WO2024131136A1 - 具有鉴定dna功能的试剂盒和鉴定dna的方法 - Google Patents

具有鉴定dna功能的试剂盒和鉴定dna的方法 Download PDF

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WO2024131136A1
WO2024131136A1 PCT/CN2023/116518 CN2023116518W WO2024131136A1 WO 2024131136 A1 WO2024131136 A1 WO 2024131136A1 CN 2023116518 W CN2023116518 W CN 2023116518W WO 2024131136 A1 WO2024131136 A1 WO 2024131136A1
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conjugated polymer
photothermal
kit
primer pair
seq
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French (fr)
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王树
李明宇
吕凤婷
申奇
黄一鸣
白昊天
刘礼兵
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中国科学院化学研究所
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • C—CHEMISTRY; METALLURGY
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    • 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
    • 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 nucleic acid detection, and in particular to a kit with a DNA identification function and a DNA identification method.
  • Bacteria are ubiquitous in the environment and human body. Some of them are harmless to humans and even establish beneficial relationships with the host. However, pathogenic strains that cause human diseases do exist and pose a serious threat to public health.
  • the American Association of Clinical Medicine proposed the concept of "point-of-care testing", which means that it is necessary to research and develop detection methods that are short in time, easy to operate, and portable.
  • Traditional detection methods are usually divided into three categories: bacterial culture, serological testing, and molecular testing.
  • the gold standard for bacterial diagnosis is still bacterial culture, but it takes a long time, usually 2-3 days; on the other hand, it may be dormant under culture conditions, which makes growth and bacterial counting difficult.
  • Serological testing is mainly for antigen and antibody detection.
  • RPA recombinase polymerase isothermal amplification
  • the purpose of the present invention is to overcome the above problems existing in the prior art and provide a kit having the function of identifying DNA and a method for identifying DNA.
  • the kit and the method realize the identification of pathogenic bacteria genome. Rapid, visual detection of DNA.
  • the first aspect of the present invention provides a kit having the function of identifying DNA, the kit comprising a primer pair, photothermal conjugated polymer nanoparticles and fluorescently labeled dNTPs;
  • the photothermal conjugated polymer nanoparticles include D-A photothermal conjugated polymers
  • the second aspect of the present invention provides a method for identifying DNA, which comprises: under illumination conditions, mixing the DNA to be tested with the primer pair, photothermal conjugated polymer nanoparticles and fluorescently labeled dNTPs in the above-mentioned kit for amplification.
  • the present invention can achieve DNA amplification using a conjugated polymer with good photothermal properties, without the need for temperature control using instruments and equipment, and can achieve temperature control at a specific wavelength of light and illumination time, and has high sensitivity. More preferably, using another conjugated polymer to report the test results can achieve rapid, visual, and controllable pathogen detection, overcoming the problems of time-consuming and space-limited existing detection methods. In a harsh environment with limited resources, it can be separated from the laboratory environment and the dependence on PCR instruments, and the presence or absence of pathogenic microorganisms can be determined only by laser, which greatly improves the detection efficiency and saves valuable time for subsequent targeted treatment.
  • Figure 1 is the agarose gel electrophoresis result of the amplified product of Staphylococcus aureus
  • Fig. 2 is the sequencing result of Staphylococcus aureus amplification product
  • FIG3 is a fluorescence spectrum when the amplification template is Staphylococcus aureus genomic DNA
  • FIG4 is a FRET ratio when the amplification template is Staphylococcus aureus genomic DNA
  • Figure 5 shows the amplification templates of Candida albicans and Pseudomonas aeruginosa. FRET ratio for Pseudomonas aeruginosa and Enterococcus faecalis genomic DNA;
  • FIG6 shows the color when the amplification template is Staphylococcus aureus genomic DNA.
  • the first aspect of the present invention provides a kit having the function of identifying DNA, the kit comprising a primer pair, photothermal conjugated polymer nanoparticles and fluorescently labeled dNTPs;
  • the photothermal conjugated polymer nanoparticles include D-A photothermal conjugated polymers (D-A type conjugated polymers);
  • the particle size of the photothermal conjugated polymer nanoparticles is 30-50 nm.
  • the photothermal conjugated polymer nanoparticles further include an encapsulating agent, and the structural formula of the encapsulating agent is shown in formula (b).
  • Y is an alkali metal, preferably sodium; more preferably, the weight ratio of the DA photothermal conjugated polymer to the encapsulating agent is 1:8-12, which can be 1:8, 1:9, 1:10, 1:11, 1:12 or any two of the above. The range formed by the values and the values within the range.
  • C 10 H 21 may refer to a straight chain or a branched chain
  • C 12 H 25 may refer to a straight chain or a branched chain
  • C 17 H 35 may refer to a straight chain or a branched chain.
  • the preparation method of the photothermal conjugated polymer nanoparticles can be: mixing a conjugated polymer with an encapsulating agent, the weight ratio of the conjugated polymer to the encapsulating agent is preferably 0.5-1.5:10, preferably, the mixing method is ultrasound at 35-38°C, and the mixing time is 7-13 minutes. More preferably, the mixture also includes a solvent, and the amount of the solvent is 2-6mL relative to 1mg of the conjugated polymer.
  • the solvent can be tetrahydrofuran (THF). More preferably, the solvent, conjugated polymer and encapsulating agent can all pass through a 0.2-0.25 ⁇ m organic phase filter membrane.
  • the mixed product is mixed with water and continued to be cleaned in an ultrasonic cleaning machine.
  • the solvent is removed by rotary evaporation, and the rotary evaporation gradually reduces the pressure from 250-350kPa to 60-90kPa at 35-38°C, and maintains for 25-35 minutes.
  • the rotary evaporation product is filtered through a 0.2-0.25 ⁇ m organic phase filter membrane to cut off the rotary evaporation product with a molecular weight of 80-120 kDa, and the filtered product is centrifuged (at a rate of 3000-4000 rpm for 3-8 min).
  • the precipitate after centrifugation is mixed with water, resuspended, and centrifuged again, and repeated 7-12 times to remove the free encapsulating agent. It can also be concentrated as needed.
  • the kit further includes a cationic conjugated polymer.
  • R is When X is a halogen, n is 10-20, and n is an integer, the signal can be further amplified to improve the detection sensitivity; more preferably, X is fluorine, chlorine, bromine or iodine, more preferably fluorine, chlorine or bromine, and further preferably bromine.
  • the kit in order to enable rapid amplification of the primer pair and the DNA to be tested, also includes at least one of a recombinase and a polymerase.
  • the kit when the recombinase and the polymerase act together, the effect is better, therefore, preferably, the kit includes the recombinase and the polymerase.
  • the recombinase includes a recombinase that can bind to single-stranded nucleic acid; preferably, the polymerase includes a single-stranded DNA binding protein and/or a strand displacement DNA polymerase.
  • the primer pair is a primer pair for amplifying at least one specific gene fragment in Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa and Enterococcus faecalis, and each primer pair is stored separately.
  • the Staphylococcus aureus 16s rRNA gene can be used as a specific gene fragment. Therefore, when detecting Staphylococcus aureus, the primer pair includes the single-stranded DNA shown in SEQ ID No.1 and the single-stranded DNA shown in SEQ ID No.2.
  • SEQ ID No.1 is AGTAACACGTGGATAACCTACCTATAAGAC.
  • SEQ ID No.2 is CTTCCCTAATAACAGAGTTTTACGATCCGAAG.
  • the Candida albicans its gene can be used as a specific gene fragment. Therefore, when detecting Candida albicans, the primer pair includes the single-stranded DNA shown in SEQ ID No.3 and the single-stranded DNA shown in SEQ ID No.4.
  • SEQ ID No.3 is CCCAGCCTGCCGCCAGAGGTCTAAACTTACAAC.
  • SEQ ID No.4 is CAGCGATCCCGCCTTACCACTACCGTCTTTC.
  • the Pseudomonas aeruginosa eta gene can be used as a specific gene fragment. Therefore, when detecting Pseudomonas aeruginosa, the primer pair includes the single-stranded DNA shown in SEQ ID No.5 and the single-stranded DNA shown in SEQ ID No.6.
  • SEQ ID No.5 is CAGTTCATAAATCCCATAAAAGCCCTCTTC.
  • SEQ ID No.6 is CTGATCGAGCGGTTGGTTTTTCTTGTC.
  • the Enterococcus faecalis esp gene can be used as a specific gene fragment. Therefore, when detecting Enterococcus faecalis, the primer pair includes the single-stranded DNA shown in SEQ ID No.7 and the single-stranded DNA shown in SEQ ID No.8.
  • SEQ ID No.7 is CCTGATACTTCTAACGTTACTGATAGTACG.
  • SEQ ID No.8 is GGTTGTTGTTATAGGTACGTATGTTGCATC.
  • the introduction of fluorescently labeled dNTP makes the subsequent identification of target DNA easier.
  • the fluorescently labeled dNTP is dATP and/or dCTP; more preferably, the fluorescent label is fluorescein.
  • the kit also includes dNTPs without fluorescent labels, and the dNTPs without fluorescent labels include dATP, dCTP, dGTP and dTTP.
  • the ratio between dNTPs with fluorescent labels and dNTPs without fluorescent labels can account for 8-20% of the total molar amount of dNTPs, and those skilled in the art can choose according to needs.
  • the second aspect of the present invention provides a method for identifying DNA, which comprises: under illumination conditions, mixing the DNA to be tested with the primer pair, photothermal conjugated polymer nanoparticles and fluorescently labeled dNTPs in the above-mentioned kit for amplification.
  • DNA in the sample to be tested can be extracted first and then tested. Therefore, the method of the present invention can also include the step of extracting DNA in the sample to be tested.
  • the amplification method is recombinase polymerase isothermal amplification.
  • the amplification can be achieved using the kit described above, or the recombinase polymerase isothermal amplification kit can be directly purchased for operation.
  • the molar ratio of a single primer in the primer pair to the D-A photothermal polymer is 1:700-800, which can be 1:700, 1:710, 1:720, 1:730, 1:740, 1:750, 1:760, 1:770, 1:780, 1:790, 1:800, and the range formed by any two of the above values and the values within the range.
  • the molar ratio of a single primer in the primer pair to the fluorescently labeled dNTP is 1:60-100, and can be 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, and a range formed by any two of the above values, and a value within the range.
  • the conditions for amplification are not limited, and the target DNA can be amplified normally, preferably at 37-42°C for 8-13 minutes.
  • the temperature can be increased for a long time under light without external heating, and amplification can be completed.
  • the wavelength of the light is preferably 780-820nm, and more preferably, the light power density of the light is 0.3-2W/ cm2 .
  • the method can also include agarose gel electrophoresis and/or sequencing of the amplified product to further identify the presence of the target DNA.
  • the inventors of the present invention have found that the fluorescent DNA after amplification can undergo fluorescence resonance energy transfer with a cationic conjugated polymer. Therefore, the presence of the target DNA can be determined by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the fluorescence spectrum is characterized by a spectrophotometer.
  • the ratio of I 535nm /I 430nm can be used as the FRET detection result. When the ratio is lower than 0.12, it indicates that there is no target DNA. When the ratio is higher than 0.12, it indicates that there is a target DNA.
  • the The method further comprises: contacting the amplified product with the cationic conjugated polymer in the above-mentioned kit, more preferably, the substance amount ratio of the single primer in the primer pair to the cationic conjugated polymer is 1:10-20, which can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20 and the range formed by any two of the above values and the values within the range.
  • the contact conditions of the amplified product with the cationic conjugated polymer include: the temperature is: 37-42°C, which can be 37°C, 38°C, 39°C, 40°C, 41°C, 42°C and the range formed by any two of the above values and the values within the range, and the time is 10-12 minutes.
  • the inventors of the present invention have found that the fluorescent DNA after amplification can undergo fluorescence resonance energy transfer with the cationic conjugated polymer, and presents a color change visible to the naked eye under ultraviolet light. When it is green, it indicates the presence of target DNA, and when it is blue-purple, it indicates the absence of target DNA.
  • the method further comprises: contacting the amplified product with the cationic conjugated polymer in the kit according to claim 3, placing the contact product under ultraviolet light, and observing the color change; more preferably, the molar ratio of a single primer in the primer pair to the cationic conjugated polymer is 1:30-45, which can be 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45 and any range formed by any two of the above values and values within the range; more preferably, the contact conditions of the amplification product and the cationic conjugated polymer include: temperature: 37-42°C, which can be 37°C, 38°C, 39°C, 40°C, 41°C, 42°C and any range formed by any two of the above values and values within the range, and the time is 10-12 minutes; further preferably, the wavelength of
  • the amplification product in order to prevent the free dNTP with fluorescent label from undergoing fluorescence resonance energy transfer with the cationic conjugated polymer, thereby affecting the test result, can be digested before contacting with the cationic conjugated polymer.
  • the digestion agent can be shrimp alkaline phosphatase, which is carried out in SAP buffer.
  • the digestion conditions include: 30-90° C., 20-40 min.
  • the reaction can be carried out at 30-40° C. for 10-20 min, and then at 80-90° C. for 10-20 min.
  • n is an integer of 20; Wherein X is bromine.
  • the PFP can be referred to Conducting Polymers-Thylakoid Hybrid Materials for Water Oxidation and Photoelectric Conversion, Zhou X, Wang S et al, Adv. Electron. Mater. 2019, 5(3), 1800789.
  • Staphylococcus aureus source China General Microbiological Culture Collection Center, collection number: CGMCC1.879;
  • Candida albicans source China General Microbiological Culture Collection Center, accession number: ATCC10231;
  • Pseudomonas aeruginosa source China General Microbiological Culture Collection Center, accession number: ATCC29853;
  • the concentration of extracted genomic DNA was determined by absorbance at 260 nm using a Nanodrop instrument.
  • the specific sequences of each bacterial genome were queried in NCBI, and RPA primers were designed for each gene fragment using Primer3Plus.
  • the primer design sequences are as follows:
  • Staphylococcus aureus-F is shown in SEQ ID No.1;
  • Staphylococcus aureus-R is shown as SEQ ID No. 2;
  • Candida albicans-F is shown as SEQ ID No.3;
  • Candida albicans-R is shown as SEQ ID No.4;
  • Pseudomonas aeruginosa-F is shown as SEQ ID No.5;
  • Pseudomonas aeruginosa-R is shown as SEQ ID No.6;
  • Enterococcus faecalis-F is shown as SEQ ID No.7;
  • Enterococcus faecalis-R is shown as SEQ ID No.8.
  • the photothermal D-A conjugated polymer described in formula (a) can be prepared by referring to the method disclosed in Near-Infrared-Light Remote-Controlled Activation of Cancer Immunotherapy Using Photothermal Conjugated Polymer Nanoparticles, Fu XC, Wang S et al, Adv. Mater. 2021, 33, 2102570.
  • m is 3-20, and m is a positive integer, the number average molecular weight is 97.3 kDa, and the long-chain alkyl groups are all straight-chain alkyl groups.
  • the synthesis method of photothermal conjugated polymer nanoparticles (CPNs) is as follows:
  • the resulting liquid was filtered with a 0.22 ⁇ m aqueous phase filter membrane and transferred to a 50 mL ultrafiltration tube with a molecular weight cutoff of 100 kDa. Centrifuge at 3500 rpm for 5 min to enrich, add ultrapure water, resuspend, continue centrifugation to enrich, and repeat the above operation 10 times to remove DSPE-PEG2000. Finally, it was concentrated to 2 mL, that is, the concentration of the obtained DA photothermal conjugated polymer was 500 ⁇ g/mL.
  • Reaction system 1 includes a recombinase polymerase isothermal amplification kit (Hangzhou Zhongce Technology Co., Ltd.), which includes a dry powder tube (the dry powder tube contains UvsX recombinase, Bsu DNA polymerase, single-stranded binding protein, nuclease, and recombinase auxiliary protein lyophilized reagent), A buffer, and B buffer.
  • a recombinase polymerase isothermal amplification kit Hangzhou Zhongce Technology Co., Ltd.
  • the dry powder tube contains UvsX recombinase, Bsu DNA polymerase, single-stranded binding protein, nuclease, and recombinase auxiliary protein lyophilized reagent
  • a buffer and B buffer.
  • a buffer 25 ⁇ L of A buffer, 6 ⁇ L of D-A photothermal conjugated polymer nanoparticles (CPNs) (concentration of 500 ⁇ g/mL), 2 ⁇ L of primers Staphylococcus aureus-F (10 ⁇ M) and Staphylococcus aureus-R (10 ⁇ M), 2 ⁇ L of dNTP Mix (4 mM, FL-dATP and FL-dCTP are 400 ⁇ M each), Staphylococcus aureus genomic DNA (10 ng/ ⁇ L), 2.5 ⁇ L of B buffer, and ultrapure water are added to the dry powder tube;
  • CPNs D-A photothermal conjugated polymer nanoparticles
  • Reaction system 2 included the recombinase polymerase isothermal amplification kit (Hangzhou Zhongce Technology Co., Ltd.), which included a dry powder tube (the dry powder tube contained UvsX recombinase, Bsu DNA polymerase, single-stranded binding protein, nuclease, and recombinase auxiliary protein lyophilized reagent), A buffer, and B buffer.
  • the dry powder tube contained UvsX recombinase, Bsu DNA polymerase, single-stranded binding protein, nuclease, and recombinase auxiliary protein lyophilized reagent
  • a buffer and B buffer.
  • a buffer 25 ⁇ L of A buffer, 6 ⁇ L of D-A photothermal conjugated polymer nanoparticles (CPNs) (concentration of 500 ⁇ g/mL), 2 ⁇ L of primers Staphylococcus aureus-F (10 ⁇ M) and Staphylococcus aureus-R (10 ⁇ M), 2 ⁇ L of dNTP Mix (4 mM, FL-dATP and FL-dCTP were 400 ⁇ M each), 2 ⁇ L of B buffer, and ultrapure water were added to the dry powder tube.
  • CPNs D-A photothermal conjugated polymer nanoparticles
  • the reaction conditions are as follows: 808nm laser irradiates the reaction system, the light power density is 1W/ cm2 , the temperature rise during illumination is controlled at 38 ⁇ 1°C (no other external force increases the temperature, the temperature is measured by infrared thermal imager), the illumination time is 10min, the reaction system is recovered, and the amplified product is obtained.
  • Agarose gel electrophoresis was used to verify whether the product was the target DNA fragment. As shown in Figure 1, the agarose gel electrophoresis results are shown, where the band in lane 1 represents the amplified product of Staphylococcus aureus. Lane 2 is the amplification result of the blank group (i.e., no target template), and there is no corresponding target band. The above results show that the extracted DNA combined with specific primers can obtain the target DNA fragment using this photothermal amplification method.
  • the amplified product was further subjected to DNA sequencing.
  • the sequencing results are shown in FIG2 , which further confirmed that the amplified product was consistent with the target DNA sequence.
  • Example 1 The method of Example 1 is the same, except that the primers for the Candida albicans its gene are used for amplification, and the Staphylococcus aureus genomic DNA is replaced with the Candida albicans genomic DNA.
  • the results show that the method of the present invention can specifically identify the Candida albicans its gene with high sensitivity.
  • Example 1 The method of Example 1 was followed, except that primers targeting the Pseudomonas aeruginosa eta gene were used for amplification, and the Staphylococcus aureus genomic DNA was replaced with the Pseudomonas aeruginosa genomic DNA.
  • the results showed that the method of the present invention can specifically identify the Pseudomonas aeruginosa eta gene with high sensitivity.
  • Example 1 The method of Example 1 is the same, except that primers for the esp gene of Enterococcus faecalis are used for amplification, and the genomic DNA of Staphylococcus aureus is replaced with the genomic DNA of Enterococcus faecalis.
  • the results show that the method of the present invention can specifically identify the esp gene of Enterococcus faecalis with high sensitivity.
  • the two groups of amplification products obtained by the reaction system in the above Examples 1-4 were digested.
  • the digestion reaction system was shrimp alkaline phosphatase (rSAP).
  • 10X SAP buffer and amplification products were mixed, reacted at 37°C for 15 min, reacted at 85°C for 15 min, and finally stored at 4°C.
  • the PFP stock solution (10 mM, solvent is dimethyl sulfoxide, DMSO) was diluted to 1 mM with ultrapure water and filtered through a 0.22 ⁇ m aqueous filter. 15 ⁇ L PFP (1 mM) was added to 500 ⁇ L N-2-hydroxyethylpiperidinium chloride.
  • the PFP working solution was prepared by adding 25 mM HEPES buffer and 485 ⁇ L DMSO.
  • Figure 3 shows the fluorescence spectrum when the template is Staphylococcus aureus genomic DNA, and the results show that fluorescence resonance energy transfer can occur. When the template is only water, fluorescence resonance energy transfer does not occur.
  • the fluorescent marker is fluorescein, and the detection result is judged by the FRET ratio, and the ratio of I 535nm /I 430nm is selected as the FRET detection result.
  • Figure 4 shows the FRET ratio when the template is Staphylococcus aureus genomic DNA
  • Figure 6 shows the FRET ratio when the template is Candida albicans, Pseudomonas aeruginosa, and Enterococcus faecalis genomic DNA. The results show that the FRET ratio of DNA without target template is lower than 0.12, and the FRET ratio of DNA to be tested is higher than 0.12.
  • Figure 5 shows the color when the template is Staphylococcus aureus genomic DNA.
  • the results show that the color of the template DNA is green, and the color of the blank group without template DNA is close to the color of PFP, which is blue-purple.
  • the presence or absence of pathogens can be determined by the change in color.
  • the above results show that the method of the present invention can perform visual detection.

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Abstract

涉及核酸检测领域，公开了一种具有鉴定DNA功能的试剂盒，该试剂盒包括引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP；所述光热共轭聚合物纳米粒子包括为D-A光热共轭聚合物。该试剂盒利用良好光热性质的共轭聚合物即可实现DNA扩增，无需利用仪器设备进行控温，实现了致病菌基因组DNA快速、可视化检测。

Description

具有鉴定DNA功能的试剂盒和鉴定DNA的方法
相关申请的交叉引用
本申请要求2022年12月22日提交的中国专利申请202211659318.1的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及核酸检测领域，具体涉及一种具有鉴定DNA功能的试剂盒和鉴定DNA的方法。
背景技术
细菌在环境和人体中无处不在，其中部分对人体无害，甚至与宿主建立了有益的关系。然而，导致人类疾病的致病菌株确实存在，并对公众健康构成严重威胁。美国临床医学会提出“即时检测”概念，即需要研究开发时间短、操作简便、便携式的检测手段。传统的检测方法通常分为三大类：细菌培养、血清学检测以及分子学检测。如今对于细菌诊断金标准仍为细菌培养，但耗时长，通常需2-3天；另一方面其在培养条件下可能处于休眠状态，这使得生长和细菌计数变得困难。血清学检测主要针对抗原抗体检测，此方式虽时间短，但无法达到疾病潜伏期检测的目标，不利于疾病的早期诊断。对于分子学检测，目前临床手段仍以PCR为主，该方法灵敏度高，但通常需要2-3h，时间较长，难以实现即时检测。
为满足即时检测需求，已出现利用重组酶聚合酶等温扩增(RPA)方法代替传统的PCR，该方法快速，将扩增时间缩短至10-30min，同时无需高温变性，仅37-42℃的条件即可实现扩增，条件温和，但此方法灵敏度相对较低。除此之外，目前大多数扩增过程仍需利用仪器设备进行控温，不利于在资源有限环境中进行检测。
发明内容
本发明的目的是为了克服现有技术存在的上述问题，提供一种具有鉴定DNA功能的试剂盒和鉴定DNA的方法，该试剂盒和方法实现了致病菌基因组 DNA快速、可视化检测。
为了实现上述目的，本发明第一方面提供一种具有鉴定DNA功能的试剂盒，该试剂盒包括引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP；
所述光热共轭聚合物纳米粒子包括为D-A光热共轭聚合物；
所述D-A光热聚合物的结构式如式(a)所示其中，m＝3-20，且m为整数。
本发明第二方面提供一种鉴定DNA的方法，该方法包括：在光照条件下，将待测DNA与上述的试剂盒中的引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP混合进行扩增。
通过上述技术方案，本发明利用具有良好光热性质的共轭聚合物即可实现DNA扩增，无需利用仪器设备进行控温，在特定波长的光照和光照时间即可实现控温，并且灵敏度高。更优的，利用另一共轭聚合物进行检测结果的报告，可实现快速、可视化、可控的病原菌检测，克服了现有检测方法耗时、空间局限性等问题，在资源有限的恶劣环境中，可脱离实验室环境以及PCR仪器的依赖，仅通过激光即可判断病原微生物的有无，大大提高检测效率，为后续开展针对性治疗节约宝贵的时间。
附图说明
图1是金黄葡萄球菌(Staphylococcus aureus)扩增产物琼脂糖凝胶电泳结果；
图2是金黄葡萄球菌扩增产物测序结果；
图3是当扩增模板为金黄葡萄球菌基因组DNA时的荧光光谱；
图4是当扩增模板为金黄葡萄球菌基因组DNA时FRET比值；
图5是当扩增模板为白色念珠菌(Candida albicans)、铜绿假单胞菌 (Pseudomonas aeruginosa)、粪肠球菌(Enterococcus faecalis)基因组DNA时FRET比值；
图6是当扩增模板为金黄葡萄球菌基因组DNA时的颜色。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
本发明第一方面提供一种具有鉴定DNA功能的试剂盒，该试剂盒包括引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP；
所述光热共轭聚合物纳米粒子包括为D-A光热共轭聚合物(D-A型共轭聚合物)；
所述D-A光热聚合物的结构式如式(a)所示，其中，m＝3-20，且m为整数。
优选的，所述光热共轭聚合物纳米粒子的粒径为30-50nm。
优选的，所述光热共轭聚合物纳米粒子还包括包裹剂，所述包裹剂的结构式如式(b)所示，其中Y为碱金属，优选为钠；更优选的，所述D-A光热共轭聚合物与包裹剂的重量比为1：8-12，可以为1：8、1：9、1：10、1：11、1：12以及以上任意两 个数值形成的范围以及范围内的数值。
本发明涉及的结构式中，C10H21可以指直链，也可以指支链；C12H25可以指直链，也可以指支链；C17H35可以指直链，也可以指支链。
本发明中，所述光热共轭聚合物纳米粒子的制备方法可以为：将共轭聚合物与包裹剂混合，所述共轭聚合物与包裹剂的重量比优选为0.5-1.5：10，优选的，所述混合的方式为在35-38℃下超声，混合的时间为7-13分钟，更优选的，所述混合物中还包括溶剂，相对于1mg的共轭聚合物，溶剂的用量为2-6mL。所述溶剂可以为四氢呋喃(THF)。更优选的，所述溶剂、共轭聚合物和包裹剂均可通过0.2-0.25μm有机相滤膜，更优选的，将混合后的产物与水混合继续在超声清洗机中清洗，清洗后进行旋蒸去除溶剂，旋蒸在35-38℃下由250-350kPa逐渐降低压强直至60-90kPa，并维持25-35分钟。将旋蒸产物通过0.2-0.25μm有机相滤膜截取分子量为80-120kDa的旋蒸产物，将过滤后的产物进行离心(速率为3000-4000rpm，时间为3-8min)，取离心后的沉淀与水混合重悬继续离心，重复7-12次，以去除游离的包裹剂。后续还可以根据需求进行浓缩。
本发明的发明人发现，阳离子共轭聚合物可以放大受体荧光信号，优选的，所述试剂盒中还包括阳离子共轭聚合物。
本发明的发明人发现，当所述阳离子共轭聚合物的结构式如式(c)，其中，R为X为卤素，n为10-20，且n为整数时，可以进一步的放大信号，提高检测灵敏度；进一步优选的，所述X为氟、氯、溴或碘，更优选为氟、氯或溴，进一步优选为溴。
本发明中，为了可以使得引物对与待测DNA可以快速进行扩增，所述试剂盒中还包括重组酶和聚合酶中的至少一种。
本发明中，当重组酶和聚合酶共同作用时，效果更佳，因此，优选的，所述试剂盒中包括重组酶和聚合酶。
本发明中，所述重组酶包括能结合单链核酸的重组酶；优选的，所述聚合酶包括单链DNA结合蛋白和/或链置换DNA聚合酶。
本发明的发明人发现，本发明的试剂盒更有利于金黄葡萄球菌、白色念珠菌、铜绿假单胞菌或粪肠球菌的检测，因此，所述引物对为扩增金黄葡萄球菌、白色念珠菌、铜绿假单胞菌和粪肠球菌中至少一种特异性基因片段的引物对，每个引物对单独保存。
本发明中，可使用金黄葡萄球菌16s rRNA基因作为特异性基因片段，因此，当检测金黄葡萄球菌时，引物对包括SEQ ID No.1所示的单链DNA和SEQ ID No.2所示的单链DNA。
SEQ ID No.1为AGTAACACGTGGATAACCTACCTATAAGAC。
SEQ ID No.2为CTTCCCTAATAACAGAGTTTTACGATCCGAAG。
本发明中，可使用白色念珠菌its基因作为特异性基因片段，因此，当检测白色念珠菌时，引物对包括SEQ ID No.3所示的单链DNA和SEQ ID No.4所示的单链DNA。
SEQ ID No.3为CCCAGCCTGCCGCCAGAGGTCTAAACTTACAAC。
SEQ ID No.4为CAGCGATCCCGCCTTACCACTACCGTCTTTC。
本发明中，可使用铜绿假单胞菌eta基因作为特异性基因片段，因此，当检测铜绿假单胞菌时，引物对包括SEQ ID No.5所示的单链DNA和SEQ ID No.6所示的单链DNA。
SEQ ID No.5为CAGTTCATAAATCCCATAAAAGCCCTCTTC。
SEQ ID No.6为CTGATCGAGCGGTTGGTTTTTCTTGTC。
本发明中，可使用粪肠球菌esp基因作为特异性基因片段，因此，当检测粪肠球菌，引物对包括SEQ ID No.7所示的单链DNA和SEQ ID No.8所示的单链DNA。
SEQ ID No.7为CCTGATACTTCTAACGTTACTGATAGTACG。
SEQ ID No.8为GGTTGTTGTTATAGGTACGTATGTTGCATC。
本发明中，带有荧光标记的dNTP(脱氧核糖核苷三磷酸)的引入使得后续目标DNA的鉴定更简便，优选的，所述带有荧光标记的dNTP为dATP和/或dCTP；更优选的，所述荧光标记为荧光素。
本发明中，所述试剂盒中还包括不带有荧光标记的dNTP，所述不带有荧光标记的dNTP包括dATP、dCTP、dGTP和dTTP。可以理解，对于带有荧光标记 的dNTP与不带有荧光标记的dNTP之间的比例没有限制，只要在扩增后的DNA中引入带有荧光标记的dNTP即可，例如带有荧光标记的dNTP的摩尔量可以占dNTP总摩尔量的8-20％，本领域技术人员可以根据需求进行选择。
本发明第二方面提供一种鉴定DNA的方法，该方法包括：在光照条件下，将待测DNA与上述的试剂盒中的引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP混合进行扩增。
本发明中，可以先提取待测样品中的DNA再进行检测，因此，本发明的方法还可以包括提取待测样品中的DNA的步骤。
本发明中，所述扩增的方式没有限制，可以使得目标DNA正常扩增即可，为了可以快速扩增，优选的，所述扩增的方式为重组酶聚合酶等温扩增，在实际操作中，可使用如前所述的试剂盒实现扩增，也可直接购买重组酶聚合酶等温扩增试剂盒进行操作。
优选的，所述引物对中的单个引物与D-A光热聚合物的物质的量比为1：700-800，可以为1：700、1：710、1：720、1：730、1：740、1：750、1：760、1：770、1：780、1：790、1：800以及以上任意两个数值形成的范围以及范围内的数值。
优选的，所述引物对中的单个引物与荧光标记的dNTP的物质的量比为1：60-100，可以为1：60、1：65、1：70、1：75、1：80、1：85、1：90、1：95、1：100以及以上任意两个数值形成的范围以及范围内的数值。
本发明中，所述扩增的条件没有限制，可以使得目标DNA正常扩增即可，优选为37-42℃，时间8-13分钟。本发明中，可以不用通过外部进行增温，在光照下即可时间增温，完成扩增，所述光照波长优选为780-820nm，更优选的，所述光照的光功率密度为0.3-2W/cm2。所述方法还可以包括对扩增产物进行琼脂糖凝胶电泳和/或测序从而进一步鉴定目标DNA的存在。
本发明的发明人发现，上述扩增后带有荧光的DNA可以与阳离子共轭聚合物发生荧光共振能量转移，因此，可以通过荧光能量共振转移(FRET)来测定是否存在目标DNA，通过分光光度计表征荧光光谱，在实际操作中，可以通过I535nm/I430nm的比值作为FRET检测结果，当比值低于0.12表示没有目标DNA，当比值高于0.12时，表示有目标DNA，因此，在一种优选的实施方式中，所述 方法还包括：将扩增产物与上述试剂盒中的阳离子共轭聚合物接触，更优选的，所述引物对中的单个引物与阳离子共轭聚合物的物质的量比为1：10-20，可以为1：10、1：11、1：12、1：13、1：14、1：15、1：16、1：17、1：18、1：19、1：20以及以上任意两个数值形成的范围以及范围内的数值。进一步优选的，所述扩增产物与阳离子共轭聚合物接触条件包括：温度为：37-42℃，可以为37℃、38℃、39℃、40℃、41℃、42℃以及以上任意两个数值形成的范围以及范围内的数值，时间为10-12分钟。
本发明的发明人发现，上述扩增后带有荧光的DNA可以与阳离子共轭聚合物发生荧光共振能量转移，在紫外灯照射呈现肉眼可视的颜色变化，当为绿色时，表示有目标DNA，当为蓝紫色时，表示没有目标DNA，因此，在另一种优选的实施方式中，所述方法还包括：将扩增产物与权利要求3所述的试剂盒中的阳离子共轭聚合物接触，将接触产物置于紫外光照射下，观察颜色变化；更优选的，所述引物对中的单个引物与阳离子共轭聚合物的物质的量比为1：30-45，可以为1：30、1：31、1：32、1：33、1：34、1：35、1：36、1：37、1：38、1：39、1：40、1：41、1：42、1：43、1：44、1：45以及以上任意两个数值形成的范围以及范围内的数值；更优选的，所述扩增产物与阳离子共轭聚合物接触条件包括：温度为：37-42℃，可以为37℃、38℃、39℃、40℃、41℃、42℃以及以上任意两个数值形成的范围以及范围内的数值，时间为10-12分钟；进一步优选的，所述紫外光的波长为365nm，光功率密度为0.12-0.15W/cm2。
本发明中，为了防止游离的带有荧光标记的dNTP与阳离子共轭聚合物发生荧光共振能量转移，从而影响测试的结果，可以将扩增产物与阳离子共轭聚合物接触前进行消化，所述消化剂可使用虾碱性磷酸酶，在SAP缓冲液中进行，消化条件包括：30-90℃，20-40min，优选的，可以先在30-40℃下反应10-20min，在再80-90℃下反应10-20min。
以下将通过实施例对本发明进行详细描述。以下实施例中，所述PFP结构如式(c)所示，
n为20的整数；其中，X为溴。所述PFP可参照 Conducting Polymers–Thylakoid Hybrid Materials for Water Oxidation and Photoelectric Conversion,Zhou X,Wang S et al,Adv.Electron.Mater.2019,5(3),1800789.中公开的方法制备得到；
金黄葡萄球菌来源：中国普通微生物菌种保藏管理中心，保藏号：CGMCC1.879；
白色念珠菌来源：中国普通微生物菌种保藏管理中心，保藏号：ATCC10231；
铜绿假单胞菌来源：中国普通微生物菌种保藏管理中心，保藏号：ATCC29853；
粪肠球菌来源：中国普通微生物菌种保藏管理中心，保藏号：ATCC29212。
制备例
(1)待测病原菌的DNA提取
在培养基中培养四种病原菌金黄葡萄球菌、白色念珠菌、铜绿假单胞菌以及粪肠球菌，利用细菌基因组DNA提取试剂盒(离心柱型)(天根生化科技有限公司)提取DNA，具体步骤如下：
取细菌培养液4mL(菌落数为108cfu/mL)，7300rpm离心3min，倒掉上清。向菌体沉淀中加200μL TE缓冲液、100μL溶菌酶(50mg/mL)，37℃摇床放置1h，后加入4μL RNase A(100mg/mL)，振荡15s，室温放置5min。向管中加入20μL Proteinase K，混匀。加入300μL GB缓冲液，振荡15s，70℃放置20min，溶液变清亮。向其中加入220μL无水乙醇，充分振荡15s，出现絮状沉淀。将上述溶液和絮状沉淀转移至吸附柱CB3中，吸附柱放入收集管中，12000rpm离心30s，倒废液，将吸附柱放入收集管。向CB3中加入500μL GD缓冲液，12000rpm离心30s，倒废液，吸附柱放入收集管中。向CB3中加入600μL PW漂洗液，12000rpm离心30s，倒废液，吸附柱放入收集管中。重复向CB3中加入600μL PW漂洗液，12000rpm离心30s，倒废液，吸附柱放入收集管中，12000rpm离心2min，倒废液，将吸附柱置于室温放置2h，以彻底晾干吸附柱中残余的漂洗液。将吸附柱CB3转移至干净离心管中，向吸附膜中间部位悬空滴加100μL TE洗脱缓冲液，室温放置5min，12000rpm离心2min，将溶液收集到离心管中，重复此步骤2次，得到所需基因组DNA。
使用Nanodrop仪器根据260nm吸光度测定提取的基因组DNA浓度。
(2)扩增所需引物的设计和使用
在NCBI中查询各菌基因组的特异性序列，利用Primer3Plus针对各基因片段设计RPA引物。引物设计序列如下：
金黄葡萄球菌-F如SEQ ID No.1所示；
金黄葡萄球菌-R如SEQ ID No.2所示；
白色念珠菌-F如SEQ ID No.3所示；
白色念珠菌-R如SEQ ID No.4所示；
铜绿假单胞菌-F如SEQ ID No.5所示；
铜绿假单胞菌-R如SEQ ID No.6所示；
粪肠球菌-F如SEQ ID No.7所示；
粪肠球菌-R如SEQ ID No.8所示。
(3)D-A光热共轭聚合物纳米粒子合成
其中Y为钠
式(a)所述光热D-A共轭聚合物可参照Near-Infrared-Light Remote-Controlled Activation of Cancer Immunotherapy Using Photothermal Conjugated Polymer Nanoparticles,Fu XC,Wang S et al,Adv.Mater.2021,33,2102570.中公开的方法制备得到，m为3-20，且m为正整数，数均分子量为97.3kDa，长链烷基均为直链烷基。光热共轭聚合物纳米粒子(CPNs)的合成方法如下：
所有玻璃器皿使用前均用1mM铬酸浸泡30分钟洗净除去杂质。THF用0.22μm有机相滤膜过滤备用。将D-A共轭聚合物使用THF制备成0.5mg/mL溶液，超声10min，过0.22μm有机相滤膜得到母液，放置棕色玻璃瓶备用。利 用上述同样方法制备5mg/mL的DSPE-PEG2000包裹剂式(b)(艾拓伟(上海)医药科技有限公司)母液。取5mL棕色玻璃瓶，加入D-A共轭聚合物母液和包裹剂母液各2mL，超声10min充分混合均匀。取一只50mL的单口圆底烧瓶，加入18mL的超纯水，将其敞口放入超声清洗机中，在超声状态下，将混合后的母液用1mL移液器加入烧瓶中，继续超声10min混合均匀。旋蒸除去有机溶剂THF。将所得液体用0.22μm水相滤膜过滤后转移至50mL截取分子量为100kDa的超滤管中。3500rpm离心5min富集，加入超纯水，重悬，继续离心富集，重复上述操作10次从而去除DSPE-PEG2000。最终浓缩至2mL，即所得D-A光热共轭聚合物浓度为500μg/mL。
实施例1
针对金黄葡萄球菌16s rRNA基因的引物进行扩增，反应体系分为2个进行：
反应体系1包括重组酶聚合酶等温扩增试剂盒(杭州众测科技有限公司)，试剂盒中包括干粉管(干粉管中为UvsX重组酶、Bsu DNA聚合酶、单链结合蛋白、核酸内切酶、重组酶辅助蛋白冻干试剂)、A缓冲液、B缓冲液，向干粉管中加入A缓冲液25μL，D-A光热共轭聚合物纳米粒子(CPNs)(浓度为500μg/mL)6μL，引物金黄葡萄球菌-F(10μM)和金黄葡萄球菌-R(10μM)各2μL，dNTP Mix(4mM，其中FL-dATP和FL-dCTP各为400μM)2μL，金黄葡萄球菌基因组DNA(10ng/μL)，B缓冲液2.5μL，超纯水补至50μL；
反应体系2(空白组)包括重组酶聚合酶等温扩增试剂盒(杭州众测科技有限公司)，试剂盒中包括干粉管(干粉管中为UvsX重组酶、Bsu DNA聚合酶、单链结合蛋白、核酸内切酶、重组酶辅助蛋白冻干试剂)、A缓冲液、B缓冲液，向干粉管中加入A缓冲液25μL，D-A光热共轭聚合物纳米粒子(CPNs)(浓度为500μg/mL)6μL，引物金黄葡萄球菌-F(10μM)和金黄葡萄球菌-R(10μM)各2μL，dNTP Mix(4mM，其中FL-dATP和FL-dCTP各为400μM)2μL，B缓冲液2.5μL，超纯水补至50μL。
反应条件为：808nm激光器照射反应体系，光功率密度为1W/cm2，光照升温控制在38±1℃(没有其它外力增温，温度通过红外热成像仪测定)，光照时间为10min，回收反应体系，得到扩增产物。
利用琼脂糖凝胶电泳验证产物是否是目标DNA片段。如图1所示为琼脂糖凝胶电泳结果，其中泳道1的条带代表金黄葡萄球菌扩增产物。泳道2为空白组(即无目标模板)的扩增结果，并没有相应的目标条带。以上结果说明：提取的DNA结合特异性引物利用此光热扩增方法可得到目标DNA片段。
进一步将扩增产物进行DNA测序。测序结果如图2所示，结果进一步证实扩增产物与目标DNA序列一致。
实施例2
按照实施例1的方式，不同的是，使用针对白色念珠菌its基因的引物进行扩增，将金黄葡萄球菌基因组DNA更换为白色念珠菌基因组DNA。结果显示，本发明的方法能够特异性地鉴定白色念珠菌its基因且灵敏度较高。
实施例3
按照实施例1的方式，不同的是，使用针对铜绿假单胞菌eta基因的引物进行扩增，将金黄葡萄球菌基因组DNA更换为铜绿假单胞菌基因组DNA。结果显示，本发明的方法能够特异性地鉴定铜绿假单胞菌eta基因且灵敏度较高。
实施例4
按照实施例1的方式，不同的是，使用针对粪肠球菌esp基因的引物进行扩增，将金黄葡萄球菌基因组DNA更换为粪肠球菌基因组DNA。结果显示，本发明的方法能够特异性地鉴定粪肠球菌esp基因且灵敏度较高。
测试例1
基于阳离子共轭聚合物PFP进行检测
按照上述实施例1-4中反应体系得到的2组扩增产物进行消化，消化反应体系为虾碱性磷酸酶(rSAP)，10X SAP缓冲液和扩增产物混合，37℃反应15min，85℃反应15min，最后4℃保存。
将PFP储存液(10mM，溶剂为二甲基亚砜，DMSO)，用超纯水稀释至1mM，过0.22μm水相滤膜。将15μL PFP(1mM)加入到500μL的N-2-羟乙基哌 嗪-N’-2-乙磺酸(HEPES)缓冲液(25mM)和485μL DMSO中，得到PFP工作液。
(1)将保存的消化后扩增产物(其中扩增产物50μL)、20μL PFP工作液(15μM)和530μL HEPES缓冲液(25mM)混合均匀转移至微量比色皿中，放入荧光分光光度计中进行检测，激发波长为380nm，发射波长为400nm-700nm，检测实验组与空白组荧光光谱差异。
结果：图3为当模板为金黄葡萄球菌基因组DNA时的荧光光谱，结果显示可发生荧光共振能量转移，模板仅为水时，则不会发生荧光共振能量转移。
荧光标记为荧光素，检测结果通过FRET比值来判断，选用I535nm/I430nm的比值作为FRET检测结果。如图4为当模板为金黄葡萄球菌基因组DNA时FRET比值，图6为当模板为白色念珠菌、铜绿假单胞菌、粪肠球菌基因组DNA时FRET比值。结果显示：无目标模板DNA的FRET比值低于0.12，存在待测DNA的FRET比值高于0.12。
(2)将保存的消化后扩增产物(其中扩增产物30μL)和30μL PFP工作液(15μM)混合均匀转移PCR管中，放入暗箱式多功能紫外灯分析仪中，使用波长为365nm的紫外灯照射混合后的溶液，紫外灯光功率密度为0.13W/cm2，肉眼观察溶液颜色差异，拍照记录结果，并使用Photoshop软件针对各个PCR管中的溶液的颜色计算RGB值。
结果：图5为当模板为金黄葡萄球菌基因组DNA时的颜色，结果显示：有模板DNA为绿色，无模板DNA的空白组颜色与PFP颜色接近，为蓝紫色。通过颜色的变化可判断病原菌有无，上述结果说明本发明的方法能够进行可视化检测。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 一种具有鉴定DNA功能的试剂盒，其特征在于，该试剂盒包括引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP；
    所述光热共轭聚合物纳米粒子包括为D-A光热共轭聚合物；所述D-A光热聚合物的结构式如式(a)所示，
    其中，m为3-20，且m为整数。
  2. 根据权利要求1所述的试剂盒，其中，所述光热共轭聚合物纳米粒子的粒径为30-50nm；
    和/或，所述光热共轭聚合物纳米粒子还包括包裹剂，所述包裹剂的结构式如式(b)所示，
    其中Y为碱金属，优选为钠；
    优选的，所述D-A光热共轭聚合物与包裹剂的重量比为1∶8-12。
  3. 根据权利要求1或2所述的试剂盒，其中，所述试剂盒中还包括阳离子共轭聚合物；
    优选的，所述阳离子共轭聚合物的结构式如式(c)所示，
    其中，R为X为卤素，n为10-20，且n为整数；
    优选的，所述X为氟、氯、溴或碘，更优选为氟、氯或溴，进一步优选为溴。
  4. 根据权利要求1或2所述的试剂盒，其中，所述试剂盒中还包括重组酶和聚合酶中的至少一种；
    优选的，所述试剂盒中还包括重组酶和聚合酶；
    优选的，所述重组酶包括能结合单链核酸的重组酶；
    优选的，所述聚合酶包括单链DNA结合蛋白和/或链置换DNA聚合酶。
  5. 根据权利要求1或2所述的试剂盒，其中，所述引物对为扩增金黄葡萄球菌、白色念珠菌、铜绿假单胞菌和粪肠球菌中至少一种特异性基因片段的引物对，每个引物对单独保存；
    和/或，所述带有荧光标记的dNTP为dATP和/或dCTP；
    优选的，所述荧光标记为荧光素。
  6. 根据权利要求5所述的试剂盒，其特征在于，所述金黄葡萄球菌特异性基因片段为16s rRNA基因；
    和/或，所述白色念珠菌特异性基因片段为its基因；
    和/或，所述铜绿假单胞菌特异性基因片段为eta基因；
    和/或，所述粪肠球菌特异性基因片段为esp基因。
  7. 根据权利要求5所述的试剂盒，其中，扩增所述金黄葡萄球菌特异性基因片段的引物对包括SEQ ID No.1所示的单链DNA和SEQ ID No.2所示的单链DNA；
    和/或，扩增所述白色念球菌特异性基因片段的引物对包括SEQ ID No.3所示的单链DNA和SEQ ID No.4所示的单链DNA；
    和/或，扩增所述铜绿假单胞菌特异性基因片段的引物对包括SEQ ID No.5所示的单链DNA和SEQ ID No.6所示的单链DNA；
    和/或，扩增所述粪肠球菌特异性基因片段的引物对包括SEQ ID No.7所示的单链DNA和SEQ ID No.8所示的单链DNA。
  8. 一种鉴定DNA的方法，其特征在于，该方法包括：在光照条件下，将待测DNA与权利要求1-7中任意一项所述的试剂盒中的引物对、光热共轭聚合物纳米粒子和荧光标记的dNTP混合进行扩增。
  9. 根据权利要求8所述的方法，其中，所述扩增的方式为重组酶聚合酶等温扩增；
    和/或，所述引物对中的单个引物、D-A光热聚合物和荧光标记的dNTP的物质的量比为1∶700-800∶60-100；
    和/或，所述扩增的条件包括：时间为8-13分钟；
    和/或，所述光照波长为780-820nm；
    和/或，所述光照的光功率密度为0.3-2W/cm2。
  10. 根据权利要求8或9所述的方法，其中，所述方法还包括：将扩增产物与权利要求3所述的试剂盒中的阳离子共轭聚合物接触；优选的，所述引物对中的单个引物与阳离子共轭聚合物的物质的量比为1∶10-20；优选的，所述扩增产物与阳离子共轭聚合物接触条件包括：温度为：37-42℃，时间为10-12分钟；
    和/或，所述方法还包括：将扩增产物与权利要求3所述的试剂盒中的阳离子共轭聚合物接触，将接触产物置于紫外光照射下，观察颜色变化；优选的，所述引物对中的单个引物与阳离子共轭聚合物的物质的量比为1∶30-45；优选的，所述扩增产物与阳离子共轭聚合物接触条件包括：温度为：37-42℃，时间为10-12分钟；优选的，所述紫外光的波长为365nm，光功率密度0.12-0.15W/cm2。
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