated multiplex real-time PCR kits for rapid detection of carbapenemase genes in gram-negative clinical isolates
CN113667718B (zh) 利用双链核酸检测器进行靶核酸检测的方法
WO2022012423A1 (zh) 利用含有无碱基间隔物的核酸检测器检测靶核酸的方法
CN104928355A (zh) 检测braf基因突变的方法及其试剂盒
CN116732212B (zh) 一种引物组合及在制备盖尔森基兴诺卡菌复合群检测试剂盒中的应用
CN110804652B (zh) 一种快速检测dna的实时定量pcr的添加剂、试剂盒及反应方法
WO2012068956A1 (zh) 尿液样本细菌性病原体检测试剂盒及其检测方法
CN109457020B (zh) 耐药性检测组合物、耐药性检测试剂盒及耐药性检测方法
CN117210587A (zh) 检测血流感染多种病原体的检测试剂盒及其应用
CN111334592A (zh) 一种用于检测幽门螺杆菌耐药基因的核酸组合物及其试剂盒和应用
GB2549799A (en) A multiplex assay for the sensitive and specific detection and differentiation of Clostridium difficile
CN114277169B (zh) 一种用于检测奶牛乳房炎致病菌的试剂盒及其方法
CN114657272A (zh) 用于肺炎链球菌rpa-lfs检测法的引物探针组合及其应用
CN110129460B (zh) 超级细菌两种耐药基因的双重qPCR试剂盒及检测方法
CN109306373B (zh) 用于人类基因组成熟tRNA谱检测的接头、引物组和试剂盒
CN116179739B (zh) 鉴定荚膜组织胞浆菌的pcr引物探针组合物及试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23858722

Country of ref document: EP

Kind code of ref document: A1