CN113140256A

CN113140256A - Substance DNA tracing method

Info

Publication number: CN113140256A
Application number: CN202010065176.0A
Authority: CN
Inventors: 丁远彤; 潘光明
Original assignee: MGI Tech Co Ltd
Current assignee: MGI Tech Co Ltd
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2021-07-20

Abstract

The invention discloses a substance DNA tracing method. The method is an additive method and comprises the following steps: designing and synthesizing specific DNA and a privacy primer, and adding the specific DNA at a sample source; carrying out DNA amplification sequencing and tracing on the sample according to the confidential primer; or a direct measurement method, comprising the following steps: establishing a specific DNA database D2 of a sample source; and performing DNA sequencing and tracing on the sample. The method can be used for content anti-counterfeiting in a low-cost mode, and meanwhile, the block chain technology is used for information management anti-counterfeiting tracing, so that the method is a comprehensive tracing anti-counterfeiting method combining management anti-counterfeiting and technical anti-counterfeiting, and the characteristic of high information content of DNA is utilized, and the tracing anti-counterfeiting safety is greatly improved.

Description

Substance DNA tracing method

Technical Field

The invention relates to the technical field of biology, in particular to a substance DNA tracing method.

Background

The tracing anti-counterfeiting method can be generally divided into two modes of management and technology, wherein the management anti-counterfeiting is also called soft anti-counterfeiting, which means that the anti-counterfeiting tracing purpose is achieved by a certain management means, but the steps are complicated and the management cost is high. The technical anti-counterfeiting is also called hard anti-counterfeiting, which means that the anti-counterfeiting tracing purpose is realized by a certain technical means, and the technical anti-counterfeiting can be divided into form anti-counterfeiting and content anti-counterfeiting. At present, most of anti-counterfeiting technologies and methods belong to form anti-counterfeiting, such as anti-counterfeiting packages, passwords, marks or trademarks, but the situation that the package is true but the content is false cannot be identified usually. Content anti-counterfeiting generally depends on high technical content of products, and although the content is most concealed, the content anti-counterfeiting is only suitable for a small amount of products. At present, the traceability anti-counterfeiting technology is developed in a more comprehensive and more comprehensive direction, and new technologies such as biological characteristics with high information content, material chemistry and the like are adopted.

There are reports related to substance DNA source tracing. For example, the Chinese patent application 201310191588.9 discloses a method for preparing a DNA gelatin nano microgel anti-counterfeiting coating, which proposes to produce a glue containing DNA to mark a product, and to perform extended sequencing on the DNA during verification so as to judge the authenticity. The method can only realize the anti-counterfeiting of the product adopting the glue, but cannot realize the traceability of each product including batch information and other personalized information, and cannot realize the traceability of the product content when the coating is attached to the product package instead of the product content. Chinese patent application 200880018420.5, a method and system for identifying individual samples from a multiplex mixture, proposes to identify individual samples by sequencing, but cannot realize traceability including batch information and other personalized information for each product. Chinese patent application 201680091177.4, a gene label for nucleic acid sample identification, a kit and applications thereof, proposes to use nucleic acid to manufacture a gene label, but cannot realize the traceability of each product including batch information and other personalized information, and cannot realize the traceability of the product content itself when the label is attached to the product package rather than the product content itself. The Chinese patent application 201610226723.2, a DNA bar code compiling method and a tracing method for pork tracing, proposes a method for tracing by designing a DNA as a pig feed to make a pig carry the DNA to generate a DNA bar code, and is characterized in that a tracing sample is a living animal and is not suitable for non-living substances. The Chinese patent application 201711387781.4, a method for tracing the anti-counterfeiting of the whole chain of agricultural products by using gene labels, proposes to accurately identify and determine individuals and their varieties by using the genetic information of the individuals, thereby ensuring the traceability and fidelity in the field of production and sale of agricultural and pastoral products. The invention provides a Chinese patent application 201810432497.2, an Internet of things anti-counterfeiting traceability system and an anti-counterfeiting traceability method for rapidly identifying codonopsis pilosula DNA, wherein the anti-counterfeiting traceability of codonopsis pilosula is realized through a cloud end, a DNA chip, an identification device, a traceability label, a DNA retrieval terminal and a traceability retrieval terminal. The chinese patent application 201811191487.0, a method and system for tracing object identity, proposes to use a block chain method to verify DNA codes of biological products and GGT codes of biological products, but does not provide a scheme for how to design DNA labels and a method for tracing. In summary, the deficiencies of the prior art are reflected in the following aspects: the anti-counterfeiting mark technology based on the packaging materials (such as wine bottles) is easy to be manufactured and counterfeited by collecting and adopting the genuine packaging materials. It is inconvenient to trace the source and prevent the forgery of each small package (such as each bottle of wine) product. The anti-counterfeiting tracing cost is high, and the anti-counterfeiting and tracing cost is not suitable to be reduced.

Disclosure of Invention

In order to solve the technical problems, the invention provides an improved new method for product traceability and anti-counterfeiting. The invention aims to provide a substance DNA tracing method, which comprises a method for adding specific DNA into a sample substance and utilizing the DNA carried in the substance sample, and the method is used for interfering the design of the DNA through the design of the DNA and a secret primer and recording information by utilizing a block chain so as to achieve the aims of specific identification, decoding prevention, anti-counterfeiting and anti-tampering.

At present, the traceability and anti-counterfeiting of common products are generally realized by a mode of printing anti-counterfeiting marks on outer packages, but the condition that the packages are true but the contents are false cannot be identified. The method provides a method for tracing and anti-counterfeiting by adding a specific DNA marker into an actual product such as wine. The added specific DNA molecule consists of a specific secret primer area and an encrypted coding area, so that each bottle of products is guaranteed to correspond to a unique code, and each code section is invalid after one-time use. The confidential primer library and coding library information are held by the manufacturer or a third party organization. During checking, only DNA is extracted from residual liquor, specific security primers are used for amplification and sequencing to obtain a coding sequence, and the coding sequence is compared with a coding database to check the authenticity. If the code does not exist in the code library, the code is a counterfeit product, and if the code exists in the code library but is inquired, the code can be judged to be a counterfeit product, namely, each code is used once. Only if the code exists in the code library and is not queried is it judged to be true. Meanwhile, the block chain is used for storing and verifying all information such as secret primers, codes, products and the like so as to ensure that the information cannot be tampered.

In addition to the organization that owns the confidential primer library and the coding library information, a barrier to third parties attempting to copy the coding information from the product is that the coding information cannot be amplified from the product without holding a specific confidential primer sequence. In addition, the random amplification sequencing can be prevented by adding interfering DNA to obtain specific secret primers or coding information.

Specifically, the invention provides a substance DNA tracing method, namely the method A and/or the method B.

The method A comprises the following steps: an additive process comprising the steps of: designing and synthesizing specific DNA and a privacy primer, and adding the specific DNA at a sample source; and carrying out DNA amplification sequencing and tracing on the sample according to the confidential primer.

The method B comprises the following steps: the direct measurement method comprises the following steps: establishing a specific DNA database D2 of a sample source; and performing DNA sequencing and tracing on the sample.

In the method A, the step of "designing and synthesizing specific DNA and privacy primer, and adding the specific DNA at the sample source" can be carried out according to a method comprising the following steps:

(A1) designing a core coding segment of a specific DNA according to the attributes of the sample, and synthesizing the specific DNA according to the core coding segment.

(A2) Designing and synthesizing the corresponding privacy primer according to the core coding segment of the specific DNA and the privacy primer design rule.

(A3) And establishing a database D1 of the corresponding relation among the sample, the specific DNA, the confidential primer and the source information and/or batch information of the sample for later reference.

(A4) Adding the specific DNA corresponding to the sample into the sample according to a determined proportion before the sample leaves a factory; or, the specific DNA corresponding to the sample is added to the inner package of the sample.

The core coding segment of the specific DNA may be multiple segments, for example, a segment of core coding segment is designed for each source information and/or batch information (specifically, product type, production place, production date, production batch, product serial number, etc.) of the product. The security primer consists of a forward security primer and a reverse security primer. The specific DNA is formed by connecting the forward secrecy primer, a core coding segment (if a plurality of segments are connected in sequence) of the specific DNA and the reverse secrecy primer in sequence.

In the method A, the step of performing DNA amplification sequencing and tracing on the sample according to the confidential primers can be performed according to a method comprising the following steps:

(A5) and (2) carrying out pollution-free sampling on the sample obtained in the market or the inner package of the sample, and separating the DNA molecules from the sample by a centrifugal mode, a filtering mode and a purifying mode.

(A6) And acquiring a secret primer corresponding to the sample in the database D1, and amplifying by using a PCR (polymerase chain reaction) mode in the presence of the secret primer to complete DNA sequencing and obtain the sequence information of the coding segment.

In the step, agarose gel electrophoresis detection can be carried out after PCR amplification, and if a target band does not exist, sequencing is not needed, and tracing failure can be directly judged. If the target band exists, further sequencing is needed to judge the result through sequence information.

(A7) Tracing and comparing according to the following steps:

a first step comprising:

if the confidential primer cannot be obtained or the amplification of the confidential primer fails, tracing the source fails; if the confidential primer is obtained and amplified successfully, entering the next step and tracing further;

a second step comprising:

comparing the sequence of the coding segment obtained from the sample or the inner package of the sample obtained from the market with the coding segment in the database D1, wherein if the coding segment exists in the database D1, the source information and/or batch information of the sample corresponding to the coding segment is the source information and/or batch information of the sample, and the tracing is successful; if the encoded segments do not exist in the database D1, tracing fails.

In the step (a7), a step of outputting the tracing result may be further included.

In the second step of (a7), if the code segments have been aligned before, outputting repeated alignment information.

In the method A, a step of keeping the specific DNA and the privacy primer secret may be further included.

The users of the method A comprise manufacturers and source tracing users.

In the method B, the establishing of the specific DNA database D2 of the source of the sample may be performed according to a method comprising the following steps:

(B1) and (2) carrying out pollution-free sampling in the sample source, and separating DNA molecules from the sampling in a centrifugal mode and a filtering mode.

(B2) And selecting coding segments from the DNA molecules according to the source characteristics of the sample.

(B3) And establishing a database D2 of the corresponding relation between the samples and the coding segments and the source information of the samples for future reference.

The method B may further comprise the step of publishing or not publishing the selected code segment in (B2) on the inner package of the sample.

In the method B, the "DNA sequencing and tracing the sample" can be performed according to a method comprising the following steps:

(B4) obtaining the sample in the market for sampling without pollution, and separating DNA molecules from the sample by a centrifugal mode and a filtering mode

(B5) Sequencing the DNA molecules, and tracing the source in the sequencing result according to the sequence information of the coding segment corresponding to the sample in the database D2; if the coding segment is found in the sequencing result, tracing is successful, otherwise, tracing fails.

Sequencing the DNA molecules, and tracing the source in the sequencing result according to the coding segments published on the sample; if the coding segment is found in the sequencing result, tracing is successful, otherwise, tracing is failed; the unpublished code segment will be recorded in the database D2.

In the method B, the following steps may be further included: designing a unique code for each minimum package of the sample sold, identifying the unique code on the minimum package, recording the unique code into the batch information of the database D2, and checking the unique code when tracing the source.

In the method a and/or the method B, a step of adding interfering DNA at the source of the sample may be further included.

In the method a and/or the method B, the "adding interfering DNA at the source of the sample" may be performed according to a method comprising the steps of:

(a1) designing, depending on the properties of the specific DNA, an interfering DNA that does not include the coding sequence of the specific DNA and the coding sequence of the privacy primer and does not amplify the specific DNA and the privacy primer.

(a2) Adding the specific DNA and the interfering DNA corresponding to the sample into the sample according to a determined proportion before the sample leaves a factory; or, the specific DNA and the interfering DNA corresponding to the sample are added to the inner package of the sample.

The method may further comprise the step of determining a method of tracing from the characteristics of the sample according to any of the following steps:

(C1) for a sample (such as a part of white spirit) which does not contain any DNA, adopting the method A (addition method), and screening the specific DNA and/or the interference DNA which does not generate dissolution or any other reaction with the sample for tracing;

(C2) tracing the source of a sample (such as red wine, yellow wine, beer, wine, fruit wine and the like) which contains DNA by itself by adopting the method B (direct measurement method);

(C3) for a sample containing DNA itself, the method A and/or the method B is used, and the specific DNA and/or the interfering DNA which do not react with any reaction with the sample and the DNA contained in the sample itself are screened for tracing.

The method A can also comprise the step of introducing more than one section of specific DNA and more than one matched secret primer and/or more than one interference DNA aiming at a single independent package of the sample so as to increase the difficulty of cracking and counterfeiting prevention.

The method specifically comprises the following steps: and determining the combination CBn of the coding fragment DNAn of the specific DNA and the secret primer PMn matched with the coding fragment DNAn as more than one group of different combinations, wherein n is the number of the combination and is a natural number. And designing privacy primers PMn synthesizing BP number, ratio of guanine and cytosine, GC content and DNA dissolution temperature TM value of different base pair length to fit different DNAn.

In addition, the step of establishing database D1 in the additive method to the traceability alignment can be updated according to the sample and method selection steps.

The method may further include the step of the manufacturer managing the user using blockchain technology.

The "manufacturer manages users by using a block chain technology" may specifically include:

the manufacturer establishes the database D1 or the database D2 at the sample source and manages the block chain of the user;

selecting a cloud storage as the user node, selecting an encryption and decryption algorithm and a consensus mechanism;

the sample is subjected to unique identification code making and registration before leaving a factory by taking a single package or sample variety as a unit;

setting a source tracing frequency record for the sample of the identification code, and outputting the source tracing frequency to a verification report;

expanding the contents of the databases D1 and D2, and incorporating the sample sales records, the user traceability records, traceability success and failure records and the user internet addresses to establish big data analysis and provide anti-counterfeiting early warning reports.

The method may further comprise the step of establishing an application system of the internet, internet of things based on the database D1 or the database D2.

The method specifically comprises the following steps:

establishing a sample information management system, and supporting the online query step of the comparison and source tracing steps;

the temperature and time query steps supporting the amplification process including denaturation, annealing and extension cycles;

outputting a query report according to the query result;

recording and incorporating the user query behavior into a big data system, and establishing a big data analysis system.

In the method of the present invention, the sample may be wine (e.g., white spirit containing no DNA itself) or food or other substances.

When the sample is a food such as wine or food, the specific DNA and/or the interfering DNA added to the source of the sample is required to comply with food hygiene regulations without affecting the quality, taste, etc. of the food.

Compared with the prior art, the invention has the following beneficial effects: the adoption of biological DNA tracing technology greatly increases the difficulty of counterfeit. The DNA coding space is huge, different codes and source records are carried out on each product by adopting the technology based on the Internet and the Internet of things, and the uniqueness tracing and anti-counterfeiting of each product are supported. The secret primers and the products are distributed by channels, and the difficulty for obtaining the secret primers by a counterfeiter is very high. By adopting the block chain technology, the verification of each product cannot be tampered, and each product can be verified once, so that the reliability of anti-counterfeiting traceability is improved.

In a word, the method provides a mode of adding a specific DNA marker into an actual product, such as wine, for tracing and anti-counterfeiting. The method can be widely used for content anti-counterfeiting in a low-cost mode, and meanwhile, the block chain technology is used for information management anti-counterfeiting tracing, so that the method is a comprehensive tracing anti-counterfeiting method combining management anti-counterfeiting and technical anti-counterfeiting, and the characteristic of high information content of DNA is utilized, and the tracing anti-counterfeiting safety is greatly improved.

Drawings

FIG. 1 is a step diagram of a wine tracing method.

FIG. 2 shows the steps in more detail.

Fig. 3 shows the steps of the encoding process.

Fig. 4 shows the steps of the marking process.

Fig. 5 shows the verification process steps.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 wine tracing method

As is well known, wines, especially famous brands of white spirits, red wines, beer, wine, fruit wine and the like, are very important for effectively preventing counterfeiting, and have higher commercial value. In the retrieved anti-counterfeiting method, the traceability of each product is an effective good method. Therefore, the invention adopts the DNA technology to realize the traceability, which is an important intention of the invention. The DNA is deoxyribonucleic acid.

In the present invention, the producer of the specimen using the method of the present invention is referred to as a manufacturer, and the consumer using the specimen is referred to as a user.

The specific scheme is as follows:

fig. 1 is a step diagram of a wine tracing method, fig. 2 is a step diagram of specific details, and fig. 3, fig. 4 and fig. 5 are a coding process step, a marking process step and a verification process step, respectively.

The specific method comprises an addition method and a direct measurement method, and comprises the following specific steps:

an adding method step, namely, steps such as 1001, 1002, 1003, 1004 and 1005 in the figure 1, designing and synthesizing specific DNA and a confidential primer, adding the specific DNA at a sample source, and carrying out DNA amplification sequencing and tracing on the sample according to the confidential primer.

Referring to fig. 2, a wine tracing method includes designing step 2001, synthesizing step 2002, synthesizing step 2003, interfering DNA step 2004, privacy primers step 2005, adding step 2006 to a finished wine package or a label or an inner package of a wine bottle, adding step 2007 to a finished wine product such as white wine, red wine, beer, yellow wine, fruit wine and the like, database D1 or database D2 step 2008, marketing step 2009, bottling or other packaged wine step 2010, sampling wine step 2011, DNA amplification step 2012, DNA sequencing step 2013, comparison step 2014 according to the database 2008, and generating a tracing conclusion step 2015.

The steps in fig. 2 may be subdivided into the encoding process of fig. 3, the labeling process of fig. 4, and the verification process of fig. 5.

The sample herein refers to an alcoholic beverage product using the present invention and refers to an alcoholic beverage before filling at the time of production. Adding trace amount of specific DNA into the wine, sampling the wine after the wine is sold to extract the specific DNA, carrying out sequencing after the amplification of the secret primer, and then searching the source of the wine by comparing the database of a wine manufacturer to confirm the production batch of the wine and confirm whether the wine is a counterfeit product.

The difference between the addition method and the direct measurement method is that in the addition method, not only specific DNA but also matched secret primers need to be designed, and in the direct measurement method, secret primers are not needed, but insecure primers can be provided by a tracing user or the sample manufacturer. It should be noted that, since the specific DNA is added to the alcoholic beverage, it should be noted that, when designing the specific DNA, the specific DNA is in accordance with the food hygiene regulations, and the specific DNA should not affect the quality of the alcoholic beverage, the taste, or the harm to the human body, and the addition ratio should be as small as possible as long as the specific DNA can be detected in the alcoholic beverage.

As another example, the specific DNA may be used on the inner packaging of the liquor, and any place where the specific DNA may be used, such as on a wine bottle, a cap, a label of a wine bottle, etc., including a label printing pigment, the specific DNA may be used instead of being directly added to the liquor.

The privacy primer is designed to target a specific DNA in order to be able to amplify the specific DNA. For sequencing a particular DNA, amplification cannot be achieved without the secret primers. Therefore, the sequencing difficulty of the specific DNA is increased, and even the sequencing cannot be realized, and the tracing cannot be realized. For the wine products using the method of the invention, manufacturers need to strictly control the circulation of the confidential primer, for example, the confidential primer cannot be circulated along with the wine products, so that counterfeiters cannot obtain the confidential primer, the difficulty of counterfeit activities is increased, and the anti-counterfeiting traceability capability is improved.

And (3) a direct measurement method step, namely establishing a specific DNA database D2 of a sample source, and performing DNA sequencing and tracing on the sample.

The direct measurement method is different from an addition method, does not need to add extra DNA, but directly traces the source according to the inherent DNA in the wine product using the invention, does not need confidential primers, and can provide non-confidential primers by a tracing user or the sample manufacturer, wherein the wine is red wine, yellow wine, beer, wine, fruit wine and the like.

The direct measurement method has simpler steps and lower cost, but the anti-counterfeiting performance is different from that of an addition method, and a user can select the anti-counterfeiting performance according to the characteristics of the product.

On the basis of the above technical solutions, in other aspects of the present invention, one or more of the following measures for local improvement may be adopted:

the adding method steps include the encoding process of fig. 3, the marking process of fig. 4 and the verification process of fig. 5, and specifically include but are not limited to:

designing more than one core coding segment of specific DNA according to the sample property, and synthesizing the specific DNA.

The sample is the traceable product wine of the embodiment.

Some design examples of specific DNA core coding segments are given below (table 1), with emphasis on: the user may define other design solutions at his or her discretion.

TABLE 1 specific DNA core coding fragment design examples

Note: type I white spirit has been determined to be DNA free itself.

The complete example of the coding sequence is obtained according to the rules as above:

it should be noted here that the core coding segment of the specific DNA includes one or more segments, different coding designs may be used between the segments, and for different batches of wine products, different segments and specific DNA synthesized by the combination of the segments may be added and recorded in the database D1 as the basis for judging the wine products and the batches thereof in the future.

Designing and synthesizing a corresponding privacy primer according to the core coding segment of the specific DNA and the privacy primer design rule.

The secret primer design rules include, but are not limited to, the number of BP base pairs of the length of the secret primer, the GC content of the ratio of guanine and cytosine, the Tm value of DNA dissolution temperature, the base requirement of 3 'end, the base requirement of 5' end, the Δ G value of free energy required for DNA double strand formation, the constraints of the numbers of hairpin structure, dimer, mismatch, cross-dimer, etc.

Some examples of the design of privacy primers are given below (table 2), with emphasis on: the user may design the privacy primers in other ways.

Randomly generating a primer sequence with the length of 18bp, analyzing the quality of the primer by using software such as OligoEvaluator and the like, and reserving the primer meeting the following conditions: the GC content is between 40 and 60 percent, the Tm value is between 55 and 80 ℃, the 3' end base is not A, and secondary structure and dimer do not exist.

TABLE 2 confidential primer design example

Number of secret primers	Sequence (5 '-3')
		Forward primer 1	5’-CACACGTCAGCTCTCAGT-3’
Reverse primer 1	5’-AGGTGAGTCGACGAGACT-3’

The coding fragment sequence is designed according to the table 1, and a security primer sequence or a complementary sequence is added at two ends, so that the security primer can amplify the coding fragment in the subsequent step, and the complete sequence (SEQ ID No.1) of the specific DNA is obtained as follows:

it should be noted that the correspondence between the security primer and the core coding segment of the specific DNA needs to be recorded in the database D1 for future reference.

And establishing a database D1 for later reference, wherein the database includes but is not limited to the corresponding relation among the sample, the specific DNA, the confidential primer, the sample source information and/or the batch information.

Some design examples of database records are given below (table 3), with emphasis on: the user may design the D1 database in other ways.

TABLE 3D 1 database design example

Note: the D1 database shown in table 3 was determined based on the relevant contents of tables 1 and 2.

Database D1 may also include, but is not limited to, lot information for the sample production, which may include different specific DNAs, as well as specific DNA core coding fragments produced from different recipes for coding fragments and combinations of coding fragments. In fact, in said batch information, a unique identification can be made for each product's smallest package, for example each bottle of wine.

Adding the specific DNA corresponding to the sample at a predetermined ratio before the sample is shipped from the factory, or adding the specific DNA corresponding to the sample to an internal package of the sample.

In this embodiment, two methods of adding the specific DNA are actually included, the first is to add the specific DNA directly to the sample (i.e., alcoholic product), and the second is to add the specific DNA to the printed material of the inner package of the sample (i.e., alcoholic product) product or the label attached to the wine bottle. In the source tracing, the two addition methods are respectively sampled, amplified and DNA sequenced and entered into a database D1 for comparison.

Examples of adding specific DNA are given below, it being emphasized that the user may choose other concentrations and the like to add specific DNA to the vector:

the specific DNA sequence (SEQ ID No.1) was synthesized by the solid phase phosphoramidite method and added to 100mL of the distillate spirit sample A (i.e., the distillate spirit type I in Table 1) so that the DNA concentration was 10-8 mol/L. As a control, another 100mL of the distillate sample B was prepared without the addition of the specific DNA (SEQ ID No.1), and a mock sample was simulated.

It is emphasized here that the specific DNA of the first method, since it is added directly to wine, must be taken care of in compliance with relevant standards of food hygiene.

And (2) carrying out pollution-free sampling on the sample obtained in the market or the inner package of the sample, and separating the DNA molecules from the sampling by the methods including but not limited to centrifugation, filtration and purification.

An example of a DNA extraction procedure is given below, it being emphasized that the user may choose other ways to extract DNA:

respectively carrying out the following extraction steps on the white spirit samples A and B: taking 50ml of sample, and centrifuging the sample for 30 minutes at 2500g at 4 ℃; discarding the supernatant; to the pellet was added 750. mu.L of CTAB buffer (formulation: 25mM EDTA; 1M Tris-HCl, pH 8; 2M NaCl, 3% (w/v) CTAB); after fully mixing, transferring the mixture into 0.2 percent of 2-mercaptoethanol and 1 percent of polyvinylpyrrolidone, and inoculating the mixture in a water bath at 65 ℃ for 60 minutes; adding a volume of phenol-chloroform-isoamyl alcohol (25: 24: 1, volume ratio) and 0.6 volume of 2-propanol, incubating at-20 ℃; 13000g at 10 ℃ for 30 minutes; discarding the supernatant; washing the precipitate with 70% ethanol; the precipitate was dried under vacuum at room temperature for 30 minutes; add 50. mu.L deionized water to obtain template DNA solution. There are also a number of studies showing that DNA can be extracted from commercial wine, vinegar, etc., indicating that DNA can be stored in such solutions for a long period of time.

Obtaining the corresponding security primer of the sample in the database D1, amplifying by using a mode including but not limited to PCR in the presence of the security primer to complete DNA sequencing, and obtaining the sequence information of the coding segment.

An example of amplifying a particular DNA is given below, it being emphasized that the user may choose other ways to obtain the coding segment:

the database D1 was queried to obtain the confidential primer sequences, which were synthesized using the solid phase phosphoramidite Trilipid method.

Taking the DNA molecules separated in the previous step and the confidential primers to carry out PCR reaction, wherein the reaction system and conditions are as follows: a 50 μ L system comprising: 10 XPCR buffer 5. mu.L; 1 mu L template DNA solution; 5.0U/. mu.L TaqDNA polymerase 0.25. mu.L; 2.5mmol/L dNTP 4. mu.L; 1 mu L of forward and reverse primers of 20 mu mol/L respectively; make up to 50 μ L of deionized water. Reaction conditions are as follows: denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 35 seconds, annealing at 55.2 ℃ for 35 seconds, extension at 72 ℃ for 45 seconds, 25 cycles; the reaction was terminated by extension at 72 ℃ for 5 minutes.

After amplification of the white spirit samples a and B described above, the products were sequenced using a BGISEQ50 instrument, and the results are shown in table 4.

TABLE 4 sequencing results of Baijiu sample A and Baijiu B

In fact, the confidential primer is designed to be matched with the added specific DNA and has certain confidential property, so that the confidential primer can be issued along with the sample wine product or can be issued independently. The winery user can select according to the confidentiality of the source tracing. The PCR is a polymerase chain reaction.

In addition, when the coding segment includes, but is not limited to, the core coding segment of a specific DNA, the core coding segment of the specific DNA needs to be determined in the alignment.

The first comparison and source tracing step specifically includes but is not limited to:

if the confidential primer cannot be obtained or the confidential primer fails to be amplified, tracing the source fails; and if the confidential primer is obtained and amplified successfully, entering the next step for further tracing.

The following gives an example of the first comparison step for the aforementioned white spirit a and B samples:

the liquor B sample is a counterfeit product because the specific DNA sequence cannot be detected due to unsuccessful amplification in the liquor B sample; and (5) if the liquor A sample is successfully amplified, entering a further tracing step.

This step is to ensure the correspondence between the confidential primers and the specific DNA, and if someone tries to counterfeit the wine product, the amplification fails because he cannot obtain the exact confidential primers. Even if a counterfeiter tries to guess the secret primer formula or obtains the genuine secret primer issued by the genuine manufacturer, amplification can not be successful because the batch correspondences do not match.

The second alignment and tracing step specifically includes, but is not limited to:

comparing the code segment obtained from the sample or the inner package of the sample obtained from the market with the code segment in the database D1, if the code segment exists in the database D1, the source information of the sample corresponding to the code segment is the source information of the sample, and tracing successfully; if the coded segment does not exist in the database D1, tracing fails; and if the code segments have been compared before, outputting repeated comparison information. Since the code segment is unique to the piece of product, if duplication occurs, the piece of product may be counterfeit.

The following gives an example of the second alignment procedure for the aforementioned sample of Baijiu A and sample of Baijiu B:

and (3) removing forward and reverse primer sequences at two ends after the liquor A sample is successfully amplified, further querying the residual sequences in a D1 database, successfully querying to obtain a record, wherein the query history number is 0, and no repeated comparison information exists, so that the product is true.

As mentioned above, the source information of the sample in the database D1 also includes the batch information, specific DNA information and confidential primer information of the product, so-called tracing success includes the method of increasing difficulty, which is to amplify not only a single specific DNA by a single confidential primer, but also the corresponding compound specific DNA by designing the confidential primers with different batches of information.

A step of outputting a tracing result and a step of keeping secret for the specific DNA and the secret primer.

In order to enhance the difficulty of breaking the tracing method, the specific DNA and the security primer need to be kept secret, and particularly the coding formula needs to be kept secret. The method of achieving this step allows for the secure storage of the coding formulations of the specific DNA and the secure primers in a block chain.

Those who use the method include, but are not limited to, the manufacturer and the user. Wherein the manufacturer is a producer of the sample, and the user is a user or a consumer of the sample.

And outputting a tracing result to the manufacturer and the user according to the tracing condition, wherein the tracing result at least comprises successful tracing, failed tracing and repeated tracing, and also comprises specific tracing details, such as product batch, production time, product type and the like.

the direct measurement method at least comprises the following steps:

a non-contaminating sample is taken from the source of the sample, and the DNA molecules are separated from the sample by means including, but not limited to, centrifugation, filtration.

For some white spirits, this method is not suitable because there is no DNA present. However, this method is suitably used for red wine, yellow wine, beer, wine, fruit wine, and the like.

And selecting a coding segment from the DNA according to the source characteristics of the sample.

Since red wine, yellow wine, beer, wine, etc. may contain many DNAs, it is necessary to design and select distinctive, representative DNA coding fragments.

Establishing a database D2 for future reference of the corresponding relation among the sample, the coding segments and the source information of the sample, and publishing or not publishing the coding segments on the inner package of the sample.

When selecting a published coding fragment, it should be noted that the coding fragment is a DNA coding fragment specific to the sample and difficult for a counterfeiter to obtain, otherwise, it would facilitate a counterfeit behavior. The difficulty of counterfeiting is increased when the code segments are not published, but for the sake of fairness, it is proposed to introduce a block chain technique to the database D2 to implement a de-centering and anti-repudiation function, so as to increase the reliability.

Obtaining the sample in the market for non-pollution sampling, and separating the DNA molecules from the sample by a centrifugal mode and a filtering mode.

Sequencing the DNA, and tracing the source in the sequencing according to the coding segment corresponding to the sample in the database D2; wherein if the coding segment is found in the sequencing result, tracing is successful, otherwise tracing is failed.

Sequencing the DNA, and tracing the source in the sequencing according to the coded fragments which are published or not published packaged in the sample; wherein if the coding segment is found in the sequencing result, tracing is successful, otherwise tracing is failed. The unpublished code segment will be recorded in the database D2.

For DNA fragments that are not published within the package within the sample, the source is searched and aligned in the database D2.

Designing a unique code for each minimum package of the sample sold, identifying the unique code on the minimum package, recording the unique code into the batch information of the database D2, and checking the unique code at the same time of tracing the source.

The intention of this step will be to enable the direct survey method to support a refined traceability of the single piece of the smallest package (such as a wine bottle), the traceability content including but not limited to the batch of the product, the information of the shelf life and the like, the traceability times and the like, and the uniqueness traceability. The uniqueness tracing means that if the product is traced and verified before, the tracing and verifying information is output, and because the uniqueness code is uniquely corresponding to the product, if the uniqueness code is repeated, the product is possibly counterfeited.

the step of the addition method further comprises but is not limited to a step of adding interfering DNA, and specifically comprises but is not limited to:

designing one or more interfering DNAs that do not include the coding sequence of the specific DNA and the coding sequence of the privacy primer and do not amplify the specific DNA and the privacy primer, depending on the properties of the specific DNA.

The intention here to increase the interfering DNA is to increase the difficulty of counterfeit prevention. It should be noted that care must be taken for the interfering DNA to be added directly to the wine product in order to meet relevant standards for food hygiene.

A step of adding the interfering DNA to the specific DNA.

Adding the specific DNA and the interfering DNA corresponding to the sample into the sample according to a determined ratio before the sample leaves the factory, or adding the specific DNA and the interfering DNA corresponding to the sample into an internal package of the sample.

for a sample which does not contain any DNA, such as part of white spirit, the addition method is adopted and the specific DNA which does not dissolve or react with the sample is screened for tracing.

For samples which contain DNA, such as red wine, yellow wine, beer, wine, fruit wine and the like, the direct measurement method is adopted to carry out the tracing step.

For a sample that itself contains DNA, a step of screening for the addition of the specific DNA that does not synthesize and produce any reaction with the sample.

introducing more than one section of specific DNA and more than one matched confidential primer, namely a plurality of specific DNAs and a plurality of matched confidential primers, so as to increase the difficulty of cracking and anti-counterfeiting; specifically, the method includes but is not limited to:

aiming at the step of designing a specific DNA core code and a primer, determining the code fragment DNAn of the specific DNA and the combination CBn of the secret primer PMn matched with the code fragment DNAn of the specific DNA into more than one group of different combinations, wherein n is the number of the combination and is a natural number.

Here, the group number is associated to the production lot of the product and recorded in the database D1 for easy query and comparison at the time of tracing.

Aiming at the step of designing a specific DNA core code and a primer, designing a step of synthesizing a privacy primer PMn with different base pair length BP numbers, GC content of the ratio of guanine and cytosine and TM value of DNA dissolving temperature to be suitable for different DNNs.

Updating a database D1 established in the addition method to a tracing comparison step according to the sample and method selection step;

the step of managing the user by the manufacturer by using a block chain technology specifically includes but is not limited to:

the manufacturer establishes the databases D1 and D2 at the sample source and the step of blockchain management of the user.

The selecting includes, but is not limited to, cloud storage as the step for the user node, the step of selecting an encryption/decryption algorithm, and the step of a consensus mechanism.

The user node records a distributed account book and stores records verified by users in the databases D1 and D2. The encryption and decryption algorithms must comply with the specifications of the blockchain technique. The consensus mechanisms include, but are not limited to, validity confirmation of records and tamper-proof measures.

And the sample is subjected to unique identification code making and registration before leaving the factory by taking a single package or sample variety as a unit.

The unique identifier and the unique registration are recorded in the databases D1 and D2, and the node where the user is verified to be present is recorded in the distributed ledger.

And setting a source tracing frequency record for the sample of the identification code, and outputting the source tracing frequency to a verification report.

And establishing big data analysis, wherein the step of providing the anti-counterfeiting early warning report expands the contents of the databases D1 and D2, and includes but is not limited to the sample sales record, the user traceability record, traceability success and failure records and the user internet address, so as to establish the big data analysis and provide the anti-counterfeiting early warning report.

Therefore, a big data information management system based on sample product sales and traceability is established, analysis steps and an artificial intelligence analysis method are designed according to historical records, and a big data analysis system is provided for manufacturers adopting the method.

establishing an application system based on the database D1 and the database D2 of the Internet and the Internet of things; specifically, the method includes but is not limited to:

and establishing a sample information management system to support the online query step of the second comparison and source tracing step.

Temperature and time interrogation steps supporting the aforementioned amplification protocols including, but not limited to, denaturation, annealing and extension cycles.

And outputting a query report according to the query result.

Embodiment 2 food tracing method

The embodiment is a food tracing method of the invention. The same points as those in example 1 are not described, except that:

1. the sample comprises a food product.

2. The specific DNA and the interference DNA must not produce any chemical reaction with the food adopting the method and influence the taste.

3. For food packaged in a disposable form, such as plastic bag packaging, paper bag packaging, etc., the specific DNA, interfering DNA may be marked by mixing into the package or by printing on the package with a pigment.

4. In the database D1 or the database D2, the validity period of the food is recorded into the database D1 and the uniquely encoded batch information, and the source tracing includes checking the validity period of the food.

5. When the direct measurement method is adopted for tracing, the unique code including the two-dimensional code is designed for the minimum package of each product, the batch information of the product including the validity period information is recorded in the database D2, and the verification is carried out together.

Embodiment 3 extensive material tracing method

The embodiment is a ubiquitous substance tracing method. Compared with the embodiment 1 and the embodiment 2, the same points are not described, and the difference is that:

1. the sample includes various products of matter.

2. The specific DNA and the interference DNA must not produce any chemical reaction with the product of the generic substance by the method and influence the performance of the generic substance.

3. For general-substance products using disposable packaging, such as plastic bag packaging, paper bag packaging, etc., the specific DNA, interfering DNA may be marked by mixing into the packaging, or printing pigment of the packaging.

4. In the database D1 or the database D2, the individual characteristic information of the pan material product is recorded into the database D1 and the uniquely coded batch information, and the tracing includes the verification of the individual characteristic information of the pan material product.

5. When the direct measurement method is adopted for tracing, the unique code including the two-dimensional code is designed for the minimum package of each product, the batch information of the product including the individual characteristic information is recorded in the database D2, and the verification is carried out together.

<110> Shenzhen Hua Dazhi science and technology Limited

<120> substance DNA tracing method

<130> GNCLN200227

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 132

<212> DNA

<213> Artificial sequence

<400> 1

cacacgtcag ctctcagtaa ggcagcctta acgatatcgg ggacttgcga tgtacgtgct 60

ttggttcaat acatacgtgg cccagtagtt atccaatatc ggaacatcaa ttgtagtctc 120

gtcgactcac ct 132

Claims

1. A substance DNA tracing method is a method A and/or a method B:

the method A comprises the following steps: an additive process comprising the steps of: designing and synthesizing specific DNA and a privacy primer, and adding the specific DNA at a sample source; carrying out DNA amplification sequencing and tracing on the sample according to the confidential primer;

2. The method of claim 1, wherein: in the method A, the step of designing and synthesizing specific DNA and privacy primer, and adding the specific DNA at the sample source is carried out according to the method comprising the following steps:

(A1) designing a core coding segment of a specific DNA according to the attributes of the sample, and synthesizing the specific DNA according to the core coding segment;

(A2) designing and synthesizing a corresponding secret primer according to the core coding segment of the specific DNA and a secret primer design rule;

(A3) establishing a database D1 of the corresponding relation among the sample, the specific DNA, the confidential primer and the source information and/or batch information of the sample for later reference;

3. The method of claim 2, wherein: in the method A, the step of performing DNA amplification sequencing and tracing on the sample according to the confidential primers is performed according to a method comprising the following steps of:

(A5) obtaining said sample in the market or making a non-contaminating sample on the inner packaging of said sample, isolating DNA molecules from said sample;

(A6) obtaining a confidential primer corresponding to the sample in the database D1, amplifying the DNA molecule separated in the step (A5) under the participation of the confidential primer, completing DNA sequencing, and obtaining sequence information of a coding fragment;

(A7) tracing and comparing according to the following steps:

a first step comprising:

a second step comprising:

comparing the sequence of the coding segment obtained from the sample or the internal package of the sample with the coding segment in the database D1, wherein if the coding segment exists in the database D1, the source information and/or batch information of the sample corresponding to the coding segment is the source information and/or batch information of the sample, and the tracing is successful; if the encoded segments do not exist in the database D1, tracing fails.

4. The method of claim 1, wherein: in the method B, the step of establishing the specific DNA database D2 of the sample source is carried out according to a method comprising the following steps:

(B1) performing a non-contaminating sampling in said sample source, isolating DNA molecules from said sampling;

(B2) selecting a coding segment from the DNA molecule according to the source characteristics of the sample;

5. The method according to claim 1 or 4, characterized in that: in the method B, the "DNA sequencing and tracing the sample" is performed according to a method comprising the following steps:

(B4) obtaining the sample in the market for pollution-free sampling, and separating DNA molecules from the sampling;

6. The method according to any one of claims 1-5, wherein: in the method a and/or the method B, a step of adding interfering DNA at the source of the sample is further included.

7. The method of claim 6, wherein: in the method a and/or the method B, the "adding interfering DNA at the source of the sample" is performed according to a method comprising the steps of:

(a1) designing, depending on the properties of the specific DNA, an interfering DNA that does not include the coding sequence of the specific DNA and the coding sequence of the privacy primer and does not amplify the specific DNA and the privacy primer;

8. The method according to any one of claims 1-7, wherein: the method further comprises the step of determining a tracing method according to the characteristics of the sample according to any one of the following steps:

(C1) for a sample which does not contain any DNA, adopting the method A, and screening the specific DNA and/or the interfering DNA which does not generate dissolution or any other reaction with the sample for tracing;

(C2) for a sample containing DNA, tracing by using the method B;

9. The method according to any one of claims 1-8, wherein: the method also comprises the step that a manufacturer manages the user by adopting a block chain technology; and/or

Establishing an application system of the Internet, the Internet of things and based on the database D1 or the database D2.

10. The method according to any one of claims 1-9, wherein: the sample is wine or food or other substances.