WO1998006084A1 - A method of nucleic acid code analystic technique used in falseproof label - Google Patents

A method of nucleic acid code analystic technique used in falseproof label Download PDF

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Publication number
WO1998006084A1
WO1998006084A1 PCT/CN1997/000078 CN9700078W WO9806084A1 WO 1998006084 A1 WO1998006084 A1 WO 1998006084A1 CN 9700078 W CN9700078 W CN 9700078W WO 9806084 A1 WO9806084 A1 WO 9806084A1
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WIPO (PCT)
Prior art keywords
nucleic acid
dna
label
solid
counterfeiting
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PCT/CN1997/000078
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French (fr)
Chinese (zh)
Inventor
Su Han
Xinghua Guo
Xueyuan Jin
Xiaozhou Shen
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Beijing Sanzhu Xinda Biological Probe Co., Ltd.
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Application filed by Beijing Sanzhu Xinda Biological Probe Co., Ltd. filed Critical Beijing Sanzhu Xinda Biological Probe Co., Ltd.
Priority to AU37648/97A priority Critical patent/AU3764897A/en
Publication of WO1998006084A1 publication Critical patent/WO1998006084A1/en

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    • GPHYSICS
    • G09EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
    • G09FDISPLAYING; ADVERTISING; SIGNS; LABELS OR NAME-PLATES; SEALS
    • G09F3/00Labels, tag tickets, or similar identification or indication means; Seals; Postage or like stamps
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention applies the nucleic acid code analysis technology to the invisible anti-counterfeiting mark of goods or other objects, and belongs to the technical field of cryptography methods.
  • anti-counterfeiting marks are mostly products of physical or chemical nature, such as laser holograms, optical discoloration films, image scramblers, thermally variable inks, magnetic stripes, and software.
  • anti-counterfeiting products made by biotechnology have begun to appear internationally. These products mainly adopt the principle of antigen-antibody reaction, which is more accurate and sensitive than ordinary anti-counterfeiting products and methods.
  • the antigen antibody is a protein, which has poor stability, and is easy to inactivate especially in a high temperature environment, which will reduce the sensitivity and reliability of anti-counterfeiting.
  • the antigen-antibody response changes little, and once one of the components is known, it is easy to be counterfeited.
  • the purpose of the present invention is to overcome the defect that the existing mark is easy to be counterfeited in the technical field of anti-counterfeiting, and to design an invisible labeling method in which the nucleic acid code analysis technology in genetic engineering is applied to anti-counterfeiting.
  • the present invention is based on the principle of genetic engineering, and proposes a new invisible anti-counterfeiting design and technology, the core of which is the biological macromolecule-nucleic acid.
  • the genetic material (genes) of all life are nucleic acids, that is, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • Nucleic acids are composed of four kinds of alternating arrangements of bases. The difference in the arrangement and combination of these forms of life on earth There are many types. A DNA with only 1 000 base pairs (1 kb) may have 10 6 ° two permutations and combinations.
  • Species existing in nature are numerous security genome length of each species is generally greater than 104 to 1 0 6 kb. If a nucleic acid with a length of 1 kb is set as an anti-counterfeiting mark, the available nucleic acid code is 10 6 X (1 0 4-1 0 6 ) ⁇ IO 1 0- ] 0 1 2 as many. Therefore, biodiversity provides an inexhaustible source of anti-counterfeiting technology. With the huge amount of information such as DNA, hundreds of millions of passwords can be used for anti-counterfeiting designs.
  • the present invention is a nucleic acid fragments preselected some (mainly DNA fragments, because very stable) in an amount (as little to 10--8 g) was dissolved in the solution and applied to a variety of solid support, After evaporative drying, the nucleic acid fragment is attached to the carrier.
  • the solid support can be a variety of materials, such as paper or films made of various natural or synthetic fibers, porous particles or powders, natural or synthetic leather, plastics, or various organic or inorganic polymers and resins, Solid paraffin, natural or synthetic inorganic substances such as ceramics, glass, crystal, metal, diatomaceous earth, etc.
  • the nucleic acid fragment can also be directly added to the liquid product as a solution as an invisible anti-counterfeiting mark, or the nucleic acid fragment can also be placed in a variety of liquid carriers such as ink, ink, paint or adhesive to form an anti-counterfeiting mark. Adhere to merchandise or other packaging.
  • a protective layer is added on the surface of the carrier containing the nucleic acid fragment, for example, a plastic film,
  • the invisible anti-counterfeit of nucleic acid made of fucose and the like can be fixed on a commodity or an object or on a package and an accessory.
  • nucleic acid anti-counterfeiting mark When inspection is required, remove the anti-counterfeiting mark, remove the protective layer, and dissolve the nucleic acid fragments attached to the carrier with a buffer solution.
  • Detecting personnel are aware of the types of nucleic acid anti-counterfeiting marks on goods or objects, and can use specific nucleic acid molecular hybridization or polymerase chain reaction (PCR) specific primers for detection. If the hybridization signal or PCR product with the correct length (PCR specific primer method) can be displayed, it can be judged that the detected commodity or object is genuine, otherwise it is a fake or fake.
  • PCR specific primer method polymerase chain reaction
  • nucleic acid code is colorless and hard to see with the naked eye.
  • code comes from the huge gene bank in nature. Even if you know the code of the gene but do n’t know which one, the counterfeiter cannot decipher and forge it. For inspectors, as long as they are tested with a dedicated analysis method, the authenticity is clear at a glance.
  • nucleic acid codes can be easily designed as anti-counterfeiting markers, each of which has its own corresponding probes and primers, which are mutually exclusive. Therefore, the nucleic acid code can provide a unique anti-counterfeiting marking system for a variety of goods and objects.
  • the D N A password is stable, especially the dry D N A is stored for a long time, it is not easy to decompose, and it is suitable for the security of some long-term storage objects.
  • the detection and detection method used to distinguish the authenticity of the object is an inseparable technical content of the nucleic acid code anti-counterfeiting method.
  • the present invention uses a specific nucleic acid molecule hybridization method or a polymerase chain reaction (P C R) method for detection and detection.
  • Figure 1 is a schematic diagram of nucleic acid molecular hybridization method to verify the authenticity of DNA anti-counterfeiting marks.
  • A is blank (no DNA);
  • B is other non-specific DNA, 10 micrograms;
  • C is C ⁇ T coding gene, 0.0 2 microgram.
  • FIG. 2 is a schematic diagram of the P C R method to verify the authenticity of the D N A anti-counterfeiting mark.
  • A is blank (without D N A template);
  • B is non-specific D N A, 1 microgram;
  • C is lambda phage D N A template '. 0 1 microgram
  • Example 1 The reliability and specificity of anti-counterfeiting marks depends on accurate methods of forgery detection.
  • the present embodiment uses a method of hybridizing nucleic acid molecules.
  • DN A is a double-helix structure of two nucleotide strands that are coiled together in opposite directions.
  • the two nucleotide chains depend on the hydrogen bonds on the four bases of adenine (A) and thymine (T) and guanine (G) and cytosine (C). Therefore, the binding between DNA-DNA or DNA-RN A chains is called "molecular hybridization", and its stability depends entirely on the strict complementarity between the double-stranded bases.
  • a and T are complementary (in DNA-RNA) hybrids
  • a and U are complementary
  • G and C are complementary.
  • Single-stranded DNA or RNA molecular probes hybridize only to their DNA strands that are complementary to their homology.
  • Solid-phase hybridization The denatured DNA membrane is placed in the hybridization solution, and a nucleic acid probe (isotopic probe or non-isotopic chemiluminescence probe) is added to the hybridization solution for 4 hours or overnight in a 65'C hybridization solution. Washing to remove non-specifically bound probes, and finally exposing them with X-ray film;
  • P CR Polymerase chain reaction
  • T aq DNA polymerase thermostable bacteria DNA polymerase
  • the PCR reaction consists of a specific one or a pair of primers and a template DNA. A very small amount of DNA is placed on the mark of the anti-counterfeit object. The DNA is removed during the detection and dissolved in a certain amount of buffer solution.
  • T aq DA polymerase and other reaction solutions Use PCR to perform amplification reactions.
  • the DNA amplification reaction is the key to anti-counterfeiting technology. Enzymes and other methods that can make DNA amplified can also achieve the purpose.
  • a part of the reaction solution was taken out for electrophoresis analysis.
  • the product should be an amplified D N A fragment of a predetermined size, which can be separated by agarose gel electrophoresis, then stained with ethidium bromide, observed and identified under ultraviolet light, and the operation process is briefly described as follows:
  • reaction components (1) DNA template (about 0. () micrograms), (2) one or more specific primers, (3) T aq DNA polymerase or other DNA polymerase; (4) four Deoxynucleoside triphosphates (d AT P, d TTP, d CT P, and d GT P), (5) a reaction buffer, 2. Reaction temperature and procedure:
  • Electrophoretic separation Take 10 microliters of the reaction solution, place it in a 2% agarose gel (containing ethidium bromide dye), and perform electrophoresis with a DC voltage of 100 V. After electrophoresis, remove the agarose gel and place it in Observe and photograph under a UV light of 2 50-3 0 0 m.
  • Embodiment 2 is only a general example, and is not a fixed operation procedure. In practice, it often varies with the design of the D N A die and the primer, otherwise the expected results cannot be achieved. This strict operating condition is also very beneficial to anti-counterfeiting, because the anti-counterfeit cannot know the reaction conditions and cannot decode the password.
  • the nucleic acid anti-counterfeiting label of the present invention has imitation, reliability, diversity, stability, and wide applicability, and has high industrial applicability.

Abstract

To overcome the disadvantage that prior labels are readily falsified, a method of nucleic acid code analystic technique used in an invisible falseproof label of goods or items is disclosed, which belongs to the field of the code technique. A selected nucleic acid fragment (especially DNA fragment) is dissolved in a solution and applied to various solid supports, then attached on the support after being dried. As a falseproof label, it is labelled directly on various solid goods, items or packages and auxiliaries thereof. Nucleic acid can be also incorporated directly into liquid goods or various liquid supports to form an invisible falseproof label. The detection method is nucleic acid molecule hybridization probe assays or polyase chain reaction specific primer assays.

Description

核酸密码分析技术应用于防伪的方法 技术领域  Method for applying nucleic acid code analysis technology to anti-counterfeiting
本发明是把核酸密码分析技术应用在商品或其他物件的隐形防伪标记上, 属于 密码术方法的技术领域。  The invention applies the nucleic acid code analysis technology to the invisible anti-counterfeiting mark of goods or other objects, and belongs to the technical field of cryptography methods.
背景技术 Background technique
传统的防伪标记大多是物理或化学性质的制品, 如激光全息、 光学变色膜、 图 象扰频、 热变油墨、 磁条、 软件等。 目前国际上开始出现用生物技术制作的防伪产 品, 这些产品主要采用抗原抗体反应原理, 比一般的防伪产品和方法准确、 灵敏。 但抗原抗体是蛋白质, 稳定性较差, 尤其在高温环境中容易失活, 从而将降低防伪 的灵敏度和可靠性。 此外, 抗原抗体反应变化少, 一旦知道其中一种成分, 就容易 被伪制。  Traditional anti-counterfeiting marks are mostly products of physical or chemical nature, such as laser holograms, optical discoloration films, image scramblers, thermally variable inks, magnetic stripes, and software. At present, anti-counterfeiting products made by biotechnology have begun to appear internationally. These products mainly adopt the principle of antigen-antibody reaction, which is more accurate and sensitive than ordinary anti-counterfeiting products and methods. However, the antigen antibody is a protein, which has poor stability, and is easy to inactivate especially in a high temperature environment, which will reduce the sensitivity and reliability of anti-counterfeiting. In addition, the antigen-antibody response changes little, and once one of the components is known, it is easy to be counterfeited.
发明内容 Summary of the Invention
本发明的目的就是在防伪的技术领域克服已有标记容易被伪制的缺陷, 设计出 将基因工程中的核酸密码分析技术应用于防伪的隐形标记方法。  The purpose of the present invention is to overcome the defect that the existing mark is easy to be counterfeited in the technical field of anti-counterfeiting, and to design an invisible labeling method in which the nucleic acid code analysis technology in genetic engineering is applied to anti-counterfeiting.
本发明是基于基因工程原理, 提出全新的隐形防伪设计和技术, 其核心是生物 大分子-核酸。 人们知道, 一切生命的遗传物质 (基因) 都是核酸, 也就是脱氧核 糖核酸 (D N A ) 或核糖核酸 (R N A ) , 核酸由四种碱基交互排列组成, 其排列 组合的差异, 形成地球上生命类型的多种多样。 一条仅仅 1 0 0 0 碱基对 (1 k b ) 的 D N A即可能有 1 0 6 ° 2 种排列组合方式。 现存自然界的生物种类 (动植物和 微生物) 在一百万种以上, 因此可以利用作防伪的天然生物基因多不胜 每个物 种基因组的长度一般均大于 1 0 4 至 1 0 6 k b。 假若设定 1 k b 长度的核酸作防 伪标记, 则可供选择的核酸密码就有 1 0 6 X (1 0 4 - 1 0 6 ) ^ I O 1 0 - ] 0 1 2 之多。 因此, 生物多样性为防伪技术提供了取之不尽的来源。 利用 D N A这 样巨大的信息量可以有数以亿万计的密码可用于防伪设计。 The present invention is based on the principle of genetic engineering, and proposes a new invisible anti-counterfeiting design and technology, the core of which is the biological macromolecule-nucleic acid. It is known that the genetic material (genes) of all life are nucleic acids, that is, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids are composed of four kinds of alternating arrangements of bases. The difference in the arrangement and combination of these forms of life on earth There are many types. A DNA with only 1 000 base pairs (1 kb) may have 10 6 ° two permutations and combinations. Species existing in nature (animals, plants and microorganisms) in one million kinds of the above, it is possible to make use of natural biological gene are numerous security genome length of each species is generally greater than 104 to 1 0 6 kb. If a nucleic acid with a length of 1 kb is set as an anti-counterfeiting mark, the available nucleic acid code is 10 6 X (1 0 4-1 0 6 ) ^ IO 1 0- ] 0 1 2 as many. Therefore, biodiversity provides an inexhaustible source of anti-counterfeiting technology. With the huge amount of information such as DNA, hundreds of millions of passwords can be used for anti-counterfeiting designs.
本发明是将预先选定的某种核酸片段 (主要是 D N A片段, 因其非常稳定) 以 一定量 (可少到 1 0 - 8 克) 溶于溶液中并加到各种固相载体上, 待蒸发干燥后核 酸片段即附着在载体上。 固相载体可以是各种各样的材料, 如各种天然或人造纤维 制成的纸张或薄膜、 多孔颗粒或粉末, 天然或合成的皮革、 塑料或各种有机或无机 多聚体和树脂、 固体石腊、 天然或合成的无机物质如陶瓷、 玻璃、 水晶、 金属、 硅 藻土等。 另外, 核酸片段也可以溶液形式直接加入液态商品中作为隐形防伪标记, 或者核酸片段也可置于各种墨水、 印油、 涂料或粘着剂等液态载体中形成防伪标记, 通过各种液态载体印刷、 粘附在商品或其它包装上。 为了保护固相载体上作为防伪 标志的核酸片段, 在含有核酸片段的载体表面加上保护层, 例如可用塑料薄膜, 海 藻糖等, 制成的这种核酸隐形防伪记可固定在商品或物件上或 包装物及附属物上。 当需要检验时, 将防伪标记取下, 揭去保护层, 用缓冲液溶解附着在载体上的核酸 片段。 检测人员已知检测商品或物件上核酸防伪标记的种类, 就可用特定的核酸分 子杂交或聚合酶链反应 (P C R ) 特定引物进行探测。 如能显出杂交信号或具有正 确长度的 P C R产物 (P C R特定引物法) , 即能判定检测商品或物件为真品, 否 则为假冒的伪品或膺品。 利用核酸密码作隐形防伪标记有如下优点: The present invention is a nucleic acid fragments preselected some (mainly DNA fragments, because very stable) in an amount (as little to 10--8 g) was dissolved in the solution and applied to a variety of solid support, After evaporative drying, the nucleic acid fragment is attached to the carrier. The solid support can be a variety of materials, such as paper or films made of various natural or synthetic fibers, porous particles or powders, natural or synthetic leather, plastics, or various organic or inorganic polymers and resins, Solid paraffin, natural or synthetic inorganic substances such as ceramics, glass, crystal, metal, diatomaceous earth, etc. In addition, the nucleic acid fragment can also be directly added to the liquid product as a solution as an invisible anti-counterfeiting mark, or the nucleic acid fragment can also be placed in a variety of liquid carriers such as ink, ink, paint or adhesive to form an anti-counterfeiting mark. Adhere to merchandise or other packaging. In order to protect the nucleic acid fragment on the solid-phase carrier as an anti-counterfeiting mark, a protective layer is added on the surface of the carrier containing the nucleic acid fragment, for example, a plastic film, The invisible anti-counterfeit of nucleic acid made of fucose and the like can be fixed on a commodity or an object or on a package and an accessory. When inspection is required, remove the anti-counterfeiting mark, remove the protective layer, and dissolve the nucleic acid fragments attached to the carrier with a buffer solution. Detecting personnel are aware of the types of nucleic acid anti-counterfeiting marks on goods or objects, and can use specific nucleic acid molecular hybridization or polymerase chain reaction (PCR) specific primers for detection. If the hybridization signal or PCR product with the correct length (PCR specific primer method) can be displayed, it can be judged that the detected commodity or object is genuine, otherwise it is a fake or fake. The use of nucleic acid code as an invisible anti-counterfeiting mark has the following advantages:
1 、 不可仿制性: 首先核酸密码无色, 肉眼难以看到, 其次, 密码来自自然界 庞大的基因库, 即使知道是基因的密码但不知是哪一种, 仿冒者也无法破译和伪造。 而对检验人员来说, 只要以专用的分析方法测试, 真伪则一目了然。  1. Imitation: Firstly, the nucleic acid code is colorless and hard to see with the naked eye. Secondly, the code comes from the huge gene bank in nature. Even if you know the code of the gene but do n’t know which one, the counterfeiter cannot decipher and forge it. For inspectors, as long as they are tested with a dedicated analysis method, the authenticity is clear at a glance.
2 、 可靠性: 只有用按预定的核酸密码设计的探针或 P C R引物进行检验, 才 有可能探测出密码的存在与密码的种类。 所以核酸密码具有极高的专一性。  2. Reliability: Only by using probes or PCR primers designed according to the predetermined nucleic acid code to test, it is possible to detect the existence of the code and the type of the code. Therefore, the nucleic acid code is highly specific.
3 、 多样性: 可以很容易设计出千万种核酸密码作为防伪标记, 每种都有自身 相应的探针和引物, 互不通用。 所以核酸密码可为多种多样的商品和物件分别提供 独特的防伪标记体系。  3. Diversity: Tens of thousands of nucleic acid codes can be easily designed as anti-counterfeiting markers, each of which has its own corresponding probes and primers, which are mutually exclusive. Therefore, the nucleic acid code can provide a unique anti-counterfeiting marking system for a variety of goods and objects.
4 、 稳定性: D N A密码稳定, 特别是干燥的 D N A存放经久, 不易分解, 适 于一些长期保存物件的防伪。  4. Stability: The D N A password is stable, especially the dry D N A is stored for a long time, it is not easy to decompose, and it is suitable for the security of some long-term storage objects.
5 、 广泛的适用性; 核酸密码只需极微量 (1 0 - 8 - 1 0 — 1 ° 克) 存在于 商品或物件中即可探测出来, 因此适用于各类贵重的商品和物件, 如科学仪器和家 用电器; 高档服装、 家具及文体、 文娱用品; 高级烟酒、 食品及营养品; 农业和园 艺的珍奇或重要种子; 重要的证件和文件; 经鉴定的珍贵文件和艺术品等等, 几乎 所有固态的物件上都可适用。 5, a wide range of applicability; just a trace amount of nucleic acid code (1 0 - 8 - 1 0 - 1 ° gram) present in the article of merchandise or to detect it, and therefore applicable to all types of valuable articles and goods, as SCIENCES Instruments and household appliances; high-end clothing, furniture and sports, cultural and recreational supplies; high-end tobacco, alcohol, food and nutrition; rare or important seeds for agriculture and horticulture; important documents and documents; valuable documents and artworks that have been identified, etc. Applicable to almost all solid objects.
探测检验手段用来辨别物件的真伪是核酸密码防伪方法不可分割的技术内容。 已如前述, 本发明采用特定的核酸分子杂交法或聚合酶链反应 (P C R ) 法进行探 测检验。  The detection and detection method used to distinguish the authenticity of the object is an inseparable technical content of the nucleic acid code anti-counterfeiting method. As mentioned above, the present invention uses a specific nucleic acid molecule hybridization method or a polymerase chain reaction (P C R) method for detection and detection.
附图简述 Brief description of the drawings
图 1 为核酸分子杂交法检验 D N A防伪标记真伪的示意图, 图中, A为空白 ( 无 D N A ) ; B为其他非特异 D N A , 1 0 微克; C为 C Λ T编码基因, 0 . 0 2 微克。  Figure 1 is a schematic diagram of nucleic acid molecular hybridization method to verify the authenticity of DNA anti-counterfeiting marks. In the figure, A is blank (no DNA); B is other non-specific DNA, 10 micrograms; C is C Λ T coding gene, 0.0 2 microgram.
图 2 为 P C R法检验 D N A防伪标记真伪的示意图, 图中, A为空白 (无 D N A模板) ; B为非特异 D N A, 1 微克; C为 lambda噬菌体 D N A模板' 0 . 0 1 微克  Figure 2 is a schematic diagram of the P C R method to verify the authenticity of the D N A anti-counterfeiting mark. In the figure, A is blank (without D N A template); B is non-specific D N A, 1 microgram; C is lambda phage D N A template '. 0 1 microgram
本发明的最佳实施方式  Best Mode of the Invention
下面结合附图和实施例更详细地描述核酸密码分析防伪技术。  The anti-counterfeiting technology of nucleic acid cryptanalysis is described in more detail below with reference to the drawings and examples.
实施例 1 防伪标记是否可靠专一, 取决于精确的辩伪检验方法。 本卖施例采用核酸分子 杂交的方法。 DN A是两条走向相反而盘绕在一起的核苷酸链, 为双螺旋结构。 两 条核苷酸链之间依赖腺嘌呤 (A) 与胸腺嘧啶 (T ) 以及鸟嘌呤 (G) 与胞嘧啶 ( C) 四种碱基上的氢键维系。 因此, DN A - DN A或 DN A -RN A链之间的结 合即所谓的 "分子杂交" , 其稳定性完全取决于双链碱基之间严格的互补性。 即 A 与 T互补 (在 DNA - RNA) 杂交中为 A与 U (尿嘧啶) 互补) , G与 C互补。 单链 D N A或 R N A分子探针, 只与其同源互补的 DN A链杂交。 当在某防伪标记 内加入某种特定的 D N A片段时, 在辩伪过程中将 D N A从防伪标记上洗脱下来, 用该种 D N A探针探测检验即可辩出真伪。 其具体操作步骤如下: Example 1 The reliability and specificity of anti-counterfeiting marks depends on accurate methods of forgery detection. The present embodiment uses a method of hybridizing nucleic acid molecules. DN A is a double-helix structure of two nucleotide strands that are coiled together in opposite directions. The two nucleotide chains depend on the hydrogen bonds on the four bases of adenine (A) and thymine (T) and guanine (G) and cytosine (C). Therefore, the binding between DNA-DNA or DNA-RN A chains is called "molecular hybridization", and its stability depends entirely on the strict complementarity between the double-stranded bases. That is, A and T are complementary (in DNA-RNA) hybrids, A and U (uracil) are complementary, and G and C are complementary. Single-stranded DNA or RNA molecular probes hybridize only to their DNA strands that are complementary to their homology. When a certain DNA fragment is added to a certain anti-counterfeiting mark, the DNA is eluted from the anti-counterfeiting mark during the counterfeiting process, and the DNA probe can be used to detect the authenticity. The specific steps are as follows:
1 、 样品处理: 从防伪记洗脱 D N A (约 0 · 0 2 微克) , 浓缩后点在杂交用 的硝基纤维素膜或尼龙膜上;  1. Sample processing: D N A (approximately 0. 02 micrograms) was eluted from the security record, and concentrated on the nitrocellulose membrane or nylon membrane for hybridization;
2 、 变性、 中和: 膜上的 DNA经 0 . 5 mol/L氢氧化钠变性, 再用 1 mol/L缓 冲液中和;  2. Denaturation and neutralization: The DNA on the membrane is denatured with 0.5 mol / L sodium hydroxide, and then neutralized with 1 mol / L buffer solution;
3 、 固相杂交: 经变性的 DN A膜置于杂交液中, 加入核酸探针 (同位素探针 或非同位素化学发光探针) , 在 6 5 'C杂交液中 4 小时或过夜, 再经过洗涤去除非 特异结合的探针, 最后用 X光底片曝光;  3. Solid-phase hybridization: The denatured DNA membrane is placed in the hybridization solution, and a nucleic acid probe (isotopic probe or non-isotopic chemiluminescence probe) is added to the hybridization solution for 4 hours or overnight in a 65'C hybridization solution. Washing to remove non-specifically bound probes, and finally exposing them with X-ray film;
4 、 鉴定: X光底片显影后, 如在杂交膜点样的位置上存在显影斑点如图 1 ( C) 所示, 即证明样品中含有预设的防伪 DNA, 若如图 1 (A, B ) 的对照所示 未出现斑点, 则证明其为假冒的伪品或赝品。  4. Identification: After the X-ray film is developed, if there is a developing spot on the spot where the hybridization membrane is spotted, as shown in Figure 1 (C), it means that the sample contains a preset anti-counterfeit DNA. If shown in Figure 1 (A, B ) Shows no spots, which proves that it is a fake or fake.
实施例 2  Example 2
聚合酶链反应 (P CR) 特定的引物同样可以用来对核酸防伪标记进行探测。 P CR的原理为: 在两个专一性引物的引导下, 经耐热菌 DN A聚合酶 (T a q DNA聚合酶) 催化, 沿着专一性 DN A模板大量拷贝新生的 DN A链, 产物可用 琼脂糖凝胶电泳分离, 染色显示。 P CR反应是由专一性的一个或一对引物、 一个 模板 DNA组成。 极微量的 DN A模扳置于防伪物件的标记上, 探测检验时取下 D N A并溶于一定量的缓冲液中, 在加人其专一性引物和 T a q D A 聚合酶及 其他反应液后, 用 P C R依进行扩增反应。 DN A扩增反应是防伪技术的关键, 能 使 DN A扩增的酶类和其他可以使 DN A扩增的方法也可达到目的。 反应结束, 取 出一部分反应液进行电泳分析。 其产物应是一个预定大小的扩增 D N A片段, 可用 琼脂糖凝胶电泳分离, 然后用溴乙锭染色, 紫外灯下观察和鉴定, 其操作过程简述 如下:  Polymerase chain reaction (P CR) -specific primers can also be used to detect nucleic acid security labels. The principle of P CR is: Under the guidance of two specific primers, catalyzed by thermostable bacteria DNA polymerase (T aq DNA polymerase), a large number of newly-generated DNA chains are copied along the specific DNA template. The products can be separated by agarose gel electrophoresis and stained. The PCR reaction consists of a specific one or a pair of primers and a template DNA. A very small amount of DNA is placed on the mark of the anti-counterfeit object. The DNA is removed during the detection and dissolved in a certain amount of buffer solution. After adding its specific primer, T aq DA polymerase and other reaction solutions Use PCR to perform amplification reactions. The DNA amplification reaction is the key to anti-counterfeiting technology. Enzymes and other methods that can make DNA amplified can also achieve the purpose. After the reaction was completed, a part of the reaction solution was taken out for electrophoresis analysis. The product should be an amplified D N A fragment of a predetermined size, which can be separated by agarose gel electrophoresis, then stained with ethidium bromide, observed and identified under ultraviolet light, and the operation process is briefly described as follows:
1 、 反应成分: (1 ) DNA模板 (约 0 . () 〗 微克) , (2 ) 一个或一个以 上专一性引物, (3 ) T a q D N A聚合酶或其他 D N A聚合酶; (4 ) 四种脱 氧核苷三磷酸 (d AT P , d T T P, d CT P和 d GT P ) , (5 ) 反应缓冲液, 2、 反应温度和程序: 1. Reaction components: (1) DNA template (about 0. () micrograms), (2) one or more specific primers, (3) T aq DNA polymerase or other DNA polymerase; (4) four Deoxynucleoside triphosphates (d AT P, d TTP, d CT P, and d GT P), (5) a reaction buffer, 2. Reaction temperature and procedure:
9 4 °Cl 0秒 6 0 eC3 0秒 或 9 4 ° Cl 0 seconds 6 0 e C3 0 seconds or
9 4 'C 6 0秒 5 5 'C 6 0秒 7 2 'C 1 2 0秒  9 4 'C 6 0 seconds 5 5' C 6 0 seconds 7 2 'C 1 2 0 seconds
重复 2 0 - 3 0 次, 最后延伸 1 0分钟。 Repeat 20-30 times, lasting for 10 minutes.
3 、 电泳分离: 取 1 0微升反应液, 置 2 %琼脂糖凝胶 (含溴乙锭染料) 中, 用 1 0 0 V直流电压进行电泳, 电泳后将琼脂糖凝胶取出, 置于 2 5 0 - 3 0 0 η m波长紫外灯下观察并摄影。  3. Electrophoretic separation: Take 10 microliters of the reaction solution, place it in a 2% agarose gel (containing ethidium bromide dye), and perform electrophoresis with a DC voltage of 100 V. After electrophoresis, remove the agarose gel and place it in Observe and photograph under a UV light of 2 50-3 0 0 m.
4、 鉴定: 如图 2 (C ) 所示显示预定大小的 D N A带, 以此条状带是 否存在以及大小是否正确来判别鉴定物件的真伪。 图 2 (A , B ) 未显示条状带则 可认定为伪品。  4. Identification: As shown in Fig. 2 (C), a D N A belt of a predetermined size is displayed, and the authenticity of the identification object is determined based on the existence of the strip and the correct size. Figure 2 (A, B) If the strip is not shown, it can be regarded as a fake.
需要说明的是实施例 2 只是一个通例, 不是固定的操作程序。 实际操作中, 常 随 D N A模扳和引物设计上的不同而有所变化, 否则不能达到预期结果。 这种严格 的操作条件对于防伪又极为有利, 因防冒者不知反应条件, 无从破译密码。  It should be noted that Embodiment 2 is only a general example, and is not a fixed operation procedure. In practice, it often varies with the design of the D N A die and the primer, otherwise the expected results cannot be achieved. This strict operating condition is also very beneficial to anti-counterfeiting, because the anti-counterfeit cannot know the reaction conditions and cannot decode the password.
工、 Ik应用性 Ik applicability
本发明的核酸防伪标记具有不可仿制性、 可靠性、 多样性、 稳定性和广泛的适 用性, 在工业上有很高的实用性。  The nucleic acid anti-counterfeiting label of the present invention has imitation, reliability, diversity, stability, and wide applicability, and has high industrial applicability.

Claims

权 利 要 求 Rights request
1 、 一种应用生物遗传密码进行防伪的方法, 其特征在于将核酸片段、 主要是 脱氧核糖核酸 (D N A ) 片段用作为隐形防伪标记, 而后采用核酸分子探针法或聚 合酶链反应 (P C R ) 引物法检验该隐形防伪标记的真伪。 1. A method for preventing forgery using a biological genetic code, which is characterized in that a nucleic acid fragment, mainly a DNA fragment, is used as an invisible anti-counterfeiting marker, and then a nucleic acid molecular probe method or a polymerase chain reaction (PCR) is used. The primer method checks the authenticity of the invisible security mark.
2、 根据权利要求 1 所述的方法, 其特征在于所述的核酸片段通过溶于溶液中 并附着在固相载体上而形成隐形防伪标记, 或者所述的核酸片段直接以溶液形式置 于液态商品中作为隐形防伪标记, 或者所述的核酸片段置于液态载体中形成隐形防 伪标记。  2. The method according to claim 1, characterized in that the nucleic acid fragment is dissolved in a solution and attached to a solid-phase carrier to form an invisible security mark, or the nucleic acid fragment is directly placed in a liquid form in a liquid form. The product is used as a stealth security mark, or the nucleic acid fragment is placed in a liquid carrier to form a stealth security mark.
3 、 根据权利要求 2 所述的方法, 其特征在于核酸片段的固相载体为选自天然 或人造纤维, 纸张、 薄膜、 皮革、 塑料、 多孔颗粒、 粉末、 树脂、 固体石蜡、 陶瓷、 玻璃的有机或无机固态材料。  3. The method according to claim 2, characterized in that the solid phase carrier of the nucleic acid fragment is selected from the group consisting of natural or artificial fiber, paper, film, leather, plastic, porous particles, powder, resin, solid paraffin, ceramic, and glass. Organic or inorganic solid materials.
4、 根据权利要求 2或 3 所述的方法, 其特征在于附着在固相载体上的隐形防 伪记直接固定在任何固态商品、 物件或其包装物及附属物上。  4. The method according to claim 2 or 3, characterized in that the invisible security mark attached to the solid-phase carrier is directly fixed to any solid commodity, article or its packaging and appendages.
5 、 根据权利要求 4 所述的方法, 其特征在于附着有核酸片段的固相载体表面 加覆塑料薄膜或海藻糖保护层。  5. The method according to claim 4, characterized in that the surface of the solid phase carrier to which the nucleic acid fragments are attached is coated with a plastic film or a trehalose protective layer.
6 、 根据权利要求 2 所述的方法, 其特征在于核酸片段的液态载体为墨水、 印 油、 涂料或粘着剂。  6. The method according to claim 2, wherein the liquid carrier of the nucleic acid fragment is ink, ink, paint or adhesive.
7 、 根据权利要求 1 所述的方法, 其特征在于所采用的检验防伪标记真伪的核 酸探针法的步骤依次是: 样品处理, 变性, ' 中和, 固相杂交, X光显影鉴定。  7. The method according to claim 1, wherein the steps of the nucleic acid probe method for checking the authenticity of the anti-counterfeiting label are: sample processing, denaturation, 'neutralization, solid phase hybridization, and X-ray development identification.
8 、 根据权利要求 1 或 7 所述的方法, 其特征在于核酸探针法中分子杂交所用 的探针为同位素标记物或其他类型的非同位素标记物。  8. The method according to claim 1 or 7, characterized in that the probe used for molecular hybridization in the nucleic acid probe method is an isotope label or another type of non-isotopic label.
9 、 根据权利要求 1 所述的方法, 其特征在于所采用的检验防伪标记真伪的聚 合酶链反应 (P C R ) 引物法包括如下步骤: 取下 D N A并溶于一定量缓冲液中, 再加入专一性引物和 T a q D N A聚合酶或其他聚合酶及其他反应液, 用 P C R 仪进行扩增反应, 取出反应液进人电泳分析, 然后用溴乙锭染色, 在紫外光下观察 辩别鉴定 D N A产物是否存在及大小是否正确。  9. The method according to claim 1, characterized in that the polymerase chain reaction (PCR) primer method used to check the authenticity of the anti-counterfeiting label comprises the following steps: removing the DNA and dissolving it in a certain amount of buffer solution, and then adding Specific primers and Taq DNA polymerase or other polymerases and other reaction solutions were amplified by a PCR instrument, and the reaction solution was taken for electrophoresis analysis, and then stained with ethidium bromide, and identified by UV observation The presence and size of the DNA product is correct.
1 0、 根据权利要求 9 所述的方法, 其特征在于 P C R反应还可使用除 T a q D N A聚合酶之外的其他任何一种 D N A聚合酶或使 D N A扩增的任何技术。  10. The method according to claim 9, characterized in that the PCR reaction can also use any D N A polymerase other than T a q D N A polymerase or any technique for amplifying D N A.
PCT/CN1997/000078 1996-08-02 1997-08-01 A method of nucleic acid code analystic technique used in falseproof label WO1998006084A1 (en)

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