WO1998006084A1

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

Info

Publication number: WO1998006084A1
Application number: PCT/CN1997/000078
Authority: WO
Inventors: Su Han; Xinghua Guo; Xueyuan Jin; Xiaozhou Shen
Original assignee: Beijing Sanzhu Xinda Biological Probe Co., Ltd.
Priority date: 1996-08-02
Filing date: 1997-08-01
Publication date: 1998-02-12
Also published as: CN1148227A; AU3764897A

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

Method for applying nucleic acid code analysis technology to anti-counterfeiting

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.

Background technique

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.

Summary of the Invention

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. 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.

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. 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. 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. 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. 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, 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.

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.

Brief description of the drawings

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.

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

Best Mode of the Invention

The anti-counterfeiting technology of nucleic acid cryptanalysis is described in more detail below with reference to the drawings and examples.

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. 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. 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. 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. 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.

Example 2

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. 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 seconds 6 0 ^e C3 0 seconds or

9 4 'C 6 0 seconds 5 5' C 6 0 seconds 7 2 'C 1 2 0 seconds

Repeat 20-30 times, lasting for 10 minutes.

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. 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.

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.

Ik applicability

The nucleic acid anti-counterfeiting label of the present invention has imitation, reliability, diversity, stability, and wide applicability, and has high industrial applicability.

Claims

Rights request

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. 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. 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. 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. 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. The method according to claim 2, wherein the liquid carrier of the nucleic acid fragment is ink, ink, paint or adhesive.

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. 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. 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.

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.