WO2024078420A1 - 一种用于检测耳念珠菌的恒温核酸扩增raa引物探针组合及其应用 - Google Patents

一种用于检测耳念珠菌的恒温核酸扩增raa引物探针组合及其应用 Download PDF

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WO2024078420A1
WO2024078420A1 PCT/CN2023/123418 CN2023123418W WO2024078420A1 WO 2024078420 A1 WO2024078420 A1 WO 2024078420A1 CN 2023123418 W CN2023123418 W CN 2023123418W WO 2024078420 A1 WO2024078420 A1 WO 2024078420A1
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primer
raa
candida auris
reaction
probe
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PCT/CN2023/123418
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English (en)
French (fr)
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杨英
张欣然
马腾
王宇辰
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2024078420A1 publication Critical patent/WO2024078420A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—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
    • 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
    • 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 invention belongs to the technical field of pathogenic microorganism detection, and specifically relates to a constant temperature nucleic acid amplification RAA primer probe combination for detecting Candida auris and an application thereof.
  • Candida auris is a pathogenic fungus first discovered in the external auditory canal secretions of a Japanese patient in 2009. So far, Candida auris has been reported in more than 40 countries on six continents except Antarctica. Candida auris infection can occur in people of all ages, among which it is most common in the elderly, and newborns and children are occasionally infected. Common hosts of Candida auris can be divided into three categories: one is people with severe immunodeficiency diseases or weak autoimmunity, the second is patients undergoing open surgery or using invasive catheters, and the third is people treated with broad-spectrum antibiotics. The fungus mainly causes persistent and invasive infections, and is multidrug-resistant to commonly used antifungal drugs such as fluconazole.
  • Candida auris has large genomic diversity, multidrug resistance and high mortality. It is very easy to spread and infect hospital patients, causing outbreaks of hospital infections, and the infection is often candidemia. Since the common phenotypic identification and microbial identification systems are unable to accurately identify Candida auris, clinical microbiology laboratories often misidentify the strain, which leads to misdiagnosis, making it extremely difficult to prevent and control the spread of Candida auris strains in a timely manner in clinical practice.
  • Candida auris has general similarities with other Candida species in terms of phenotype and physiological and biochemical aspects, which makes it easy to be misdiagnosed in the clinical diagnosis process.
  • MALDI-TOF MS matrix-assisted laser desorption/ionization time of flight mass spectrometry
  • ITS internal transcribed spacer
  • 28S rDNA ribosomal DNA
  • Candida chromogenic culture media and commercial microbiological detection systems are not only time-consuming and labor-intensive, but also have low positive rates and high error rates.
  • the most commonly used clinical microbiological identification systems such as VITEK2, API-20C AUX and BD yeast identification systems, often misdiagnose Candida auris as other closely related Candida species such as C. haemulonii, C. duobushaemulonii, C. pseudohaemulonii, C. guilliermondii, C. parapsilosis, etc., resulting in missed diagnosis and extremely high misdiagnosis rates for patients with Candida auris infection.
  • Isothermal nucleic acid amplification technology is a general term for a class of molecular biology technologies that have been newly developed in recent years. It can amplify specific DNA or RNA at a specific temperature. It is simpler and more convenient than PCR technology in terms of both actual operation and instrument requirements. It gets rid of the dependence on sophisticated equipment, greatly shortens the reaction time, and shows good application prospects in clinical and on-site rapid diagnosis.
  • RAA isothermal amplification technology that can perform single-molecule nucleic acid detection at room temperature within 20 minutes.
  • the object of the present invention is to provide a constant temperature nucleic acid amplification RAA primer probe combination for detecting Candida auris and its application.
  • a first aspect of the present invention provides a constant temperature nucleic acid amplification RAA primer-probe combination for Candida auris detection.
  • the primer-probe combination includes an upstream primer, a downstream primer and a probe
  • the nucleotide sequence of the upstream primer is F10’-1: 5’-AAGGATCATTATTGATATTTTGCATACACA-3’;
  • the nucleotide sequence of the downstream primer is R10': 5'-Biotin-TTCAAAGATTCGATGATTCACGTCTGCAAG-3';
  • the nucleotide sequence of the probe is P: 5’-FAM-ACTGATTTGGATTTTAAAACTAACCCAACG[THF]TAAGTTCAACTAAAC-C3spacer-3’.
  • the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:22
  • the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:26, and is modified with biotin at its 5' end
  • the nucleotide sequence of the probe is as shown in SEQ ID NO:21, and is modified with a fluorescent group (FAM) at its 5' end, the 3' end is blocked with a blocking group (C3-spacer), and the 31st base is replaced with tetrahydrofuran ([THF]).
  • FAM fluorescent group
  • primers and/or probes corresponding to sequences having more than 70% homology with the primer and/or probe sequences described in the present invention are also included in the protection scope of the present invention, that is, it can be understood that the primers and/or probes obtained after changes are made on the basis of the primers and/or probes provided by the present invention also fall within the protection scope of the present invention.
  • the present invention has no particular restrictions on the modification groups (e.g., biotin) contained in the primers, the fluorescent groups (e.g., FAM), the blocking groups (e.g., C3-spacer), and the replacement groups (e.g., tetrahydrofuran) contained in the probes.
  • modification groups e.g., biotin
  • FAM fluorescent groups
  • blocking groups e.g., C3-spacer
  • the replacement groups e.g., tetrahydrofuran
  • the modification groups for modifying the primers include but are not limited to: biotin, phosphorylation, digoxigenin, thiol, Spacer, thio, deoxyuracil, deoxyhypoxanthine, etc.;
  • the fluorescent groups in the probes include but are not limited to: FAM, HEX, VIC, ROX, Cy5, TET, etc.;
  • the blocking groups in the probes include but are not limited to: C3-spacer, phosphate, biotin-TEG, etc.
  • the Candida auris is not particularly limited, including but not limited to: C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonis, C. rugosa, C. albicans, C. neoformans, C. parapsilosis, C. glabrata, C. guilliermondii, E. coli, S. aureus, E. faecalis, C. tropicalis, C. krusei, C. dubliniensis, K. pneumoniae, etc.
  • Candida auris of any strain source and/or any strain type is within the protection scope of the present invention.
  • a second aspect of the present invention provides a kit for detecting Candida auris.
  • the kit comprises a primer-probe mixture consisting of the primer-probe combination described in the first aspect of the present invention.
  • the kit also comprises A Buffer, B Buffer, RAA reaction dry powder reagent, and ddH2O ; preferably, the A Buffer is 20% PEG; preferably, the B Buffer is 280mM MgAc; preferably, the RAA reaction dry powder reagent comprises the following components: dNTP, SSB protein, recA recombinase protein or Rad51, Bsu DNA polymerase, Tricine, PEG, dithiothreitol, creatine kinase, and Nfo nuclease; more preferably, the concentration of each component in the RAA reaction dry powder reagent is as follows: 1mmol/L dNTP, 90ng/ ⁇ L SSB protein, 120ng/ ⁇ L recA recombinase protein or 30ng/ ⁇ L Rad51, 30ng/ ⁇ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/ ⁇ L creatin
  • the kit also comprises a lateral flow chromatography test strip.
  • the primers and probes disclosed in the present invention can be provided in the form of a kit for detecting Candida auris.
  • a suitable amount of one or more probes and/or primers e.g., primers and probes disclosed in the first aspect of the present invention
  • Nucleic acid probes and/or primers can be provided as suspensions in an aqueous solution, or, for example, as freeze-dried or lyophilized powders.
  • the container in which the nucleic acid is provided can be any conventional container capable of accommodating the provided form, such as a microcentrifuge tube, an ampoule or a bottle.
  • the kit can include labeled or unlabeled probes for detecting Candida auris nucleotide sequences.
  • one or more primers such as primer pairs, can be provided in separate, typically disposable tubes or equivalent containers in pre-measured single-use amounts. Using such a setup, samples for testing the presence of Candida auris nucleic acids can be added to separate tubes and directly amplified.
  • the amount of nucleic acid primers provided in the kit can be any suitable amount and can depend on the target market for the product. For example, if the kit is suitable for research or clinical applications, the amount of each nucleic acid primer provided can be an amount sufficient to initiate several PCR amplification reactions. General guidance on determining suitable amounts can be found in the literature of Innis et al., Sambrook et al., and Ausubel et al.
  • the kit can contain more than two primers to facilitate PCR amplification of larger amounts of Candida auris nucleotide sequences.
  • the kit may contain the necessary reagents for performing the PCR amplification reaction, including DNA sample preparation reagents, suitable buffers (e.g., polymerase buffers), salts (e.g., magnesium chloride), and deoxyribonucleotides (dNTPs).
  • suitable buffers e.g., polymerase buffers
  • salts e.g., magnesium chloride
  • dNTPs deoxyribonucleotides
  • the kit obtained by simply replacing the types of these conventional reagents is also included in the protection scope of the present invention.
  • one or more control sequences for the PCR reaction may also be provided in the kit.
  • the third aspect of the present invention provides a method for detecting Candida auris based on RAA-lateral flow chromatography technology.
  • the method comprises the following steps:
  • nucleotide sequence of the upstream primer of RAA amplification is F10'-1: 5'-AAGGATCATTATTGATATTTTGCATACACA-3';
  • nucleotide sequence of the downstream primer is R10': 5'-Biotin-TTCAAAGATTCGATGATTCACGTCTGCAAG-3';
  • nucleotide sequence of the probe is P: 5'-FAM-ACTGATTTGGATTTTAAAACTAACCCAACG[THF]TAAGTTCAACTAAAC-C3spacer-3';
  • the process of the amplification reaction in step (2) includes the following steps: adding upstream primers, downstream primers, probes, A Buffer, ddH 2 O, and templates to the detection unit tube containing the RAA reaction dry powder reagent, adding B Buffer to the tube cover, covering the tube cover, and continuing the amplification reaction after low-speed centrifugation to obtain an amplified product.
  • the amounts of each substance used are: 2 ⁇ M upstream primer 2 ⁇ L, 2 ⁇ M downstream primer 2 ⁇ L, 2 ⁇ M probe 0.6 ⁇ L, A Buffer 25 ⁇ L, 15.9 ⁇ L ddH 2 O, 2 ⁇ L template, B Buffer 2.5 ⁇ L.
  • the reaction conditions of the amplification reaction in step (2) are: reaction at 37°C for 15 minutes.
  • reaction conditions and/or the amount of each substance in the amplification reaction in step (2) are not intended to limit the scope of protection of the present invention.
  • the adjusted reaction conditions and/or the amount of each substance will also fall within the scope of protection of the present invention.
  • the amplification principle of the RAA technology is as follows: RAA technology mainly uses recombinase, single-strand binding protein and DNA polymerase to amplify the target gene in large quantities.
  • the recombinase obtained from bacteria or fungi can bind tightly to the primer DNA at room temperature to form a recombinase/primer complex, invade the DNA double-stranded nucleic acid template, and the recombinase opens the double strand at the invasion site.
  • the single strand binds to the single strand opened by the recombinase, maintaining the double-stranded template in an open-strand state.
  • the recombinase/primer complex begins to scan the double strand.
  • the primer When the target gene searches for a complementary sequence that matches it perfectly on the template, the recombinase/primer complex disintegrates, and the DNA polymerase binds to the 3' end of the primer to start synthesizing a new chain.
  • the synthesized new chain can be used as a template again, and the final amplification product grows exponentially to complete the amplification of the target gene.
  • the downstream primer and probe are modified with biotin and carboxyfluorescein (FAM) (Tsingke Biotechnology Co., Ltd., Beijing, China) at the 5' end, respectively.
  • FAM carboxyfluorescein
  • the fluorescently labeled probe binds to the amplified product, and the endonuclease in the reaction system recognizes and cuts the [THF] site without purine and pyrimidine.
  • the probe is cleaved by the endonuclease, it is amplified together with the biotin-labeled primer to form a fragment with fluorescent labels and biotin labels at both ends, which can be read using a lateral flow chromatography test strip.
  • the lateral flow chromatography test strip is a universal nucleic acid detection test strip that only requires a small amount of product for detection, and the product needs to be appropriately diluted when the concentration is too high.
  • both ends of the amplified product are labeled with biotin and FAM, and FAM binds to AuNPs.
  • biotin binds to streptavidin, and the other end presents a positive signal through gold nanoparticles (AuNPs).
  • Insert the lateral flow chromatography test strip (Hangzhou Zhongce Bio-Sci&Tech Co., Ltd., Hangzhou, China) with the sample pad facing downward into the diluted reaction solution for 2 minutes and then visually read the test results.
  • the present invention further optimizes the reaction conditions and screens out the optimal temperature and time of the amplification reaction in the RAA method to be 37°C and 15 minutes, respectively.
  • the method exhibits extremely high specificity and good sensitivity, and can detect Candida auris genomic DNA as low as 10 1 fg/reaction, and the detection sensitivity is not affected by the presence of other fungal DNA.
  • the sample to be tested is derived from a clinical sample of a subject in need thereof, including but not limited to: cells, tissues, body fluids, such as: skin; mucosa; blood; blood derivatives or fractions, such as serum; extracted bile; tissues removed by biopsy or surgery, including, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; breast milk; shavings; surface washings; urine; sputum; cerebrospinal fluid; prostatic fluid; pus; bone marrow aspirate; middle ear effluent, bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngeal aspirate, oropharyngeal aspirate or saliva.
  • cells, tissues, body fluids such as: skin; mucosa; blood; blood derivatives or fractions, such as serum
  • extracted bile tissues removed by biopsy or surgery, including, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears
  • the subject includes humans and non-human animals.
  • Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians and reptiles.
  • the subject is preferably a human.
  • the fourth aspect of the present invention provides use of the primer-probe combination described in the first aspect of the present invention and the kit described in the second aspect of the present invention in the preparation of a product for detecting Candida auris.
  • the fifth aspect of the present invention provides the use of the primer-probe combination described in the first aspect of the present invention and the kit described in the second aspect of the present invention in the preparation of a product for differential diagnosis of Candida auris and C. persedohaemulonii, C. duobushaemulonis, and C. haemulonii.
  • the sixth aspect of the present invention provides use of the primer-probe combination described in the first aspect of the present invention and the kit described in the second aspect of the present invention in detecting Candida auris.
  • the seventh aspect of the present invention provides the use of the primer-probe combination described in the first aspect of the present invention and the kit described in the second aspect of the present invention in differential diagnosis of Candida auris and C. persedohaemulonii, C. duobushaemulonis, and C. haemulonii.
  • the primer-probe combination provided by the present invention can accurately and effectively identify and distinguish Candida auris from three closely related bacteria that are often misdiagnosed in clinical microbial identification systems: C. persedohaemulonii, C. duobushaemulonis, and C. haemulonii.
  • the eighth aspect of the present invention provides a method for diagnosing whether a subject suffers from a Candida auris infection disease, the method comprising the following steps: using the method described in the third aspect of the present invention to detect a sample from the subject.
  • the present invention has the following advantages and beneficial effects:
  • the present invention provides a constant temperature nucleic acid amplification RAA primer-probe combination for rapid detection of Candida auris, the primer-probe combination comprising a primer pair (as shown in SEQ ID NO: 22 and SEQ ID NO: 26) and a probe (as shown in SEQ ID NO: 21), the primer pair can specifically amplify Candida auris, and does not cross-react with other pathogens except Candida auris, that is, the primer pair provided by the present invention has a strong specific amplification characteristic, and can clearly distinguish Candida auris from closely related bacteria that are often misdiagnosed in clinical microbiological identification systems—C. haemulonii, C. pseudohaemulonii, and C.
  • the present invention proves through comparative experiments that the RAA primer-probe combination has the highest amplification efficiency, the strongest specificity, and no obvious false positives, and is significantly better than the other three RAA primer-probe combinations that are also designed for the ITS sequence of the Candida auris rDNA gene. This effect is a technical effect that is unexpected by those skilled in the art.
  • the present invention also provides a POCT (point-of-care test) method for rapid detection of Candida auris, which is a detection method combining lateral flow strip detection (LFS) and recombinase aided amplification (RAA) (RAA-LFS).
  • DNA is extracted and purified by a simple Chelex-100 boiling method, which saves the time and effort of the detection system.
  • the traditional detection time is shortened.
  • the test no longer requires fluorescence detection equipment and electrophoresis devices. It only needs a constant temperature water bath to provide the temperature required for the reaction. Even body temperature can be used to heat the test to complete effective amplification.
  • the test results can be obtained within 15 minutes.
  • the present invention adopts RAA-lateral flow chromatography technology for the first time to establish a method for rapid detection of Candida auris, and through specificity and sensitivity evaluation, it can be used in the detection of actual blood samples, providing a sensitive, reliable and effective new method for on-site rapid detection of Candida auris.
  • the primer pairs selected by the present invention are obtained through a large number of experimental screenings, have good specificity, and have no cross-reaction with other pathogens.
  • the primer-probe combination used in the present invention has high amplification efficiency, strong band specificity, and can form a high concentration of primer-probe heterodimers in the detection area, so that the test strip presents a strong positive reaction, increasing the sensitivity of the detection.
  • the method for detecting Candida auris by combining RAA technology and lateral flow chromatography technology established by the present invention has the advantages of high sensitivity and high throughput of molecular biological detection, good specificity and simple operation of immunological detection, and does not require complex instruments, and has a fast detection speed. It is particularly suitable for rapid screening and detection of Candida auris in grassroots laboratories and quarantine sites.
  • Figure 1 is a schematic diagram of RAA-LFS, wherein Figure A: RAA-nfo probe design, Figure B: fluorescently labeled probe binding to template, and Figure C: Schematic diagram of lateral flow chromatography test strip (LFS) operation;
  • Figure A RAA-nfo probe design
  • Figure B fluorescently labeled probe binding to template
  • Figure C Schematic diagram of lateral flow chromatography test strip (LFS) operation
  • Fig. 2 is a schematic diagram of a method for interpreting test results
  • FIG3 is a graph showing the agarose gel electrophoresis results of the primer pairs screened by the amplification performance of RAA, wherein the NTC lane is the no-template control of the corresponding primer pair, the band size of the DNA ladder is shown on the left, and the primer dimer is indicated by a white arrow;
  • FIG4 is a graph showing the results of a lateral flow chromatography test strip (LFS) using recombinase-mediated isothermal nucleic acid amplification (RAA) with different primer-probe sets, wherein NTC is a no-template control of the corresponding primer-probe set, the positions of the test line and the control line are marked on the right, the template is Candida auris genomic DNA, and the reaction conditions are 15 minutes at 39° C.;
  • LFS lateral flow chromatography test strip
  • RAA recombinase-mediated isothermal nucleic acid amplification
  • FIG5 is a diagram showing the result of a phylogenetic tree of the whole genomes of Candida auris and other common pathogenic Candida species;
  • Figure 6 is a map of the primer-probe combination targeting fragments, comparing the ITS sequence fragments of three closely related strains, C. persedohaemulonii, C. duobushaemulonis and C. haemulonii, with the ITS sequence section of Candida auris, with the strain name shown at the beginning of each sequence, the sequences corresponding to the primers and probes written below the positions in the alignment, arrows indicating the direction of primer and probe extension, and tetrahydrofuran (THF) sites indicated by " ⁇ ";
  • FIG7 is a graph showing the detection results of RAA-LFS under different reaction temperatures and reaction times, wherein the temperature of the RAA reaction is shown at the top of each strip, the RAA reaction time is shown on the left side of each line, the amplification template is Candida auris genomic DNA, and the positions of the control line and the test line are shown on the right side of the image;
  • FIG8 is a graph showing the test results of different reference strains, wherein the strain name is shown on the top of each strip, NTC is a no-template control, the positions of the control line and the test line are shown on the right side of the image, and the reaction is carried out at 37° C. for 15 min;
  • FIG9 is a graph showing the detection results of other closely related fungi and pathogenic bacteria, wherein, FIGA: the result graph of RAA-LFS detection of C. haemulonii, C. Perudohaemulonii and C. Duobushaemulonis strains, FIGB: the result graph of RAA-LFS detection of other related Candida and common pathogens, the strain type is shown at the top of each strip, NTC is a no-template control, the positions of the control line and the test line are shown on the right side of the image, and the reaction is carried out at 37° C. for 15 min;
  • FIG10 is a graph showing the sensitivity of the RAA-LFS technique for detecting Candida auris, wherein FIGA shows the LFS results of RAA amplification and different numbers of Candida auris cultures, and FIGB shows the LFS results of RAA amplification using different numbers of Candida auris genomic DNA;
  • FIG11 is a graph showing the sensitivity of RAA-LFS technology in detecting Candida auris under the interference of other fungi, wherein, FIGA: LFS result of RAA amplification of different Candida auris cultures after adding 10 5 CFU/ ⁇ L Candida albicans culture medium, FIGB: LFS result of RAA amplification of 1ng Candida albicans genomic DNA added to the reaction in addition to Candida auris genomic DNA;
  • Figure 12 shows the test results of the detection performance of RAA-LFS technology on human blood samples, where Figure A: the result of RAA-LFS detection of Candida auris solution of different concentrations mixed into human blood samples, and Figure B: the result of RAA-LFS detection of Candida auris genomic DNA and DNA extracted from human blood mixed at a ratio of 1:10, where NTC is a no-template control, the positions of the control line and the test line are shown on the right side of the image, and the reaction was carried out at 37°C for 15 minutes.
  • kits contains a primer-probe mixture consisting of the primer-probe combination
  • primer-probe mixture consisting of the primer-probe combination
  • amplification refers to increasing the copy number of a nucleic acid molecule.
  • the amplified product obtained is referred to as an "amplicon".
  • the amplification of a nucleic acid molecule refers to the use of technology to increase the copy number of a nucleic acid molecule in a sample.
  • An example of amplification is the polymerase chain reaction (PCR), in which a sample is contacted with a pair of oligonucleotide primers under conditions that allow the primers to hybridize with a nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, reannealed, extended, and dissociated to amplify the copy number of the nucleic acid.
  • the cycle can be repeated.
  • the amplified product can be characterized by techniques such as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or connection, and/or nucleic acid sequencing.
  • Examples of other in vitro amplification techniques include quantitative real-time PCR, reverse transcriptase PCR (RT-PCR), real-time PCR, real-time reverse transcriptase PCR (rt RT-PCR), nested PCR, strand displacement amplification (see U.S. Patent No. 5,744,311), non-transcriptional isothermal amplification (see U.S. Patent No. 6,033,881), repair chain reaction amplification (see WO 90/01069), ligase chain reaction amplification (see European Patent Publication EP-A-320 308), gap-filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889), and NASBATM RNA non-transcriptional amplification (see U.S. Patent No. 6,025,134), etc.
  • RT-PCR reverse transcriptase PCR
  • rt RT-PCR real
  • primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, such as a sequence of at least 15 nucleotides, which can be annealed with a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid chain.
  • the primer can be extended along the target nucleic acid molecule by a polymerase.
  • primers can be used to amplify target nucleic acid molecules (such as Candida auris nucleic acid sequences), wherein the sequence of the primer is specific for the target nucleic acid molecule, so for example, the primer will hybridize with the target nucleic acid molecule under very high stringency hybridization conditions.
  • the specificity of the primer increases with its length. Therefore, for example, a primer containing 30 continuous nucleotides will anneal with a target sequence with a higher specificity compared to a corresponding primer with only 15 nucleotides. Therefore, in order to obtain higher specificity, probes and primers containing at least 15, 20, 25, 30, 35, 40, 45, 50 or more continuous nucleotides can be selected.
  • probe refers to an isolated nucleic acid that can hybridize with a target nucleic acid (e.g., a Candida auris nucleic acid molecule).
  • a detectable label or reporter molecule may be attached to the probe.
  • Typical labels include radioactive isotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
  • the length of the probe is generally at least 20 nucleotides, e.g., at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60 or more consecutive nucleotides that are complementary to the target nucleic acid molecule, e.g., 20-60 nucleotides, 30-60 nucleotides, 20-50 nucleotides, 30-50 nucleotides, 20-40 nucleotides, or 20-30 nucleotides.
  • homology refers to the identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have relatively high levels of sequence identity when aligned using standard methods. Sex/similarity.
  • Primer and probe design Primer 5.0;
  • Nucleotide and amino acid comparison tool an online tool from the National Center for Biotechnology Information.
  • genomic DNA of all strains was extracted using the Chelex-100 boiling method. All extracted DNA was quantitatively measured using a Qubit 2 fluorometer (Thermo Fisher Scientific) and stored in a -20°C refrigerator until use.
  • the specific steps for extracting genomic DNA are as follows:
  • the ITS sequence of the Candida auris rDNA gene was retrieved from the NCBI nucleic acid database GenBank (http://www.ncbi.nlm.nih.gov).
  • the ITS1 and ITS2 of the Candida auris rDNA gene are conserved sequences with specificity for Candida auris as target sequences.
  • Primers and probes were designed according to the primer design principles using Primer Premier 5.0 software (Premier Biosoft International, CA, USA).
  • a total of 10 candidate primer pairs (ITS-1—ITS-10) were designed, as shown in Table 4.
  • the designed primer pairs were screened using the BLAST tool on NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • the primer pairs and probes used were synthesized by Qingke Company.
  • a total of 50 ⁇ L of RAA primer screening system add 2 ⁇ L of 10 ⁇ M upstream and downstream primers, 25 ⁇ L of A Buffer, 13.5 ⁇ L of double distilled water, and 5 ⁇ L of Candida auris genomic DNA to the detection unit tube containing the reaction dry powder.
  • After the above system is prepared and 47.5 ⁇ L in total, mix and centrifuge, and add 2.5 ⁇ L of B Buffer to the tube cap. Cover the tube cap, turn it upside down and shake it gently for 5-6 times to mix thoroughly, and add it to the system by centrifugation to catalyze the reaction.
  • Primers and probes were designed using Primer Premier 5.0 software (Premier Biosoft International, CA, USA) according to the manufacturer's instructions in the RAA-nfo nucleic acid amplification reagent (test strip type) kit (Hangzhou Zhongce Bio-Sci&Tech Co., Ltd., Hangzhou, China).
  • the forward primer F10 was extended by 16bp at the 3' end to obtain a candidate probe for RAA-nfo detection. All possible cross-dimers generated by the probe and the reverse primer were predicted. Finally, four new upstream primers for RAA-nfo detection were designed in the upstream sequence of the probe, and the 5' end of the original downstream primer sequence was modified with biotin as the downstream primer for RAA-nfo detection.
  • the primer probe sequences designed above are shown in Table 5. Among them, FAM is a fluorescent group; THF is tetrahydrofuran; C3-spacer is a blocking group.
  • FIG1 is a schematic diagram of RAA-LFS.
  • the RAA-nfo nucleic acid amplification reagent (test strip type) kit Hangzhou Zhongce Bio-Sci&Tech Co., Ltd., Hangzhou, China was used for screening according to the manufacturer's instructions.
  • the RAA reaction process is as follows: the reaction system is 50 ⁇ L in total, 2 ⁇ L of 2 ⁇ M upstream and downstream primers, 0.6 ⁇ L of 2 ⁇ M probe, 25 ⁇ L of A Buffer (20% PEG), 15.9 ⁇ L of double distilled water (ddH 2 O), 2 ⁇ L of Candida auris genomic DNA, and 2.5 ⁇ L of B Buffer (280 mM MgAc) are added to the tube cap. Cover the tube cap, turn it upside down and shake it gently to mix it thoroughly 5-6 times, and add it to the system by centrifugation to catalyze the reaction.
  • a PCR instrument or a constant temperature metal bath or water bath.
  • 50 ⁇ L of the amplified product is added to 300 ⁇ L of sterile water or 1 ⁇ PBS solution for dilution, and a disposable nucleic acid test strip (lateral flow chromatography test strip) (Hangzhou Zhongce Bio-Sci&Tech Co., Ltd., Hangzhou, China) is inserted into the diluted reaction solution with the sample pad facing down for 2 minutes, and the sample is absorbed into the absorption pad, and then the result is visually judged.
  • a disposable nucleic acid test strip lateral flow chromatography test strip
  • the 10 candidate primer pairs designed are shown in Table 4.
  • the candidate primer pairs were tested by amplifying the target gene fragment with a no-template control. After preliminary screening, the amplification products were electrophoresed on agarose gel to compare the amplification performance of the target and primer dimer formation in the template-free control. The results are shown in Figure 3. According to the electrophoresis results, the band of the primer pair ITS-10 is the brightest, the amplification efficiency is the highest, and there is no obvious primer dimer. Therefore, the probe can be designed according to the position of F10 and R10. According to the above probe design principles, the candidate probe was obtained by extending the forward primer F10 by 16bp at the 3' end. All possible cross-dimers generated by the probe and the reverse primer were predicted.
  • a phylogenetic tree was made for the whole genome of Candida auris and other common pathogenic Candida (as shown in FIG5 ), and the ITS sequence alignment with the three most closely related Candida species showed that the primer-probe combination F10′-1/R10′/P obtained by screening can detect and distinguish Candida auris (C. auris) from C. persedohaemulonii, C. duobushaemulonis, and C. haemulonii (as shown in FIG6 ), further indicating that the primer-probe combination obtained by design and screening of the present invention has high specificity.
  • the template concentration in the reaction was 10 3 fg/reaction of Candida auris genomic DNA.
  • the reaction conditions were explored and verified.
  • the reaction temperature was set to 35-45°C (one gradient per 2°C), and the reaction time was set to 5-35 min (one gradient per 5 min).
  • the test strip results were incubated separately to analyze the suitable reaction temperature range.
  • the experimental method was the same as 1.2.3.4 in Example 1.
  • the amplification results were analyzed using LFS.
  • the experimental results showed that the pink color on the test line did not appear under the reaction condition of 45°C, which may be due to the inactivation of the enzyme due to the high temperature.
  • the pink color on the test lines of the other paper strips appeared at 39°C and 10 minutes. At 15 minutes, the colors of the strips began to darken, among which the 37°C and 39°C strips were the most obvious. After 20 minutes, the darkness of the strips did not change significantly (as shown in Figure 7). Therefore, 37°C and 15 minutes were selected as the optimal reaction temperature and time for RAA.
  • RAA-LFS amplification tests were performed on 12 reference strains of Candida auris, 4 strains of C. haemulonii, 3 strains of C. pseudohaemulonii, 4 strains of C. duobushaemulonis and other closely related Candida species and common pathogenic bacteria.
  • the experimental method was the same as 1.2.4.4 in Example 1, that is, RAA-nfo nucleic acid amplification reagent (Hangzhou Zhongce Bio-Sci&Tech Co., Ltd., Hangzhou, China) was used for RAA reaction according to the manufacturer's instructions.
  • the RAA reaction process is as follows: the reaction system is 50 ⁇ L in total, 2 ⁇ L of 2 ⁇ M upstream and downstream primers, 0.6 ⁇ L of 2 ⁇ M probe, 25 ⁇ L of A Buffer, 15.9 ⁇ L of double distilled water (ddH 2 O), 2 ⁇ L of DNA template, and 2.5 ⁇ L of B Buffer is added to the tube cap. Cover the tube cap, shake it upside down and mix it thoroughly 5-6 times, and add it to the system by centrifugation to catalyze the reaction. After low-speed centrifugation for 10 seconds, incubate at 37°C in a PCR instrument (or a constant temperature metal bath or water bath) for 15 minutes.
  • the reaction After the reaction is completed, 50 ⁇ L of the amplified product is added to 300 ⁇ L of sterile water or 1 ⁇ PBS solution for dilution, and the disposable nucleic acid test strip (lateral flow chromatography test strip) (Hangzhou Zhongce Bio-Sci&Tech Co., Ltd., Hangzhou, China) is inserted into the diluted reaction solution with the sample pad facing down for 2 minutes, and the sample is absorbed into the absorption pad, and then the result is visually interpreted.
  • the result interpretation method is the same as described in Example 1.
  • Candida auris of the 12 reference strains were positive (as shown in Figure 8), and other closely related fungi and pathogenic bacteria were negative (as shown in Figures 9A and 9B), indicating that the F10'-1/R10'/P primer-probe set showed good tolerance and specificity for Candida auris, and did not cross-react with other pathogenic bacteria and fungi.
  • All C. haemulonii, C. pseudohaemulonii and C. duobushaemulonis were negative in the test (as shown in Figure 9A), indicating that the system can detect Candida auris and accurately distinguish it from C. haemulonii, C. pseudohaemulonii and C. duobushaemulonis, with excellent specificity.
  • this example tested 10-fold serial dilutions of the Candida auris bacterial solution, ranging from 10 5 to 10 0 CFU/ ⁇ L (reaction volume: 50 ⁇ L, 2 ⁇ L of the Candida auris genome was added to each reaction). Although the color was lighter, a pink band still appeared on the test line of 100 CFU/ ⁇ L. In addition, as the concentration of the Candida auris bacterial solution increased, the pink band darkened (as shown in Figure 10A). In a similar manner, a 10-fold serial dilution of purified Candida auris genomic DNA was performed.
  • Candida auris genomic DNA as low as 10 1 fg/reaction can be detected (as shown in Figure 10B).
  • 10 5 CFU/ ⁇ L of Candida albicans bacterial solution or 1ng/ ⁇ L genomic DNA was added to 10-fold diluted Candida auris bacterial solution (10 5 -10 0 CFU/ ⁇ L) or genomic DNA (10 7 fg/reaction-10 0 fg/reaction).
  • the results showed that C.albicans bacterial solution (as shown in Figure 11A) or genomic DNA (as shown in Figure 11B) did not interfere with the detection of Candida auris by RAA-LFS.
  • the detection limit of the RAA-LFS system is 2 CFU per reaction, or 10fg genomic DNA/50 ⁇ L. The detection sensitivity is not affected by the presence of other fungal DNA.
  • the Candida auris genomic DNA was mixed with DNA extracted from human blood at a ratio of 1:10 and then diluted 10 times in a series to make the final concentration of the Candida auris genome 10 7 fg/reaction-10 0 fg/reaction for RAA-LFS detection to observe the change in the detection limit.
  • the experimental method was the same as the RAA reaction process described in Example 3.
  • DNA was directly extracted from whole blood spiked with Candida auris for RAA detection, and its application in rapid detection of clinical blood samples was preliminarily evaluated.
  • the specific steps were as follows: 10-fold serial dilutions of Candida auris were mixed into each 200 ⁇ L whole blood sample for RAA-LFS detection, with a concentration gradient of 10 5 CFU/ ⁇ L-10 0 CFU/ ⁇ L, and the experimental method was the same as the RAA reaction process described in Example 3.
  • This embodiment simulates clinical blood samples, and mixes different concentrations of Candida auris liquid into human blood samples for RAA-LFS detection.
  • the experimental results show that the method of RAA-LFS detection of Candida auris can be applied to human blood samples and the detection sensitivity remains unchanged (as shown in FIG. 12A ).
  • the Candida auris genomic DNA and DNA extracted from human blood were mixed at a ratio of 1:10 and then subjected to RAA-LFS detection.
  • the results show that human DNA has no significant inhibitory effect on the detection and does not change its detection limit (as shown in FIG. 12B ).

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Abstract

一种用于检测耳念珠菌的恒温核酸扩增RAA引物探针组合及其应用，所述引物探针组合的扩增效率高、特异性强且无明显的假阳性，此外，其能够将耳念珠菌与临床微生物鉴定系统中常常误诊的三种近缘菌有效区分开，本发明基于RAA-侧流层析技术建立了一种快速检测耳念珠菌的方法，并通过特异性和灵敏度评价，可用于实际样品的检测中。

Description

一种用于检测耳念珠菌的恒温核酸扩增RAA引物探针组合及其应用
相关申请的交叉引用
本申请要求享有以下申请文件的优先权：2022年10月09日提交的申请号为2022112254681、名称为“一种用于检测耳念珠菌的恒温核酸扩增RAA引物探针组合及其应用”的发明专利申请，其内容以全文引用的方式并入本文。
技术领域
本发明属于病原微生物检测技术领域，具体涉及一种用于检测耳念珠菌的恒温核酸扩增RAA引物探针组合及其应用。
背景技术
耳念珠菌(Candida auris)是于2009年在一名日本患者的外耳道分泌物中首次发现的病原真菌，至今为止，耳念珠菌已经在除南极洲以外其他六大洲的40多个国家被报道。耳念珠菌感染可以发生在各个年龄段的人群，其中，在老年人中最为常见，新生儿和儿童也偶有感染。耳念珠菌常见的宿主可以分为三类：一是患有严重免疫缺陷疾病或自身免疫能力较弱的人群，二是开放性手术患者或侵入性导管使用患者，三是使用广谱抗菌药物治疗的人群。该真菌主要引起持续性、侵袭性感染，对氟康唑等常用抗真菌药物呈多重耐药性，感染患者的病死率高达30％-60％，可快速播散并能引起医院感染的暴发流行，故被称为“超级真菌”。耳念珠菌作为一种新出现的多重耐药的酵母菌，具有较大的基因组多样性、多重耐药性和高致死亡率，其极易传播并感染院内患者，从而造成医院内爆发式感染，且感染多见于念珠菌血症。由于目前常见的表型鉴定及微生物鉴定系统均无法准确鉴定耳念珠菌，因此，常导致临床微生物学实验室对该菌株的鉴定错误，进而导致误诊，为临床上及时预防和控制耳念株菌的传播带来了极大困难。
病原体的准确鉴定是任何传染病治疗的前提条件，及时准确的诊断才能保证实施最快的合理治疗，并有效控制进一步传染的风险，从而降低传染性疾病在环境中爆发的机会。耳念珠菌在表型及生理生化方面与其他念珠菌具有普遍的相似性，因此导致其在临床诊断过程中容易被误诊。因此该真菌感染的诊断需要使用相对特殊的生化检测或分子生物学检测手段，例如，基于基质辅助激光解吸电离飞行时间质谱仪(matrix-assisted laser desorption/ionization time of flight mass spectrometry，MALDI-TOF MS)检测特异性蛋白或基于核糖体内转录间隔区(internal transcribed spacer，ITS)及28S rDNA(ribosomal DNA)D1/D2区的序列分析鉴定。其中，虽然MALDI-TOF MS可用于鉴定耳念珠菌，但是该方法依赖于数据库的更新，并非所有的MALDI-TOF MS设备都可以用于耳念珠菌的检测，并且其必须基于纯培养物，可用于菌株特异性鉴定的蛋白质种类有限，因此在实际应用中往往受到限制。此外，上述用于耳念珠菌鉴别诊断的方法均需要配合相应的仪器使用，通常需要高成本的额外设备和经过专业培训的技术人员，鉴定成本高，很多医疗机构难以负担。
此外，常规真菌鉴定手段，包括念珠菌显色培养基和微生物商业检测系统，不仅耗时费力，而且阳性率低、错误率高。其中，最常用的临床微生物鉴定系统，例如VITEK2、API-20C AUX和BD酵母鉴定系统经常将耳念珠菌误诊为其他近缘念珠菌如C.haemulonii、C.duobushaemulonii、C.pseudohaemulonii、C.guilliermondii、C.parapsilosis等，导致耳念珠菌感染患者漏诊、误诊率极高。韩国于2011年报告3例由耳念珠菌引起的医院内真菌感染，同时该研究表明耳念珠菌通常被VITEK和API-20C AUX等商业鉴定系统错误地鉴定为希木龙念珠菌和黏红酵母菌。印度的一项综合研究经ITS测序证实，先前用VITEK系统鉴定为希木龙念珠菌或无名假丝酵母菌的102株菌株中有90株应为耳念珠菌，错误鉴定的比例高达88.2％。可见现有的诊断工具成本高、准确度低，可能导致耳念珠菌以及其近缘种的发病率在全球范围内被低估，尤其是医疗资源受限地区(如非洲和东南亚)。这也一定意义上解释了现有的爆发流行以及感染个案均来自于发达国家(如美国，英国和德国)或低收入地区的医疗发达城市(如新德里，印度)的原因。
等温核酸扩增技术是近年来新发展起来的一类分子生物学技术的总称。其可在特定温度下扩增特定的DNA或RNA。无论是在实际操作还是仪器要求方面，都比PCR技术更为简单方便，它摆脱了对精良设备的依赖，反应时间大大缩短，在临床和现场快速诊断中显示出了良好的应用前景。目前已开发出了多种等温扩增方法，如依赖核酸序列扩增(Nucleic acid sequence-based amplification，NASBA)、链替代扩增(Strand displacement amplification，SDA)、环介导等温扩增技术(Loop-mediated isothermal amplification，LAMP)、解链酶扩增(Helicase-dependent amplification，HAD)、重组酶聚合酶等温扩增技术(Recombinase polymerase amplification，RPA)、重组酶介导的等温扩增技术(Recombinase aided amplification，RAA)、转录依赖的扩增系统(Transcription-based Amplification System，TAS)、滚环扩增(Rolling circleamplification，RCA)等。其中，RAA是一种新型核酸恒温扩增技术，能够在20分钟内进行常温下的单分子核酸检测。其不依赖于昂贵的实验室设备和专业操作人员，对于在经济卫生条件不发达的地区以及疾病爆发现场开展耳念珠菌的监测和鉴定工作具有重要的意义和良好的应用前景。然而，目前尚未见RAA技术用于耳念珠菌检测的相关报道，更无合适高效的RAA引物探针组能够用于耳念珠菌的检测。
因此，如何克服现有技术存在的上述不足，建立一种特异性高、准确、灵敏、快速、方便、成本低廉的耳念珠菌检测方法是目前本领域亟需解决的技术问题之一。
发明内容
针对现有技术存在的上述不足，本发明的目的在于提供一种用于检测耳念珠菌的恒温核酸扩增RAA引物探针组合及其应用。
本发明的上述目的通过以下技术方案得以实现：
本发明的第一方面提供了一种用于耳念珠菌检测的恒温核酸扩增RAA引物探针组合。
进一步，所述引物探针组合包括上游引物、下游引物和探针；
所述上游引物的核苷酸序列为F10’-1：5’-AAGGATCATTATTGATATTTTGCATACACA-3’；
所述下游引物的核苷酸序列为R10’：5’-Biotin-TTCAAAGATTCGATGATTCACGTCTGCAAG-3’；
所述探针的核苷酸序列为P：5’-FAM-ACTGATTTGGATTTTAAAACTAACCCAACG[THF]TAAGTTCAACTAAAC-C3spacer-3’。
在本发明的具体实施方案中，所述上游引物的核苷酸序列如SEQ ID NO:22所示，所述下游引物的核苷酸序列如SEQ ID NO:26所示，并在其5’端用生物素(Biotin)进行修饰；所述探针的核苷酸序列如SEQ ID NO:21所示，并在其5’端用荧光基团(FAM)修饰，3’端用阻断基团(C3-spacer)进行阻断，第31个碱基用四氢呋喃([THF])替换。
在一些实施方案中，与本发明所述的引物和/或探针序列具有70％以上同源性的序列对应的引物和/或探针也均包含在本发明的保护范围内，也即可以理解为：在本发明提供的引物和/或探针的基础上进行改变之后得到的引物和/或探针同样落入本发明的保护范围。
在一些实施方案中，本发明对引物中包含的修饰基团(例如：生物素)、探针中包含的荧光基团(例如：FAM)、阻断基团(例如：C3-spacer)、替换基团(例如：四氢呋喃)均无特别限制，将上述基团中的一个或多个替换为本领域技术人员公知的其它可替代基团之后得到的引物和/或探针同样包含在本发明的保护范围内。
在一些实施方案中，所述对引物进行修饰的修饰基团包括但不限于：生物素、磷酸化、地高辛、巯基、Spacer、硫代、脱氧脲嘧啶、脱氧次黄嘌呤等；所述探针中的荧光基团包括但不限于：FAM、HEX、VIC、ROX、Cy5、TET等；所述探针中的阻断基团包括但不限于：C3-spacer、磷酸盐、生物素-TEG等。
在本发明中，所述耳念珠菌并无特别限制，包括但不限于：C.auris、C.haemulonii、C.pseudohaemulonii、C.duobushaemulonis、C.rugosa、C.albicans、C.neoformans、C.parapsilosis、C.glabrata、C.guilliermondii、E.coli、S.aureus、E.faecalis、C.tropicalis、C.krusei、C.dubliniensis、K.pneumoniae等。任何菌株来源和/或任何菌株类型的耳念珠菌均在本发明的保护范围内。
本发明的第二方面提供了一种用于检测耳念珠菌的试剂盒。
进一步，所述试剂盒包含由本发明第一方面所述的引物探针组合组成的引物探针混合液。
进一步，所述试剂盒还包含A Buffer、B Buffer、RAA反应干粉试剂、ddH2O；优选地，所述A Buffer为20％PEG；优选地，所述B Buffer为280mM MgAc；优选地，所述RAA反应干粉试剂中包含如下的组分：dNTP、SSB蛋白、recA重组酶蛋白或Rad51、Bsu DNA聚合酶、Tricine、PEG、二硫苏糖醇、肌酸激酶、Nfo核酸内切酶；更优选地，所述RAA反应干粉试剂中各组分的浓度如下：1mmol/L dNTP、90ng/μL SSB蛋白、120ng/μL recA重组酶蛋白或30ng/μL Rad51、30ng/μL Bsu DNA聚合酶、100mmol/L Tricine、20％PEG、5mmol/L二硫苏糖醇、100ng/μL肌酸激酶、Nfo核酸内切酶。
进一步，所述试剂盒还包含侧流层析试纸条。
在一些实施方案中，本发明公开的引物和探针可以用于检测耳念珠菌的试剂盒的形式提供。在这样的试剂盒中，适合量的一种或多种探针和/或引物(例如本发明第一方面公开的引物和探针)，被提供在一个或多个容器中，或固定在基质上。核酸探针和/或引物可以被提供成悬浮在水溶液中，或例如作为冷冻干燥的或冻干的粉末。其中提供有核酸的容器可以是任何能够容纳所提供的形式的常规容器，例如微量离心管、安瓿瓶或瓶。试剂盒可以包含标记的或未标记的、用于检测耳念珠菌核苷酸序列的探针。在某些应用中，一个或多个引物，例如引物对，可以预先测量好的单次使用量提供在独立的、典型情况下用后即可抛弃的管或相当的容器中。使用这样的设置，用于测试耳念珠菌核酸的存在的样品，可以加入到独立的管中，直接进行扩增。
试剂盒中提供的核酸引物的量，可以是任何适合的量，可以依赖于产品所针对的靶市场。例如，如果试剂盒适用于研究或临床应用，提供的每种核酸引物的量，可以是足够引发几个PCR扩增反应的量。确定适合的量的一般性指导，可以在Innis等、Sambrook等和Ausubel等的文献中发现。试剂盒可以包含两个以上的引物，以便于较大量耳念珠菌核苷酸序列的PCR扩增。
在一些实施方案中，试剂盒可以含有执行PCR扩增反应所必需的反应试剂，包括DNA样品制备试剂、适合的缓冲液(例如聚合酶缓冲液)、盐(例如氯化镁)和脱氧核糖核苷酸(dNTPs)，对这些常规反应试剂的类型进行简单替换之后得到的试剂盒同样包含在本发明的保护范围内。此外，在试剂盒中也可以提供一个或多个用于PCR反应的对照序列。
本发明的第三方面提供了一种基于RAA-侧流层析技术检测耳念珠菌的方法。
进一步，所述方法包括如下步骤：
(1)提取待测样本DNA；
(2)以待测样本DNA为模板，进行RAA扩增反应，得到扩增产物，其中，RAA扩增的上游引物的核苷酸序列为F10’-1：5’-AAGGATCATTATTGATATTTTGCATACACA-3’；下游引物的核苷酸序列为R10’：5’-Biotin-TTCAAAGATTCGATGATTCACGTCTGCAAG-3’；探针的核苷酸序列为P：5’-FAM-ACTGATTTGGATTTTAAAACTAACCCAACG[THF]TAAGTTCAACTAAAC-C3spacer-3’；
(3)应用侧流层析试纸条对扩增产物进行检测，当试纸条出现两条红色条带，一条位于质控区，一条位于检测区，则结果为阳性，表明样本中含有耳念珠菌；当试纸条只有质控区出现一条红色条带，检测区没有条带，则结果为阴性，表明样本中不含耳念珠菌。
进一步，步骤(2)中所述扩增反应的过程包括如下步骤：在装有RAA反应干粉试剂的检测单元管中加入上游引物、下游引物、探针、A Buffer、ddH2O、模板，于管盖上添加B Buffer，盖上管盖，低速离心后继续进行扩增反应，得到扩增产物。进一步，所述各物质的用量分别为：2μM上游引物2μL，2μM下游引物2μL，2μM探针0.6μL，A Buffer 25μL，15.9μL ddH2O，2μL模板，B Buffer 2.5μL。进一步，步骤(2)中所述扩增反应的反应条件为：37℃的条件下反应15min。
在本发明中，本领域技术人员了解，对步骤(2)中所述扩增反应的反应条件和/或各物质的用量进行适当的调整后，仍然能够发挥对受试者来源的待测样本中的耳念珠菌进行检测的目的，因此，如前所述的扩增反应的反应条件和/或各物质的用量并不旨在对本发明的保护范围进行限制，对其进行适当调整后，只要能够发挥检测受试者来源的待测样本中的耳念珠菌的这一目的，这样经调整后的反应条件和/或各物质的用量也将落入本发明的保护范围内。
在本发明的具体实施方案中，所述RAA技术的扩增原理如下：RAA技术主要利用重组酶、单链结合蛋白和DNA聚合酶对目的基因进行大量的扩增。从细菌或真菌中获得的重组酶在常温下可与引物DNA紧密结合，形成重组酶/引物复合体，侵入DNA双链核酸模板，在侵入位点重组酶将双链打开，同时单链结合到重组酶打开的单链上，维持双链模板处于开链的状态。重组酶/引物复合体开始对双链进行扫描，当引 物在模板上搜索到与之完全匹配的互补序列时，重组酶/引物复合体解体，DNA聚合酶结合到引物3’端，开始合成新链。合成的新链又可以作为模板，最终扩增产物以指数级增长，完成靶标基因的扩增。
在本发明的具体实施方案中，下游引物和探针在5’末端分别用生物素(Biotin)和羧基荧光素(FAM)(Tsingke Biotechnology Co.,Ltd.,Beijing,China)修饰。经过荧光标记的探针与扩增产物结合，反应体系中的核酸内切酶识别并切割无嘌呤和无嘧啶的[THF]位点，当探针被核酸内切酶酶切后，与生物素标记的引物共同扩增形成两端有荧光标记及生物素标记的片段，可采用侧流层析试纸条进行判读。所述侧流层析试纸条是一种通用型核酸检测试纸条，只需少量产品即可检测，产品浓度过高时需适当稀释。当稀释的产物滴加到样品垫上时，扩增产物的两端都用生物素和FAM标记，FAM与AuNPs结合，通过链霉亲和素检测线时，生物素与链霉亲和素结合，另一端通过金纳米颗粒(AuNPs)呈阳性信号。将侧流层析试纸条(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)样品垫朝下插入稀释后的反应液中2分钟即可目测读取检测结果。
在本发明的具体实施方案中，本发明通过进一步反应条件的优化，筛选得到所述RAA方法中扩增反应的最佳温度和时间分别为37℃、15min。并且该方法表现出极高的特异性和良好的灵敏度，可以检测到低至101fg/reaction的耳念珠菌基因组DNA，且检测灵敏度不会受其他真菌DNA存在的影响。
在一些实施方案中，所述待测样本来源于有需要的受试者的临床样本，包括但不限于：细胞、组织、体液，例如：皮肤；黏膜；血液；血液衍生物或级份，例如血清；提取的胆汁；活组织检查或手术取出的组织，包括例如未固定的、冷冻的、固定在福尔马林和/或包埋在石蜡中的组织；泪液；乳汁；皮屑；表面清洗液；尿液；痰液；脑脊液；前列腺液；脓；骨髓吸出物；中耳流出物，支气管肺泡灌洗物，气管吸出物，痰液，鼻咽吸出物，口咽吸出物或唾液。
在一些实施方案中，所述受试者包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。在某些具体的实施方案中，所述受试者优选为人。
本发明的第四方面提供了本发明第一方面所述的引物探针组合、本发明第二方面所述的试剂盒在制备用于检测耳念珠菌的产品中的应用。
本发明的第五方面提供了本发明第一方面所述的引物探针组合、本发明第二方面所述的试剂盒在制备用于鉴别诊断耳念珠菌和C.persedohaemulonii、C.duobushaemulonis、C.haemulonii的产品中的应用。
本发明的第六方面提供了本发明第一方面所述的引物探针组合、本发明第二方面所述的试剂盒在检测耳念珠菌中的应用。
本发明的第七方面提供了本发明第一方面所述的引物探针组合、本发明第二方面所述的试剂盒在鉴别诊断耳念珠菌和C.persedohaemulonii、C.duobushaemulonis、C.haemulonii中的应用。
在本发明的具体实施方案中，本发明经验证发现，本发明提供的引物探针组合能够准确有效鉴别区分耳念珠菌与临床微生物鉴定系统中常常误诊的三种近缘菌C.persedohaemulonii、C.duobushaemulonis、C.haemulonii。
本发明的第八方面提供了一种用于诊断受试者是否患有耳念珠菌感染疾病的方法，所述方法包括如下步骤：采用本发明第三方面所述的方法对受试者来源的待测样本进行检测。
相对于现有技术，本发明具有的优点和有益效果：
(1)本发明提供了一种快速检测耳念珠菌的恒温核酸扩增RAA引物探针组合，所述引物探针组合包括引物对(如SEQ ID NO:22和SEQ ID NO:26所示)和探针(如SEQ ID NO:21所示)，所述引物对能够特异性扩增耳念珠菌，除耳念珠菌外没有与其他病原菌产生交叉反应，也即本发明提供的引物对具有较强的特异性扩增特点，并且可以明显将耳念珠菌与临床微生物鉴定系统中常常误诊的近缘菌—C.haemulonii、C.pseudohaemulonii和C.duobushaemulonis区分开。此外，本发明通过对比实验证明了所述RAA引物探针组合的扩增效率最高、特异性最强且无明显的假阳性，显著优于同样是针对耳念珠菌rDNA基因的ITS序列设计的其他三种RAA引物探针组合，这一效果属于本领域技术人员所预料不到的技术效果。
(2)本发明还提供了一种快速检测耳念珠菌的POCT(现场快速检测)方法，即将侧流层析试纸条(Lateral flow strip detection，LFS)和重组酶介导等温核酸扩增技术(Recombinase aided amplification，RAA)结合的检测方法(RAA-LFS)，通过简易的Chelex-100煮沸方法进行DNA的提取和纯化节省了该检测系 统的检测时间，该检测不再需要荧光检测设备及电泳装置，仅需要一台恒温水浴锅即可提供反应所需温度，甚至用体温供热便可以完成有效扩增，能够在15min内获得检测结果，且特异性高、灵敏度高、仪器依赖性低、不需要经过专业培训的实验室人员、可操作性强。可用于现场满足床边诊断的需要或条件较弱的偏远医院的需要，对耳念珠菌的快速检测具有重要意义。
(3)本发明首次采用RAA-侧流层析技术建立了一种快速检测耳念珠菌的方法，并通过特异性和灵敏度评价，可用于实际血液样品的检测中，为耳念珠菌的现场快速检测提供一种灵敏、可靠、有效的新方法。本发明选用的引物对是经大量实验筛选获得的，特异性好，且与其它病原菌无交叉反应。本发明所使用的引物探针组合扩增效率高、条带特异性强、能够在检测区形成较高浓度的引物-探针异二聚体，因而使试纸条呈现强阳性反应，增加了检测的灵敏度。本发明建立的RAA技术和侧流层析技术结合检测耳念珠菌的方法，既具有分子生物学检测的高灵敏、高通量，又具有免疫学检测的特异性好、操作简便的优点，还不需复杂仪器，且检测速度快，尤其适用于基层实验室和检疫现场的耳念珠菌快速筛查检测。
附图说明
图1为RAA-LFS示意图，其中，A图：RAA-nfo探针设计，B图：荧光标记的探针与模板结合，C图：侧流层析试纸条(LFS)工作示意图；
图2为检测结果判读方法示意图；
图3为通过RAA的扩增性能筛选引物对的琼脂糖凝胶电泳结果图，其中，NTC泳道是相应引物对的无模板对照，DNA ladder的条带大小显示在左侧，引物二聚体由白色箭头表示；
图4为使用不同引物-探针组的重组酶介导的等温核酸扩增(RAA)的侧流层析试纸条(LFS)的结果图，其中，NTC是相应引物-探针组的无模板对照，测试线和对照线的位置标在右侧，模板是耳念珠菌基因组DNA，反应条件为在39℃下反应15分钟；
图5为对耳念珠菌和其他常见病原念珠菌的全基因组做系统发育树的结果图；
图6为引物-探针组合靶向片段图，将三个密切相关的菌株C.persedohaemulonii、C.duobushaemulonis和C.haemulonii的ITS序列片段与耳念珠菌的ITS序列切片进行比较，并在每个序列的开头显示菌株名称，对应于引物和探针的序列写在比对中的位置下，箭头表示引物和探针延伸的方向，四氢呋喃(THF)位点用“□”表示；
图7为不同反应温度和反应时间条件下的RAA-LFS的检测结果图，其中，RAA反应的温度显示在每个条带的顶部，RAA反应时间显示在每行左侧，扩增模板为耳念珠菌基因组DNA，对照线和测试线的位置显示在图像的右侧；
图8为不同参考菌株的检测结果图，其中，菌株名称显示在每个条带的顶部，NTC为无模板对照，对照线和测试线的位置显示在图像的右侧，反应在37℃的条件下进行15min；
图9为其他近缘真菌和致病性细菌的检测结果图，其中，A图：RAA-LFS检测C.haemulonii、C.Perudohaemulonii和C.Duobushaemulonis菌株的结果图，B图：RAA-LFS检测其他相关念珠菌和常见病原菌的结果图，菌株的种类显示在每个条带的顶部，NTC为无模板对照，对照线和测试线的位置显示在图像的右侧，反应在37℃的条件下进行15min；
图10为RAA-LFS技术检测耳念珠菌灵敏性的结果图，其中，A图：RAA扩增与不同数量的耳念珠菌培养物的LFS结果图，B图：用不同数量的耳念珠菌基因组DNA进行RAA扩增的LFS结果图；
图11为在其他真菌干扰下RAA-LFS技术检测耳念珠菌灵敏性的结果图，其中，A图：不同耳念珠菌培养物加入105CFU/μL白色念珠菌培养基后RAA扩增的LFS结果图，B图：除了耳念珠菌基因组DNA外，还向反应中添加了1ng白色念珠菌基因组DNA进行RAA扩增的LFS结果图；
图12为RAA-LFS技术对人体血液样本的检测性能测试结果图，其中，A图：在人血液样本中混入不同浓度的耳念珠菌液进行RAA-LFS检测的结果图，B图：将耳念珠菌基因组DNA与人血提取的DNA按1：10混匀后进行RAA-LFS检测的结果图，其中，NTC为无模板对照，对照线和测试线的位置显示在图像的右侧，反应在37℃的条件下进行15min。
具体实施方式
除非另有指明，本文中涉及的技术术语通常按照其常规用法使用。分子生物学常用术语的定义可以在 Benjamin Lewin的《基因VII》(Genes VII，由Oxford University Press出版，1999)；Kendrew等主编的《分子生物学百科全书》(The Encyclopedia of Molecular Biology，由BlackwellScience Ltd.出版，1994)；和Robert A.Meyers主编的《分子生物学与生物技术：综合桌面参考》(Molecular Biology and Biotechnology：aComprehensive Desk Reference，由VCH Publishers，Inc.出版，1995)；以及其它类似参考书中发现。
本文使用的术语“包含”或“包括”，是指包含指定元件，但也不排除其它元件，例如：“试剂盒包含由所述引物探针组合组成的引物探针混合液”意味着“包含由所述引物探针组合组成的引物探针混合液”而不排除其它元件。
本文使用的术语“扩增”，是指为了增加核酸分子的拷贝数。获得的扩增产物被称为“扩增子”。核酸分子(例如DNA或RNA分子)的扩增，是指使用技术增加样品中核酸分子的拷贝数。扩增的一个例子是聚合酶链反应(PCR)，其中将样品与一对寡核苷酸引物，在允许引物与样品中的核酸模板杂交的条件下相接触。引物在适合的条件下延伸，从模板解离，重新退火、延伸并解离，以扩增核酸的拷贝数。该循环可以重复。扩增的产物可以通过例如电泳、限制性内切酶裂解图、寡核苷酸杂交或连接，和/或核酸测序等技术来表征。
其它的体外扩增技术的例子包括定量实时PCR，反转录酶PCR(RT-PCR)，实时PCR，实时反转录酶PCR(rt RT-PCR)，巢式PCR，链置换扩增(参见美国专利No.5,744,311)，无转录恒温扩增(参见美国专利No.6,033,881)，修复链反应扩增(参见WO 90/01069)，连接酶链反应扩增(参见欧洲专利公开EP-A-320 308)，缺口填补连接酶链反应扩增(参见美国专利No.5,427,930)；偶联的连接酶检测和PCR(参见美国专利No.6,027,889)，以及NASBATM RNA无转录扩增(参见美国专利No.6,025,134)等。
本文使用的术语“引物”，是指短的核酸分子，例如DNA寡核苷酸，例如至少15个核苷酸的序列，可以通过核酸杂交与互补的靶核酸分子退火，在引物和靶核酸链之间形成杂交体。引物可以通过聚合酶沿着靶核酸分子延伸。因此，引物可用于扩增靶核酸分子(例如耳念珠菌核酸序列)，其中引物的序列对于靶核酸分子是特异性的，因此例如引物将在非常高严紧性杂交条件下与靶核酸分子杂交。引物的特异性随着其长度增加。因此，例如，含有30个连续核苷酸的引物，将以与只有15个核苷酸的相应引物相比更高的特异性与靶序列退火。因此，为了获得更高的特异性，可以选择含有至少15、20、25、30、35、40、45、50个或以上连续核苷酸的探针和引物。
其中，制备和使用引物的方法描述在例如Sambrook等《分子克隆实验指南》((1989)Molecular Cloning：A Laboratory Manual，Cold SpringHarbor，New York)；Ausubel等《分子生物学现代方法》((1987)CurrentProtocols in Molecular Biology，Greene Publ.Assoc.&Wiley-Intersciences)中。
本文使用的术语“探针”，是指包含能够与靶核酸(例如耳念珠菌核酸分子)杂交的分离的核酸。探针上可以连接可检测标记物或报告分子。典型的标记物包括放射性同位素、酶的底物、辅因子、配体、化学发光或荧光试剂、半抗原和酶。
用于标记的方法和选择适合于各种不同目的的标记物的准则，讨论在Sambrook等《分子克隆实验指南》(Molecular Cloning：ALaboratory Manual，Cold Spring Harbor Laboratory Press(1989))和Ausubel等《分子生物学现代方法》(Current Protocols in MolecularBiology，Greene Publishing Associates and Wiley-Intersciences(1987))中。
探针的长度一般为至少20个核苷酸，例如至少20、至少21、至少22、至少23、至少24、至少25、至少26、至少27、至少28、至少29、至少30、至少31、至少32、至少33、至少34、至少35、至少36、至少37、至少38、至少39、至少40、至少41、至少42、至少43、至少44、至少45、至少46、至少47、至少48、至少49、至少50、至少51、至少52、至少53、至少54、至少55、至少56、至少57、至少58、至少59、至少60个或以上与靶核酸分子互补的连续的核苷酸，例如20-60个核苷酸、30-60个核苷酸、20-50个核苷酸、30-50个核苷酸、20-40个核苷酸或20-30个核苷酸。
本文使用的术语“同源性”，是指两个或多个核酸序列，或两个或多个氨基酸序列之间的同一性/相似性，根据序列之间的同一性或相似性来表示。序列同一性可以根据百分同一性来度量；百分率越高，序列越一致。当使用标准方法比对时，核酸或氨基酸序列的同源物或直系同源物，具有相对高水平的序列同一 性/相似性。
用于比对序列进行比较的方法在本技术领域的众所周知的。各种不同的程序和比对算法描述在：Smith&Waterman，Adv.Appl.Math.2：482，1981；Needleman&Wunsch，J.Mol.Biol.48：443，1970；Pearson&Lipman，Proc.Natl.Acad.Sci.USA 85：2444，1988；Higgins&Sharp，Gene，73：237-44，1988；Higgins&Sharp，CABIOS 5：151-3，1989；Corpet等，Nuc.Acids Res.16：10881-90，1988；Huang等，Computer Appls.in theBiosciences 8，155-65，1992；以及Pearson等，Meth.Mol.Bio.24：307-31，1994中。Altschul等，J.Mol.Biol.215：403-10，1990，提出了对序列比对方法和同源性计算的详细考虑。
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、生物材料等，如无特殊说明，均可从商业途径得到。
实施例1 RAA-LFS系统引物探针组的设计和筛选
1.1材料与方法
1.1.1菌株来源
本发明实施例中涉及到的实验菌株来源如表1所示。
表1实验材料

1.1.2主要实验仪器
本发明实施例中涉及到的主要实验仪器如表2所示。
表2主要实验仪器
1.1.3主要试剂耗材
本发明实施例中涉及到的主要试剂耗材如表3所示。
表3主要试剂耗材
1.1.4生物信息学软件
引物和探针设计：Primer 5.0；
序列比对、拼接处理软件：MEGA7.0、BoEidt软件；
核苷酸与氨基酸对比工具：美国生物技术信息中心在线工具。
1.2实验方法
1.2.1菌株复苏
取甘油保种的菌株，按照无菌操作原则，将真菌涂布于SDA固体培养基，细菌涂布于LB固体培养基，于培养箱中倒置培养。于生物安全柜中挑取单克隆于液体培养基中培养。
1.2.2菌株DNA提取
使用Chelex-100煮沸法提取所有菌株基因组DNA，使用所有提取的DNA均用Qubit 2荧光计(Thermo Fisher Scientific)进行定量测量，并保存在-20℃冰箱中直至使用。提取基因组DNA具体步骤如下：
(1)培养好的菌液离心后去上清收集菌体；
(2)在菌体沉淀中加入Chelex-100裂解液，置于金属浴或者恒温水浴锅中煮沸；
(3)上述加热煮沸后的粗提取液充分离心，取上清加入等体积的DNA提取酚试剂(Beijing Solarbio Science&Technology Co.,Ltd.,Beijing,China)混匀，充分离心后吸取上清，用Qubit 2.0定量后，标记好后-20℃冻存备用。
1.2.3 RAA检测方法建立
1.2.3.1引物设计
从NCBI的核酸数据库GenBank(http://www.ncbi.nlm.nih.gov)检索获得耳念珠菌rDNA基因的ITS序列(NCBI Reference Sequence:NR_154998.1)，耳念珠菌rDNA基因ITS1和ITS2为耳念珠菌具有特异性的保守序列作为靶序列，根据引物设计原则用Primer Premier 5.0软件(Premier Biosoft International，CA，USA)设计引物及探针，共设计出10对候选引物对(ITS-1—ITS-10)，如表4所示。并且使用NCBI上的BLAST工具(http://blast.ncbi.nlm.nih.gov/Blast.cgi)对所设计的引物对进行筛选。所用引物对和探针均由擎科公司合成。
表4设计得到的10对候选引物对
1.2.3.2引物筛选体系及反应步骤
首先在NCBI对已经设计的10对候选引物进行初步验证，后根据制造商说明书，用基础型RAA核酸扩增试剂(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)进行引物筛选，这些引物通过用无模板对照扩增靶基因片段来初步筛选。扩增产物在琼脂糖凝胶上进行电泳，以比较无模板对照中靶标和引物二聚体形成的扩增性能。选择了显示最佳扩增性能且没有交叉二聚体形成迹象的引物对。
RAA引物筛选体系共50μL：在装有反应干粉的检测单元管中加入10μM的上下游引物各2μL，A Buffer 25μL，双蒸水13.5μL，耳念珠菌基因组DNA 5μL，将以上共计47.5μL体系配制完成后混匀离心，于管盖上添加B Buffer 2.5μL。盖上管盖，上下颠倒轻甩充分混匀5-6次，通过离心的方式将其加入到体系中催化反应的发生。低速离心10s后在PCR仪(或恒温金属浴或水浴锅)中39℃孵育30min。反应结束后，在检测单元管中加入50μL DNA提取酚试剂(Beijing Solarbio Science&Technology Co.,Ltd.,Beijing,China)，充分混匀后12000rpm/min离心5min，取6μL上清与1μL 6×上样缓冲液(Loading Buffer)混匀后经2.5％琼脂糖凝胶电泳，电泳结束后用凝胶成像系统观察结果。
1.2.3.3适用于RAA-nfo的引物及探针设计
根据RAA-nfo核酸扩增试剂(试纸条型)试剂盒(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)中的制造商说明书，使用Primer Premier 5.0软件(Premier Biosoft International，CA，USA)进行引物及探针的设计。
1.2.3.4引物-探针组的筛选体系及RAA反应步骤
基于上述引物筛选结果，通过将正向引物F10在3’端延伸16bp获得作为用于RAA-nfo检测候选探针。预测了该探针和反向引物产生的所有可能的交叉二聚体，最后在探针上游序列设计四条新的用于RAA-nfo检测上游引物，在原有下游引物序列的5’端用生物素修饰作为用于RAA-nfo检测下游引物。上述设计的引物探针序列如表5所示。其中，FAM为荧光基团；THF为四氢呋喃；C3-spacer为阻断基团。
表5设计得到的4对候选引物-探针组合
图1为RAA-LFS示意图。在本实施例中，为筛选最佳的引物-探针组，根据制造商说明书，使用RAA-nfo核酸扩增试剂(试纸条型)试剂盒(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)进行筛选。RAA反应过程如下：反应体系共50μL，在装有RAA反应干粉试剂的检测单元管中加入2μM的上下游引物各2μL，2μM的探针0.6μL，A Buffer(20％PEG)25μL，双蒸水(ddH2O)15.9μL，耳念珠菌基因组DNA 2μL，于管盖上添加B Buffer(280mM MgAc)2.5μL。盖上管盖，上下颠倒轻甩充分混匀5-6次，通过离心的方式将其加入到体系中催化反应的发生。低速离心10s后在PCR仪(或恒温金属浴或水浴锅)中39℃孵育15min。反应结束后，将50μL扩增产物添加到300μL的无菌水或1×PBS溶液中稀释，将一次性核酸检测试纸条(侧流层析试纸条)(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)样品垫朝下插入稀释后的反应液中2分钟，待样品吸收至吸收垫处，然后目测对结果判读。
上述结果判读方法如下：试纸条出现两条红色条带，一条位于质控区(C线)，一条位于检测区(T线)。阳性结果为C线与T线同时出现，表明样本中含有耳念珠菌；阴性结果为只出现C线，不出现T线，表明样本中不含耳念珠菌或数量低于最低检出限；当C线与T线均未出现表明结果无效。如图2所示。
1.3实验结果
1.3.1耳念珠菌引物探针设计结果
设计得到的10对候选引物对如表4所示，通过用无模板对照扩增靶基因片段来对上述候选引物对进行 初步筛选，扩增产物在琼脂糖凝胶上进行电泳，以比较无模板对照中靶标和引物二聚体形成的扩增性能，结果如图3所示，根据电泳结果可知：引物对ITS-10的条带最亮，扩增效率最高，且无明显的引物二聚体，因此，可根据F10、R10的位置设计探针。根据上述探针设计原则，通过将正向引物F10在3’端延伸16bp获得候选探针。预测了该探针和反向引物产生的所有可能的交叉二聚体。在探针上游设计了4个新的上游引物，并对其进行测试筛选。在探针的5’端用FAM修饰，3’端用C3-spacer进行阻断，第31个碱基用[THF]替换。在探针上游序列设计四条新的用于RAA-nfo检测上游引物，在原有下游引物序列的5’端用生物素修饰作为用于RAA-nfo检测下游引物。适用于RAA-nfo的引物及探针设计结果如表5所示。
1.3.2最佳引物-探针组筛选结果
为筛选最佳引物-探针组，对表5中所述的4种组合(F10’-1/R10’/P、F10’-2/R10’/P、F10’-3/R10’/P、F10’-4/R10’/P)进行了测试筛选，结果如图4所示，RAA-LFS结果显示只有F10’-1/R10’/P在四种组合里扩增效率最高，且NTC结果无假阳性，即F10’-1/R10’/P组合扩增效率最高且无明显的假阳性，满足检测要求，这一结果属于本领域技术人员在未经实验验证之前所预料不到的技术效果，因此在随后的实验中使用了F10’-1/R10’/P为最佳引物-探针组合。
此外，对耳念珠菌和其他常见病原念珠菌的全基因组做系统发育树(如图5所示)，与其中最近缘的三种念珠菌的ITS序列比对表明，经筛选得到的引物探针组合F10’-1/R10’/P能检测区分耳念珠菌(C.auris)和C.persedohaemulonii、C.duobushaemulonis、C.haemulonii(如图6所示)，进一步表明了本发明经设计筛选得到的引物-探针组合具有较高的特异性。
实施例2耳念珠菌RAA检测最佳反应条件的筛选
1、实验方法
为了筛选本次实验的最佳反应参数，选用103fg/reaction的基因组浓度，将相同的反应体系置于不同的反应温度进行测试。具体筛选方法如下：
反应中模板浓度选取103fg/reaction的耳念珠菌基因组DNA，对反应条件的摸索验证，将反应温度设置为35～45℃(每2℃设置为一梯度)，将反应时间设置为5～35min(每5min一梯度)分别孵育结合试纸条结果以分析适合反应温度范围，实验方法同实施例1中的1.2.3.4。
2、实验结果
扩增结果采用LFS分析，实验结果显示，测试线上的粉红色在45℃的反应条件下一直未出现，可能是因温度过高导致酶失活。其余纸条测试线上的粉红色在39℃、10min时出现，15min时各条带颜色开始变暗，其中37℃与39℃条带最为明显。20分钟后，条带的暗度没有显著变化(如图7所示)。因此，选择37℃和15min作为RAA的最佳反应温度和时间。
实施例3 RAA-LFS技术检测耳念珠菌特异性评价
1、实验方法
为了确认引物-探针组的包容性和特异性，对12株耳念珠菌参考菌株、4株C.haemulonii、3株C.pseudohaemulonii、4株C.duobushaemulonis和其他近缘念珠菌及常见致病性细菌进行RAA-LFS扩增测试，实验方法同实施例1中的1.2.4.4，即根据制造商的说明，使用RAA-nfo核酸扩增试剂(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)进行RAA反应。RAA反应过程如下：反应体系共50μL，在装有RAA反应干粉试剂的检测单元管中加入2μM的上下游引物各2μL，2μM的探针0.6μL，A Buffer 25μL，双蒸水(ddH2O)15.9μL，DNA模板2μL，于管盖上添加B Buffer 2.5μL。盖上管盖，上下颠倒轻甩充分混匀5-6次，通过离心的方式将其加入到体系中催化反应的发生。低速离心10s后在PCR仪(或恒温金属浴或水浴锅)中37℃孵育15min。反应结束后，将50μL扩增产物添加到300μL的无菌水或1×PBS溶液中稀释，将一次性核酸检测试纸条(侧流层析试纸条)(Hangzhou Zhongce Bio-Sci&Tech Co.,Ltd.,Hangzhou,China)样品垫朝下插入稀释后的反应液中2分钟，待样品吸收至吸收垫处，然后目测对结果判读。结果判读方法同实施例1中所述。
2、实验结果
12株参考菌株的耳念珠菌结果呈阳性(如图8所示)，其他近缘真菌和致病性细菌为阴性(如图9A和9B所示)，表明F10’-1/R10’/P引物-探针组对耳念珠菌表现出良好的包容性和特异性，并且不会与其他病原细菌和真菌发生交叉反应。所有C.haemulonii、C.pseudohaemulonii和C.duobushaemulonis在测试中均为阴性(如图9A所示)，表明了该系统可以检测到耳念珠菌的同时可以准确的区分开其与C.haemulonii、C.pseudohaemulonii和C.duobushaemulonis，特异性极好。
实施例4 RAA-LFS技术检测耳念珠菌灵敏性评价
1、实验方法
为了确定RAA-LFS检测系统对耳念珠菌的检测限，测试了耳念珠菌基因组和菌液的10倍系列稀释度，基因组浓度梯度为107fg/reaction-100fg/reaction，菌液浓度梯度为105CFU/μL-100CFU/μL(反应体积：50μL，2μL C.auris基因组被添加到每个反应中)。同时为了确定其他菌株的污染是否会干扰检测灵敏度，将白念珠菌105CFU/μL处理后的培养物或1ng/μL基因组DNA添加到10倍稀释的耳念珠菌培养物(105-100CFU/μL)或基因组DNA(107fg/reaction-100fg/reaction)，实验方法同实施例3所述的RAA反应过程。
2、实验结果
为了确定RAA-LFS系统对耳念珠菌的检测限，本实施例测试了耳念珠菌菌液的10倍系列稀释度，范围从105到100CFU/μL(反应体积：50μL，2μL耳念珠菌基因组被添加到每个反应中)。尽管颜色较浅，但在100CFU/μL的测试线上仍出现粉红色条带。此外，随着耳念珠菌菌液浓度的增加，粉红色带变暗(如图10A所示)。以类似的方式，对纯化的耳念珠菌基因组DNA进行了10倍系列稀释。可以检测到低至101fg/reaction的耳念珠菌基因组DNA(如图10B所示)。为了测试系统是否能够抵抗其他常见病原真菌DNA的干扰，将白念珠菌105CFU/μL的菌液或1ng/μL基因组DNA添加到10倍稀释的耳念珠菌菌液(105-100CFU/μL)或基因组DNA(107fg/reaction-100fg/reaction)，结果表明C.albicans菌液(如图11A所示)或基因组DNA(如图11B所示)不会干扰RAA-LFS对耳念珠菌的检测。可见RAA-LFS系统的检测限为每反应2CFU，或10fg基因组DNA/50μL。检测灵敏度不受其他真菌DNA存在的影响。
实施例5 RAA-LFS技术检测耳念珠菌应用性评价
1、实验方法
(1)血液DNA中掺入不同浓度耳念珠菌基因组DNA
为了确定人类DNA对耳念珠菌检测的影响，将耳念珠菌基因组DNA与人血提取的DNA按1:10混匀后进行10倍系列稀释梯度，使耳念珠菌基因组最终浓度为107fg/reaction-100fg/reaction进行RAA-LFS检测，观察检测限变化，实验方法同实施例3所述的RAA反应过程。
(2)直接检测血液中耳念珠菌
从掺入了耳念珠菌的全血中直接提取DNA进行RAA检测，初步评价其对临床血液样本快速检测的应用。具体步骤如下：每200μL全血样本中混入耳念珠菌的10倍系列稀释菌液进行RAA-LFS检测，菌液浓度梯度为105CFU/μL-100CFU/μL，实验方法同实施例3所述的RAA反应过程。
2、实验结果
本实施例模拟了临床血液样本，在人血液样本中混入不同浓度的耳念珠菌液进行RAA-LFS检测，实验结果表明：RAA-LFS检测耳念珠菌的方法可以应用于人血液样本且检测的灵敏度不变(如图12A所示)；同时为了确定人DNA对耳念珠菌检测的影响，将耳念珠菌基因组DNA与人血提取的DNA按1：10混匀后进行RAA-LFS检测，结果显示，人DNA对检测没有显著的抑制作用且不改变其检测限(如图12B所示)。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (17)

  1. 一种用于耳念珠菌检测的恒温核酸扩增RAA引物探针组合，其特征在于，所述引物探针组合包括上游引物、下游引物和探针；
    所述上游引物的核苷酸序列为F10’-1：5’-AAGGATCATTATTGATATTTTGCATACACA-3’；
    所述下游引物的核苷酸序列为R10’：5’-Biotin-TTCAAAGATTCGATGATTCACGTCTGCAAG-3’；
    所述探针的核苷酸序列为P：5’-FAM-ACTGATTTGGATTTTAAAACTAACCCAACG[THF]TAAGTTCAACTAAAC-C3spacer-3’。
  2. 一种用于检测耳念珠菌的试剂盒，其特征在于，所述试剂盒包含由权利要求1所述的引物探针组合组成的引物探针混合液。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述试剂盒还包含A Buffer、B Buffer、RAA反应干粉试剂、ddH2O。
  4. 根据权利要求3所述的试剂盒，其特征在于，所述A Buffer为20％PEG。
  5. 根据权利要求3所述的试剂盒，其特征在于，所述B Buffer为280mM MgAc。
  6. 根据权利要求3所述的试剂盒，所述RAA反应干粉试剂中包含如下的组分：dNTP、SSB蛋白、recA重组酶蛋白或Rad51、Bsu DNA聚合酶、Tricine、PEG、二硫苏糖醇、肌酸激酶、Nfo核酸内切酶。
  7. 根据权利要求6所述的试剂盒，所述RAA反应干粉试剂中各组分的浓度如下：1mmol/L dNTP、90ng/μL SSB蛋白、120ng/μL recA重组酶蛋白或30ng/μL Rad51、30ng/μL Bsu DNA聚合酶、100mmol/L Tricine、20％PEG、5mmol/L二硫苏糖醇、100ng/μL肌酸激酶。
  8. 根据权利要求3所述的试剂盒，其特征在于，所述试剂盒还包含侧流层析试纸条。
  9. 一种基于RAA-侧流层析技术检测耳念珠菌的方法，其特征在于，所述方法包括如下步骤：
    (1)提取待测样本DNA；
    (2)以待测样本DNA为模板，进行RAA扩增反应，得到扩增产物，其中，RAA扩增的上游引物的核苷酸序列为F10’-1：5’-AAGGATCATTATTGATATTTTGCATACACA-3’；下游引物的核苷酸序列为R10’：5’-Biotin-TTCAAAGATTCGATGATTCACGTCTGCAAG-3’；探针的核苷酸序列为P：5’-FAM-ACTGATTTGGATTTTAAAACTAACCCAACG[THF]TAAGTTCAACTAAAC-C3spacer-3’；
    (3)应用侧流层析试纸条对扩增产物进行检测，当试纸条出现两条红色条带，一条位于质控区，一条位于检测区，则结果为阳性，表明样本中含有耳念珠菌；当试纸条只有质控区出现一条红色条带，检测区没有条带，则结果为阴性，表明样本中不含耳念珠菌。
  10. 根据权利要求9所述的方法，其特征在于，步骤(2)中所述扩增反应的过程包括如下步骤：在装有RAA反应干粉试剂的检测单元管中加入上游引物、下游引物、探针、A Buffer、ddH2O、模板，于管盖上添加B Buffer，盖上管盖，充分混匀，低速离心后继续进行扩增反应，得到扩增产物。
  11. 根据权利要求10所述的方法，其特征在于，所述各物质的用量分别为：2μM上游引物2μL，2μM下游引物2μL，2μM探针0.6μL，A Buffer 25μL，15.9μL ddH2O，2μL模板，B Buffer 2.5μL。
  12. 根据权利要求11所述的方法，其特征在于，步骤(2)中所述扩增反应的反应条件为：37℃的条件下反应15min。
  13. 权利要求1所述的引物探针组合、权利要求2-8中任一项所述的试剂盒在制备用于检测耳念珠菌的产品中的应用。
  14. 权利要求1所述的引物探针组合、权利要求2-8中任一项所述的试剂盒在制备用于鉴别诊断耳念珠菌和C.persedohaemulonii、C.duobushaemulonis、C.haemulonii的产品中的应用。
  15. 权利要求1所述的引物探针组合、权利要求2-8中任一项所述的试剂盒在检测耳念珠菌中的应用。
  16. 权利要求1所述的引物探针组合、权利要求2-8中任一项所述的试剂盒在鉴别诊断耳念珠菌和C.persedohaemulonii、C.duobushaemulonis、C.haemulonii中的应用。
  17. 一种用于诊断受试者是否患有耳念珠菌感染疾病的方法，其特征在于，所述方法包括如下步骤：采用权利要求9-12中任一项所述的方法对受试者来源的待测样本进行检测。
PCT/CN2023/123418 2022-10-09 2023-10-08 一种用于检测耳念珠菌的恒温核酸扩增raa引物探针组合及其应用 WO2024078420A1 (zh)

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