WO2020063714A1 - 一种同时检测多种喷施雾滴飘失或沉积检测试剂盒及检测方法 - Google Patents

一种同时检测多种喷施雾滴飘失或沉积检测试剂盒及检测方法 Download PDF

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WO2020063714A1
WO2020063714A1 PCT/CN2019/108036 CN2019108036W WO2020063714A1 WO 2020063714 A1 WO2020063714 A1 WO 2020063714A1 CN 2019108036 W CN2019108036 W CN 2019108036W WO 2020063714 A1 WO2020063714 A1 WO 2020063714A1
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probe
transition
fixed
detection
solution
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PCT/CN2019/108036
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English (en)
French (fr)
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宋坚利
刘杨
何雄奎
张振华
逄森
徐少卿
王光宇
李宗洋
吴学民
李学锋
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中国农业大学
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Priority to CN201980063340.XA priority Critical patent/CN113039255A/zh
Publication of WO2020063714A1 publication Critical patent/WO2020063714A1/zh
Priority to US17/214,743 priority patent/US20210214777A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G2/00—Vegetative propagation
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention relates to the technical field of agricultural spray droplet detection, in particular to a qualitative and quantitative simultaneous detection of spray droplet deposition of various pesticides, fertilizers, water, and the like, a reverse dot hybridization technology kit and a detection method developed.
  • Plant protection integration is one of the latest planting schemes in agriculture, which refers to the selection and germination of seeds during the entire planting and cultivation process, and then to watering, fertilization, and pesticide application at the end. The harvesting is carried out in a reasonable, programmed and monitored manner, so as to produce high-quality agricultural products.
  • mist droplet detection methods mainly include three methods: direct measurement method, tracer method and direct droplet observation method.
  • the direct observation method refers to the collection of samples after the spraying process, and then through a series of steps such as extraction, purification, concentration, etc., to detect the amount of the original drug on the sample by HPLC-MS, GC-MS, etc. To calculate the deposition amount.
  • this method has high accuracy and can detect multiple original drugs at one time, it cannot be used for certain inorganic fertilizers.
  • this method can only be used to detect the amount of sediment, but it cannot give fog droplets. Particle size and distribution.
  • the tracer method is to add a tracer to the spray solution. After spraying, the sample is recovered, and the content of the tracer on the target is determined by instrumental analysis, and the amount of pesticide deposited on the target is calculated.
  • Commonly used tracers include water-soluble dyes such as lemon yellow, temptation red, and fluorescent tracers such as Brilliant sulphoflavine (BSF) and Pyranin. This method is fast, low-cost, and has low requirements for drug storage. It is currently used for pesticide deposition detection.
  • the detection accuracy of this method is affected by the properties of the tracer, the accuracy is poor, and because the tracer is a colored dye, it is easy to cause color pollution to crops and inspectors during spraying.
  • the above tracer has no specific detection method and low recognition, it is impossible to detect multiple different spray solutions or mixed solutions at the same time.
  • the direct observation method of mist droplets is to receive the deposition mist droplets through water-sensitive paper, oil-sensitive paper, carromite paper card, etc., and observe the particle diameter and distribution of the droplets in the deposition area by image processing; or Direct observation instruments such as laser particle sizers are used to directly observe the properties of the droplets.
  • the water-sensitive paper test method is one of the more commonly used spray detection methods in the industry. This method can visually observe the droplet deposition and distribution status in the field, but the water-sensitive paper changes color when exposed to water, and is easily affected by the environment. It cannot be used in the case of high humidity, and the droplets cannot be quantitatively analyzed.
  • This method is used to detect the sprayed dispersion (water or oil). It has no specificity, and it can not simultaneously detect multiple different samples.
  • Reverse dot blot is a commonly used DNA detection technology, which refers to the use of the specific binding of DNA sequences to fix the complementary strand of the target DNA to be detected on the substrate material.
  • the function of the DNA test sample is to capture the amplified test DNA with a biotin label to realize detection of the test object.
  • reverse dot hybridization mainly detects natural nucleic acid short strands, but artificially designed and synthesized single-stranded DNA has not been used in this technology.
  • field spraying detection process due to the complexity of the field environment, natural short nucleic acid chains may interfere, leading to false positives or false negatives in the test, and it will affect the quantitative determination. It is not suitable for field spraying. Shi detection.
  • the purpose of the present invention is to develop a kit that can be used to simultaneously detect the deposition characteristics of multiple agricultural spray mist droplets by using reverse dot hybridization technology, so as to simultaneously realize fast, high accuracy, easy operation, low cost, and simultaneously Detection of droplet distribution characteristics of each test liquid.
  • the present invention first provides a method that can be used to simultaneously detect the deposition amount of multiple spray droplets.
  • the entire process can be divided into film formation, spray solution preparation, spray application, standard curve establishment, color development, etc. 5 processes.
  • the specific process is as follows: Utilizing the specificity of single-stranded DNA binding with different characteristic sequences, a series of single-stranded DNAs with different characteristic sequences are designed as fixed probes to be fixed on a substrate material to make different target membranes. Next, the corresponding single-stranded DNA with different characteristic sequences is added as a tracer to different spraying solutions. After spraying, the base material is recovered, and the signal is amplified by the amplified signal to obtain different spraying solutions. Droplet size, droplet distribution, and more. Finally, the corresponding deposition amount can be calculated by computer image processing software.
  • the present invention provides a method for simultaneously detecting the loss or deposition of multiple spray mist droplets, including the following steps:
  • transition probes are added to multiple spraying solutions as tracers, and only one type of transition probe is added to each spraying solution;
  • the transition probe in the spraying liquid can specifically bind to the corresponding fixed probe on the detection film, which is fixed with the fixed probe Base material
  • the transition probe is not biotin-modified, and has a nucleotide sequence capable of complementary pairing with its corresponding fixed probe and chromogenic probe, respectively, and the fixed probe and the chromogenic probe do not specifically bind, and different transition probes No specific binding between the needles.
  • the transition probe and the fixed probe are single-stranded DNAs with characteristic sequences; wherein the length of the transition probe is 24-50 nt, the length of the fixed probe is 12-25 nt, and one end of the fixed probe is amino-modified, The other end is covalently bonded to the carboxyl exposed end of the base material.
  • the complementary pairing base of the chromogenic probe and the transition probe is 15-40 nt; if the fixed probe is 5 ′ labeled, the chromogenic probe is 3 ′ biotin labeled, and if the fixed probe is 3 'label, the chromogenic probe is 5' biotin label.
  • the length of the fixed probe is preferably 18-20 nt, and the complementary pairing base of the transition probe and the fixed probe is preferably 15-25 nt, such as 15-20 nt.
  • the reagents are mainly used: 0.1-0.3M (preferably 0.1M) HCl solution, 10-20% (preferably 15%) EDC solution (1- (3-dimethylaminopropyl) ) -3-ethylcarbodiimide), 0.025-0.2 ⁇ M (preferably 0.03 ⁇ M) fixed probe solution, 0.3-1.0M (preferably 0.5M) NaHCO 3 solution, 0.05-0.5M (preferably 0.2) M) NaOH solution.
  • the detection membrane in step (2) is prepared by: cutting the base material according to the required size, cleaning after 0.1-0.3M HCl treatment; adding 10-20% EDC solution to soak and then cleaning; adding the containing 0.025-0.2 ⁇ M fixed probe is immersed in 0.3-1.0M NaHCO 3 solution; then add NaOH solution to soak the treated base material, wash, and dry.
  • the detection membrane is prepared by: cutting the base material according to the required size, cleaning after 0.1M HCl treatment; adding 15% EDC to soak for 0.5-1h and then cleaning; adding a fixed probe containing 0.03 ⁇ M Soak in 0.5M NaHCO 3 solution for 10-20min; add 0.05-0.5M NaOH solution to soak the membrane for 5-15min, wash, and dry.
  • the base material is a nitrocellulose film, a nylon film, a carboxylated modified organic glass or a carboxylated modified polypropylene plastic film.
  • the spraying liquid in step (2) is a pesticide formulation, a liquid fertilizer, other liquid formulations, or water.
  • the spraying liquid formula is: In the spraying liquid preparation process, the spraying liquid mainly contains: 0-60% of a pesticide formulation or a liquid fertilizer (water can also be used directly), 0.025-0.1 ⁇ M (preferably 0.060 ⁇ M). Transition probe, 0-0.045mol / L ion buffer solution, 0-0.15% surfactant (if using pesticide or fertilizer preparation, because it contains surfactant and ion buffer solution, you can directly add the transition probe; If water is used as the spray solution directly, a certain amount of ionic buffer and surfactant should be added).
  • the main dosage forms are water-based preparations and oil-based preparations.
  • the pesticide formulation includes water, oil, wettable powder, microcapsule, water suspension, oil suspension, etc .; the pesticide types include pesticides, fungicides, herbicides, acaricides, nematicides Wait.
  • the liquid fertilizer includes a clear liquid type, a suspension type, a foliar fertilizer, and the like; wherein the type of the fertilizer is one or two or more compound fertilizers of nitrogen fertilizer, phosphate fertilizer, and potassium fertilizer.
  • the transition probe is a single-stranded DNA with a characteristic sequence of 24-50 nt (preferably 36-40 nt).
  • the ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like.
  • the surfactant is sodium alkyl sulfonate, pulverized powder, tea withered powder, saponin powder, SDS (sodium lauryl sulfate), Morwet EFW (sodium butyl naphthalene sulfonate), TERWET 1004, etc. One or more.
  • the reagents used are: hybridization solution, washing solution, 0.05-0.20 ⁇ M color development probe solution, catalase solution, and TMB single-component solution.
  • the main components of the hybridization solution are 0.02-0.045mol / L ion buffer solution, 0.06-0.15% surfactant.
  • the main components of the washing solution are 5.0-10.0 mol / L ion buffer solution, and 0.02-0.20% surfactant.
  • the ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like.
  • the surfactant is sodium alkyl sulfonate, pulverized powder, tea withered powder, saponin powder, SDS (sodium lauryl sulfate), Morwet EFW (sodium butyl naphthalene sulfonate), TERWET 1004, etc. One or more.
  • the chromogenic probe is a single-stranded DNA with a characteristic sequence of 12-25nt (preferably 18-20nt).
  • the main components of the TMB single component solution are: 0.5-2.0mM (preferably 1.0mM) TMB (3,3 ', 5,5'-tetramethylbenzidine), 0.5-2.0mM (preferably 1.0) mM) oxidant, 150-300 mM (preferably 200 mM) ion buffer, 0.1-0.5 mM stabilizer.
  • the specific preparation process is as follows: Solution A: Weigh TMB and stabilizer, add DMSO to dissolve it; Solution B: dissolve with deionized water to prepare an ion buffer solution, add oxidant, and adjust the pH to 4.0-6.0 with hydrochloric acid. After the preparation, the two liquids a and b are prepared according to a certain ratio before use to obtain a TMB single-component liquid.
  • the oxidant is one or more of hydrogen peroxide, urea hydrogen peroxide, peroxyacetic acid, t-butyl hydroperoxide, dimethyldioxane, and the like.
  • the ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like.
  • the stabilizer is one or more of sodium borohydride, sodium cyanoborohydride, tetrabutylammonium borohydride (TBABH), tri-sec-butyl lithium borohydride, lithium borohydride and the like.
  • detection membrane Cut the carboxylized modified nylon membrane according to the required size, clean after 0.1M HCl treatment; add 15% EDC to soak for 1h before cleaning; add 0.5M NaHCO 3 solution containing 0.03 ⁇ M fixed probe Soak for 20min in medium; add 0.2M NaOH solution to soak the membrane for 5-15min, wash, and dry. In this step, detection membranes containing different fixed probes can be separately prepared according to different fixed probes.
  • Spraying process spraying solution preparation, adding pesticide formulation, liquid fertilizer or water to the medicine box, then adding 0.025-0.1 ⁇ M transition probe, and finally adding 0-0.15% of surface according to the needs of pesticide formulation or spraying equipment
  • the active agent and 0-0.045mol / L ion buffer solution are formulated into a transition probe spray solution.
  • the transition probe to be added must be selected in combination with the fixed probe.
  • the detection films containing different fixed probes are laid on the target crops to be sprayed. After spraying, the detection films are recovered separately, and color development is to be performed.
  • a picture file is obtained by taking a picture or scanning, and a gray value per unit area is obtained through image processing software (for example: Photoshop, Image, etc.), and the total gray value under the selected area is calculated. The amount of deposition was converted by a standard curve.
  • the present invention also provides a kit capable of simultaneously detecting the loss or deposition of multiple spray mist droplets, comprising a detection film, a transition probe, and a color development probe; and a detection film, a transition probe
  • a kit capable of simultaneously detecting the loss or deposition of multiple spray mist droplets comprising a detection film, a transition probe, and a color development probe; and a detection film, a transition probe
  • the number of needles and chromogenic probes are both ⁇ 2 and are not the same;
  • the detection membrane is a base material to which a fixed probe is fixed.
  • the length of the fixed probe is 12-25 nt.
  • One end is modified with an amino group, and the other end is covalently bonded to the carboxyl exposed end of the base material. material;
  • the length of the transition probe is 24-50 nt; the 3 'or 5' end of the color development probe is labeled with biotin, and the color development probe can specifically bind to the transition probe and cannot specifically bind to the fixed probe.
  • the length of the chromogenic probe is 12-25 nt.
  • the catalase is a solution of streptavidin-labeled horseradish peroxidase.
  • the main components of the TMB single component solution are: 0.5-2.0mM (preferably 1.0mM) TMB (3,3 ', 5,5'-tetramethylbenzidine), 0.5-2.0mM (preferably 1.0) mM) oxidant, 150-300 mM (preferably 200 mM) ion buffer, 0.1-0.5 mM stabilizer.
  • the specific preparation is as follows: Solution A: Weigh TMB and stabilizer, add DMSO to dissolve it; Solution B: Dissolve with deionized water to prepare an ion buffer solution. After adding an oxidant, adjust the pH to 4.0-6.0 with hydrochloric acid. After the configuration, the two liquids a and b are prepared according to a certain ratio before use to obtain a TMB single-component liquid.
  • the oxidant is one or more of hydrogen peroxide, urea hydrogen peroxide, peroxyacetic acid, t-butyl hydroperoxide, dimethyldioxane, and the like.
  • the ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like.
  • the stabilizer is one or more of sodium borohydride, sodium cyanoborohydride, tetrabutylammonium borohydride (TBABH), tri-sec-butyl lithium borohydride, lithium borohydride and the like.
  • the method of the invention overcomes the limitation that the existing spray droplet detection method cannot test the spray condition of multiple spray liquids at the same time. Utilizing the specificity of single-stranded DNA binding of different characteristic sequences, when spray droplets containing different transition probes are sprayed on the detection membrane, they will only be combined with their corresponding complementary fixed probes, rather than Will be combined with other detection membranes. Next, we use different color-probe probes to perform color-detection on different detection films, and then use computer software to simultaneously obtain information about different droplet properties.
  • the beneficial effects of the detection method of the present invention are mainly reflected in: (1) it can solve the problem of lack of specificity and selectivity of existing tracers; (2) it can simultaneously spray multiple pesticides, mix pesticides and fertilizers, etc.
  • the method of the present invention introduces a transition probe as a tracer, which is colorless and odorless, and solves the problem of color pollution of the environment by water-soluble dyes and fluorescent tracers without pollution; (4) the method of the present invention can The qualitative and quantitative information of the deposition characteristics of the sprayed droplets was obtained through only one inspection.
  • the present invention can solve the complex situation of water, fertilizer, and medicine spray detection in the modern agricultural production process.
  • the specificity of single-stranded DNA binding of different characteristic sequences all spray mists can be obtained in one test.
  • the characteristic information of the droplets are processed by computer software, quantitative deposition detection is realized.
  • FIG. 1 is a principle flow chart of three-stage reverse dot blot hybridization for detecting the spray deposition amount of various spray droplets.
  • FIG. 2A is a result of detection pictures of 5 detection films containing a fixed probe
  • FIG. 2B is a result of establishing a standard curve of the detection method of the present invention.
  • FIG. 3 is an experiment of simultaneously detecting the properties of mist droplets by two probes of Example 2.
  • FIG. 4 is an experiment of simultaneously detecting the properties of fog droplets by using three kinds of probes in Example 3.
  • FIG. 4 is an experiment of simultaneously detecting the properties of fog droplets by using three kinds of probes in Example 3.
  • FIG. 5 is an experiment of simultaneously detecting the properties of fog droplets using the four probes of Example 4.
  • Embodiment 1 Method for Simultaneously Detecting Lost or Deposited Amount of Spraying Drops
  • the entire process can be divided into five processes: film formation, spray solution preparation, spray application, standard curve establishment, and color development.
  • the specific process is as follows: using single-stranded DNA binding with different characteristic sequences to have specificity, a series of single-stranded DNAs with different characteristic sequences are designed as fixed probes to be fixed on a base material (the base material used in this embodiment is nylon Film) to make different target films. Next, the corresponding single-stranded DNA with different characteristic sequences is added as a tracer to different spraying solutions. After spraying, the base material is recovered, and the signal is amplified by the amplified signal to obtain different spraying solutions. Droplet size, droplet distribution, and more. Finally, the corresponding deposition amount can be calculated by computer image processing software.
  • the length of the fixed probe is 12-25 nt, preferably 18-20 nt.
  • One end of the fixed probe is amino modified, and the other end is covalently bound to the carboxyl exposed end of the base material.
  • the transition probe is a single-stranded DNA with a characteristic sequence of 24-50 nt (preferably 36-40 nt) and is not modified by biotin.
  • the complementary base of the transition probe and the fixed probe is 15-25 nt.
  • the chromogenic probe is a single-stranded DNA with a characteristic sequence of 12-25 nt (preferably 18-20 nt).
  • the complementary pairing base of the chromogenic probe and the transition probe is 15-40nt; if the fixed probe is 5 'labeled, the chromogenic probe is 3' biotin labeled, and if the fixed probe is 3 'labeled, the The color probe is 5 'biotin labeled.
  • the chromogenic probe can specifically bind to the transition probe and cannot specifically bind to the fixed probe.
  • the three probe sequences in Table 1 are the probe sequences used in the examples.
  • any single-stranded DNA sequence that meets the above requirements can be used. Probes in this application.
  • transition probe spray solutions (0.02-0.045mol / L ion buffer, 0.06-0.15% surfactant).
  • the main dosage forms are water-based preparations and oil-based preparations.
  • Example 1 In the spraying liquid preparation process, the spraying liquid composition was 30 mM trisodium citrate, 3 mM SDS, and 0.06 ⁇ M transition probe.
  • the probe combination can be selected as the combination 1 in Table 1, that is, the transition probe 1. After spraying, the test films are recovered separately and wait for color development.
  • the hybridization solution is an aqueous solution containing 30 mmol / L trisodium citrate, 26 mmol / L SDS
  • the hybridization solution is an aqueous solution containing 30 mmol / L trisodium citrate, 26 mmol / L SDS
  • the hybridization solution re-add 50 ml of hybridization solution to wash the detection membrane for 2 min
  • Example 1 The method of Example 1 was used to prepare detection films (hereinafter referred to as detection film A and detection film B) containing fixed probe 1 and fixed probe 2 (combination 1 and combination 4), and the spray solution preparation method according to embodiment 1 was prepared. , Prepare the corresponding transition probe spray solution (spray solution A and spray solution B) containing the fixed probe.
  • a petri dish was placed on an iron stand below the traveling track of the spray crane, and each of the Petri dishes contained two detection membranes (one each of A and B), which was in the middle of the traveling track of the spray crane.
  • a spray crane (speed: 5km / h, height: 0.5m) was installed with a Lechler ST110-03 conventional fan-shaped spray nozzle to spray the test detection film (spray liquids A and B once).
  • the experimental materials are collected separately, and the detection film is developed with the corresponding color development probe according to the above-mentioned color development method.
  • the coverage area of the droplets on the detection film and water-sensitive paper is read out by the instrument, the number of droplets and the coverage ratio are calculated.
  • image processing software for example: Photoshop, Image, etc.
  • the amount of deposition was converted by a standard curve.
  • the specific results are shown in Figure 3 and Table 2. From the results shown in Figure 3, there is no interference between the two groups of probes, and the droplets of different spraying methods can be detected at the same time; at the same time, the results of Table 2 show the deposition amount Perform accurate quantification.
  • detection membranes A, B, and C detection membranes containing fixed probe 1, fixed probe 2, and fixed probe 3 (combinations 1, 4, and 7) were prepared, and prepared in accordance with the above-mentioned spray solution.
  • Method: Prepare the corresponding transition probe spray solution (spray solution A, B, C) containing the fixed probe.
  • a petri dish was placed on an iron stand below the running track of the spray crane, and each petri dish contained three detection membranes (one each for A, B, and C). It is in the middle of the running track of the spray crane.
  • Probe A B C Deposition amount ⁇ L / cm 2 2.23 1.97 1.94 Theoretical deposition amount ⁇ L / cm 2 2.86 2.86 2.86 Ratio (calculated value / theoretical value) 0.78 0.69 0.68
  • the detection membranes (hereinafter referred to as the detection membranes A, B, C, and C) containing the fixed probe 1, the fixed probe 2, the fixed probe 3, and the fixed probe 4 (combination 1, 4, 7, 10) are prepared.
  • D) According to the spraying solution preparation method, a corresponding transition probe spraying solution (spraying liquids A, B, C, and D) containing the fixed probe is prepared.
  • a petri dish is placed on an iron stand below the traveling track of the spray crane, and each petri dish contains 4 detection membranes (one for each of A, B, C, and D). It is in the middle of the running track of the spray crane.
  • Probe A B C D Deposition amount ⁇ L / cm 2 2.06 1.97 2.23 2.40 Theoretical deposition amount ⁇ L / cm 2 2.86 2.86 2.86 2.86 Ratio (calculated value / theoretical value) 72% 69% 78% 84%

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Abstract

提供一种同时检测多种喷施雾滴飘失或沉积特性检测的试剂盒及检测方法，本发明试剂盒含有固化有固定探针的检测膜、能够与固定探针特异结合的过渡探针、能够与过渡探针特异结合且生物素标记的显色探针。将过渡探针加到喷施液中作为示踪剂，喷施后过渡探针与检测膜上的固定探针特异性结合；将带有生物素标记的显色探针通过杂交技术结合在过渡探针上，经过显色处理后，根据颜色深浅测定雾滴量，根据显色点的位置和大小确定雾滴沉积参数。

Description

一种同时检测多种喷施雾滴飘失或沉积检测试剂盒及检测方法
本申请要求2018年9月26日向中国国家知识产权局提交的专利申请号为2018111209358，发明名称为“一种同时检测多种喷施雾滴飘失或沉积检测试剂盒及检测方法”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
发明涉及农业喷施雾滴检测技术领域，具体地，涉及针对多种农药、肥料、水等喷施雾滴沉积定性和定量同时检测，开发的反向斑点杂交技术试剂盒以及检测方法。
背景技术
如何利用最少的耕地面积去生产更多优质的作物是农业发展过程中一直存在的问题，尤其是在耕地面积逐渐减少的今天。根据统计，仅仅从2015到2016年一年的时间，全国耕地面积净减少115.3万亩。为了保证农产品稳产增收，合理的浇水、施肥和施药是现代农业中最为重要的环节。“植保一体化”是现在农业中最新提出的种植方案之一，其是指在整个作物的种植栽培过程中，从种子的选育、萌发，再到后期的浇水、施肥、施药，最后的采摘都采用合理的、程序化的、可监控的方式进行，从而优质农产品生产。如何通过简单高效的方法同时地监控浇水、施肥、施药等复杂的过程，对种植有着指导意义；而以上所述的三个过程，多数是通过农用植保机械喷洒雾滴的方式进行，那么对于雾滴的粒径大小、分布情况、飘失/沉积量的监控测定方法有很高的要求。常用的雾滴检测方式主要包括直接测定法、示踪剂法和雾滴直接观测法三类。
其中，直接观测法是指在经过喷施过程后，对样品进行采集，然后通过萃取、净化、浓缩等一系列步骤后，通过HPLC-MS、GC-MS等器对样品上的原药量检测，从而计算出沉积量。虽然该方法精度高，并且可以一次性检测多个原药，但是对于某些无机的肥料却无法用该方式检测，另外，该方法也只能用于沉积量的检测，却无法给出雾滴的粒径、分布情况等信息。
示踪剂法就是在喷施液中加入示踪剂，在喷施结束后，对样品进行回收，通过仪器分析测定靶标上的示踪物含量，推算出靶标上的农药沉积量。常用的示踪剂包括柠檬黄、诱惑红 等水溶性染料以及Brilliant sulphoflavine(BSF)、Pyranin等荧光示踪剂，该方法测试快速，费用低，对药品保存要求不高，是目前农药沉积检测的常用方法之一，但该方法检测准确性受到示踪剂性质的影响，精准度差，且由于示踪剂为有色染料，喷施过程中易对农作物和检测人员造成颜色污染。与此同时，由于以上的示踪剂没有特异性检测方法，识别度低，无法实现同时检测多个不同的喷施液或是混合液。
雾滴直接观测法是通过水敏纸、油敏纸、卡罗米特纸卡等接收沉积雾滴，通过图像处理的方法观测在沉积区域中的雾滴的粒径、分布情况等信息；或者在利用激光粒径仪等直接观测仪器对雾滴性质进行直接观测。其中，水敏纸测试法是目前行业中应用较多的检测喷雾方法之一，该方法能够在现场直观观测雾滴沉积分布状态，但水敏纸遇水变色，易受环境影响，雨天或空气湿度很大的情况下不能使用，且无法对雾滴进行定量分析。该方法是检测喷施的分散液(水或油)，没有特异性，同样无法实现多个不同样品的同时检测。
通过上述三种方法不难看出，在现有的方法中没有任何一种方法可以即可以得到雾滴的粒径、分散情况等信息的同时，又可以准确定量飘失/沉积量，同时还能实现多种样本同时检测。
反向斑点杂交(reverse dot blot，RDB)是常用的DNA检测技术，是指利用DNA序列特异性结合的特点，将目标待测DNA的互补链固定在基底材料上，与提取并扩增后的DNA待测样本作用，捕获扩增后带有生物素标记的待测DNA从而实现对待测物检测。反向斑点杂交在现有的应用中主要检测的是天然核酸短链，而人工设计合成的特征序列的单链脱氧核糖核酸却并未被使用在该技术中。在田间喷施检测过程中，由于田间环境的复杂性，天然的核酸短链可能出现干扰，导致检测中出现假阳性或假阴性的情况，并且会影响定量的测定，并不能够适用于田间喷施检测。
发明内容
本发明的目的在于利用反向斑点杂交技术开发一种可用于同时检测多种农业喷施雾滴沉积特性的试剂盒，从而同时实现快速、准确性高、易于操作、成本低廉、并且同时对多个待测药液雾滴分布特性检测。
本发明首先提供一种可用于同时检测多种喷施雾滴沉积量的方法，如图1所示，整个流程可以分为制膜、喷施液配制、喷施、标准曲线建立、显色等5个流程。具体流程如下：利用不同特征序列的单链脱氧核糖核酸结合具有的特异性，设计出一系列不同特征序列的单链脱氧核糖核酸作为固定探针固定在基底材料上制成不同的靶标膜。接着，将对应的不同特征 序列的单链脱氧核糖核酸作为示踪剂加入到不同的喷施液中去，在喷施后，回收基底材料，通过放大信号显色后，得到不同的喷施液的雾滴粒径、雾滴分布等信息。最后通过计算机图像处理软件处理即可计算出对应的沉积量。
具体地，本发明提供的一种同时检测多种喷施雾滴飘失或沉积量的方法，包括以下步骤：
(1)将不同的过渡探针分别加到多种喷施液中作为示踪剂，每种喷施液中只加一种过渡探针；
(2)喷施有过渡探针的喷施液后，喷施液中的过渡探针能与检测膜上的所对应的固定探针特异性结合，所述检测膜为固定有固定探针的基底材料；
(3)将带有生物素标记的显色探针通过杂交技术结合在对应的过渡探针上，显色处理后，根据颜色深浅测定雾滴量，根据显色点的位置和大小确定多种雾滴飘失或沉积量。
所述过渡探针不经生物素修饰，具有能够分别与其对应的固定探针和显色探针互补配对的核苷酸序列，并且固定探针与显色探针不特异结合，不同的过渡探针之间不特异结合。
所述过渡探针和固定探针均为特征序列的单链脱氧核糖核酸；其中过渡探针的长度为24-50nt，固定探针的长度为12-25nt，固定探针的一端为氨基修饰，另一端与基底材料的羧基裸露端共价结合。
本发明的方法中，显色探针与过渡探针的互补配对碱基为15-40nt；若固定探针是5’标记，则显色探针为3’生物素标记，若固定探针是3’标记，则显色探针为5’生物素标记。
本发明的方法中，固定探针的长度优选为18-20nt，过渡探针与固定探针的互补配对碱基优选为15-25nt，例如15-20nt。
步骤(2)中，主要用到试剂为：0.1-0.3M(优选为0.1M)的HCl溶液，10-20％(优选为15％)EDC溶液(1-(3-二甲基氨基丙基)-3-乙基碳二亚胺)，0.025-0.2μM(优选为0.03μM)固定探针溶液，0.3-1.0M(优选为0.5M)的NaHCO 3溶液，0.05-0.5M(优选为0.2M)的NaOH溶液。
步骤(2)所述检测膜，是通过以下方式制备得到的：根据需要的大小裁剪基底材料，0.1-0.3M的HCl处理后清洗；加入10-20％的EDC溶液浸泡后再清洗；加入含有0.025-0.2μM固定探针的0.3-1.0M NaHCO 3溶液中浸泡处理；再加入NaOH溶液浸泡处理后的基底材料，清洗，晾干即得。
优选地，所述检测膜是通过以下方式制备得到的：根据需要的大小裁剪基底材料，0.1M的HCl处理后清洗；加入15％EDC浸泡0.5-1h后再清洗；加入含有0.03μM固定探针的0.5M NaHCO 3溶液中浸泡10-20min；再加入0.05-0.5M的NaOH溶液浸泡膜5-15min，清洗， 晾干即得。所述基底材料为硝酸纤维素膜、尼龙膜、羧基化改性的有机玻璃或羧基化改性的聚丙烯塑料薄膜。
步骤(2)的喷施液为农药制剂、液体肥料、其他液体制剂或水。喷施液配方为：在喷施液配制流程中，喷施液中主要含有：0-60％的农药制剂或液体肥料(也可直接使用水)，0.025-0.1μM(优选为0.060μM)的过渡探针，0-0.045mol/L的离子缓冲液，0-0.15％的表面活性剂(若是使用农药或肥料制剂，由于本身含有表面活性剂和离子缓冲液，直接添加过渡探针即可；若是直接用水作为喷施液，则需添加一定量的离子缓冲液和表面活性剂)，主要剂型为水基制剂和油基制剂。
所述的农药剂型包括水剂、油剂、可湿性粉剂、微胶囊剂、水悬浮剂、油悬浮剂等；其中农药类型包括杀虫剂、杀菌剂、除草剂、杀螨剂、杀线虫剂等。
所述的液体肥料包括清液型、悬浮型、叶面肥料等；其中肥料类型为氮肥、磷肥、钾肥中的一种或者两种、多种复合肥料。
所述的过渡探针为24-50nt(优选为36-40nt)的特征序列的单链脱氧核糖核酸。
所述的离子缓冲液是一种或多种无机盐、有机盐配成的缓冲溶液，其中溶液阴离子为碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、柠檬酸根、柠檬酸二氢根等的一种或几种，阳离子为钾离子、钠离子、锂离子、钙离子等的一种或几种。
所述的表面活性剂是烷基磺酸钠、拉开粉、茶枯粉、皂角粉、SDS(十二烷基硫酸钠)、Morwet EFW(丁基萘磺酸钠)、TERWET 1004等的一种或几种。
本发明方法的步骤(3)中，使用的试剂有：杂交液、洗液、0.05-0.20μM显色探针溶液、过氧化氢酶液、TMB单组分液。
所述的杂交液主要成分为0.02-0.045mol/L的离子缓冲液，0.06-0.15％的表面活性剂。
所述的洗液主要成分为5.0-10.0mol/L的离子缓冲液，0.02-0.20％的表面活性剂。
所述的离子缓冲液是一种或多种无机盐、有机盐配成的缓冲溶液，其中溶液阴离子为碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、柠檬酸根、柠檬酸二氢根等的一种或几种，阳离子为钾离子、钠离子、锂离子、钙离子等的一种或几种。
所述的表面活性剂是烷基磺酸钠、拉开粉、茶枯粉、皂角粉、SDS(十二烷基硫酸钠)、Morwet EFW(丁基萘磺酸钠)、TERWET 1004等的一种或几种。
所述的显色探针为12-25nt(优选为18-20nt)的特征序列的单链脱氧核糖核酸。
所述的TMB单组分液的主要成分为：0.5-2.0mM(优选为1.0mM)TMB(3,3',5,5'-四甲基联苯胺)，0.5-2.0mM(优选为1.0mM)氧化剂，150-300mM(优选为200mM)离子缓冲液， 0.1-0.5mM稳定剂。具体配制过程如下：a液：称取TMB和稳定剂，加入DMSO将其溶解；b液：用去离子水溶解配成离子缓冲液，加入氧化剂后,用盐酸调节pH至4.0-6.0。配制好后，使用前按一定比例将a、b两液配制得到TMB单组分液。
所述的氧化剂是过氧化氢、尿素过氧化氢、过氧乙酸、叔丁基过氧化氢、二甲基二氧杂环丙烷等一种或几种。
所述的离子缓冲液是一种或多种无机盐、有机盐配成的缓冲溶液，其中溶液阴离子为碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、柠檬酸根、柠檬酸二氢根等的一种或几种，阳离子为钾离子、钠离子、锂离子、钙离子等的一种或几种。
所述的稳定剂是硼氢化钠、氰基硼氢化钠、四丁基硼氢化铵(TBABH)、三仲丁基硼氢化锂、硼氢化锂等的一种或几种。
本发明的实施例中，同时检测多种喷施雾滴飘失或沉积量的方法具体流程如下：
1)制备检测膜：根据需要的大小裁剪羧基化改性的尼龙膜，0.1M的HCl处理后清洗；加入15％EDC浸泡1h后再清洗；加入含有0.03μM固定探针的0.5M NaHCO 3溶液中浸泡20min；再加入0.2M的NaOH溶液浸泡膜5-15min，清洗，晾干即得。该步骤根据固定探针的不同可分别制备含有不同固定探针的检测膜。
2)喷施流程：喷施液配制，在药箱中加入农药制剂、液体肥料或者水，然后加入0.025-0.1μM过渡探针，最后根据农药制剂或喷施设备需要，加入0-0.15％表面活性剂和0-0.045mol/L离子缓冲液配制成过渡探针喷施液。所加入的过渡探针，需结合固定探针进行选择。在待喷施的靶标作物上布好含有不同固定探针的检测膜，经过喷施后，分别回收检测膜，待进行显色处理。
3)显色流程：喷施有过渡探针喷施液的检测膜在杂交液中30-40℃孵育25-40min，加入50ml杂交液清洗2min；将检测膜取出加入到含有对应的显色探针的杂交液30-40℃条件下反应5-15min；将检测膜取出加入50ml洗液清洗检测膜3次；将检测膜取出加入50mL杂交液中清洗2min；取15μL链霉亲和素标记的辣根过氧化氢酶液加入到杂交液中配制酶液，37℃与检测膜酶联反应15-20min；用杂交液冲洗检测膜，将检测膜加入TMB单组分液进行显色反应，TMB单组分显色液会被检测膜上结合的辣根过氧化氢酶催化显色，从而在检测膜上显色。3min后清水冲洗，终止反应，将膜晾干。通过显色可以直接观测到雾滴覆盖密度、雾滴粒径大小等信息。最后利用拍照或扫描得到图片文件，通过图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。通过标准曲线换算出沉积量。
4)标准曲线建立：选5张含有固定探针的检测膜，用移液枪吸取0.5μL的过渡探针喷施液，依次分别在5张检测膜上点1个、2个、3个、4个、5个点，5张检测膜片上探针溶液体积分别为0.5μL，1.0μL，1.5μL，2.0μL，2.5μL。另取一张检测膜，作为空白背景。利用拍照或扫描得到图片文件，最后通过图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。最后以过渡探针溶液体积作为横坐标，总灰度值作为纵坐标，绘制标准曲线，并计算出相应的线性方程。
基于本发明的检测方法，本发明还提供一种能够同时检测多种喷施雾滴飘失或沉积量的试剂盒，含有检测膜，过渡探针、显色探针；且检测膜，过渡探针、显色探针的数量均≥2，且均不相同；
所述检测膜为固定有固定探针的基底材料，固定探针长度为12-25nt，其一端加氨基修饰，另一端与基底材料的羧基裸露端共价结合，所述基底材料为羧基裸露的材料；
所述过渡探针的长度为24-50nt；所述显色探针3’或5’端标记生物素，且显色探针能够与过渡探针特异结合，不能与固定探针特异结合。
所述的显色探针的长度为12-25nt。
所述的过氧化氢酶为链霉亲和素标记的辣根过氧化物酶溶液。
所述的TMB单组分液的主要成分为：0.5-2.0mM(优选为1.0mM)TMB(3,3',5,5'-四甲基联苯胺)，0.5-2.0mM(优选为1.0mM)氧化剂，150-300mM(优选为200mM)离子缓冲液，0.1-0.5mM稳定剂。具体配制如下：a液：称取TMB和稳定剂，加入DMSO将其溶解；b液：用去离子水溶解配成离子缓冲液，加入氧化剂后,用盐酸调节pH至4.0-6.0。配置好后，使用前按一定比例将a、b两液配制得到TMB单组分液。
所述的氧化剂是过氧化氢、尿素过氧化氢、过氧乙酸、叔丁基过氧化氢、二甲基二氧杂环丙烷等一种或几种。
所述的离子缓冲液是一种或多种无机盐、有机盐配成的缓冲溶液，其中溶液阴离子为碳酸根、碳酸氢根、磷酸根、磷酸氢根、磷酸二氢根、柠檬酸根、柠檬酸二氢根等的一种或几种，阳离子为钾离子、钠离子、锂离子、钙离子等的一种或几种。
所述的稳定剂是硼氢化钠、氰基硼氢化钠、四丁基硼氢化铵(TBABH)、三仲丁基硼氢化锂、硼氢化锂等的一种或几种。
本发明方法克服了现有的喷施雾滴检测方法中无法实验同时对多个喷施液喷施情况进行检测的局限性。利用不同特征序列的单链脱氧核糖核酸结合的特异性，当含有不同的过渡探针喷施液雾滴作用检测膜上时，仅仅会和其对应的互补的固定探针的相结合，而不会同其他 的检测膜结合。接着我们利用不同的显色探针对不同的检测膜进行显色检测后，通过计算机软件即可同时得到不同雾滴性质的各个信息。本发明检测方法的有益效果主要体现在：(1)能解决现有示踪剂没有特异性和选择性问题；(2)能同时对多个农药依次喷施、农药和肥料混用等多种复杂情况；(3)本发明方法引入过渡探针为示踪剂，无色无味，解决了水溶性染料、荧光示踪剂对环境的颜色污染问题，对环境无污染；(4)本发明方法可以仅通过一次检测，同时获取喷施雾滴的沉积特性定性、定量信息。
总之，本发明可以解决现代农业生产过程水、肥、药喷施的复杂情况的检测，利用不同特征序列的单链脱氧核糖核酸结合的特异性，通过一次检测即可获取到所有的喷施雾滴的特性信息，并且通过计算机软件处理后，实现沉积定量检测。
附图说明
图1为本发明的三段式反向斑点杂交用于检测多种喷施雾滴喷施沉积量的原理流程图。
图2A为5张含有固定探针的检测膜的检测图片结果，图2B为本发明检测方法标准曲线建立结果。
图3为实施例2两种探针同时检测雾滴性质实验。
图4为实施例3三种探针同时检测雾滴性质实验。
图5为实施例4四种探针同时检测雾滴性质实验。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1同时检测多种喷施雾滴飘失或沉积量的方法
如图1所示，整个流程可以分为制膜、喷施液配制、喷施、标准曲线建立、显色等5个流程。具体流程如下：利用不同特征序列的单链脱氧核糖核酸结合具有特异性，设计出一系列不同特征序列的单链脱氧核糖核酸作为固定探针固定在基底材料(本实施例所用的基底材料为尼龙膜)上制成不同的靶标膜。接着，将对应的不同特征序列的单链脱氧核糖核酸作为示踪剂加入到不同的喷施液中去，在喷施后，回收基底材料，通过放大信号显色后，得到不同的喷施液的雾滴粒径、雾滴分布等信息。最后通过计算机图像处理软件处理即可计算出对应的沉积量。
1、探针的确定
固定探针的长度为12-25nt，优选18-20nt，固定探针的一端为氨基修饰，另一端与基底材料的羧基裸露端共价结合。
过渡探针为24-50nt(优选为36-40nt)的特征序列的单链脱氧核糖核酸，不经生物素修饰。过渡探针与固定探针的互补配对碱基为15-25nt。
显色探针为12-25nt(优选为18-20nt)的特征序列的单链脱氧核糖核酸。显色探针与过渡探针的互补配对碱基为15-40nt；若固定探针是5’标记，则显色探针为3’生物素标记，若固定探针是3’标记，则显色探针为5’生物素标记。
显色探针能够与过渡探针特异结合，不能与固定探针特异结合。表1中的三种探针序列为实施例所用的探针序列，作为示例，除了表1中探针的核苷酸序列外，凡是能满足上述要求的单链脱氧核糖核酸序列都可以用于本申请中的探针。
表1
Figure PCTCN2019108036-appb-000001
Figure PCTCN2019108036-appb-000002
2、制备检测膜
根据需要的大小裁剪表面富含羧基的尼龙膜，0.1M的HCl处理后清洗；加入15％EDC溶液浸泡1h后再清洗；加入含有0.03μM固定探针(例如表1的探针组合1)的0.5M NaHCO 3溶液中浸泡20min；再加入0.2M的NaOH溶液浸泡膜15min，清洗，晾干即得。制备好的检测膜置于待喷施的靶标上，用于收集喷施雾滴和后续检测。
3、喷施液的配制和喷施
在药箱中加入待喷施的制剂(农药制剂、液体肥料、其他液体制剂或者水)，然后加入过渡探针(溶剂为水)，使喷施液中的过渡探针终浓度为0.60μM。不同的喷施液只加入一种过渡探针，且各个过渡探针均不相同。最后根据农药制剂、液体肥料或喷施设备需要，加入表面活性剂和离子缓冲液配制成过渡探针喷施液(0.02-0.045mol/L的离子缓冲液，0.06-0.15％的表面活性剂)，主要剂型为水基制剂和油基制剂。实施例1在喷施液配制流程中，喷施液成分为30mM柠檬酸三钠，3mM SDS，0.06μM过渡探针。探针组合可以选择为表1的组合1，即过渡探针1。经过喷施后，分别回收检测膜后，待显色。
4、本发明标准曲线的建立
选5张含有固定探针的检测膜，用移液枪吸取0.5μL的过渡探针喷施液，依次分别在5张检测膜上点1-5个点，5张检测膜片上过渡探针溶液体积分别为0.5μL，1.0μL，1.5μL，2.0μL，2.5μL。另取一张检测膜，作为背景。利用拍照或扫描得到图片文件(图2A)，最 后通过图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。最后以药液体积作为横坐标，总灰度值作为纵坐标，绘制标准曲线，并计算出相应的线性方程，结果于图2B所示。
5、显色
将喷施有过渡探针喷施液的检测膜收集后，加入50mL杂交液(杂交液成分为含有30mmol/L柠檬酸三钠，26mmol/L SDS的水溶液)中，37℃孵育40min；去掉孵育杂交液后，重新加入50ml杂交液洗涤检测膜2min；取出检测膜加入到含有显色探针的杂交液中37℃条件下反应15min后，取出检测膜用50ml洗液(洗液成分为含有7.5mmol/L柠檬酸三钠，6mmol/L SDS的水溶液)中清洗3次，50mL杂交液冲洗1次。取15μL链霉亲和素标记的辣根过氧化氢酶液加入到杂交液中配置酶液，加入到之前清洗好的检测膜中，37℃酶联反应20min；接着用50mL杂交液冲洗检测膜，将检测膜加入到TMB单组分液中进行显色反应，TMB单组分显色液会被检测膜上结合的辣根过氧化氢酶催化显色，从而在检测膜上显色。3min后清水冲洗，终止反应，将膜晾干。通过显色可以直接观测到雾滴覆盖密度、雾滴粒径大小等信息。最后利用拍照或扫描得到图片文件，通过图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。
实施例2天车模拟田间喷施——两种探针同时检测实验
采用实施例1的方法分别制备含有固定探针1和固定探针2(组合1和组合4)的检测膜(以下称为检测膜A和检测膜B)，按照实施例1喷施液配制方法，配制含有上述固定探针的对应过渡探针喷施液(喷施液A和喷施液B)。在喷雾天车运行轨迹下方的铁架台上放置培养皿，每个培养皿中含有2片检测膜(A和B各1片)，处于喷雾天车运行轨迹正中间。在压力3bar下，使用喷雾天车(速度：5km/h、高度：0.5m)安装Lechler ST110-03常规扇形雾喷头对供试检测膜进行喷雾(喷施液A和B各喷1次)。待喷雾结束后分别收集实验材料，按照上述的显色方法对检测膜分别用配套的显色探针进行显色。通过仪器分别读出检测膜和水敏纸上的雾滴覆盖面积后，计算出雾滴数和覆盖率。通过拍照或扫描等方法得到数字化的图像后，利用图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。通过标准曲线换算出沉积量。具体结果见图3、表2，从图3的结果来看，2组探针之间不存在干扰，可以同时检测多种不同喷施方式的雾滴；同时表2结果显示对雾滴沉积量进行准确定量。
表2两种探针同时检测雾滴性质实验
探针 A B
沉积量μL/cm 2 2.40 2.06
理论沉积量μL/cm 2 2.86 2.86
比率(计算值/理论值) 0.84 0.72
实施例3天车模拟田间喷施——三种探针同时检测实验
如上所述分别制备含有固定探针1、固定探针2和固定探针3(组合1、4、7)的检测膜(以下称为检测膜A、B、C)，按照上述喷施液配制方法，配制含有上述固定探针的对应过渡探针喷施液(喷施液A、B、C)。在喷雾天车运行轨迹下方的铁架台上放置培养皿，每个培养皿中含有3片检测膜(A、B、C各一片)。处于喷雾天车运行轨迹正中间。在压力3bar下，使用喷雾天车(速度：5km/h、高度：0.5m)安装Lechler ST110-03常规扇形雾喷头对供试检测膜进行喷雾(喷施液A、B、C各喷一次)。待喷雾结束后分别收集实验材料，按照上述的显色方法对检测膜分别用配套的显色探针进行显色。通过仪器分别读出检测膜和水敏纸上的雾滴覆盖面积后，计算出雾滴数和覆盖率。通过拍照或扫描等方法得到数字化的图像后，利用图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。通过标准曲线换算出沉积量。(具体结果见图4，表3)，从图4的结果来看，3组的探针之间不存在干扰，可以同时检测多种不同喷施方式的雾滴；同时表2结果显示对雾滴沉积量进行准确定量。
表3三种探针同时检测雾滴性质实验
探针 A B C
沉积量μL/cm 2 2.23 1.97 1.94
理论沉积量μL/cm 2 2.86 2.86 2.86
比率(计算值/理论值) 0.78 0.69 0.68
实施例4天车模拟田间喷施——四种探针同时检测实验
如上所述分别制备含有固定探针1、固定探针2、固定探针3和固定探针4(组合1、4、7、10)的检测膜(以下称为检测膜A、B、C、D)，按照上述喷施液配制方法，配制含有上述固定探针的对应过渡探针喷施液(喷施液A、B、C、D)。在喷雾天车运行轨迹下方的铁架台上放置培养皿，每个培养皿中含4片检测膜(A、B、C、D各一片)。处于喷雾天车运行轨迹正中间。在压力3bar下，使用喷雾天车(速度：5km/h、高度：0.5m)安装Lechler ST110-03常规扇形雾喷头对供试检测膜进行喷雾(喷施液A、B、C、D各喷一次)。待喷 雾结束后分别收集实验材料，按照上述的显色方法对检测膜分别用配套的显色探针进行显色。通过仪器分别读出检测膜和水敏纸上的雾滴覆盖面积后，计算出雾滴数和覆盖率。通过拍照或扫描等方法得到数字化的图像后，利用图像处理软件(例如：Photoshop、Image J等)获取单位面积的灰度值，并计算选取面积下的总灰度值。通过标准曲线换算出沉积量。(具体结果见图5，表4)从图5的结果来看，4组的探针之间不存在干扰，可以同时检测多种不同喷施方式的雾滴；同时表3结果显示对雾滴沉积量进行准确定量。
表4四种探针同时检测雾滴性质实验
探针 A B C D
沉积量μL/cm 2 2.06 1.97 2.23 2.40
理论沉积量μL/cm 2 2.86 2.86 2.86 2.86
比率(计算值/理论值) 72％ 69％ 78％ 84％

Claims (10)

  1. 一种同时检测多种喷施雾滴飘失或沉积量的方法，其特征在于，包括以下步骤：
    (1)将不同的过渡探针分别加到多种喷施液中作为示踪剂，每种喷施液中只加一种过渡探针，得到含过渡探针的喷施液；
    (2)喷施含过渡探针的喷施液后，喷施液中的过渡探针能与检测膜上的所对应的固定探针特异性结合，所述检测膜为固定有固定探针的基底材料；
    (3)将带有生物素标记的显色探针通过杂交技术结合在对应的过渡探针上，显色处理后，根据颜色深浅测定雾滴量，根据显色点的位置和大小确定多种雾滴飘失或沉积量；
    所述过渡探针不经生物素修饰，具有能够分别与其对应的固定探针和显色探针互补配对的核苷酸序列，并且固定探针与显色探针不特异结合，不同的过渡探针之间不特异结合。
  2. 如权利要求1所述的方法，其特征在于，所述过渡探针和固定探针均为特征序列的单链脱氧核糖核酸；其中过渡探针的长度为24-50nt，固定探针的长度为12-25nt，固定探针的一端为氨基修饰，另一端与基底材料的羧基裸露端共价结合。
  3. 如权利要求1所述的方法，其特征在于，显色探针与过渡探针的互补配对碱基为15-40nt；若固定探针是5’标记，则显色探针为3’生物素标记，若固定探针是3’标记，则显色探针为5’生物素标记。
  4. 如权利要求1所述的方法，其特征在于，固定探针的长度为18-20nt，过渡探针与固定探针的互补配对碱基为15-25nt。
  5. 如权利要求1-4任一所述的方法，其特征在于，步骤(2)所述检测膜，是通过以下方式制备得到的：根据需要的大小裁剪基底材料，0.1-0.3M的HCl处理后清洗；加入10-20％的EDC溶液浸泡后再清洗；加入含有0.025-0.2μM固定探针的0.3-1.0M NaHCO 3溶液中浸泡处理；再加入NaOH溶液浸泡处理后的基底材料，清洗，晾干即得。
  6. 如权利要求5所述的方法，其特征在于，所述检测膜是通过以下方式制备得到的：根据需要的大小裁剪基底材料，0.1M的HCl处理后清洗；加入15％EDC浸泡0.5-1h后再清洗；加入含有0.03μM固定探针的0.5M NaHCO 3溶液中浸泡10-20min；再加入0.05-0.5M的NaOH溶液浸泡处理后的基底材料5-15min，清洗，晾干即得。
  7. 如权利要求5所述的喷施雾滴飘失或沉积量检测方法，其特征在于，所述基底材料为硝酸纤维素膜、尼龙膜、羧基化改性的有机玻璃或羧基化改性的聚丙烯塑料薄膜。
  8. 如权利要求1-4任一所述的喷施雾滴飘失或沉积量检测方法，其特征在于，步骤(2)含有过渡探针的喷施液中过渡探针的最终浓度为0.025-0.1μM。
  9. 一种能够同时检测多种喷施雾滴飘失或沉积量的试剂盒，其特征在于，含有检测膜，过渡探针、显色探针；且检测膜，过渡探针、显色探针的数量均≥2，且均不相同，所述显色探针3’或5’端标记生物素，且显色探针能够与过渡探针特异结合，不能与固定探针特异结合；
    优选，所述检测膜为固定有固定探针的基底材料，固定探针长度为12-25nt，其一端加氨基修饰，另一端与基底材料的羧基裸露端共价结合，所述基底材料为羧基裸露的材料；
    优选，所述过渡探针的长度为24-50nt。
  10. 如权利要求9所述的试剂盒，其特征在于，所述试剂盒还含有3,3',5,5'-四甲基联苯胺TMB单组分液，链霉亲和素标记的辣根过氧化物酶。
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