WO2024083104A1 - 一种核酸适配体试纸条、其制备方法及应用 - Google Patents

一种核酸适配体试纸条、其制备方法及应用 Download PDF

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WO2024083104A1
WO2024083104A1 PCT/CN2023/124890 CN2023124890W WO2024083104A1 WO 2024083104 A1 WO2024083104 A1 WO 2024083104A1 CN 2023124890 W CN2023124890 W CN 2023124890W WO 2024083104 A1 WO2024083104 A1 WO 2024083104A1
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aptamer
nucleic acid
detection
pad
acid aptamer
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PCT/CN2023/124890
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English (en)
French (fr)
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吴薇
王芹志
侯秀丹
吕良涛
杨庆利
赵方圆
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青岛农业大学
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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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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 pathogen detection, and specifically relates to a nucleic acid aptamer test strip, a preparation method and application thereof.
  • Listeria monocytogenes is a zoonotic pathogen that mainly transmits food. After infection, the main symptoms are sepsis, meningitis and monocytosis. It is widely present in nature and can still grow and reproduce in an environment of 4°C. It is one of the main pathogens that threaten human health in refrigerated food. Staphylococcus aureus (S. aureus) is a common facultative anaerobic Gram-positive pathogen that can produce a variety of toxins and enzymes, which can easily cause suppurative infections in humans and animals, human food poisoning, and can contaminate food during food processing, packaging and transportation.
  • Escherichia coli 0157:H7 (E.coli) is a common foodborne disease outbreak culprit around the world. It is widely distributed in nature and can cause gastrointestinal infections in humans or animals. It can cause severe spasmodic abdominal pain and recurrent hemorrhagic diarrhea, accompanied by fever, vomiting and other symptoms. Foods of animal origin, such as beef, chicken, milk, and dairy products, are the main factors for the transmission of Escherichia coli through food.
  • the main detection methods for foodborne pathogens include traditional culture method, molecular detection method, immunoassay method and biosensor method.
  • the detection time of traditional culture method is long and the detection process is complicated.
  • Molecular detection method such as the common PCR detection method, has high detection sensitivity and can realize automated detection, but it needs to rely on expensive instruments.
  • Immunoassay method such as enzyme-linked immunosorbent assay, uses the interaction between antigen and antibody to achieve specific detection of foodborne pathogens, but it is prone to false positive results.
  • the biosensor method uses sensors and other analytical equipment to achieve rapid and portable detection of foodborne pathogens. Biosensor detection is an emerging technology in recent years.
  • the invention provides a nucleic acid aptamer test strip, comprising a sample pad, a conjugation pad, a nitrocellulose membrane, a water absorbent pad and a pad; the conjugation pad contains one or more capture aptamers; the nitrocellulose membrane contains one or more detection lines and a quality control line, the detection line contains the detection aptamer, and the quality control line contains the quality control aptamer; wherein the capture aptamer on the conjugation pad is modified on silver core gold shell nanoparticles.
  • the capture aptamer is used to capture pathogens;
  • the detection aptamer can form a sandwich structure with the capture aptamer that captures pathogens, so that the capture aptamer that captures pathogens is retained on the detection line;
  • the quality control aptamer is the complementary chain of the capture aptamer, which can combine with the empty capture aptamer through complementary action, so that the empty capture aptamer is retained on the quality control line.
  • the present invention provides the use of the above-mentioned nucleic acid aptamer test strip in the detection of pathogens for non-diagnostic purposes.
  • the nucleic acid aptamer test strip is used for the detection of pathogens in food; the food includes but is not limited to milk, cheese, cooked meat products, beef, cold meats, and leftovers that are not thoroughly heated.
  • the above-mentioned pathogens are preferably selected from food-borne pathogens, including but not limited to one or more of Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli O157:H7.
  • nucleic acid aptamer test strip preferably:
  • the capture aptamer is selected from nucleic acid aptamer A1, nucleic acid aptamer A2 or nucleic acid aptamer A3; the detection aptamer is selected from nucleic acid aptamer B1, nucleic acid aptamer B2 or nucleic acid aptamer B3; the quality control aptamer is selected from nucleic acid aptamer C; wherein, the sequence of nucleic acid aptamer A1 is shown in SEQ ID NO:1; the sequence of nucleic acid aptamer A2 is shown in SEQ ID NO:2; the sequence of nucleic acid aptamer A3 is shown in SEQ ID NO:3; the sequence of nucleic acid aptamer B1 is shown in SEQ ID NO:4; the sequence of nucleic acid aptamer B2 is shown in SEQ ID NO:5; the sequence of nucleic acid aptamer B3 is shown in SEQ ID NO:6; the sequence of nucleic acid aptamer
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugation pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugation pad contains nucleic acid aptamer A1; the nitrocellulose membrane contains a detection line and a quality control line, the detection line contains nucleic acid aptamer B1, and the quality control line contains nucleic acid aptamer C.
  • the nucleic acid aptamer test strip can be used for the detection of Listeria monocytogenes for non-diagnostic purposes.
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugation pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugation pad contains nucleic acid aptamer A2; the nitrocellulose membrane contains a detection line and a quality control line, the detection line contains nucleic acid aptamer B2, and the quality control line contains nucleic acid aptamer C.
  • the nucleic acid aptamer test strip can be used for the detection of Staphylococcus aureus for non-diagnostic purposes.
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugate pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugate pad contains nucleic acid aptamer A3; the nitrocellulose membrane contains a detection line and a quality control line, the detection line contains nucleic acid aptamer B3, and the quality control line contains nucleic acid aptamer C.
  • the nucleic acid aptamer test strip can be used for the detection of Escherichia coli O157:H7 for non-diagnostic purposes.
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugation pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugation pad contains nucleic acid aptamer A1 and nucleic acid aptamer A2; the nitrocellulose membrane contains two detection lines and a quality control line, wherein one detection line contains nucleic acid aptamer B1, the other detection line contains nucleic acid aptamer B2, and the quality control line contains nucleic acid aptamer C.
  • the nucleic acid aptamer test strip can be used for the non-diagnostic purpose of detecting Listeria monocytogenes and Staphylococcus aureus.
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugation pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugation pad contains nucleic acid aptamer A1 and nucleic acid aptamer A3; the nitrocellulose membrane contains two detection lines and a quality control line, wherein one detection line contains nucleic acid aptamer B1 and the other detection line contains nucleic acid aptamer B2. It contains aptamer B3, and the quality control line contains aptamer C.
  • the aptamer test strip can be used for the non-diagnostic detection of Listeria monocytogenes and Escherichia coli O157:H7.
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugation pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugation pad contains nucleic acid aptamer A2 and nucleic acid aptamer A3; the nitrocellulose membrane contains two detection lines and a quality control line, wherein one detection line contains nucleic acid aptamer B2, the other detection line contains nucleic acid aptamer B3, and the quality control line contains nucleic acid aptamer C.
  • the nucleic acid aptamer test strip can be used for the non-diagnostic purpose of detecting Staphylococcus aureus and Escherichia coli O157:H7.
  • the nucleic acid aptamer test strip of the present invention comprises a sample pad, a conjugation pad, a nitrocellulose membrane, a water-absorbing pad and a pad; the conjugation pad contains nucleic acid aptamers A1, A2 and A3; the nitrocellulose membrane contains three detection lines and one quality control line, wherein one detection line contains nucleic acid aptamer B1, the other two detection lines contain nucleic acid aptamer B2 and nucleic acid aptamer B3 respectively, and the quality control line contains nucleic acid aptamer C.
  • the nucleic acid aptamer test strip can be used for the non-diagnostic purpose detection of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7.
  • nucleic acid aptamer A1 nucleic acid aptamer A1
  • nucleic acid aptamer A2 nucleic acid aptamer A3 are all modified on silver core-gold shell nanoparticles.
  • the above detection aptamer can be modified with biotin and streptavidin according to actual conditions.
  • the above quality control aptamer can be modified with biotin according to actual conditions.
  • the present invention provides a method for preparing the above-mentioned nucleic acid aptamer test strip, the steps of which are as follows:
  • the glass cellulose membrane was cut into strips, soaked in Tris-HCl buffer containing NaCl, Tween-20, bovine serum albumin, sucrose and Triton X-100, and dried to make a sample pad and a conjugate pad; then the silver core-gold shell nanoparticles modified with the capture aptamer were sprayed on the conjugate pad and dried for use; the detection aptamer was sprayed on the nitrocellulose membrane to form a detection line; the quality control aptamer was drawn on the side close to the absorbent pad to form a quality control line; the sprayed nitrocellulose membrane was dried for use; the nitrocellulose membrane, sample pad, conjugate pad and absorbent pad were sequentially glued to the backing plate, with an overlap of 2 mm between adjacent pads, and after the assembly was completed, they were cut into 4 mm wide test strips and stored in a dry and dark place.
  • the present invention provides a method for modifying a capture aptamer on a silver core-gold shell nanoparticle, the steps of which are as follows:
  • the capture aptamer is added to the TCEP solution for activation; the activated capture aptamer is placed in the silver core gold shell nanoparticle solution for coupling reaction; NaCl solution is added dropwise to the reaction system, mixed thoroughly, and aged at room temperature; after the reaction is completed, the reaction solution is centrifuged, the supernatant is discarded, and the solution is washed to obtain the silver core gold shell nanoparticles modified with the capture aptamer.
  • the silver core-gold shell nanoparticles can be prepared by the following method:
  • the present invention uses silver core gold shell nanoparticles as signal amplification elements, replacing the colloidal gold used in traditional test strips, and prepares a new type of nucleic acid aptamer test strip.
  • silver core gold shell nanoparticles can further amplify Raman signals to obtain more sensitive signals, and have better SERS enhancement characteristics than colloidal gold, and can be combined with Raman imaging spectrometers for quantitative analysis.
  • the nucleic acid aptamer test strip of the present invention uses nucleic acid aptamers of foodborne pathogens instead of antibodies, and uses a sandwich method to capture pathogens, thereby improving detection sensitivity and reducing detection costs. It is simple to operate, can be detected and observed by the naked eye, has high stability, and is suitable for rapid on-site detection.
  • FIG1 is a diagram showing the composition of a nucleic acid aptamer test strip, wherein the strip comprises a sample pad 1, a binding pad 2, a nitrocellulose membrane 3, a detection line 4, a quality control line 5, a water-absorbing pad 6 and a pad 7;
  • FIG2 is a specificity evaluation of nucleic acid aptamer test strips
  • FIG3 is a sensitivity evaluation of the nucleic acid aptamer test strip; wherein FIGA is a detection standard curve of Listeria monocytogenes; FIGB is a detection standard curve of Staphylococcus aureus; and FIGC is a detection standard curve of Escherichia coli O157:H7;
  • FIG4 is a stability evaluation of nucleic acid aptamer test strips
  • FIG5 is an affinity comparison experiment between nucleic acid aptamers and antibodies.
  • the present invention first provides a nucleic acid aptamer test strip, which is composed of a sample pad 1, a conjugation pad 2, a nitrocellulose membrane 3, a water-absorbing pad 6 and a pad 7; the nitrocellulose membrane 3, the sample pad 1, the conjugation pad 2 and the water-absorbing pad 6 are sequentially adhered to the pad 7, and the overlap between two adjacent pads is 2 mm; wherein the nitrocellulose membrane 3 contains a detection line 4 and a quality control line 5, the detection line 4 is close to one side of the conjugation pad 2, and the quality control line 5 is close to one side of the water-absorbing pad 6. As shown in FIG1 .
  • one or more capture aptamers may be sprayed on the conjugate pad, wherein the capture aptamers on the conjugate pad are modified on silver core gold shell nanoparticles; one or several detection lines may be sprayed on the side of the nitrocellulose membrane close to the conjugate pad, and the detection lines contain detection aptamers; a quality control line may be sprayed on the side of the nitrocellulose membrane close to the absorbent pad, and the quality control line contains quality control aptamers.
  • the detection principle of the present invention is as follows:
  • the silver core gold shell nanoparticles themselves are dark red.
  • the capture aptamers in the sample pad will specifically recognize and bind to the pathogenic bacteria, and flow forward under the push of the liquid.
  • the detection aptamers When flowing to the detection line, the detection aptamers will form a sandwich structure with the capture aptamers that have captured the pathogenic bacteria, causing the capture aptamers to remain on the detection line, causing the detection line to change color and appear red.
  • the capture aptamers that have not bound to pathogenic bacteria, that is, the unloaded capture aptamers cannot form a sandwich structure with the detection aptamers, will cross the detection line, continue to flow forward, and eventually interact with the quality control aptamers on the quality control line.
  • the ions are combined with the ions through the complementary reaction, and thus stay on the quality control line, causing the quality control line to change color and appear red.
  • the quality control line changes color, the test result is credible; if the quality control line does not change color, it means that the test strip is invalid and the test result is unreliable.
  • the present invention provides a method for preparing silver core-gold shell nanoparticles, the steps of which are as follows:
  • the present invention provides a method for modifying a capture aptamer on a silver core-gold shell nanoparticle, the steps of which are as follows:
  • the present invention provides the following nucleic acid aptamers:
  • the capture aptamer is selected from nucleic acid aptamer A1, nucleic acid aptamer A2 or nucleic acid aptamer A3, wherein nucleic acid aptamer A1 can be used to capture Listeria monocytogenes (ATCC19115), nucleic acid aptamer A2 can be used to capture Staphylococcus aureus (ATCC6538), and nucleic acid aptamer A3 can be used to capture Escherichia coli O157:H7 (ATCC8739); the detection aptamer is selected from nucleic acid aptamer B1, nucleic acid aptamer B2 or nucleic acid aptamer B3, wherein nucleic acid aptamer B1 can form a sandwich structure with nucleic acid aptamer A1 capturing Listeria monocytogenes, nucleic acid aptamer B2 can form a sandwich structure with nucleic acid aptamer A2 capturing Staphylococcus aureus, and nu
  • nucleic acid sequence of the above-mentioned nucleic acid aptamer is as follows:
  • the silver core-gold shell nanoparticles modified with the capture aptamer were evenly sprayed on the conjugate pad at 1 ⁇ L/cm using a gold spray film scribing instrument, and dried at 25°C for use.
  • the capture aptamer is selected from Listeria monocytogenes capture aptamer, Staphylococcus aureus capture aptamer or Escherichia coli O157:H7 capture aptamer, and the 5' end is modified with a "HS SH C6" thiol structure.
  • the purpose of modifying the nucleic acid aptamer with "HS SH C6" is to connect the aptamer to the nanoparticles through the action of the Au-S bond.
  • the Au-S bond (gold-sulfur bond) is a common self-assembly technology, which mainly occurs between the thiol group and the gold atom, and realizes the chemical covalent bond between the nucleic acid aptamer and the gold nanoparticles.
  • Staphylococcus aureus capture aptamer
  • Escherichia coli O157:H7 capture aptamer
  • the detection aptamer was streaked on the nitrocellulose membrane using a gold spraying membrane streaking instrument to form a detection line (T line); the quality control aptamer was streaked on the side close to the absorbent pad to form a quality control line (C line); the sprayed nitrocellulose membrane was dried at 25°C for 2h and stored in a dry environment at room temperature for future use.
  • the above detection aptamer is modified with biotin and streptavidin; the above quality control aptamer is modified with biotin.
  • the detection aptamer is modified with biotin, and before spraying on the nitrocellulose membrane, streptavidin needs to be added to the detection aptamer to allow biotin to react with streptavidin, so as to fix the detection aptamer on the nitrocellulose membrane, so that it is not easily washed away by the liquid.
  • streptavidin modification method for detecting aptamers is as follows:
  • the detection aptamer is selected from a Listeria monocytogenes detection aptamer, a Staphylococcus aureus detection aptamer or an Escherichia coli O157:H7 detection aptamer, which can form a sandwich structure with a Listeria monocytogenes capture aptamer, a Staphylococcus aureus capture aptamer or an Escherichia coli O157:H7 capture aptamer, respectively, and its nucleic acid sequence after modification with biotin is as follows:
  • the spike recovery experiment was conducted using milk samples to measure bacterial suspensions of different concentrations.
  • the bacterial suspensions of three foodborne pathogens (Staphylococcus aureus, Escherichia coli O157:H7, and Listeria monocytogenes) were added to the milk samples to make the final bacterial solution concentration reach the added concentration in Table 1.
  • the final concentrations of Staphylococcus aureus were 1.14 ⁇ 10 4 CFU/mL, 1.14 ⁇ 10 5 CFU/mL, and 1.14 ⁇ 10 6 CFU/mL, respectively.
  • the milk containing different concentrations of bacterial solution was dripped onto the test strip. After the reaction was complete, the actual detection amount was measured using a Raman spectrometer, and the recovery rate was calculated.
  • the spike recovery experiment can be used to analyze the feasibility of the detection experiment and simulate the situation of the detection method in actual application. The higher the recovery rate, the smaller the error of the detection method and the higher the relative accuracy.
  • the nucleic acid aptamer test strip of the present invention has a high recovery rate of 71.3% to 88.6% for Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7. This shows that the nucleic acid aptamer test strip of the present invention has a small error and a high accuracy rate in detecting foodborne pathogens.
  • a single bacterial solution was selected for the test strip specificity test. Taking Listeria monocytogenes as an example, the bacterial solution concentration was 2 ⁇ 10 6 CFU/mL, and the same concentration of Yersinia enterocolitica, Salmonella enteritidis, and Vibrio parahaemolyticus were used to verify the specificity of the test strip.
  • the C-line area of the test strips all showed color reaction, and only the test strip for detecting Listeria monocytogenes showed a red strip on its T line, while the T lines on the other test strips did not show color, which shows that the nucleic acid aptamer test strip of the present invention has good specificity.
  • Figure 3 shows the standard curves of three bacteria.
  • the Raman intensity is proportional to the target concentration in the range of 10 1 to 10 6 CFU/mL.
  • the detection limit of Listeria monocytogenes by naked eye was 1.88 ⁇ 10 3 CFU/mL
  • the detection limit of Staphylococcus aureus by naked eye was 1.14 ⁇ 10 3 CFU/mL
  • the detection limit of Escherichia coli O157:H7 by naked eye was 1.42 ⁇ 10 3 CFU/mL.
  • the nucleic acid aptamer test strip After the nucleic acid aptamer test strip was placed for 7 days, 14 days, 21 days, and 28 days, the color development of the T line was observed by taking Listeria monocytogenes as an example, and the results are shown in Figure 4. As can be seen from Figure 4, after being placed for 7 days, 14 days, and 21 days, the color development of the T line of the test strip is still obvious, and the color development of the T line becomes weaker after 28 days. Therefore, the nucleic acid aptamer test strip of the present invention still maintains a clear and visible color within 21 days of color development, and its stability is good.
  • Silver core-gold shell nanoparticles modified with Listeria monocytogenes capture aptamers, Staphylococcus aureus capture aptamers and Escherichia coli O157:H7 capture aptamers were respectively sprayed on the conjugate pad at 1 ⁇ L/cm using a gold spray film instrument, and dried at 25°C for use.
  • the Listeria monocytogenes detection aptamers, Staphylococcus aureus detection aptamers and Escherichia coli O157:H7 detection aptamers were streaked on the nitrocellulose membrane using a gold spray streaking instrument to form three detection lines (T lines); the quality control aptamer was streaked on the side close to the absorbent pad to form a quality control line (C line); the sprayed nitrocellulose membrane was dried at 25°C for 2h and stored in a dry environment at room temperature for future use.
  • the antibody and nucleic acid aptamer were modified with FITC fluorescence, they were incubated with Listeria monocytogenes for 1 hour, and then the average fluorescence intensity was measured using a FACSCalibur flow cytometer.
  • the antibody used was Anti-Listeria Antibody, genus specific, FITC labeled (produced by KPL, USA), purchased from Shanghai Jinpan Biotechnology Co., Ltd.; the nucleic acid aptamer used was Listeria monocytogenes capture aptamer.
  • the average fluorescence intensity of antibody binding is 42.4 (a.u.), while the average fluorescence intensity of aptamer binding is 152 (a.u.). It can be seen that the affinity of the aptamer selected in the present invention is 3.6 times that of the antibody, which shows that the aptamer has higher sensitivity and is more advantageous in preparing test strips.

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Abstract

一种核酸适配体试纸条、其制备方法及应用，属于病菌检测技术领域。核酸适配体试纸条包括：样品垫（1）、结合垫（2）、硝酸纤维素膜（3）、吸水垫（6）和垫板（7）；结合垫（2）上含有一种或多种捕获适配体；硝酸纤维素膜（3）上含有一条或几条检测线（4）和一条质控线（5），检测线（4）上含有检测适配体，质控线（5）上含有质控适配体；其中，结合垫（2）上的捕获适配体被修饰在银核金壳纳米粒子上。该核酸适配体试纸条，采用核酸适配体代替抗体，利用三明治夹心法捕获致病菌，同时以银核金壳纳米粒子为信号放大元件，代替了传统试纸条所采用的胶体金，提高了检测灵敏度，降低了检测成本，操作简单，可以实现肉眼检测和观察，稳定性高，适合现场快速检测。

Description

一种核酸适配体试纸条、其制备方法及应用 技术领域
本发明属于病菌检测技术领域，具体涉及一种核酸适配体试纸条、其制备方法及应用。
背景技术
单增李斯特菌(Listeria monocytogenes)是一种人畜共患病的病原菌，主要以食物为传染媒介，感染后主要表现为败血症、脑膜炎和单核细胞增多；它广泛存在于自然界中，在4℃的环境中仍可生长繁殖，是冷藏食品威胁人类健康的主要病原菌之一。金黄色葡萄球菌(S.aureus)是常见的兼性厌氧革兰氏阳性致病菌，它可产生多种毒素及多种酶类，易引起人和动物化脓感染、人食物中毒，能够在食品加工、包装及运输过程污染食品，其分泌的毒素对热不敏感，即使加热煮沸30分钟仍可致病。大肠埃希氏菌0157:H7(E.coli)是世界各地常见的食源性疾病暴发元凶，在自然界分布广泛，能够引起人体或动物胃肠道感染，会发生严重的痉挛性腹痛和反复发作的出血性腹泻，同时伴有发热、呕吐等表现；动物来源的食物，如牛肉、鸡肉、牛奶、奶制品等是大肠埃希氏菌经食物传播的主要因素。
目前食源性致病菌的主要检测方法有传统培养法、分子检测法、免疫检测法以及生物传感器法等。传统培养法的检测时间长，检测流程复杂。分子检测法，例如常见的PCR检测法，其检测灵敏度较高能实现自动化检测，但需要依赖贵重仪器。免疫检测法例如酶联免疫法，是利用抗原抗体间的相互作用，实现食源性致病菌的特异检测，但容易出现假阳性结果。生物传感器法是利用传感器等分析设备实现快速便携检测食源性致病菌，生物传感器法检测是近些年的新兴技术，能实现高灵敏快速检测食源性致病菌，但其检测也需要依赖相应的仪器，不够便携。为此，提供一种便携、高效、稳定，以及可肉眼观察的检测体系，并能够实现上述三种致病菌的同时检测，具有重要意义。
发明内容
本发明提供了一种核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有一种或多种捕获适配体；所述硝酸纤维素膜上含有一条或几条检测线和一条质控线，检测线上含有检测适配体，质控线上含有质控适配体；其中，所述结合垫上的捕获适配体被修饰在银核金壳纳米粒子上。
上述核酸适配体试纸条中，捕获适配体用于捕获病原菌；检测适配体可与捕获有病原菌的捕获适配体形成夹心结构，以使捕获有病原菌的捕获适配体滞留在检测线上；质控适配体是捕获适配体的互补链，其可通过互补作用与空载的捕获适配体结合，以使空载的捕获适配体滞留在质控线上。
本发明提供了上述核酸适配体试纸条在病原菌非诊疗目的的检测中的应用。例如，将核酸适配体试纸条应用于食品中病原菌的检测；所述食品包括但不限于牛奶、乳酪、熟肉制品、牛肉、冷荤食品以及未彻底加热的剩饭剩菜等。上述病原菌优选自食源性致病菌，包括但不限于单增李斯特菌、金黄色葡萄球菌以及大肠埃希氏菌O157:H7中的一种或几种。
在上述核酸适配体试纸条中，优选地：
所述捕获适配体选自核酸适配体A1、核酸适配体A2或核酸适配体A3；所述检测适配体选自核酸适配体B1、核酸适配体B2或核酸适配体B3；所述质控适配体选自核酸适配体C；其中，核酸适配体A1的序列如SEQ ID NO:1所示；核酸适配体A2的序列如SEQ ID NO:2所示；核酸适配体A3的序列如SEQ ID NO:3所示；核酸适配体B1的序列如SEQ ID NO:4所示；核酸适配体B2的序列如SEQ ID NO:5所示；核酸适配体B3的序列如SEQ ID NO:6所示；核酸适配体C的序列如SEQ ID NO:7所示。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A1；所述硝酸纤维素膜上含有一条检测线和一条质控线，检测线上含有核酸适配体B1，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于单增李斯特菌的非诊疗目的的检测。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A2；所述硝酸纤维素膜上含有一条检测线和一条质控线，检测线上含有核酸适配体B2，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于金黄色葡萄球菌的非诊疗目的的检测。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A3；所述硝酸纤维素膜上含有一条检测线和一条质控线，检测线上含有核酸适配体B3，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于大肠埃希氏菌O157:H7的非诊疗目的的检测。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A1和核酸适配体A2；所述硝酸纤维素膜上含有两条检测线和一条质控线，其中一条检测线上含有核酸适配体B1，另一条检测线上含有核酸适配体B2，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于单增李斯特菌和金黄色葡萄球菌的非诊疗目的的检测。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A1和核酸适配体A3；所述硝酸纤维素膜上含有两条检测线和一条质控线，其中一条检测线上含有核酸适配体B1，另一条检测线上 含有核酸适配体B3，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于单增李斯特菌和大肠埃希氏菌O157:H7的非诊疗目的的检测。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A2和核酸适配体A3；所述硝酸纤维素膜上含有两条检测线和一条质控线，其中一条检测线上含有核酸适配体B2，另一条检测线上含有核酸适配体B3，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于金黄色葡萄球菌和大肠埃希氏菌O157:H7的非诊疗目的的检测。
在一个具体实施方案中，本发明的核酸适配体试纸条，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有核酸适配体A1、核酸适配体A2和核酸适配体A3；所述硝酸纤维素膜上含有三条检测线和一条质控线，其中一条检测线上含有核酸适配体B1，另外两条检测线上分别含有核酸适配体B2和核酸适配体B3，质控线上含有核酸适配体C。该核酸适配体试纸条可被用于单增李斯特菌、金黄色葡萄球菌和大肠埃希氏菌O157:H7的非诊疗目的的检测。
上述核酸适配体A1、核酸适配体A2和核酸适配体A3均被修饰在银核金壳纳米粒子上。
上述检测适配体可根据实际情况修饰有生物素和链霉亲和素。上述质控适配体可根据实际情况修饰有生物素。
本发明提供了上述核酸适配体试纸条的制备方法，步骤如下：
将玻璃纤维素膜裁成条状，在含有NaCl、吐温-20、牛血清白蛋白、蔗糖以及曲拉通X-100的Tris-HCl缓冲液中浸泡，干燥，制成样品垫和结合垫；然后将修饰有捕获适配体的银核金壳纳米粒子喷涂在结合垫上，烘干备用；将检测适配体喷在硝酸纤维素膜上形成检测线；将质控适配体在靠近吸水垫的一侧划线，形成质控线；将喷涂好的硝酸纤维素膜烘干备用；将硝酸纤维素膜、样品垫、结合垫和吸水垫依次粘在衬板上，相邻两个垫之间重叠2mm，组装完成后切成4mm宽的试纸条，干燥避光保存。
本发明提供了一种将捕获适配体修饰在银核金壳纳米粒子上的方法，步骤如下：
将捕获适配体加入到TCEP溶液中活化；将活化的捕获适配体置于银核金壳纳米粒子溶液中，进行偶联反应；向反应体系中滴加NaCl溶液，充分混匀，室温老化；待反应结束后，将反应溶液离心，弃上清，洗涤，获得修饰有捕获适配体的银核金壳纳米粒子。
上述银核金壳纳米粒子可由如下方法制备而成：
将水加热至沸腾，然后依次加入硝酸银溶液和柠檬酸三钠溶液，搅拌反应，生成银纳米粒子；向银纳米粒子溶液中加入盐酸羟胺溶液和氯金酸溶液中，搅拌反应，待反应结束后，生成银核金壳纳米粒子。
本发明的有益效果为：
本发明以银核金壳纳米粒子为信号放大元件，代替了传统试纸条所采用的胶体金，制备了新型核酸适配体试纸条。与普通胶体金试纸条相比，银核金壳纳米粒子能进一步放大拉曼信号，获得更灵敏的信号，且比胶体金具有更好的SERS增强特性，能结合拉曼成像光谱仪进行定量分析。此外，本发明的核酸适配体试纸条，采用食源性致病菌的核酸适配体代替抗体，利用三明治夹心法捕获致病菌，提高了检测灵敏度，降低了检测成本，操作简单，可以实现肉眼检测和观察，稳定性高，适合现场快速检测。
附图说明
图1为核酸适配体试纸条组成图；其中，样品垫1、结合垫2、硝酸纤维素膜3、检测线4、质控线5、吸水垫6和垫板7；
图2为核酸适配体试纸条的特异性评价；
图3为核酸适配体试纸条的灵敏度评价；其中，A图为单增李斯特菌的检测标准曲线；B图为金黄色葡萄球菌的检测标准曲线；C图为大肠埃希氏菌O157:H7的检测标准曲线；
图4为核酸适配体试纸条的稳定性评价；
图5为核酸适配体与抗体的亲和力对比实验。
具体实施方式
本发明首先要提供一种核酸适配体试纸条，由样品垫1、结合垫2、硝酸纤维素膜3、吸水垫6和垫板7组成；所述硝酸纤维素膜3、样品垫1、结合垫2和吸水垫6依次粘在垫板7上，相邻两个垫之间重叠2mm；其中，硝酸纤维素膜3上含有一条检测线4和一条质控线5，检测线4靠近结合垫2的一侧，质控线5靠近吸水垫6的一侧。如图1所示。
在具体地实施方式中，结合垫上可喷涂有一种或多种捕获适配体，其中，所述结合垫上的捕获适配体被修饰在银核金壳纳米粒子上；硝酸纤维素膜上靠近结合垫的一侧可喷涂一条或几条检测线，检测线上含有检测适配体；硝酸纤维素膜上靠近吸水垫的一侧喷涂有质控线，质控线上含有质控适配体。
本发明的检测原理，如下所示：
银核金壳纳米粒子自身呈现暗红色。当样品垫接触含有致病菌的溶液时，样品垫中的捕获适配体会特异性识别并结合致病菌，并在液体的推动下向前流动，流动至检测线时，检测适配体会与捕获有致病菌的捕获适配体形成夹心结构，使捕获适配体滞留在检测线上，使检测线变色，呈现红色。未结合致病菌的捕获适配体，即空载的捕获适配体，因无法与检测适配体形成夹心结构，将越过检测线，继续向前流动，并最终与质控线上的质控适配体通过互 补作用而结合，从而滞留在质控线上，使质控线变色，呈现红色。当质控线变色时，检测结果可信；如果质控线不发生变色反应，说明试纸条失效，检测结果不可信。
本发明提供了一种银核金壳纳米粒子的制备方法，步骤如下：
将100mL超纯水置于250mL锥形瓶中搅拌加热至沸腾，加入1.6mL浓度为0.1M的硝酸银标准溶液，1min后加入3mL的浓度为1％的柠檬酸三钠溶液，搅拌加热至100℃，反应30min，生成银纳米粒子，于4℃保存。取40mL上述银纳米粒子溶液，加入1mL浓度为5×10-3M的盐酸羟胺溶液和4mL浓度为4.65×10-4M的氯金酸溶液，常温下持续搅拌1h，生成银核金壳纳米粒子，于4℃保存。
本发明提供了一种将捕获适配体修饰在银核金壳纳米粒子上的方法，步骤如下：
向13μL浓度为10μM的捕获适配体溶液中加入2μL浓度为1mM的TCEP(三(2-羧乙基)膦盐酸盐)溶液，4℃活化2h。取5mL银核金壳纳米粒子溶液，8000r/min离心20min，弃上清，加5mL超纯水复溶，加入活化后的捕获适配体，涡旋混匀，常温下偶联反应16h。反应完成后加入浓度为1％的SDS溶液，使SDS的终浓度为0.01％，常温反应1h。最后向反应完成后的溶液中滴加80μL浓度为2M的NaCl溶液，滴加时分多次缓慢滴加，使NaCl的终浓度为160mM，充分混匀，4℃老化24h。将所得溶液12000r/min离心15min，弃上清，用超纯水清洗一次，获得修饰有捕获适配体的银核金壳纳米粒子。可将修饰有捕获适配体的银核金壳纳米粒子采用10mM PBS重悬液(10mM PBS溶液中添加2％蔗糖、0.5％吐温-20、0.25％BSA而得)重悬，4℃避光保存。
本发明提供了如下几种核酸适配体：
捕获适配体选自核酸适配体A1、核酸适配体A2或核酸适配体A3，其中，核酸适配体A1可用于捕获单增李斯特菌(ATCC19115)，核酸适配体A2可用于捕获金黄色葡萄球菌(ATCC6538)，核酸适配体A3可用于捕获大肠埃希氏菌O157:H7(ATCC8739)；检测适配体选自核酸适配体B1、核酸适配体B2或核酸适配体B3，其中，核酸适配体B1可与捕获有单增李斯特菌的核酸适配体A1形成夹心结构，核酸适配体B2可与捕获有金黄色葡萄球菌的核酸适配体A2形成夹心结构，核酸适配体B3可与捕获有大肠埃希氏菌O157:H7的核酸适配体A3形成夹心结构；质控适配体选自核酸适配体C；
上述核酸适配体的核酸序列，如下：
核酸适配体A1：
5'-TACTCGTTATTTCGTAGCACTTTTCCCCACCACCTTGGTGTTTTTTTTT-3'(SEQ ID NO:1)；
核酸适配体A2：
5'-CTCCCAACCGCTCCACCCTGCCTCCGCCTCTTTTTTTTT-3'(SEQ ID NO:2)；
核酸适配体A3：
5'-CAAAAGTGCACGCTACTTTGCTAATTTTTTTTT-3'(SEQ ID NO:3)；
核酸适配体B1：
5'-TGGGGGGTGGTTGGGGGTAGTATATCGGGTCAGTGGTGCG-3'(SEQ ID NO:4)；
核酸适配体B2：
5'-CCCCCCAGTCCGTCCTCCCAGCCTCACACC-3'(SEQ ID NO:5)；
核酸适配体B3：
5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCAC-3'(SEQ ID NO:6)；
核酸适配体C：
5'-AAAAAAAAA-3'(SEQ ID NO:7)。
本发明中所使用的其它术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。下面结合具体实施例，并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
实施例1
单检测线核酸适配体试纸条的制备：
(1)玻璃纤维素膜处理
将玻璃纤维素膜裁成条状，在含有1％NaCl、0.5％吐温-20、1％牛血清白蛋白、2％蔗糖以及1％曲拉通X-100的Tris-HCl缓冲液(0.1M，pH＝8.0)中浸泡2h，在37℃条件下干燥，制成样品垫和结合垫。将修饰有捕获适配体的银核金壳纳米粒子，采用喷金划膜仪，以1μL/cm均匀喷涂在结合垫上，25℃烘干备用。
上述捕获适配体选自单增李斯特菌捕获适配体、金黄色葡萄球菌捕获适配体或大肠埃希氏菌O157:H7捕获适配体，且5'端均修饰有“HS SH C6”巯基结构。在本发明中，对核酸适配体进行“HS SH C6”修饰的目的是，其可通过Au-S键的作用将适配体与纳米粒子连接到一起。Au-S键(金硫键)是一种常见的自组装技术，主要发生在巯基和金原子之间，实现核酸适配体与金纳米粒子的化学共价键结合。
单增李斯特菌捕获适配体：
5'-HS SH C6-TACTCGTTATTTCGTAGCACTTTTCCCCACCACCTTGGTGTTTTTTTTT-3'；
金黄色葡萄球菌捕获适配体：
5'-HS SH C6-CTCCCAACCGCTCCACCCTGCCTCCGCCTCTTTTTTTTT-3'；
大肠埃希氏菌O157:H7捕获适配体：
5'-HS SH C6-CAAAAGTGCACGCTACTTTGCTAATTTTTTTTT-3'。
(2)检测线和质控线的制备
利用喷金划膜仪将检测适配体划在硝酸纤维素膜上，形成检测线(T线)；将质控适配体在靠近吸水垫的一侧划线，形成质控线(C线)；将喷涂好的硝酸纤维素膜在25℃条件下烘干2h，置于室温干燥环境中保存备用。
上述检测适配体修饰有生物素和链霉亲和素；上述质控适配体修饰有生物素。在本发明中，检测适配体上修饰有生物素，且在喷涂在硝酸纤维素膜上之前，还需向检测适配体中加入链霉亲和素，让生物素与链霉亲和素反应，以用于将检测适配体固定在硝酸纤维素膜上，从而不易被液体冲走。
检测适配体的链霉亲和素修饰方法，步骤如下：
将75μL浓度为10μM的修饰有生物素的检测适配体(生工生物工程股份有限公司)与25μL浓度为1mg/mL链霉亲和素溶液混合，在室温下反应2h，生成修饰有生物素和链霉亲和素的检测适配体。待反应完成后，4℃条件下6000r/min离心20min，除去没有反应的检测适配体。然后用浓度为0.01M的PBS缓冲液洗涤2次，用PBS缓冲液定容至原体积，4℃保存备用。
上述检测适配体选自单增李斯特菌检测适配体、金黄色葡萄球菌检测适配体或大肠埃希氏菌O157:H7检测适配体，其分别能够与单增李斯特菌捕获适配体、金黄色葡萄球菌捕获适配体或大肠埃希氏菌O157:H7捕获适配体形成夹心结构，其在修饰生物素后的核酸序列如下所示：
单增李斯特菌检测适配体：
5'-biotin-TGGGGGGTGGTTGGGGGTAGTATATCGGGTCAGTGGTGCG-3'；
金黄色葡萄球菌检测适配体：
5'-biotin-CCCCCCAGTCCGTCCTCCCAGCCTCACACC-3'；
大肠埃希氏菌O157:H7检测适配体：
5'-biotin-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCAC-3'。
上述质控适配体为：
5'-biotin-AAAAAAAAA-3'。
(3)试纸条的组装
将硝酸纤维素膜、样品垫、结合垫和吸水垫依次粘在垫板上，相邻两个垫之间重叠2mm， 组装完成后切成4mm宽的试纸条，干燥避光保存。
(一)致病菌回收试验
使用牛奶样品进行加标回收实验，测定不同浓度的菌悬液。分别取三种食源性致病菌(金黄色葡萄球菌、大肠埃希氏菌O157:H7、单增李斯特菌)的菌悬液加入到牛奶样品中，使最终的菌液浓度达到表1的添加量浓度，例如，金黄色葡萄球菌的最终浓度分别为1.14×104CFU/mL，1.14×105CFU/mL，1.14×106CFU/mL。将含有不同浓度菌液的牛奶滴加在试纸条上，等待反应完全后，用拉曼光谱仪测定实际的检出量，并计算回收率。通过加标回收率实验可以分析出检测实验的可行性，模拟检测方法在实际应用中的情况。回收率越高代表检测方法的误差越小，相对准确度越高。
试验结果如表1所示：
表1牛奶样品中不同浓度致病菌的检测回收率
由表1可知，本发明的核酸适配体试纸条，对单增李斯特菌、金黄色葡萄球菌以及大肠埃希氏菌O157:H7的回收率较高，在71.3～88.6％之间。这说明，本发明的核酸适配体试纸条，在检测食源性致病菌方面，误差小，准确率高。
(二)特异性试验
为避免多种致病菌相互影响，导致结果误差大，选用单一菌液进行试纸条特异性测试。以单增李斯特菌为例，菌液浓度为2×106CFU/mL，采用同种浓度的小肠结肠炎耶尔森菌、肠炎沙门氏菌和副溶血性弧菌验证试纸条的特异性。
结果如图2所示：
试纸条的C线区域均出现显色反应，只有检测单增李斯特菌的试纸条，其T线出现红色条带，而其余试纸条上的T线均未显色，这说明，本发明的核酸适配体试纸条具有良好的特异性。
(三)灵敏度试验
将不同浓度的金黄色葡萄球菌菌液、大肠埃希氏菌菌液以及单增李斯特菌菌液，分别滴加在试纸条上，待反应完全后，用拉曼光谱仪测定结果并计算相应的回归方程。浓度分别设置为：1×101CFU/mL、1×102CFU/mL、1×103CFU/mL、1×104CFU/mL、1×105CFU/mL、1×106CFU/mL。
试验结果如图3所示：
图3展示了三种菌的标准曲线，拉曼强度与靶标浓度在101～106CFU/mL成正比，单增李斯特菌的线性方程为y＝3405.1x+501.34，R2＝0.9916(A图)；金黄色葡萄球菌的线性方程为y＝3243.6x-2658.3，R2＝0.992(B图)；大肠埃希氏菌的线性方程为y＝2402.3x-642.87，R2＝0.9922(C图)。随着菌液浓度的增加，T线显色加深。其中，肉眼检出单增李斯特菌的检测限为1.88×103CFU/mL，肉眼检出金黄色葡萄球菌的检测限为1.14×103CFU/mL，肉眼检出大肠埃希氏菌O157:H7的检测限为1.42×103CFU/mL。
(四)稳定性试验
将核酸适配体试纸条放置7天、14天、21天、28天后，以单增李斯特菌为例，观察T线显色程度，结果如图4所示。由图4可见，在放置7天、14天、21天后，试纸条的T线显色依旧明显，28天后T线显色变弱。因此，本发明的核酸适配体试纸条，在显色21天内依旧保持着清晰可见的颜色，其稳定性良好。
实施例2
多检测线核酸适配体试纸条的制备：
(1)玻璃纤维素膜处理
将玻璃纤维素膜裁成条状，在含有1％NaCl、0.5％吐温-20、1％牛血清白蛋白、2％蔗糖以及1％曲拉通X-100的Tris-HCl缓冲液(0.1M，pH＝8.0)中浸泡2h，在37℃条件下干燥，制成样品垫和结合垫。分别将修饰有单增李斯特菌捕获适配体、金黄色葡萄球菌捕获适配体以及大肠埃希氏菌O157:H7捕获适配体的银核金壳纳米粒子，采用喷金划膜仪，以1μL/cm均匀喷涂在结合垫上，25℃烘干备用。
(2)检测线和质控线的制备
利用喷金划膜仪分别将单增李斯特菌检测适配体、金黄色葡萄球菌检测适配体以及大肠埃希氏菌O157:H7检测适配体划在硝酸纤维素膜上，形成三条检测线(T线)；将质控适配体在靠近吸水垫的一侧划线，形成质控线(C线)；将喷涂好的硝酸纤维素膜在25℃条件下烘干2h，置于室温干燥环境中保存备用。
(3)试纸条的组装
将硝酸纤维素膜、样品垫、结合垫和吸水垫依次粘在垫板上，相邻两个垫之间重叠2mm，组装完成后切成4mm宽的试纸条，干燥避光保存。
实施例3
亲和力对比试验：
分别将抗体与核酸适配体修饰FITC荧光后，与单增李斯特菌孵育1h，然后使用FACSCalibur流式细胞仪测量其平均荧光强度。在该实验中，抗体选用Anti-Listeria Antibody,genus specific,FITC labeled(美国KPL公司生产)，购于上海金畔生物科技有限公司；核酸适配体选用单增李斯特菌捕获适配体。
试验结果如图5所示：
抗体结合的平均荧光强度为42.4(a.u.)，而核酸适配体结合的平均荧光强度为152(a.u.)。由此可见，本发明选用的核酸适配体亲和力是抗体的3.6倍，这说明，核酸适配体灵敏度更高，在制备试纸条方面更具优势。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (8)

  1. 一种核酸适配体试纸条，其特征在于，包括样品垫、结合垫、硝酸纤维素膜、吸水垫和垫板；所述结合垫上含有一种或多种捕获适配体；所述硝酸纤维素膜上含有一条或几条检测线和一条质控线，检测线上含有检测适配体，质控线上含有质控适配体；其中，所述结合垫上的捕获适配体被修饰在银核金壳纳米粒子上。
  2. 根据权利要求1所述的核酸适配体试纸条，其特征在于，所述捕获适配体选自SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3所示的序列；所述检测适配体选自SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6所示的序列；所述质控适配体选自SEQ ID NO:7所示的序列。
  3. 根据权利要求2所述的核酸适配体试纸条，其特征在于，所述检测适配体修饰有生物素和链霉亲和素；所述质控适配体修饰有生物素。
  4. 权利要求1～3任一项所述核酸适配体试纸条的制备方法，其特征在于，步骤如下：
    将玻璃纤维素膜裁成条状，在含有NaCl、吐温-20、牛血清白蛋白、蔗糖以及曲拉通X-100的Tris-HCl缓冲液中浸泡，干燥，制成样品垫和结合垫；然后将修饰有捕获适配体的银核金壳纳米粒子喷涂在结合垫上，烘干备用；将检测适配体喷在硝酸纤维素膜上形成检测线；将质控适配体在靠近吸水垫的一侧划线，形成质控线；将喷涂好的硝酸纤维素膜烘干备用；将硝酸纤维素膜、样品垫、结合垫和吸水垫依次粘在衬板上，相邻两个垫之间重叠2mm，组装完成后切成4mm宽的试纸条，干燥避光保存。
  5. 根据权利要求4所述的制备方法，其特征在于，所述修饰有捕获适配体的银核金壳纳米粒子由如下方法制备而成：
    将捕获适配体加入到TCEP溶液中活化；将活化的捕获适配体置于银核金壳纳米粒子溶液中，进行偶联反应；向反应体系中滴加NaCl溶液，充分混匀，室温老化；待反应结束后，将反应溶液离心，弃上清，洗涤，获得修饰有捕获适配体的银核金壳纳米粒子。
  6. 根据权利要求5所述的制备方法，其特征在于，所述银核金壳纳米粒子由如下方法制备而成：
    将水加热至沸腾，然后依次加入硝酸银溶液和柠檬酸三钠溶液，搅拌反应，生成银纳米粒子；向银纳米粒子溶液中加入盐酸羟胺溶液和氯金酸溶液中，搅拌反应，待反应结束后，生成银核金壳纳米粒子。
  7. 权利要求1～3任一项所述核酸适配体试纸条在病原菌非诊疗目的的检测中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述病原菌选自单增李斯特菌、金黄色葡萄球菌以及大肠埃希氏菌O157:H7中的一种或几种。
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