TW201634699A - In-field DNA extraction, detection and authentication methods and systems therefor - Google Patents

Abstract

The invention provides a method for in-field detection of a distinctive marker. The method includes providing a sample from an article of interest and analyzing the sample to detect the presence of the distinctive marker. The analysis is performed using an in-field detection instrument. The in-field detection instrument includes a microsystem configured to perform sample in-answer out analysis and detect the presence of the distinctive marker in the sample.

Description

Method and system for on-site deoxyribonucleic acid extraction, detection and verification

The present invention relates to the use and detection of distinctive markers for tracking and verifying items of interest. The distinctive mark may be unique to the item or commercial item to be tracked for inventory or security purposes. The present invention is also directed to methods and apparatus for sampling and detecting the distinctive landmarks, as well as management, storage, and processing of identification data obtained by such devices.

A method for labeling and tracing nucleic acid containing materials is described by Dollinger in WO 90/14441. The nucleic acid tag can be any suitable linear or circular, single or double strand DNA or RNA. The peptide or protein can be any suitable natural or synthetic peptide or protein. The photoconductive agent can be, for example, an upconversion phosphor or a fluorophore. The chemical label can be any suitable chemical label, such as a stain or pheromone that can be used to optically or chemically characterize the fingerprint of an object.

Hatfield, WO 00/55609, describes a method of forming a detectable feature fingerprint on an object or article. A method for DNA fingerprinting is described by Schulz, MM and W. Reichert in "Archived or directly swabbed latent fingerprints as a DNA source for STR typing" Forensic science international 127.1 (2002): 128-130.

The present invention provides an apparatus, system, and method for on-site detection of distinctive markers.

In an embodiment of the invention, the method for detecting a distinctive mark on the spot comprises: providing the same from an item of interest; and analyzing the sample to detect the existence of a distinctive mark in. The analysis was performed using an on-site inspection instrument. The field test instrument includes a microsystem configured to perform sample in-out analysis and to detect the presence of the distinctive marker in the sample.

The sample may include the same identifier, which may include an optical reporter, a digit code, a QR code, or a code. The photoconductive agent may include an upconverting phosphor, a fluorophore (requires a developer to exhibit fluorescence), a stain, a colorant, a phosphor, or A microdot.

The distinctive marker can comprise a biomolecule or an organic non-biological molecule. The biomolecule may be, for example, an amino acid, a peptide, a protein, a lipid, an isoprene, a monosaccharide, a polysaccharide, a fatty acid, a vitamin, a nucleic acid, a protein-DNA complex. , a peptide nucleic acid (PNA), a trace element, or an isotope.

The distinctive marker can include DNA. The distinctive marker can include one or more nucleic acid sequences that can be replicated to produce a unique amplicon profile. The unique amplicon distribution can include one or more amplicons of different lengths.

The field test instrument communicates with a server that contains authenticity data for the item of interest.

The sample can be analyzed by PCR, isothermal amplification, or nucleic acid hybridization. This sample can be analyzed using next-generation sequencing.

The field test instrument can be portable.

The presence of the distinctive marker can be quickly detected. For example, the presence of the distinctive marker can be detected virtually immediately.

According to an embodiment of the invention, the method includes providing the same from an item of interest; and analyzing the sample to detect the presence of the distinctive mark. The analysis is performed using an on-site inspection instrument. The field test instrument includes a microsystem configured to perform sample in-out analysis. The authenticity of the item of interest is verified by detecting the presence of the distinctive marker in the sample.

According to an embodiment of the invention, for collecting samples taken from items of interest The set of units includes the same collection unit configured to collect a sample comprising distinctive markers suitable for analysis by on-site instrumentation.

The kit can include a buffer solution or a solvent for extracting the distinctive marker from an item of interest.

In accordance with an embodiment of the present invention, an apparatus for in situ detection of a distinctive landmark includes an on-site inspection instrument that includes a microsystem configured to perform sample in-out analysis. The field testing instrument is configured to analyze the same to determine the presence of a distinctive marker in the sample.

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1 is a flow chart of an embodiment of the method of the present invention; and FIG. 2 is a diagram showing an instant PCR result according to an embodiment of the method of the present invention, illustrating the detection of a DNA marker from a fastener; An instant PCR map according to an embodiment of the method of the present invention, illustrating the detection of a DNA marker; FIG. 4 is a flow diagram showing an embodiment of the present invention; and FIG. 5 is a diagram showing an on-site inspection apparatus according to an embodiment of the present invention. Example.

The present invention provides an apparatus, system, and method for detecting distinctive landmarks in the field.

In accordance with an embodiment of the present invention, the method of detecting a distinctive landmark on-site includes providing the same from an item of interest; and analyzing the sample to detect the presence of the distinctive marker. The analysis is performed using an on-site inspection instrument. The field test instrument includes a microsystem configured to perform sample in-out analysis and to detect the presence of the distinctive marker in the sample. The authenticity of the item of interest can be verified by detecting the presence of the distinctive marker in the sample.

Distinctive sign

The method of the invention relates to the detection of distinctive markers on site, for example: nucleic acid markers (such as linear or circular, single or double strand DNA), protein or peptide markers, optical journals, and other purposes for safety, tracking, and anti-counterfeiting purposes for verifying, validating, and tracking marked objects or items. Chemical sign.

A distinctive logo can be any detectable marker suitable for distinguishing a unique item from other items. The distinguishing marker can be a nucleic acid (eg, DNA), a protein, a peptide, an optical reporter, or a chemical marker. For example, the distinctive marker can include one or more nucleic acids having one or more unique nucleic acid sequences. The distinctive marker can comprise a unique combination of two or more nucleic acid sequences. The distinctive marker can comprise a combination of nucleic acid sequences that, when replicated, produces a unique combination of amplicons of a particular length, that is, the combination of the amplicon produces a uniquely variable amplicon distribution. The nucleic acid of the distinctive marker can include DNA.

The distinctive marker can comprise a biomolecule or an organic non-biological molecule. The biomolecule is a molecule produced by a living organism. The biomolecule may be an amino acid, a peptide, a protein, a lipid, an isoprene, a monosaccharide, a polysaccharide, a fatty acid, a vitamin, a nucleic acid, a protein-DNA complex, Or a peptide nucleic acid (PNA).

The distinctive marker can include DNA. The distinctive marker can include one or more DNA sequences that can be replicated to produce a unique amplicon distribution. For example, a unique amplicon distribution can include a particular combination of one or more nucleic acid sequences that are unique to a particular article. For the article, one or more of the DNA sequences themselves or a combination of DNA sequences can be unique. The unique amplicon distribution can include one or more amplicons of different lengths. For example, the distinctive marker can comprise a combination of nucleic acid sequences that, when replicated, produce a unique combination of amplicons of a particular length, ie, the combination of the amplicon produces a uniquely variable amplicons distributed. In one embodiment, the distinctive marker can comprise a combination of unique DNA sequences that can be replicated to produce a unique combination of amplicons, including both a particular nucleic acid sequence and a particular amplicon length.

The nucleic acid marker can be synthesized by a nucleic acid synthesizer or by isolating a nucleic acid substance from a yeast, a human cell strain, a bacterium, an animal, a plant, or the like. In one embodiment, the nucleic acid material can be treated by restriction enzymes followed by purification to produce an acceptable nucleic acid signature. Guo describes a method for analyzing nucleic acid codes in WO 98/06084. The nucleic acid taggant may comprise a particular nucleic acid sequence or may comprise a plurality of different nucleic acid sequences. In one embodiment, a polymorphic DNA fragment comprising short tandem repeats (STRs) has been used as fragment length polymorphism and/or single nucleotide polymorphisms (SNPs). ) can be used for anti-counterfeiting nucleic acid labels. While the use of a single sequence nucleic acid marker can make the detection of a marker simpler and faster, in general, the use of a plurality of nucleic acid sequences, including STRs and SNPs, provides a higher degree of security against counterfeiters.

In one embodiment of the invention, the nucleic acid marker is derived from DNA extracted from a particular plant source and is specifically cleaved and linked to produce a unique artificial nucleic acid sequence in the world. The decomposition and linkage of the extracted DNA is accomplished by standard enzymatic and ligation techniques well known in the art of molecular biology. After the modified DNA marker is produced, the marker can be coated with a substance to resist ultraviolet light and/or degradation. The DNA coated material can be of plant origin. The DNA marker sequence can be of any suitable length, for example, the DNA marker sequence can be a sequence having from about 20 to about 1000 bases.

After the nucleic acid fragment is fabricated or isolated from a known nucleic acid sequence, the method further comprises producing a DNA marker compound comprising the selected nucleic acid fragment. The resulting marker compound can be a solid or liquid, aqueous or oily, a suspension, an aggregate, or an analog thereof. One of the hallmark compounds can be characterized by protecting the nucleic acid fragment from ultraviolet light and/or other degradation factors that can degrade the nucleic acid label over time, and the nucleic acid is a validation label as one of a particular product. In an embodiment, when the marker is DNA, the nucleic acid tag can be coated or suspended in a solvent (eg, an aqueous or organic solvent) to produce a "reservoir" DNA marker solution having a particular concentration. The stock DNA solution can be added to the labeled compound to a suitable concentration depending on the product pattern to be verified. In embodiments, the DNA identifier can be mixed with other components of the marker compound without any prior coating treatment. Process applications such as nucleic acid fragment encapsulation and other techniques to avoid nucleotide (particularly DNA) degradation will be described in more detail below.

Another feature of the marker compound mixture can be used to camouflage or "conceal" the particular nucleic acid tag with a foreign or non-specific nucleic acid oligomer/fragment, thereby rendering an unauthorized individual (eg, a counterfeiter) difficult to identify the nucleic acid tag. The sequence. In an embodiment, the marker compound comprises a specific single-stranded DNA marker derived from known sources (eg, mammals, invertebrates, plants, and the like), and a genome associated with or similar to its source. DNA. a logo The number of DNA identifiers in the product depends on the specific product to be verified, the period during which the label needs to be valid before verification (eg, 1 day, 1 month, 1 year, or many years), the expected exposure environment, The detection method used, etc.

According to an embodiment, the labeling system is a single strand of DNA, and the PCR technique is used for the detection of the marker. The copy number of the DNA identifier in the marker compound used for verification at a predetermined sample size is from about 3 to about 100,000, for example, from about 10 to about 50,000, or from about 100 to about 10,000 DNA markers. Copy number.

Combination of functional groups

In embodiments, prior to incorporation into a particular product, the nucleic acid tag is labeled with at least one compound or "detection molecule" to aid in the extraction/and detection of the nucleic acid marker of the product in the supply chain. The detection molecule is a molecule or compound having at least one detection group. For example, in certain methods of detection, a fluorescent molecule can be placed in conjunction with the nucleic acid signature and described in detail below.

In embodiments of the invention, suitable stains include, but are not limited to, coumarin stains, xanthene stains, resorufin, cyanine stains , difluoroboradiazaindacene (BODIPY) dyeing agent, ALEXA staining agent, indoles, bicyclic nitrogen heterobilybdenum (bimanes), isoindoles, dansyl staining agent, naphthoquinone Naphthalimides, phthalimides, dibenzopyran, lanthanide stains, rhodamines, and luciferin. It has been known that certain visible and near-infrared stains can be detected as a single molecule due to sufficient light and stable light. In this respect, the visible light stain BODIPY R6G (525/545) and a larger stain LI-COR's near-infrared stain IRD-38 (780/810) have a single molecular sensitivity and can be detected, and then used in this paper. Describe the implementation of the verification process. In embodiments of the invention, suitable stains include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), rose bengal, 5-(2'-aminoethyl) 5-(2'-aminoethyl)amino naphthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, chelating chelate derivative (terbium chelate) Derivatives, Reactive Red 4, BODIPY stains, and cyanine stains.

A variety of linking groups and methods are known to attach fluorophore or visible light stain groups to nucleotides, as exemplified by Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991). Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3' thiol group on oligonucleotide); Zuckerman et al, Nucleic Acids Research, 15: 5305-5321 (1987) (3' thiol group on oligonucleotide); Sharma et al, Nucleic Acids Research, 19: 3019 (1991) (3'sulfhydryl); Giusti et al, PCR Methods and pplications, 2: 223-227 (1993) and Fung et al, US Patent No. 4, 757, 141 (5' phosphoamino Group via Aminolink ^TM II available from Applied Biosystems, Foster City, CA), Stabinsky, U.S. Patent No. 4,739,044 (3' aminoalkylphosphoryl group); AP3 Labeling Technology (US Patent Nos. 5,047,519 and 5,151,507, assigned to EI DuPont de Nemours &Co); Agrawal et al, Tetrahedron Letters, 31: 1543-1546 (1990) (attachment via phosphoramidate linkages); Sproat et al, Nucleic Acids Research, 15 4837 (1987) (5 'mercapto group); Nelson et al., Nucleic Acids Research, 17: 7187-7194 (1989) (3' amino group); or a similar person.

In an embodiment, the complementary nucleic acid probe is labeled with at least one compound or molecule having a function to help detect the nucleic acid tag/marker. The technique used to label the nucleic acid tag or the complementary probe and the stain used may be the same as the nucleic acid will be bound to the probe.

The detection molecules of the invention can be bound to probe motifs, such as Held et al, Genome Res. 6: 986-994 (1996), Holland et al, Proc. Nat. Acad. Sci. USA 88: 7276- Taqman probe as described in 7280 (1991), Lee et al., Nucleic Acids Res. 21: 3761-3766 (1993); Tyagi et al, Nature Biotechnol., 16:49-53 (1998), U.S. Patent No. 5,989,823 , molecular beacons issued on November 23, 1999; scorpion probes as described by Whitcomb et al, Nature Biotechnology 17:804-807 (1999); Nazarenko et al, Sunrise probe as described in Nucleic Acids Res. 25:2516-2521 (1997); Cook, R., copending and co-assigned U.S. Provisional Application Serial No. 60/138,376, filed on Jun. 9, 1999. Assisted morphological probe; Kibista et al., WO 97/45539, a peptide nucleic acid-based luminescent probe as described in December 1997; Higuchi et al, Bio/Technology 10: 413-417 (1992), Wittwer Et al, Bio/Techniques 22: 130-138 (1997), a double-strand specific DNA stain; and the like. These and other inventions A probe mode system which can be used for the detection molecule is evaluated in Nonisotopic DNA Probe Techniques, Academic Press, Inc. 1992.

In an embodiment, the molecular beacon system is used to detect and count nucleic acid tags of the product of interest. A "molecular beacon" is a hairpin-type nucleic acid detection probe that, when bound to a target, produces a morphological transformation such that it can be detected. Generally, the loop portion of a molecular beacon is a probe nucleic acid sequence that is complementary to the nucleic acid signature. The stem portion of the molecular beacon consists of an arm sequence that links the molecular beacons present at both ends of the probe sequence. a functional group such as a fluorophore (eg, coumarin, EDNAS, luciferin, lucifier yellow, tetramethylrhodamine, Texas Red, and The analog thereof is covalently attached to the tail end of the one arm sequence, and a quencher molecule such as a non-fluorescent quencher (eg, DABCYL) is covalently attached to the end of the other arm sequence. end. When a target (nucleic acid tag) is absent, the molecular beacon maintains inhibition of the functional group because the functional group is close to the quencher molecule. However, when the molecular beacon and its specific target point, the molecular beacon will produce a morphological transformation such that the trunk and the ring structure cannot be formed, thereby increasing the distance between the functional group and the quenching body, resulting in the target Existence can be detected. When the functional group is a fluorescent group, the binding of the molecular beacon to the nucleic acid tag is detected by a fluorescence spectrometer.

In an embodiment, a plurality of nucleic acid tags having multiple sequences are used to label a particular product. Different nucleic acid tags can be quantitatively detected by a plurality of molecular beacons each having a fluorophore of a different color and a unique probe sequence complementary to at least the plurality of nucleic acid tags. The quantification of different fluorophores (ie different nucleic acid tags) provides a higher degree of validation and safety. It is to be noted that other functional groups described above for labeling the acid probe can also be used in the molecular beacon of the present invention.

Application of the distinctive mark of the item

According to an embodiment, the object to be marked may be any suitable item, such as a microchip, a label, a identification card, a logo graphic, a printed matter, a document, a textile, or a commodity. Wiggins describes biochemical marker papers in WO 02/057548. In one embodiment, the invention provides a method of in situ detection and verification of a distinctive marker, such as: DNA, a protein, a peptide, an optical guide, and a labeling chemical. According to an embodiment, the method of the invention comprises providing the same from an object of interest; using a specific Detecting the sample according to the detection method of the distinctive mark to detect a distinctive mark having unique information about the object, wherein the detecting method is performed by using a portable device; and by detecting the distinctive mark in the sample The existence of the item confirms the authenticity of the item of interest.

In embodiments, the DNA marker that can be verified by the methods of the invention can be any suitable article, such as: a high-priced item, or a unique item, or an item having an important function. The tagged item can be an electronic product, such as a microchip; or the item can be a product, cloth, textile, label, identification card, logo graphic, clothing, or accessory. In an embodiment, the marked item can be an ink, a document or a printed matter, or a commodity. In one embodiment, the labeled item can be a pharmaceutical product such as, but not limited to, a lozenge, an oval lozenge, a powder, or a buccal tablet. In one embodiment, the item is selected from the group consisting of cash, a currency note, a coin, a gemstone, a jewel, an instrument, a passport, an antique, a piece of furniture, a work of art, a property contract, a stock certificate, Or a group of bond certificates. The marked item can be a preserved item or a non-storage item in transit.

In one embodiment, the DNA marker that can be verified by the method of the invention is a microchip, a label, an identification card, a logo graphic, a printed matter, a textile, or a commodity. In one embodiment, the item is selected from the group consisting of cash, a currency note, a coin, a gemstone, a jewel, a musical instrument, a passport, an antique, a piece of furniture, a work of art, a property contract, a stock A group of certificates or a bond certificate.

The distinctive mark may be attached to an item of interest using any method that is feasible and suitable for fixing the distinctive mark on the item of interest such that the distinctive mark is not easily detached during the movement of the transaction or is intentionally damaged. Let one of the distinctive signs be taken at any point in the circulation of the transaction. In the present specification and drawings, the words "items", "objects", or "interests" may be used interchangeably. In the present specification and drawings, words such as "mark", "distinct mark", "safety mark", "tag", and "marker" may be used interchangeably. A sample may include any or all of the distinguishing signs obtained from the object of interest, the object of interest having the distinguishing sign attached thereto. For example, a sample of the distinctive marker can be obtained by any suitable physical or chemical means. A plurality of samples of the same distinctive mark of the same interest may be obtained at a plurality of points in the circulation of the transaction and/or at different points in time at which the item moves in the transaction. Multiple identical distinctive markers can be placed at multiple locations on the object of interest, or multiple different distinctive landmarks Zhi can be placed on the object of interest. For example, the distinctive marker can be placed at a location on the object of interest, and a non-discriminating marker (eg, a disguise or counterfeit marker) can also be placed in the same object of interest.

An item of interest may be any item, product, solid, liquid, or gas, such as: a microchip, a label, an identification card, a logo graphic, a printed matter, a textile, a commodity, an ink, or A solution.

The object of interest may be marked with a relatively small number of distinctive markers such that the location of the distinctive marker on the object of interest is not readily visible to the naked eye and/or may be readily detectable without visual inspection of the article. However, the amount of distinctive markers is not limited and an item can be marked for any desired amount of distinctive markers. For example, a relatively large number of distinctive markers can be used for the article, as it may be desirable for the distinctive marker to be unobtrusive. Sleat describes a marking device for a nucleic acid-labeled article in WO 03/080931. Mackay describes a method of applying DNA samples to articles in GB 2434570 A. A method for labeling solids is described by Slater in WO 95/02702. A method for labeling products for identification is described by Garner et al. in WO 95/06249. A method of marking an article or substance is described in WO 87/06383 by Le Page et al. A process for marking articles and/or documents is described by Regan et al. in WO 03/030129.

As described above, the method of binding the nucleic acid tag to a product can depend on the type of product to be verified. The nucleic acid tag can be added to a labeled compound in a "naked" or coated form at a predetermined concentration to achieve accurate detection of the nucleic acid identifier. The marker compound can be a liquid upon addition to the nucleic acid identifier and can be dried prior to attachment to a particular product (eg, a pharmaceutical lozenge, or textile). When the marker compound comprising a nucleic acid identifier is in a liquid state, the marker compound can be attached to the product in the form of a lacquer, paint, or liquid spray.

In one embodiment, the microfluidic device of the present invention utilizes an enzyme treatment to break down biomolecules, such as proteins and lipids in a sample, which may be a saliva sample or a swab, and release the DNA content. Downstream analysis and detection. The released DNA then flows into the PCR thermocycler module in the microfluidic channel for amplification reaction and is detected by another module (eg, a capillary electrophoresis module). Detection can be any number of methods, such as optical detection, radiation detection, Ion Sensitive Field Effect Transistor (ISFET), or any general knowledge in the field of oligonucleotide sequencing and detection. The method of knowing. The generated data can be analyzed by the module or transmitted by a secure server to a web server or a cloud server for comparison or further analysis with other stored data. Such a system can be designed to provide data readout within 90 minutes or 90 minutes. The application of this method is bloodline biometrics, or identity authentication for personnel or passengers.

Sample identifier

In one embodiment, the DNA marker is associated with the same identifier (eg, a test reporter), and the method further comprises positioning the photodetector that binds to the DNA marker to facilitate in the on-site detection and verification step The DNA marker was sampled. The sample identifier can be an optical reporter, a digital code, a QR code, or a code. The photoconductive agent may comprise an upconversion phosphor, a phosphor, a stain, a colorant, or a phosphor. In one embodiment, the detection reporter is selected from the group consisting of a chemical reporter, a digital reporter, and/or a peptide reporter. In one embodiment, the digital reporter can be any suitable digital reporter, such as a code, a QR code, a quantum dot, or a Radio Frequency Identification (RFID). In another embodiment, the QR code, the quantum dot, the barcode, or the RFID may include a sequence encoded by the DNA marker, such as, for example, Tran et al. in WO 2013/170009 ( Verification or physical encryption taggants using digital representatives and authentications thereof ) Said in the middle.

A sample identifier can be combined with the distinctive marker. The sample identifier can be any suitable sample identifier to facilitate localization of the distinctive marker. For example, the sample identifier can be used to identify a location that is linked to the distinctive marker of interest. Similar to the distinctive marker, the sample identifier can be visually or tactilely inspected for interest and not readily detectable. For example, the sample identifier can be an optical reporter, such as a fluorescent stain, which is only activated upon exposure to ultraviolet light. Therefore, exposing the interested substance having the distinctive mark to the photoconductive agent to the ultraviolet light can visually display the position of the distinctive mark on the object of interest. A method and ink set for marking an article is described in Degott, EP 1 403 333 A1).

The sample identifier can be a tactile identifier, such as a course pattern, and can be placed on the surface of the object of interest on the surface of the distinctive mark. The haptic identifier can be an anomalous smooth pattern formed on the surface of the object of interest. However, the distinctive marker can be applied to the article separately without the need for a sample identifier.

The DNA marker itself can be a detectable DNA marker. The detectable DNA marker can be labeled, for example, with a fluorescent label or probe.

Flag sample

The sample for on-site detection and verification of a DNA marker can be any suitable sample, for example: one solid sample taken from a plane, a chip, a cut, a powder, a liquid, a mist, or a sampling. The sample may be collected by a collection container, such as a plastic or glass test tube, a capped tube, a microcap tube, a well of an assay disk, a microfluidic channel, a receiving container, or other suitable container.

A sample comprising some or all of the distinctive markers can be collected from the object of interest and analyzed to detect the presence of the distinctive marker. The sample can be analyzed by any method suitable for detecting the distinctive marker. For example, the analysis can be performed using an on-site inspection instrument. The on-site inspection instrument is an instrument that performs the analysis without transferring the sample to the laboratory. For example, a sample of a distinctive mark can be obtained from the product before it is shipped from the factory to the consumer, and the sample can be analyzed by an on-site inspection instrument in the factory without the need to ship the sample to the laboratory for processing. test. Thus, the analysis can be performed quickly and performed substantially simultaneously with the collection of the sample or within minutes of collecting the sample. For example, the distinctive marker can be detected substantially simultaneously (eg, within 10-20 seconds), detected immediately, or detected within minutes of placing the sample in the field testing instrument. For example, analyzing and/or detecting the distinctive marker can be completed in about 5 minutes to about 2 hours. More specifically, the analysis and/or detection of the distinctive marker can be completed in about 10 minutes to about 30 minutes. The authenticity of the object of interest can be verified by detecting the presence of the distinctive marker in the sample.

According to an embodiment, the kit for collecting samples from an object of interest comprises an identical collection unit configured to collect a sample comprising a distinctive marker suitable for analysis by an on-site instrument. Different nucleic acid extraction solutions (eg, solvents or buffer solutions) can be used to extract nucleic acid sequences from an interesting sample. For example, Green, Michael R. and Joseph Sambrook describe the flow of nucleic acid extraction in Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press, 2012. In one embodiment, the sample collection kit can include a buffer solution or a solvent suitable for extracting the distinctive marker from an object of interest, and the buffer solution or solvent is compatible with the instrument for performing on-site testing.

Nucleic acid label extraction and acquisition method

In recent years, a variety of nucleic acid extraction solutions have been developed for the extraction of nucleic acid sequences from an interesting sample, for example, by Green, Michael R. and Joseph Sambrook, Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press, 2012. This method typically requires one or more steps, such as a detergent-mediated step, a protease treatment step, a phenol/and or chloroform extraction step, and/or an alcohol precipitation step. Some nucleic acid extraction solutions may comprise an ethylene glycol based reactant or an ethylene glycol derivative to increase the efficiency of nucleic acid extraction, while other methods use only grinding and/or boiling the sample in water. Other methods including solvent based systems and ultrasonic extraction can also be used in conjunction with other extraction methods.

In one embodiment, the verification procedure comprises directly obtaining the nucleic acid tag with a complementary hybridization probe attached to a solid support. Generally, a method for obtaining a nucleic acid tag includes a substance in a solid layer interacting with a reactant in a liquid layer. In an embodiment, the nucleic acid probe is attached to the solid layer. The nucleic acid can be present in the solid layer by any conventional covalent or non-covalent interaction immobilization on a solid support. In an embodiment, the support comprises an insoluble material such as a controlled pore glass, a glass or glass plate, polystyrene, acrylamide gum, and activated dextran. In an embodiment, the support has a strong or semi-solid character and can be of any shape, such as a sphere such as a bead, a square, a gel, a microsphere, or a substantially flat support. In an embodiment, it is preferred to make a physically spaced region on the support, such as a hole, a raised region, a socket, a needle, a groove, a post, a needle, an inner or outer wall of the tube, and the like. Other suitable support materials include, but are not limited to, agarose, polypropylene decylamine, polystyrene, polyacrylate, hydroxyethyl methacrylic acid, polyamidamine, polyethylene, polyoxyethylene, or copolymers of this type And a graft copolymer. Exemplary solid supports include small particles, non-porous surfaces, addressable arrays, vectors, plastids, or polynucleotide immobilization matrices.

As used in the method of obtaining the nucleic acid tag, a nucleic acid probe can be attached to the solid support by covalent bond or other affinity interaction to impart a chemical reaction function to the solid support. The nucleic acid can be attached to the solid support by its 3', 5' sugar or nucleobase. In an embodiment, it is preferred that the 3' end is attached to the support by a linker, since the selection of stable or selectively decomposable linkers is numerous, preferably between the support and the nucleic acid. Covalent price The knot is fixed. The linker unit, or linker, is designed to stabilize and facilitate contact of the immobilized nucleic acid with its complementary sequence. On the other hand, non-covalent linkages such as biotin and avidin or streptavidin can be used. Other functional group linkers include: esters, guanamines, urethanes, ureas, sulfonates, ethers, and thioesters. A 5' or 3' biotin modified nucleotide can be immobilized to avidin or streptavidin attached to a support (e.g., glass).

Depending on the starting concentration of the nucleic acid tag to which the product of interest is added, the tag can be quantified without amplification via PCR. In an embodiment, a single-stranded DNA tag labeled with a detection molecule (eg, fluorophore, biotin, etc.) can hybridize to a complementary probe attached to a solid support such that the label is placed on the label. The "detection molecule" can be specifically detected. The nucleic acid DNA tag can also be double-stranded, and at least one of the lines is labeled with a detection molecule. In the case of a double-stranded DNA tag, the nucleic acid tag must be sufficiently heated and then rapidly cooled to produce a single strand of DNA, and under suitable hybridization conditions, at least one strand of one of the detection molecules can hybridize to the complementary DNA probe. .

In an embodiment of the invention, the complementary probe is labeled with a detection molecule and can hybridize to one of the nucleic acid tags. When the product is a textile, the hybridization with the probe can be completed within the product; or, when the product is in a liquid state (eg, oil, gasoline, perfume, etc.), the hybridization with the probe can be from This is done after the product has been extracted from the nucleic acid label/marker. The method of direct detection described in this specification can be based on a high initial concentration of the nucleic acid tag embedded in the product or an accurate extraction/acquisition method that concentrates the nucleic acid tag extracted from a large volume or mass specific product.

At present, the technology of complete sequencing of human genomes (ie, whole gene amplification reaction) can be performed to identify the difference in gene codes between different individuals. The study found that each person has about one base difference per thousand bases. These differences are called genetic variations or single nucleotide polymorphisms (SNPs). In medical fields such as personalized medicine, these variations are extensively studied to determine whether a body is a predicate individual for a disease (eg, diabetes).

Sample analysis and mark detection

For example, the sample can be analyzed using PCR, thermostatic amplification, or nucleic acid hybridization. This sample can be analyzed using next generation sequencing. The method of sample analysis will be described in more detail below.

DNA amplification reaction

In an embodiment, the verification procedure comprises amplifying the nucleic acid marker by a polymerase chain reaction. During amplification, the primer dimer and other foreign nucleic acids can be amplified together with the nucleic acid analyte. In order to allow subsequent analysis of the PCR product to form a sufficient amount, an internal control competitor was used to quantify the competitive PCR amplification reaction and stopped after the logarithmic proliferation phase of product formation was completed.

In an embodiment, the PCR amplification reaction is carried out in the presence of a non-primable probe that can be detected, the non-primable probe probe being specifically bound to the PCR amplification reaction product (ie: amplified DNA detection portion) ). PCR primers can be designed according to conventional standards, and PCR can be performed by commercially available instruments. The probe system can be a DNA oligonucleotide designed to specifically bind to the amplified detection molecule. The probe can be a DNA oligonucleotide specifically designed to bind to the amplified detection molecule. Preferably, the probe has a 5' reporter stain covalently attached to the probe and a downstream 3' quencher stain that conducts fluorescence resonance energy. Suitable fluorescent reporter stains include: 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxy-fluorescin (TET), 2,7- Dimethoxy-4,5-dichloro-6,5-dichloro-6-carboxy-fluorescein (JOE), and hexachloro-6-carboxyfluorescein (hexachloro-6-carboxy-fluorescein, HEX). A suitable reporter staining agent can be 6-carboxytetramethyl-rhodamine (TAMRA). These stains are commercially available from Perkin-Elmer, Philadelphia, Pa. The PCR amplification reaction product can be detected in each PCR amplification reaction cycle. In the cycle of any PCR amplification reaction, the amount of the PCR product is proportional to the number of copies of the starting sample. The number of copies of the sample can be detected by the amount of fluorescence of the reported stain. When the probe is intact, the reporter stain inhibits its fluorescent reporter due to proximity to the quencher stain. In a PCR reaction, the DNA polymerase cleaves the reporter stain and the quencher stain in a 5' to 3' direction to increase the amount of fluorescent light of the reported stain because it is not close to the quencher The stain, the increase in the amount of fluorescence measured, is directly proportional to the PCR amplification reaction. The detection system is commercially available from Perkin-Elmer's TaqMan® PCR System for real-time PCR detection. Analysis of nucleic acid probes is described in WO 97/45539 by Kubista, Mikael et al. Whitcombe, David et al., "Detection of PCR products using self-probing amplicons and fluorescence" Nature biotechnology 17.8 (1999): 804-807, describes the detection of PCR products using self-probe amplification. Nelson, Paul S., Roy A .Frye, and Edison Liu, "Controlled pore glass" is described in "Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations" Nucleic acids research 17.18 (1989): 7187-7194" , CPG) to synthesize oligonucleotide probes. Lee, G., Linda G., Charles R. Connell, and Will Bloch, "Allelic discrimination by nick-translation PCR with fluorgenic probes" Nucleic acids research 21.16 (1993): 3761-3766" for the use of fluorescent matrix probes for nick translation A nick-translaton PCR reaction.

In an embodiment, the reporter stain and quencher staining agent can be located on two separate probes that are enlarged and adjacent to each other sufficient for the quencher stain to inhibit the reporter stain Optical PCR detects molecular hybridization. In the detection system as described above, in the 5' to 3' nuclease action, the polymerase removes a stain from the probe containing the stain to cause the reporter stain to be quenched on the proximity probe. The colorant is separated to prevent the inhibition of the reported stain. As described in the above examples, the detection of the PCR product measures the increase in fluorescence of the reported stain.

The real-time quantitative PCR reaction is applied to nucleic acid analytes or samples. In this method, PCR is used to amplify the DNA in the same format in the presence of a non-extensible double-labeled fluorescent matrix hybridization probe. A fluorescent stain acts as a reporter and its emission spectrum is inhibited by a second fluorescent dye. This method utilizes the nuclease action of the 5'-Taq polymerase to cleave a hybridized probe in an extension phase in a PCR reaction. Nuclease degradation of the hybridization probe releases the inhibited reporter stain, resulting in an increase in the emission peak of the reporter. The reaction is monitored on the fly. Heid, Christian A, et al., "Real time quantitative PCR" Genome research 6.10 (1996): 986-994", Gibson, UE, Christian A. Heid, and P. Mickey Williams, "A novel method for real time quantitative RT-PCR"" Genome research 6.10 (1996): 995-1001" describes reverse transcriptase PCR reactions (RTPCR) and real-time PCR reactions. A variety of commercial temperature cycles can be used to continuously monitor the fluorescence spectrum of multiple samples in a PCR reaction, so accumulation of PCR products can be monitored "on-the-fly" while avoiding the risk of laboratory amplicon contamination (eg Heid, CA; Stevens) J.; Livak, KL; Williams, PW, as described in "Real time quantitative PCR. Gen. Meth. 6: 986-994" (1996).

In an embodiment, a strategy for real-time PCR detection, including known Techniques such as double-stranded DNA-binding stains (such as SYBR Green, a highly sensitive fluorescent colorant) that are visible with an embedded stain (eg, phenanthrene bromide) for other purposes. Processed by FMC Biotproducts), using dual fluorescent probes (as described by Wittwer et al., BioTechniques 22: 176-181 (1997)), and using pot-like fluorescent probes (eg, molecular beacons, For example, Tyagi and Kramer are described in "Nature Biotechnology 14: 303-308 (1996)"). While intercalating stains with double-stranded DNA-binding stains can instantly quantify the accumulation of products used in PCR reactions, they may lack specificity, leading to the detection of primer dimers, as well as any non-specific amplification products. Careful sample preparation, processing, and primer design using conventional techniques can reduce the presence of matrix and contaminating DNA and prevent the formation of primer dimers. Suitable PCR analysis instrument software and thermal cracking temperature analysis can provide an accurate and specific solution for use in such embodiments.

The molecular beacon system can be implemented in conjunction with an immediate PCR reaction to specifically quantify the nucleic acid sample in the sample. For example, commercially available Roche Light Cycler ^(TM) (Roche Diagnostics Corporation, Indianapolis, Indiana) or other such instruments may be used for this purpose. The detection molecule set to the molecular beacon can be visible under daylight or conventional light sources and/or fluorescent light. It should also be noted that the detection molecule can be a radiation emitter, such as a typical isotope. Tyagi, Sanjay, Diana P. Bratu, and Fred Russel Kramer describe multicolor molecular beacons in "Multicolor molecular beacons for allele discrimination" Nature biotechnology 16.1 (1998): 49-53" by Tyagi, Sanjay, and Fred Russell Kramer. Fluorescent molecular beacons are described in Molecular beacons: probes that fluoresce upon hybridization " Nature biotechnology 14.3 (1996): 303-308".

In one embodiment, DNA can be amplified by nanopore sequencing. The nanopore sequencing method involves the use of a tiny device (e.g., a nanopore having a diameter of about 1 nm) that is immersed in a conductive liquid and has a voltage applied to the nanopore. When individual nucleotides pass through or are close to the nanopore, a measurable voltage change is produced which can be used to identify individual nucleotides of the DNA. For example, Branton, Daniel et al., "The potential and challenges of nanopore sequencing" Nature biotechnology 26.10 (2008): 1146-1153", Deamer, David W. and Mark Akeson, "Nanopores and nucleic acids: prospects for ultrarapid sequencing The nanopore sequencing method is described in " Trends in biotechnology 18.4 (2000): 147-151".

In one embodiment, the DNA can be amplified by Multiple Annealing and Looping Based Amplification Cycles (MALBAC). MALBAC can be used to amplify an entire genome. The MALBAC method can amplify a genome in a quasi-linear manner and can be applied to the amplification of a single cell, the whole genome. In the case of the MALBAC method, the amplicon can have complementary endpoints. The complementary terminus forms a loop that prevents exponential replication of the amplicon and thus prevents amplification bias. For example, the MALBAC method is described by Zong, Chenghang et al. in "Genome-wide detection of single-nucleotide and copy-number variations of a single human cell" Science 338.6114 (2012): 1622-1626.

Highly sensitive, rapid and accurate detection and quantification of biologically relevant molecules is a topic of medical technology, national security, public safety, and the diagnosis of civil and military medicine. The methods currently used, including: enzyme linked immunosorbant assays (ELISAs) and PCR systems are highly sensitive. However, the need for PCR amplification reactions may make the detection method more complicated, expensive, and time consuming. In an embodiment, the anti-counterfeiting nucleic acid tag is described by Surface Enhanced Raman Scatter (SERS) in U.S. Patent No. 6,127,120. SERS is a detection method that is sensitive to relatively low target (nucleic acid) concentrations, preferably directly to an unmagnified sample. The nucleic acid tag and/or nucleic acid probe can be labeled or modified to achieve a surface enhanced Raman scattering change when the probe hybridizes to the nucleic acid tag. Quantitative detection of nucleic acids using SERS provides a relatively rapid method of analyzing and verifying a particular product.

Another useful detection method in the present invention is the Quencher-Tether-Liagnd (QTL) system described by Whitten et al., U.S. Patent No. 6,743,640. The QTL system provides a simple, fast, and highly sensitive biomolecule detection with structural specificity. The QTL system provides a chemical body consisting of a quenching body (Q), a Tethering element (T), and a ligand (L). The system detects the target biological agent in the same manner by observing the change in fluorescence.

The QTL system can quickly and accurately detect the same objectives as quantification. Biomolecules. Examples of suitable ligands that can be used in the polymer-QTL method include: chemical ligands, hormones, antibody fragments, oligonucleotides, antigens, polypeptides, glycolipids, proteins, protein fragments, enzymes, peptide nucleic acids, And polysaccharides. Examples of quenchers for QTL include: methyl viologen, quinones, metal complexes, fluorescent stains, and electron accepting, electron donating, and energy acceptors. The linking substance may be, for example, a single bond, a single divalent atom, a divalent chemical, and a multivalent chemical. However, examples of such ligands, linking materials, and quenchers that form QTL molecules do not limit the composition of the QTL molecule, and other suitable ligands, linking materials, and quenching bodies may be used.

Traditional PCR uses a repeated temperature cycle to amplify the DNA. However, according to embodiments of the present invention, amplification of DNA may not require temperature cycling (eg, by constant temperature amplification). For example, DNA can be subjected to Loop-mediated isothermal amplification (LAMP), Strand displacement amplification (SDA), Nicking enzyme amplification reaction (NEAR), and recombinase. Recombinase Polymerase Amplification (RPA), Helicase-dependent amplification (HAD), or thermophilic helices dependent amplification (tHDA) are thermostatically amplified. For example, Oriero, EC et al., "Comparison of two isothermal amplification methods: Thermophilic Helicate dependent amplification (tHDA) and loop mediated isothermal amplification (LAMP) for detection of Plasmodium falciparum" International Journal of Infectious Diseases 21 (2014): 381 "Limited by Li, Ying et al., "Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation" The Journal of Molecular Diagnostics 15.5 (2013): 634-641".

According to an embodiment of the invention, the DNA can be amplified by circular thermostat amplification (LAMP). The LAMP method uses a single tube to amplify DNA, where all of the reactants are cultured in a single sample tube. LAMP utilizes a polymerase with strand displacement characteristics without the need for a temperature circulator. For a relatively small target DNA sequence, the LAMP method can use a primer set (eg, 4 or 6 primers) to target a set of regions (eg, 6 or 8 regions). The LAMP method can use two internal primers and two external primers, plus two additional loop primers, which can be combined at the loop structure of the LAMP amplicon. This analytical method is designed to increase the sensitivity of the amplification reaction and accelerate the reaction time. For example, Oriero, EC et al., "Comparison of two isothermal amplification methods: Thermophilic Helicate dependent amplification (tHDA) and loop mediated isothermal amplification (LAMP) for detection of Plasmodium falciparum" International Journal of Infectious Diseases 21 (2014): 381 The "LAMP-mediated isothermal amplification of DNA" Nucleic acids research 28.12 (2000): e63-e63" is described by Notomi, Tsugunori et al.

According to one embodiment of the invention, DNA can be amplified by strand displacement amplification (SDA). The SDA method is a non-sequence specific DNA amplification method. In the case of the SDA method, a random hexamer primer binds to a DNA sample strand and DNA synthesis is performed by a highly accurate DNA polymerase. For example, "U.S. Patent No. 5,455,166", "U.S. Patent No. 5,712,124", Asiello, Peter J. and Antje J. Baeumner in "Miniaturized isothermal nucleic acid amplification, a review" Lab on a Chip 11.8 (2011): 1420-1430", Walker G. Terrance et al., "Miniaturized isothermal nucleic acid amplification, a review" Lab on a Chip 11.8 (2011): 1420-1430; and Walker, G. Terrance et al. in "Strand displacement amplification-an The isothermal, in vitro DNA amplification technique " Nucleic Acids Research 20.7 (1992): 1691-1696" describes the SDS method.

According to one embodiment of the invention, DNA can be amplified by a nicking enzyme amplification reaction (NEAR). The NEAR method involves the use of a replacement DNA polymerase to synthesize DNA from a nick formed by nicking enzyme. The NEAR method can produce many short nucleic acids from a target sequence relatively quickly. The alternating cycle of nicking and DNA amplification can achieve a billion-fold amplification in about 5 to 10 minutes. For example, the NEAR method is described by Ménová et al. in "Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR" Bioconjugate Chemistry 24.6 (2013): 1081-1093.

According to one embodiment of the invention, DNA can be amplified by recombinant enzyme polymerase amplification (RPA). The RPA method is a single tube amplification technique for DNA. The RPA method can include three enzymes: a recombinase, a single-granded DNA-binding protein (SSB), and a strand-dispensing polymerase. The recombinase can pair the oligonucleotide primer with the corresponding sequence in the duplex DNA. The single-stranded DNA-binding protein binds to the DNA of the replacement strand and prevents substitution of the primer. The strand displacement polymerase can initiate DNA synthesis at the point where the primer binds to a target DNA. A reverse transcriptase can be added to an RPA reaction to detect RNA and DNA without the need to generate cDNA. For example, Lutz et al. describe the RPA method in "Microfluidic lab-ona-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA)" Lab on a Chip 10.7 (2010): 887-893".

According to one embodiment of the invention, DNA can be amplified by heat-resistant melting enzyme amplification (tHDA). The tHDA method can selectively amplify a target DNA sequence (for example, a relatively short DNA sequence of about 70 bp to 120 bp), and the target DNA sequence is defined by two primers. The tHDA method uses a melting enzyme to separate DNA instead of heating, and produces a single strand of DNA sample for primer binding and DNA polymerase amplification. For example, Oriero, EC et al., "Comparison of two isothermal amplification methods: Thermophilic Helicate dependent amplification (tHDA) and loop mediated isothermal amplification (LAMP) for detection of Plasmodium falciparum" International Journal of Infectious Diseases 21 (2014): 381 The "HDA method" is described by Li, Ying et al., "Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation" The Journal of Molecular Diagnostics 15.5 (2013): 634-641.

Next generation sequencing

According to embodiments of the invention, DNA can be sequenced and/or detected by next generation sequencing (NGS) techniques. The NGS system is a group of high-throughput sequencing techniques (eg, massively sequential sequencing). The NGS method can sequence relatively large nucleic acid sequences or the entire genome. In the NGS method, many relatively small nucleic acid sequences can be sequenced simultaneously by a DNA sample and can form a small fragment (eg, read fragment) library. The read fragment can then be recombined to recognize a large nucleic acid sequence or a complete nucleic acid sequence. For example, sequences of up to 500,000 can be sequenced synchronously. For example, the MALBC method can be used to react with conventional PCR in NGS technology. For example, Mardis is in "The impact of next-generation sequencing technology on genetics" Trends in genetics 24.3 (2008): 133-141", Metzker in "Sequencing technologies-the next generation" Nature Reviews Genetics 11.1 (2009): 31 -46" describes the next generation sequencing method.

Massively Parallel Signature Sequencing (MPSS) is an example of NGS technology. The MPSS method recognizes mRNA transcripts by generating a 17 to 20 base pair marker sequence. The MPSS method can be used to simultaneously identify and quantify mRNA transcripts in the same format. For example, Brenner, Sydney et al. describe the MPSS method in "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays" Nature biotechnology 18.6 (2000): 630-634".

The Polonni sequencing method is an example of NGS technology that fixes and synchronizes millions of DNAs. The Bologna sequencing method is extremely accurate and has a low error rate. The Polonni sequencing method is a multiplex sequencing technique that measures multiple analytes in a single reaction/cycle, or analysis. For example, Shendure et al., "Advanced sequencing technologies: methods and goals" Nature Reviews Genetics 5.5 (2004): 335-344; and Shendure et al. "Next-generation DNA sequencing" Nature biotechnol 26.10 (2008): 1135- The Bolognese ordering method is described in 1145.

Pyrosequencing is an example of NGS technology that uses luciferase to detect the addition of each nucleotide of a nascent DNA. The pyrophosphate sequencing method amplifies the DNA of water droplets present in an oily solution. A water droplet can include, for example, a DNA sample attached to a primer-coated particle. For example, Vera, J. Cristobal et al., "Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing" Molecular ecology 17.7 (2008): 1636-1647; and Ronaghi "Pyrosequencing sheds light on DNA sequencing" Genome research 11.1 (2001 ): The pyrophosphate sequencing method is described in 3-11".

The Illumina sequencing method is an example of an NGS technique that attaches DNA molecules and primers to a slide. The DNA molecule can be amplified by a polymerase to form a DNA population (eg, a DNA population). For example, Hanlee's "Next-generation DNA sequencing" Nature biotechnology 26.10 (2008): 1135-1145 and Meyer and Kircher's "Illumina sequencing library preparation for highly multiplexed target capture and sequencing" Cold Spring Harbor Protocols 2010.6 (2010) ): Illumina sequencing method described in pdb-prot 5448".

Sequencing by Oligonucleotide Ligation and Detection (SOLiD Sequenceing) is an example of an NGS technique that can simultaneously generate thousands of small sequence reads (eg, DNA fragments). The sequence reads can be immobilized on a solid support for sequencing. Usually SOLiD sequencing is a method of sequencing by bonding. For example, Hanlee Ji is in "Next-generation DNA sequencing" Nature biotechnology 26.10 (2008): 1135-1145", and Meyer, Matthia and Ansorge, Wilhelm J. in "Next-generation DNA sequencing techniques" New biotechnology 25.4 (2009) ): 195-203" describes oligonucleotide ligation and detection sequences.

Ion Torrent Semiconductor Sequencing is an example of an NGS technique that detects released hydrogen ions in a DNA polymerization reaction. The ion semiconductor sequencing method is a sequence-by-synthesis method. Deoxyribonucleotide triphosphate (dNTP) can be added to a microwell containing sample DNA. When the deoxynucleotide triphosphate is complementary to the same nucleotide, the deoxynucleotide triphosphate can bind to a complementary DNA sequence leading to the release of a hydrogen ion. For example, Quail, Maichael A. et al., "A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers" BMC genomics 13.1 (2012): 341" describes ion semiconductor sequencing .

The DNA nanosphere sequencing method is an example of an NGS technique that uses rolling circle replication to amplify small fragments of genomic DNA to form DNA nanospheres. The amplified DNA sequence can then be combined using fluoroscopy as indicated. For example, Ansorge, Wilhelm J in "Next-generation DNA sequencing techniques" New biotechnology 25.4 (2009): 195-203", Drmanac et al. in "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays" Science 327.5961 (2010): 78-81" describes the DNA nanosphere sequencing method.

Heliscope Single Molecule Sequencing is an example of NGS technology that does not require a binding reaction or a PCR amplification reaction. The Heliscope single molecule sequencing method is a direct sequencing method in which DNA can be cleaved and has a poly-A-tail hybridized to the surface of a fluid cell. A large number of molecules (eg, billions of molecules) can then be sequenced simultaneously. For example, Pushkarev et al. describe Heliscope single molecule sequencing in "Single-molecule sequencing of an individual human genome" Nature biotechnology 27.9 (2009): 847-850.

Single Molecule Real Time (SMRT) Sequencing is an example of NGS technology in which DNA can be synthesized in relatively small pore-shaped containers of zero-mode waveguides (ZMW). Unmodified polymerase can be attached to the bottom of the ZMW. The unmodified polymerase can be used to sequence the DNA as well as to sequence fluorescently labeled nucleotides that are free to flow in solution. When the nucleotide binds to the DNA strand, the fluorescent label can be released by the nucleotide. The SMRT sequencing method is an example of a synthetic sequencing method. For example, Flusberg et al. describe a single molecule immediate sequencing method in "Direct detection of DNA methylation during single-molecule, real-time sequencing" Nature methods 7.6 (2010): 461-465.

In one embodiment, the present invention provides a system for in situ detection and verification of an article delineated by one or more markers selected from a DNA molecule, a protein, a peptide, an optical journal A group of agents, a chemical marker, and a digit code. The system can include a container adapted to collect a sample obtained from an indicia of interest; one or more analytical molecules, each analytical molecule being adapted to detect at least one marker using a particular detection method defined by the marker, providing test data to provide detection data And an analyzer for receiving detection data obtained from the one or more analysis molecules and determining presence or absence of the one or more markers in the sample; wherein the analyzer is configured to confirm The presence of the one or more markers in the sample determines the authenticity of the marker.

Mark detection

Examples of useful detection systems according to embodiments of the invention include: PCR-type DNA detection (digital PCR, pyrophosphate sequencing, DNAe), electrophoretic analysis (capillary or colloidal electrophoresis microfluidics), DNA sequencing using protein pores Nano devices, as well as microarray systems. Embodiments of the invention include the use of handheld, multi-function, or instrument-free USB devices to detect DNA and RNA. Sample collection devices, wafer size, analysis and data presentation methods can be customized according to the product and user requirements. Embodiments of the present invention can be performed on a wafer system at the world's largest factory utilizing standard CMOS processes that provide power to, for example, notebook computers and smart phone devices. This CMOS technology provides the fabrication of wafers with tens to millions of sensor arrays at reduced cost, so these wafers are essentially experimental for a small, stand-alone Wafer execution and can be used to solve a wide range of applications.

A device for performing and analyzing microarray detection is disclosed in US 2008/0207461 A1, and a microarray experiment for quantifying or quantifying a specific interaction between a detection probe and a target molecule in a microtiter is disclosed. Devices, and methods of making such devices. This device can simultaneously replicate nucleic acids and characterize nucleic acids as described by Ehricht et al. in US 7,888,074 B2. The construction of the device for detecting the fluorescent signal (as described in Ermantraut et al. US 2004/0196455 A1) and the analytical device and method for detecting the analyte (as described in Ermantraut et al. US 2011/0071038 A1) can be further incorporated into storage and A device for detecting the library of substances (as described in U.S. Patent No. US 2006/0147996 A1). The microarray method and apparatus are suitable for use in the methods and systems of the present invention for detecting and verifying nucleic acids and other chemical labels.

In an embodiment, the microarray device can be placed in a reaction vial configured to receive a provided sample. The microarray device can include a customized probe microarray configured to automatically detect the presence of one or more target nucleic acid sequences in a provided sample. Customized probe microarrays can be designed to detect the presence of a unique combination of one or more nucleic acid sequences that are unique to a particular interest. Customized probe microarrays can include oligonucleotide probes that are affixed to positions on the microarray. The oligonucleotide probes can each be complementary to an oligonucleotide sequence of a particular distinctive marker. The oligonucleotide probe can hybridize to a complementary target oligonucleotide sequence to detect the presence of a target acid sequence in the provided sample. The customized probe microarray can include any suitable microarray without limitation. The customized probe microarray can be of any suitable size and can include one or more control units or comparison units as desired.

A reaction vial comprising the microarray device can be configured to record and/or transmit test data to an integrated or external device to generate a distinctive mark detection report. For example, the reaction vial can automatically determine whether a nucleic acid sequence of a particular distinctive marker is present and transmit the data to an integrated or external device to generate a report indicating whether the provided sample includes the sample Distinctive signs. If a distinctive marker is detected, a report can be generated indicating that the sample provided is not from a non-counterfeit item (eg, a counterfeit). Microarrays for detecting peptides or proteins can also be designed and implemented, and the microarrays are not limited.

In one embodiment, the microarray device can be configured to provide a signal that detects the presence of the DNA to be detected in each well or matrix of the array. The wells that produce the positive detection signal can be arranged to be unique to a particular DNA signature. This form can be random or can be a specific design, such as a logo pattern or trademark, or a message. In an embodiment, the form can be optically discerned and the data can be transmitted to a server or stored locally. Van de Rijke et al., "Up-converting phosphor reporters for nucleic acid microarrays" Nature biotechnology 19.3 (2001): 273-276", describes a conversion of a phosphor reporter over a nucleic acid microarray.

In accordance with an embodiment of the present invention, the determination of whether an item of interest is marked may be performed by a quantized thermostat system that detects the presence of a nucleic acid identifier on an item. For example, the determination of whether an item is marked may be accomplished by commercially available Alere-i ^(TM) (i-NAT) devices (available from Alere Technologies, Jena, Germany).

According to an embodiment of the invention, the determination of whether the item of interest is marked by the distinctive mark can be performed by an on-site inspection instrument that employs RT-PCR and has the ability to sample the results of the analysis. For example, a point of care test Alere ^TM q platform may be applied to detect the presence of this as the distinctive sign. The Alere ^TM q internet point of care test system available on the market (Alere Technologies, Jena, Germany) . The point of care testing Alere ^TM q is an internet-based fully automated nucleic acid detection platform, which can perform automated nucleic acid extraction, separation, amplification and detection. Data analysis, data reporting, and data storage can all be automated.

According to an embodiment, a lateral flow strip test can be utilized to determine the presence of a marker. The transverse flow strip test system is designed to detect the presence or absence of a target analyte (eg, nucleic acid). Lateral flow testing can also be considered as lateral flow immunochromatographic analysis. For example, Corstjens et al., "Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papillomavirus type 16 infection" Clinical chemistry 47.10 (2001): 1885 -1893", Postumuma-Trumpie et al., "Lateral flow (immuno)assay:its strengths, weaknesses, opportunities and threats. A literature survey" Analytical and bioanalytical chemistry 393.2 (2009): 569-582" .

In accordance with an embodiment of the present invention, a Southern dot method can be utilized to determine the presence of a marker. The Southern Ink Point method is used to detect the presence of a biomolecule (eg, protein or DNA). A mixture comprising the biomolecule of interest can be placed on a membrane without the need to separate the biomolecule from the mixture, and the biomolecule can be attached to the membrane in a single point. The biomolecule of interest can then be treated with a nucleotide probe to determine the presence or absence of the biomolecule. For example, Dubeau et al., "Southern blot analysis of DNA extracted from formalin-fixed pathology specimens" Cancer research 46.6 (1986): 2964-2969", Englerblum et al., "Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity Than ³² P-Based Hybridizations " Analytical biochemistry 210.2 (1993): 235-244" describes the Southern dot method.

In one embodiment of the detection method of the present invention, the pH-related semiconductor ISFET technique is used for DNA detection, which provides a non-optical and scalable solution for a variety of applications, for example, from single nucleotide polymorphism to A rapid sample-to-answer solution for genome-wide sequencing. Since the ion system is released in a biological reaction process, the release of the ion can be tracked in real time.

In one embodiment of the method and system of the present invention, a transistor is used to detect DNA as well as RNA. For example, during DNA synthesis, when a complementary nucleotide binds to an amplified strand, hydrogen ions are released. This can be detected as an electronic signal. Conversely, if there is no pairing, no hydrogen ions will be released and no signal will be detected. This principle can be applied to all DNA and RNA analysis without the need for fluorescent stains or markers or precision optical instruments. The pH-related semiconductor (ISFET) technology uses unmodified reactants and unmodified microchip technology (CMOS), which provides these systems as a wafer-based platform with flexible, disposable advantages, fast and simple. It can be expanded. In one embodiment, the sensor, the circuit, and the active material may be integrated into a wafer, such as a single CMOS wafer (complementary metal oxide semiconductor or non-volatile simple input/output system memory) to obtain a Multi-functional wafer.

In embodiments, the amount or concentration of the nucleic acid identifier in the collected sample can be compared to the initial amount of the nucleic acid identifier placed in the product to detect the manner in which the counterfeiter dilutes the product with a secondary product. The counterfeiting behavior that is triggered. In general, the method of quantitative detection includes an internal or external control to assess the efficiency of detection from the same/analysis to the next sample/analysis. The test The efficiency can be influenced by a number of factors, such as probe hybridization, molecules or substances in the product that may interfere with detection, and/or primer correctness, enzyme quality, temperature changes using PCR detection methods. The final concentration of an accurate nucleic acid tag in the sample can be standardized by varying the conditions of the control assay.

In one embodiment, the invention provides a method of in situ detection and verification of a DNA signature, the method comprising providing a copy from an object of interest; utilizing a membrane that forms a pore between the base and the base A pore protein is used to analyze the sample to detect the presence of a DNA marker encoding a unique information for the article, and wherein the DNA sequence is an electric field applied to a pore of the membrane porin a change in characteristics caused by DNA passing through the pores of the membrane porin; and recording/outputting the sequence data to a data storage device, thereby determining that the DNA marker is present in the analysis sample To verify the authenticity of the item. DNA can pass through the well from base to base, so that changes in the electric field properties applied to each well can be detected.

In one embodiment, the invention provides a method of detecting and verifying a DNA signature in situ, comprising: providing a copy from an interesting article; analyzing the sample using a sequence of specific DNA conjugates to detect the presence of a DNA marker The DNA marker encodes information unique to the article; and verifies the authenticity of the article by determining that the DNA marker is present in the analysis sample. The sequence-specific DNA conjugate can be any suitable sequence-specific DNA binding partner, such as a sequence of specific DNA binding proteins, a membrane porin, or an antibody.

The resulting data generated by the system utilizing the above method can be encrypted and/or password protected. The analysis of the resulting data produced by the system utilizing the above method can be analyzed in a computer network system, such as that disclosed by Grinkemeyer and Dailey in US 2011/001833 A1.

In one embodiment, the invention provides a method of detecting and verifying a DNA marker in situ, the method comprising: providing an identical article from an item of interest; analyzing the sample using a DNA sequencing method to detect a DNA signature Existing, the DNA marker encodes information unique to the article, wherein the DNA sequencing method is performed using a portable DNA sequencing device; and verified by determining that the DNA marker is present in the analysis sample The authenticity of the item. The DNA sequencing method can be any suitable method, for example: Maxam & Gilbert Chemistry Sequence method, dideoxy sequencing, Sanger sequencing, nucleic acid hybridization, nanopore sequencing, pyrophosphate sequencing, or next generation sequencing; the next generation sequencing method includes: Ion dependent detection or pH sensing. In an embodiment, the DNA sequencing is performed using microarray hybridization. In one embodiment, the DNA sequencing is performed using dot blot hybridization.

A PCR sequence detection system can include an analysis device configured to be encrypted and/or password protected against unauthorized use. The analysis device can be coupled to a server via a wireless connection or a fixed line connection. The server can be a standalone server or a network server; the network server can be any suitable network server, such as a fixed line server or a cloud server. In one embodiment, the data used to verify authenticity is processed and stored in the separate server or the web server. In one embodiment, the data verifying the authenticity step is processed and stored in the separate server or the web server. In an embodiment, the analysis device is modular.

Tomazou et al. (US 7,888,015 B2) performed real-time detection/quantification of nucleic acid amplification reactions using QPCR solid-state induction and a pH sensor containing an ion-sensitive field effect transistor (ISFET) based on the detection of proton release during the amplification period of the primer. Tomazou et al. (US 7,686,929 B2) utilizes this ISFET-type sensing device to generate an electronic output signal as an ion-charged or near-local fluctuation in the surface of the transistor, and utilizes a method of detecting the electronic output signal from the ISFET. The local fluctuation of the ion charge represents an event occurring during a chemical reaction. The combination of the two is summarized in a device for detecting chemical reactions, hybridization of nucleic acid sequences, and genetic variation disclosed in Tomazou et al. US 2010/0159461 A1, using nucleic acid detection without labeling and interpretation. Such hybridization, nucleic acid sequence sequencing, or procedures and systems for deriving PCR amplicons from nucleic acid marker sequences can be configured for use in the methods and systems of the invention for detecting and verifying nucleic acids and other chemical markers. A direct method for the detection of PCR amplified DNA described by Nazarenko et al. in "A closed tube format for amplification and detection of DNA based on energy transfer" Nucleic Acids Research 25. 12 (1997): 2516-2521.

In one embodiment, the DNA can be bound to a fluorescent probe by Fluorescent in situ Hybridization (FISH). In the case of the FISH method, a fluorescent probe can be bound to a portion of a fixed nucleic acid sequence that is complementary to the fluorescent probe. A target nucleic acid sequence can be incorporated at a position to which one of the fluorescent probes is to be bound, and can be observed using a fluorescent microscope. For example, Bianchessi et al., Fluorescence in situ hybridization as described in "Point-of-care systems for rapid DNA quantification in oncology" Tumori 94(2) (2008 Mar-Apr): 216-25"; Agrawal An oligonucleotide position-specific functionalization as described in "Site-specific functionalization of oligodeoxy-nucleotides for non-radioactive labelling" US Patent 5,321,131".

In one embodiment, detection of DNA can be performed using a BEStTM ⁽ R ⁾ (available from Biohelix Corporation, Beverly, MA). For example, Tang et al., described in "Nucleic acid assay system for tier II laboratories and moderately complex clinics to detect HIV in low-resource settings" Journal of Infectious Disease 201. Supplement 1 (2010): S46-S51" use of detecting BESt ^TM cartridge.

In one embodiment, the present invention provides a method of on-site detection, wherein the combination of the distinctive marker and the particular detection method defined by the distinctive marker is selected from the group consisting of the following markers and detection systems: a nucleic acid marker with a PCR detection system, a hybridization detection system, or a nucleic acid sequencing system; a protein marker and an HPLC chromatography detection system or an ELISA detection system; an optical reporter and a photodetection system; and a chemical marker and a Chromatography detection system. In one embodiment, one of the detection device and the analysis device is modularized. In an embodiment, both the detecting device and the analyzing device are modularized.

In one embodiment, the invention provides a system for DNA detection and verification, the system comprising: a portable detector configured to detect the presence of a DNA marker having a unique identifier for the item of interest Information obtained from a sample of the article; and an analysis device operatively coupled to the portable detector and configured to determine whether the DNA is present by the portable detector The sample is used to verify the authenticity of the item. The portable DNA detection system of the present invention is useful, for example, for: anti-counterfeiting brand protection, prevention of theft/crime, source judgment, identification, tracking and tracing, and genotyping.

In one embodiment, the present invention provides a method of in situ detection and verification of a DNA marker, wherein the method comprises: providing an identical article from an interested article: analyzing the sample using a polymerase chain reaction method to detect a DNA marker Existence, the DNA marker encodes information unique to the article, and wherein the PCR method can be performed using a portable temperature cycling device; and verifying the article by determining that the DNA marker is present in the analytical sample of Authenticity, and wherein verification of the authenticity is performed using an analysis device operatively coupled to the PCR device. Verification of the authenticity of the item can be performed using any suitable method. In one embodiment, the detection and verification of the DNA marker is performed by determining whether the DNA marker is present in the sample, the sample being analyzed by a portable detector for the presence of an amplicon, eg, by A partially amplified DNA copy of the amplified DNA signature sequence by a PCR method. The PCR method can be any suitable PCR method, such as, for example, real-time PCR, digital PCR, or qPCR. Amplicon can be determined and detected by different standards of any conventional method, including electrophoresis, such as colloidal electrophoresis or capillary electrophoresis.

Protein, antibody, and carbohydrate detection

According to an embodiment of the invention, in addition to the nucleic acid marker, a marker comprising one or more proteins and/or one or more carbohydrates can be detected.

A marker comprising one or more carbohydrates can be detected by a carbohydrate microarray. Feizi et al. describe a carbohydrate microarray in more detail in "Carbohydrate microarrays-a new set of technologies at the frontiers of glycomics" Current opinion in structural biology 13.5 (2003): 637-645".

A marker comprising one or more proteins can be detected by enzyme immunoassay (ELISA). ELISA is a relatively rapid, high-throughput quantitative immunoassay for the selective detection of target antigens. The basic principles of ELISA include antibody-related capture and antigen detection on a measurable substrate. For example, ELISA detection of proteins is described in more detail by Jia et al. in "Nano-ELISA for highly sensitive protein detection" Biosensors and Bioelectronics 24.9 (2009): 2836-2841".

According to an embodiment, the determination of whether the compound of interest is distinguished by a distinctive marker comprising an antibody or a protein can be performed using an on-site detection device having sample in-out capability. For example, a Alere Determine ^TM apparatus ^{(e.g.: Alere Determine TM HIV-1/} 2 Ag / Ab Combo) as useful for detecting the presence of this distinctive mark comprises an antibody or a protein. Alere DetermineTM devices are commercially available. The ^{Alere Determine TM HIV-1/2} Ag / Ab Combo detectable anti-HIV antibodies to HIV protein may consist of Alere Technologies, Jena, Germany commercially available.

^{Alere Determine TM HIV-1/2} Ag / Ab Combo system for detecting one of a particular antibody rapid point of care test system. The Alere Determine ^TM platform can help complete the screening and HIV, tuberculosis, B hepatitis, and syphilis detected within minutes.

Authenticity verification

In one embodiment, the invention provides a detection system for detecting and verifying DNA, comprising: a portable detector configured to detect the presence of a DNA marker obtained from a sample of the article and encoded There is information unique to the item of interest; and an analysis device, which can be any suitable analysis device, for example: operatively coupled to the portable detector and configured to determine whether the DNA marker is A separate device that exists in a sample analyzed by the portable detector to verify the authenticity of the item, such as a desktop computer, a notebook computer, a smart phone, or a tablet computer.

In one embodiment, the analysis device is configured to determine whether the DNA marker is present in a sample analyzed by the portable detector and compared to and stored in a database and accessible by the analysis device. Know the sequence to verify the authenticity of the item. In an embodiment, the authenticity verification step of the method of the invention can comprise comparing the analyzed DNA marker sequence in the sample with a known sequence stored in a library and accessible by the analysis device.

In one embodiment, the analyzing device is configured to determine whether the DNA marker is present in the sample analyzed by the portable detector and to determine the presence of the DNA marker by distinguishing a microarray pattern. Verify the authenticity of the item. In another aspect, the assay device is configured to determine whether the DNA marker molecule is present in a sample analyzed by the portable detector and to detect the presence of amplified or hybridized sequences detected by a complementary nucleic acid sequence. To verify the straightness of the item.

In one embodiment, the verification of the authenticity of the object of interest is performed using an analysis device operatively coupled to the portable DNA sequencing device. The analysis device can be any suitable analysis device, such as a desktop computer, a notebook computer, a smart phone, a tablet computer, or the like. In an embodiment, the analysis device is either encrypted or password protected against unauthorized use.

In one embodiment, the verification of authenticity includes comparing the DNA signature analyzed in the sample to a known sequence stored in a database and accessible by the analysis device. The DNA sequence can be stored in a local independent server or operatively connected via wire or wireless The network device of the analysis device. In an embodiment, the analysis device can be modularized. The result of the authenticity verification can be processed and stored by a local server or a web server. The sequence of the DNA marker can be any suitable unique sequence. In one embodiment, the sequence of the DNA marker is a sequence having from about 20 to about 1000 bases.

The known sequence of the DNA marker can be any unique sequence for uniquely identifying an article, for example: a sequence having from about 20 to about 1000 bases. Articles marked by the DNA marker can also be labeled by a detectable reporter. In one embodiment, the detection reporter and the DNA marker are in the same location on the article, such that the detection reporter can serve as the same identifier to display the location of the DNA marker. Alternatively, the detection reporter and the DNA marker can be correlated such that a method of obtaining a sample of the detection reporter can be used as a guide for obtaining a sample of the DNA marker for verification and validation. In one embodiment, the detection reporter of the method is selected from the group consisting of a chemical reporter, a digital reporter, or a peptide reporter. In one embodiment, the detection reporter is a digital reporter and is a single code.

The sequence detection system can include an encrypted and/or password protected analysis device to resist unauthorized use. The analysis device can be coupled to a server via a wireless connection or a fixed line connection. The server can be a standalone server or a web server. In an embodiment, the analysis device can be modularized. In one embodiment, the authenticity verified data is processed and stored by the independent server or the web server.

In one embodiment, the article to be inspected by the system of the present invention is selected from a microchip, a logo, a badge, a logo pattern, a print, a textile, or a merchandise. In one embodiment, the item to be verified by the system is selected from the group consisting of cash, a currency note, a coin, a gemstone, a jewel, an instrument, a passport, an antique, a piece of furniture, a work of art, a property contract. , a stock certificate, a bond certificate or a group of members. The detection system of the present invention can be configured to provide verification data directly to a user or a secure location or headquarters via a display to provide data communication with a consumer, or to store in a database, or to print a report. The manner in which the verification data can be read can be a display or a signal, or an alert.

The analysis device for the on-site detection and verification DNA method can be any suitable analysis device, such as a desktop computer, a notebook computer, a smart phone, or a tablet computer. The devices are operatively configured as a system to communicate in a network. The network The path can be any suitable network, such as but not limited to one or more of RF, Wi-Fi, Bluetooth, or a fixed line connection.

Field testing equipment

The field testing instrument can include a microsystem having sample in-out analysis capabilities. The microsystem can be a complete unit that performs all necessary analysis processes without the need for additional laboratory equipment. The microsystem can be automated and only requires adding samples to the microsystem and turning on the microsystem to perform the analysis. The field test instrument can be portable or fixed in a single location. Sample in-out analysis refers to the ability of the microsystem to perform all analytical steps and automatically provide results after transmitting the sample to the microsystem. The microsystem can be configured to provide for minimization of detection that must be monitored or adjusted by an operator. In one embodiment, the sample is added directly or from the same collection device configured to be coupled to one of the sample channels of the microsystem. The microsystem is then activated and the field test instrument provides test data that interacts with the operator. The detection data can be stored and/or output. In terms of detecting the presence of a distinctive marker, a microsystem that may include a sample of the distinctive marker to the field detection instrument can be provided and automatically determined by the microsystem whether the distinctive marker is present in the sample. An exemplary microsystem for on-site inspection will be described in more detail below.

The on-site inspection instrument can communicate with a server containing the authenticity data of the item of interest. The server may contain a library of authenticity data stored with a plurality of distinctive logo file information associated with a number of interested objects. The authenticity data can be a unique file associated with the distinctive marker. For example, the distinctive marker can include one or more unique nucleic acid sequences, and the authenticity data can be a digitized copy of one of the unique nucleic acid sequences. An example of a suitable remote authentication server including a verification database is described by Zorab in WO 01/99063.

Microfluid sample preparation device

A system utilizing an exemplary embodiment suitable as a method of the present invention is disclosed in US 7,745,207 B2 to Jovanovich et al. Further, nanofluids as described by Janovich et al. (US 2012/0115189 A1) and sample treatments as described by Green (US 8,110,397 B2) are useful for additional embodiments of the invention. A PCR device (US 7,170,594 B2) as described by Green can also be used in an exemplary embodiment of the invention. Fractionation with a microfluidic system and DNA solution or division into about one DNA molecule per drop to identify in a quantum dot Analytical methods for polymorphism are described in Lo et al., US 2009/0263580 A1. A method and apparatus for digital PCR using the "water droplets in oil" technique is disclosed in US 2010/0092973 A1, which is incorporated herein by reference. A method for detecting a ricin gene and a ricin toxin using an oligonucleotide sequence is described in Czjka, US 2006/0240447, and discloses a kit for detecting this particular DNA.

According to one embodiment of the invention, a microfluidic device can be used to extract, purify, and extend a DNA sample. For example, Benítez et al. describe a method for extracting, purifying, and extending from a single cell in "Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells" Lab on a Chip 12.22 (2012): 4848-4854". Microfluidics of human DNA. A microfluidic DNA wafer is described by Zhao et al. in "Electrochemical DNA detection using Hoechst dyes in microfluidic chips" Current Applied Physics 12.6 (2012): 1493-1496". Aboud describes a method for identifying genotyping in "The development of direct ultra-fast PCR for forensic genotyping using short channel microfluidic systems with enhanced sieving matrices" (January 1, 2012), ProQuest ETD Collection for FIU. Paper AAI3541755) Direct ultra-fast PCR.

For example, Wu, Jinbo et al., "Extraction, amplification and detection of DNA in microfluidic chip-based assays", Microchimica Acta (2013): 1-21", Liu et al., "Integrated DNA purification, PCR, sample cleanup, And capillary electrophoresis microchip for forensic human identification " Lab on a Chip 11.6 (2011): 1041-1048", Jovanovich et al., U.S. Patent No. 7,745,207 B2, and U.S. Patent Application Serial No. 2012/0115189 A1, each of which is incorporated herein by reference. With specific microfluidic capillary electrophoresis devices.

According to one embodiment of the invention, one or more samples may be provided that contain one or more nucleotide sequences of interest, and one or more non-target nucleotide sequences (eg, camouflage or decoy sequences). The nucleotide sequence of interest can be purified by isolating the target nucleotide from the camouflage nucleotide. The sample can be cleaned through a bed of steric fixed to a series of microchannels. The matrix can include one or more nucleotide probes that are complementary to the target nucleotide, the nucleotide probes being configured to capture the target nucleotide by hybridization. The substrate can be any suitable substrate. For example, the matrix can be a magnetic bead having a nucleotide probe attached thereto. The nucleotide of interest can be combined with the nucleotide probe to form a nucleotide-probe conjugate. The binding of the matrix (eg, magnetic beads) to the target nucleotide can be transmitted to a detection module to determine the presence or absence of each target nucleotide. The binding of the matrix (eg, magnetic beads) to the target nucleotide can be transmitted to an amplification device or an amplification chamber. For example, the amplified product can be transferred to a capillary electrophoresis column. The separation of nucleotides can be performed using an electric field according to conventional standard capillary electrophoresis techniques. A laser and an optical detector system can be employed to detect the presence of the target nucleotide. Capillary electrophoresis and DNA detection techniques are described, for example, in Schwartz, Guttman, " Separation of DNA by capillary electrophoresis. Beckman, 1995."

Integrated microsystem

According to an embodiment of the invention, a nucleic acid signature can be detected by an integrated microsystem. For example, an integrated microsystem can include: a microfluidic wafer, a fully integrated microdevice, or a lab on a chip configuration. The integrated microsystem can prepare the same, a large target nucleic acid sequence (if desired), and detect the presence of one or more target nucleic acid sequences in a single device. All steps can be performed by an autonomous and automated microsystem without the need for any human testing in the laboratory. For example, Wu et al., "Extraction, amplification and detection of DNA in microfluidic chip-based assays", Microchimica Acta (2013): 121", Liu et al., "Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip". For forensic human identification" Lab on a Chip 11.6 (2011): 1041-1048", Ullrich, Thomas et al., "Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization" PloS one 7.4 (2012): e35438" describes integrated microsystems.

In one embodiment, the sample containing the nucleic acid identifier can be separated from other sample components, for example, by capillary action of a microfluidic channel system in a power-free microchip, the sample containing the nucleic acid identifier can be bound by A labeled and immobilized hybridization probe is detected which is complementary to at least a portion of the nucleic acid marker sequence. The nucleic acid marker can be directly detected or amplified (for example, by constant temperature amplification or PCR) and detected by laminar flow assisted dendritic amplification (LFDA), which is used for binding to The specific hybridization probe is labeled with a specific detection antibody and detected by a second antibody in a dendritic tandem reaction as well as a conventional immunoassay reaction. For example, reference can be made to Hosokawa et al. in " DNA Detection on a Power-free Microchip with Laminar Flow-assisted Dendritic Amplification, Anal. Sci. (2010) 26: 1053-1057".

In accordance with an embodiment of the present invention, an integrated microsystem can utilize an internal microarray system without the need for automated fluorescent marking and detection. The integrated microsystem can amplify one or more target sequences derived from the provided sample, fluorescently label one or more amplified products, and detect the presence of the fluorescently labeled product by using PCR or a constant temperature amplification reaction. . For example, Liu et al. describe a microarray detection system in a tube in more detail in "Microarray-in-a-tube for detection of multiple viruses" Clinical chemistry 53.2 (2007): 188-194".

In accordance with an embodiment, an apparatus for detecting a distinctive landmark in situ includes an on-site inspection instrument that includes a microsystem configured to perform sample in-out. The field testing instrument is configured to analyze the same to determine the presence of a distinctive marker in the sample. According to an embodiment, the field detection instrument can be configured to perform any suitable chemical or physical feature description method sufficient to determine one or more unique features of the distinctive signature.

According to an embodiment, the sample collection unit can be configured to pair directly with the field detection device.

According to an embodiment, the integrated microsystem can utilize a fluorescent background displacement technique. The immobilizable oligonucleotide capture probe array can be placed in a reaction chamber containing the reaction mixture. A solution comprising a fluorescently labeled reporter oligonucleotide probe can also be provided in the reaction chamber, the fluorescently labeled reporter oligonucleotide probe system being complementary to the fixed nucleotide probe. An amplicon produced in the chamber complementary to the reporter nucleotide probe can compete with the capture probe for binding to the reporter nucleotide probe. That is, the reporter probes can each be complementary to a particular target nucleotide sequence. Thus, as the number of a particular amplicon (eg, a target nucleotide sequence) increases, the fluorescent signal detected by the corresponding fixed nucleotide capture probe decreases, as fewer reporter probes are detected. The needle is coupled to the corresponding grasping probe. Detection of the characterization and quantification of the target nucleic acid sequence sample comprising the nucleotide sequence can be performed in synchronization. The optical image of the gripping probe can be taken at any point in time, including when adding the same baseline as before the reaction mixture. Because of the hybridization specificity between the reporter probe and the target nucleic acid sequence, binding of non-target nucleic acid sequences (e.g., camouflage sequences) to the reporter probe in the sample can be avoided. The capture probe can also be configured to bind directly to the target nucleic acid sequence and the capture probe. The reaction mixture with unbound reporter probes can be excluded such that only the bound reporter probes are detected (bound to the capture probe). The reaction mixture can be eliminated by any suitable exclusion mechanism. For example, Ullrich et al. describe a mechanical fluorescent background exclusion technique in "Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization" PloS one 7.4 (2012): e35438.

An exemplary integrated microsystem will be described in greater detail below.

Information DNA coding

The enormous coding ability of nucleic acid sequences is well known in the art. In an embodiment of the invention, a nucleic acid marker for identifying an item and a nucleic acid identifier for encoding the message content of the marked item can be detected in an array. The array can be any suitable array, for example: a microarray comprising DNA strands, each point comprising a bound DNA molecule comprising a nucleic acid marker or a label, label or possibly added to the article A unique sequence of complementary nucleic acid labels that are packaged. The array can be adapted for hybridization reactions with nucleic acid markers, nucleic acid markers, or derived amplicon. The hybrid array can be of any suitable type, such as a plate, a microtiter array, a matrix with dots, a colony, or a microarray.

The hybridization array can be of any size and/or any arrangement of nucleic acid markers, nucleic acid markers complementary to DNA. For example, the array can include a 1,000 x 1,000 grid point. In other embodiments, the array can be arranged in a grid of 30,000 x 30,000 or more so that one billion or more points can be included on a single array. According to an embodiment and as detailed below, the sample point arrangement can be used to provide authenticity data for an item of interest.

In one embodiment, a plurality of "single plant" oligonucleotides can be attached to a solid surface to form a customized probe array, for example, each point or "single plant" comprises a plurality of nucleic acids A signature or a copy of a DNA sequence complementary to a nucleic acid identifier. The individual plant population can be a submicron sized colony that is amplified by PCR or thermostatically on a solid surface, such as the surface of a primary generation sequencing microfluidic wafer. The solid surface can have any size and the location of the individual plant communities on the plane can be arbitrarily arranged. For example, the individual plant community can be arranged as a 1,000 x 1,000 community grid. The solid surface can be any suitable surface, such as the surface of a microscope slide having a polyacrylamide coating. Ma et al., "Isothermal amplification method for next-generation sequencing," Proc. Natl Acad Sci 110. 35 (2013): 14320-14323, describes an example of a fixed plant population that produces a DNA sample.

According to further embodiments, the customized probe array can comprise a set of randomly aligned oligonucleotide hybridization probes that are complementary to one or more nucleic acid marker sequences or nucleic acid marker oligonucleotide sequences . A plurality of camouflaged oligonucleotide probes (eg, decoy probes) can be placed on one or more locations on the customized probe array that are not attached by the oligonucleotide hybridization probe To provide a non-specific combination to reduce any background noise. Preferably, the camouflage oligonucleotide is not complementary to any of the target nucleic acid sequences. The customized probe array can include any number of oligonucleotide sequences that are not complementary to the nucleic acid marker or the acid marker oligonucleotide sequence. For example, the camouflage probe can comprise from 1,000 to 1,000,000 or more different nucleic acid sequences that do not hybridize to any target nucleic acid marker or nucleic acid marker oligonucleotide sequence. The nucleic acid signature can hybridize to the point of the complementary sequence of the immobilized oligonucleotide hybridization probe to produce a unique "fingerprint". This unique fingerprint corresponds to a particular physical arrangement of oligonucleotide hybridization probes on a particular array or wafer. Thus, the customized probe array can provide positive identification of an item of interest in at least two ways: by positively identifying the presence of a nucleic acid marker oligonucleotide sequence or a nucleic acid identifier. The second way is by positively identifying the presence of the unique fingerprint, wherein positively identifying the nucleic acid marker oligonucleotide sequence or the presence of the nucleic acid identifier does not require knowledge of the customary associated with the unique fingerprint The physical arrangement of the probe array.

According to one embodiment, the bound oligonucleotide hybridization probes are arranged non-randomly, but form a discernible symbol or pattern. The oligonucleotide hybridization probe can be placed on the array where it is to be placed to form a customized fingerprint. The customized fingerprint can include any number of oligonucleotide hybridization probes that are placed in the customized probe array in any combination of positions. For example, the probes of the customized probe array can be arranged in a grid, and the positions of the oligonucleotide probes can be arranged as a "plus sign" at a specific position in the array or grid. A star. The position of the probe having the specific oligonucleotide hybridization probe attached to the object of interest is not attached to the customized probe array. Other oligonucleotide hybridization probes allow the array to be used to detect a variety of different nucleic acid markers or nucleic acid markers. Alternatively, the position of the probe to which the oligonucleotide hybridization probe is not attached to the customized probe array can be attached to a camouflage probe or other DNA sequence.

The camouflage probe system can be any number of oligonucleotide sequences. For example, the camouflage probe can comprise 1,000 different randomly arranged nucleic acid sequences, which preferably do not hybridize to any of the target oligonucleotide sequences. In one embodiment, the customized probe array can include an arrangement of specific oligonucleotide hybridization probes for the interest, forming a letter, text, or even a symbol or binary code.

For a particular item, more than one oligonucleotide can be used for the nucleic acid signature. The customized probe array can also include more than one oligonucleotide hybridization probe corresponding to the nucleic acid marker oligonucleotide or a nucleic acid identifier linked to a library of interest or a package thereof to provide a repeat Additional security assurance and confirmation of detection, verification, and tracking.

The arrangement of the oligonucleotide hybridization probes can also be used to encode additional information about the authenticity of the object of interest. In a plurality of manufacturing, packaging, or marketing procedures, additional nucleic acid identifiers can be added to or attached to the item of interest, each corresponding to a different oligo in the array for detecting, verifying, or tracking the item. Nucleotide hybridization probes, and can be used to represent or encode any information about an item of interest. For example, the presence or absence of hybridization of a particular position in the array with the particular oligonucleotide hybridization probe or the alignment of multiple copies of the particular oligonucleotide hybridization probe can be used to encrypt the particularity of the article. Information. The encrypted information may be any information such as, but not limited to, the model, serial number, origin, composition, use portion, production date, seller, final seller, or buyer of the item of interest.

As described below, a method of labeling an article or product with a particular nucleic acid tag and then detecting the nucleic acid tag in the article or product in an efficient manner.

Figure 1 shows a process 100 for introducing a nucleic acid tag into or on a product and detecting the nucleic acid tag or label bound to the product. As shown in FIG. 1, in step 110, the process includes providing at least one specific nucleic acid fragment as a authenticity label or logo for the product. In step 120, a nucleic acid marker compound comprising a known nucleic acid fragment is produced. The nucleic acid marker can be DNA, cDNA, or any nucleic acid fragment comprising a nucleic acid or nucleic acid derivative. In the detection process of the nucleic acid marker, the marker may be a single-stranded or double-stranded nucleic acid fragment and may be based on the labeled product and The detection methods used have different lengths.

After the nucleic acid fragment having a known nucleic acid sequence is produced or isolated and added to the labeling compound, the process of verifying the particular product by detecting the nucleic acid tag further comprises applying a predetermined number of nucleic acid markers to a particular product, such as step 130. Said. The particular product may be labeled by the nucleic acid marker to the entirety of the product or to a predetermined area on the product. When the product to be verified is a solid, a particular amount of nucleic acid label can be incorporated into the entire volume of the product, or only on the surface of the product, or as described in some embodiments, only a preselected portion of the product. When the product is a prescription drug, whether in solid or liquid form, the drug (eg, pill, gel, capsule, etc.) may have the nucleic acid tag incorporated into the entirety of the product. If the product is a prescription drug of a tablet type, the nucleic acid marker compound may be a solid and may be added to the product when the product (drug) is pressed to form a tablet. If the product is a textile garment, the logo can be a solid or liquid and attached to a predetermined area of the garment. The textile may have a label with the manufacturer's name on it that can be used as the area on which the nucleic acid marker is placed. The above examples are for illustrative purposes only and are not intended to limit the scope of the invention.

The embodiment of the method of verifying a product of Figure 1 further includes directing the marked product to a supply chain or placing the product at a service point, as shown in step 140. Counterfeiters often have the best chance of accessing the product as it moves from the manufacturer/manufacturer to a retail location. The counterfeiter may also access the product at a stage in which the particular product of interest is in service or service, and the particular product of interest may be inspected or replaced, such as an aircraft fastener. A method that enables a manufacturer to track and verify its products can provide better monitoring of the time and place at which the manufacturer may be replaced or tampered with by the counterfeiter.

In step 150, a sample is taken from a particular product containing a nucleic acid tag after the product enters the supply chain or service point. The manufacturer or verifier may obtain samples from the product for verification use at any point in the supply chain, service location, or routine maintenance of the product. In embodiments, verification may include visual inspection of the marker, and/or removal, cleavage, or dissolution of one of the marker compounds in a solvent for analysis.

The embodiment illustrated in Figure 1 further includes analyzing the presence of the nucleic acid identifier in the sample taken, as described in step 160. Analysis of samples taken from the product may not require further purification, but may require extraction, isolation, or purification of some of the nucleic acid tags obtained from the sample. The extraction, concentration, and purification techniques used in the methods of the present invention are described in more detail below and in the examples described in this specification.

Analysis of the sample can include providing a "detection molecule" configured to bind to the nucleic acid tag. A detection molecule includes, but is not limited to, a nucleic acid probe and/or primer set complementary to the nucleic acid marker sequence, configured to bind to and adhere to one of the nucleic acid identifiers, a staining label or a color generating molecule. When the detection of a nucleic acid marker comprises an amplification reaction of a nucleic acid marker (eg, using PCR), the detection molecule can be a primer that specifically binds to a particular sequence of one of the nucleic acid identifiers. When real-time PCR is used for the analysis of the sample, an identifiable nucleotide probe can also be provided to facilitate detection of the nucleic acid identifier and provide semi-quantitative or qualitative verification results. For example, when using real-time PCR, the results of the sample analysis can be completed in 30 minutes to 2 hours and include extracting or purifying the nucleic acid identifier from the sample taken. Embodiments may utilize a broader range of detection methods than PCR and real-time PCR, such as fluorescent probes, configured to be in the field of Raman spectroscopy, far infrared spectroscopy, or other nucleic acid detection when the nucleic acid tag is bound to the probe. Spectral techniques used by the average person are used for detection.

In step 170, the analysis results of the acquired samples are reviewed to determine if the particular nucleic acid identifier is detected in the sample. If the nucleic acid identifier is not found or detected in the sample taken from the item of interest, then the analysis of the product is counterfeit or falsified, as shown in step 180 of Figure 1. If the nucleic acid identifier is detected in the sample as shown in step 190, the product is confirmed to be true.

Figure 2 is a graph showing an instant PCR result showing the results of detecting a DNA marker of a cingulate in a manner associated with an embodiment of the present invention. Figure 2 shows the results of an instant PCR of a sample treated with a torque seal, which was extracted by the following method. All samples A, B, C, D, and E were dried for 20 days and then ground. Samples D and E were further boiled in water. The DNA marker positive control group 300, the replicated negative control group 310, and the torque-sealed samples are placed under the conditions of real-time PCR. The positive control group 300 displays a Cp value of 29, and the negative control group 310 exhibits only a background value. Two torque-sealed samples treated by grinding: Sample A (320) and Sample B (330), respectively, have Cp values of 37 and 33. Samples that were both ground and boiled: Both sample D (340) and sample E (350) had a Cp value of 34. The results show that after 20 days of exposure, it can be taken from the torque seal The DNA marker was detected in the sample obtained from the substance. Both the grinding and boiling methods provide qualitative results, while the grinding followed by boiling provides semi-quantitative results.

Figure 3 shows the results of an instant PCR of a torque-sealed sample which was extracted by the following method. All samples were dried for 20 days and ground and dissolved in water, and further purified using a QIAgen PCR purification kit to remove particulate matter that could interfere with the optical detection of the PCR instrument. The purification step includes centrifuging to bind the DNA present in the torque-sealed sample to a filter, which is followed by any impurities, followed by washing the DNA with water. The process is fast and simple, and the execution time takes less than 15 minutes. After washing, perform real-time PCR with Sample A and Sample B, which may be 2 μL or 4 μL of the purified torque-sealed DNA, and Sample B may be 2 μL or 4 μL of the purified torque-sealed treatment. DNA. The present invention also inhibits Sample A and Sample B with 2 μL of positive control set DNA (9543-2) to test the inhibitory effect of the PCR reaction.

The amplification curve of sample A and sample B after PCR reaction is shown in Fig. 3. The curves of 2 μL of sample A and 2 μL of sample B are curve 360 and curve 370, respectively, and each has a similar Cp value of 32. The curves of 4 μL of sample A and 4 μL of sample B are curve 380 and curve 390, respectively. The Cp value with a considerable degree of reproducibility is about 30. The amplified positive control group 400 has a Cp value of 29 as expected, and the replicated negative control group 410 does not exhibit any signal above the background value. Samples A and B of the torque-sealed treatment were inhibited by positive control groups 420 and 430, respectively, to ensure that the purification set did not affect the amplification reaction of the target DNA. The inhibited sample A and the suppressed sample B have Cp values of 27 and 29, respectively. The fluorescent signal generated by the sample processed by the purification kit is smooth and has a typical curve of real-time PCR. This experiment shows the specific DNA signature that can be obtained from the dry, torque-sealed material. The results also show that the analytical method can be semi-quantitative.

Figure 4 is a flow diagram in accordance with an embodiment of the present invention. An article having a DNA marker is sampled and the sample is transferred to an identical container that is operatively coupled to an analytical device. For a particular sample, the sample is preferably processed in a DNA extraction procedure step for the detection and verification steps. The raw data is communicated to a data processing unit. The data of the processing unit can then be relayed to a local or remote processor or library for further analysis, presentation, storage, or merging into a report, for example: a "pass/fail" read cell formula. The analysis and readout shown in the flow, rectangular area shown in the triangular area is a concealed, safe process.

Figure 5 is a field inspection instrument shown in accordance with an embodiment of the present invention. Sample 110 was taken from an item of interest. The sample 110 is transmitted to the field testing instrument. The field test instrument includes an integrated microsystem having sample in-out analysis capabilities to detect the presence of the distinctive marker. The field test instrument provides a direct result output, such as a readout on a display or printed material. Alternatively or additionally, the field test instrument can communicate with a server to compare the data generated by the field test instrument with the number of data stored in the server. The server can be a standalone server or the server can be in the cloud, and the cloud server can be connected in reverse to a separate server.

Embodiment 1: Rapid on-site detection, integrated microsystem and method for distinguishing markers

According to one embodiment of the invention, the location of the distinctive marker comprising one of the one or more nucleic acid sequences is identified in the article of interest. A sample comprising the distinctive signature of the nucleic acid sequence is obtained and transmitted to an array tube comprising a customized array wafer. The sample is selectively fluorescently labeled and analyzed by a microarray to determine the presence of the one or more nucleic acid sequences.

An array tube with a customized probe array can be used in a microreactor. The array tube can be, for example, an array tube of an Alere Technologies GmbH ArrayTube with a custom probe microarray (eg, a biochip) and incorporated into a microreactor bottle, which is commercially available (Alere Technologies, Jena). , Germany). The customized array includes an oligonucleotide that is unique to the item of interest and that is complementary to the nucleic acid sequence that constitutes the distinctive marker. The customized array wafer includes a custom complementary oligonucleotide sequence selected and placed in the array wafer in the form of dots. The oligonucleotide probe hybridizes to a complementary target nucleic acid sequence to detect a target nucleic acid molecule in the sample.

The probe microarray is customized with oligonucleotides, polynucleotides, or protein/peptide probe molecules for individual applications. Each array tube wafer has dimensions of 3mm x 3mm and can provide up to 14 x 14 features, including reaction control and reference markers.

Depending on the individual analysis, arrays based on nucleic acids and proteins as well as peptides can be made. Each array tube is labeled with a unique data matrix code (a binary barcode) to identify the array and associated analysis. The array tube test system is combined with the precipitation dyeing method and is suitable for serum. Learning and nucleic acid-based analysis. The catalyzed precipitation reaction is directly related to the number of target molecules bound to the array. The analysis of the pattern of precipitation results is done by simple transmission measurements, resulting in an effective reduction in the signal input to the optical device. Transmission measurements can be performed by an ATR 03 instrument, commercially available (Alere Technologies, Jena, Germany), or by an ARRAYMATE instrument, which is also commercially available (Alere Technologies, Jena, Germany).

The data is automatically recorded and directly analyzed from the array tube containing the customized array wafer, and the results are electronically transmitted to a computer server or stored on a local hard disk. The data obtained by the automated microarray can be processed automatically and compared to a library of known nucleic acid sequences to perform validation.

Example 2: Fast on-site detection, an integrated microsystem and method for distinguishing markers

A sample of the DNA signature of interest is provided and transmitted to the integrated microsystem device for analysis. The sample includes one or more oligonucleotide sequences. The oligonucleotide sequence may comprise one or more of the distinctive oligonucleotide sequences of the distinctive marker and one or more non-target oligonucleotide sequences as a camouflage or bait.

This sample includes several oligonucleotide sequences. The target oligonucleotide can be purified from the sample by washing the sample through a bed of magnetic beads immobilized in a series of microchannels (eg, removing unrelated or camouflaged oligonucleotide sequences). Each magnetic bead has one or more oligonucleotide probes attached thereto that are complementary to a particular oligonucleotide of interest for the article of interest. The target oligonucleotide binds to the oligonucleotide probe of the magnetic bead to form a DNA-probe conjugate. This sequence-specific DNA purification method facilitates the efficiency of downstream amplification reactions by removing background oligonucleotide sequences (eg, purification of the target oligonucleotide). The sample is moved in the grab channel using a micro pump.

The magnetic beads act as a matrix to carry the target oligonucleotide to a constant temperature amplification unit for amplification amplification. A magnetic bead comprising a DNA-probe conjugate is pushed into the thermostated amplification unit and the target oligonucleotide is amplified by a thermostatic amplification reaction.

The amplified product is pushed to a grab colloid suitable for injection into a capillary electrophoresis column after amplification. The amplified product is assembled to the grasping colloid to form a sample plug that can be pushed to a capillary electrophoresis column for separation and detection. The sample plug is released (eg, released at temperature) and injected into a capillary electrophoresis column for detection of target oligonucleotide isolation and the presence or absence of each target oligonucleotide sequence. Oligonucleotide separation is performed by an electric field according to standard capillary electrophoresis techniques known in the art, and a laser and an optical detector system are employed to detect the target oligonucleotide included in the sample plug. presence. Capillary electrophoresis and DNA detection techniques are described in " Separation of DNA by capillary electrophoresis. Beckman, 1995" by Schwartz and Guttman.

A target sequence distribution is generated based on the target oligonucleotide detected in the sample. The target sequence distribution includes only the detected target oligonucleotide sequence and excludes the non-target (camouflage) oligonucleotide sequence. The distributed data is transmitted to a remote server or stored on a local hard disk for comparison with a known sequence distribution in a database to determine whether the sample is provided from a non-counterfeit item.

Example 3: Fast on-site detection, an integrated microsystem and method for distinguishing markers

A sample of the DNA signature of interest is provided and transmitted to the integrated microsystem device for analysis. The sample includes one or more oligonucleotide sequences. The oligonucleotide sequence may comprise one or more of the distinctive oligonucleotide sequences of the distinctive marker and one or more non-target oligonucleotide sequences as a camouflage or bait.

The integrated microsystem device includes a reaction chamber, a waste liquid chamber, a reaction chamber piston, a temperature control module, an optical detection module, and a data analysis module. The integrated microsystem device utilizes a mechanical fluorescent background removal technique that utilizes the following principles. An array of immobilized oligonucleotide capture probes is disposed in the reaction chamber and a solution comprising a fluorescently labeled reporter oligonucleotide probe complementary to the immobilized oligonucleotide capture probe is also provided In the reaction chamber. An amplicon line produced in the reaction chamber that is complementary to the reporter oligonucleotide probe competes with the capture probe for binding to the reporter oligonucleotide probe. That is, the reporter probes are each also complementary to a particular target oligonucleotide sequence. Thus, as the number of a particular amplicon (eg, a target nucleotide sequence) increases, the fluorescent signal detected by the corresponding fixed nucleotide capture probe decreases, as fewer reporter probes are detected. The needle is coupled to the corresponding grasping probe. Detection of the characterization and quantification of the target nucleic acid sequence sample comprising the nucleotide sequence can be performed in synchronization.

According to this embodiment, the reporter probe can be freely coupled to the gripping probe to obtain a baseline fluorescent signal prior to adding the sample to the reaction chamber. By initiating the reaction A chamber plunger obtains a fluorescent signal by pressing the array of gripping probes against a wall of the reaction chamber. When the reaction chamber piston is not activated, the grab probe array is not pressed to the wall of the reaction chamber, so the optical detection module does not detect the fluorescent signal. Activation of the reaction chamber piston also removes the reaction mixture containing the unbound reporter probe such that only the reporter probe bound (in conjunction with the capture probe) is detected.

The provided sample is added to the reaction chamber and the target nucleotide sequence is amplified by PCR. In order to detect a fluorescent signal pattern by time, the system is arranged to continuously acquire a fluorescent signal image after each PCR amplification cycle. The temperature control module is configured to automatically heat and cool the reaction chamber for a PCR amplification reaction that is initiated upon completion of each PCR amplification cycle to obtain a fluorescent signal. However, the reaction mixture may emit a background fluorescent signal to interrupt the fluorescent signal taken from the capture probe array. Thus, when the reaction chamber piston is activated, it compresses the reaction chamber thereby removing the reaction mixture to a waste chamber, thereby removing the reaction mixture from the reaction chamber and substantially eliminating background fluorescent signals. After the fluorescent signal is taken from the capture probe array, the reaction chamber is not compressed by the reaction chamber piston, so that the reaction mixture flows back from the waste liquid chamber to the reaction chamber. This procedure is repeated during each PCR amplification reaction cycle.

The data analysis module is configured to collect optical data from the optical detection module to qualitatively determine the presence of one or more target oligonucleotide sequences in the sample and/or quantitatively determine one or more target oligonucleosides The acid sequence PCR amplifies the rate of the reaction (eg, the amount of amplicon produced). Generating a target sequence distribution from the data analysis module and transmitting the data to a remote server or storing it on a local hard disk for comparison with a known sequence distribution database to determine whether the provided sample is from On a non-counterfeit item.

Example 4: Fast field detection, an integrated microsystem and method for distinguishing markers

The integrated microsystem described in Example 3 can be retrofitted using a thermostatic amplification reaction to eliminate temperature cycling. Therefore, the temperature control module can be ignored, and the rate at which the image is taken can be increased to accommodate the faster cycle time of the constant temperature amplification reaction.

The methods and systems of the present invention can also be used to identify safety marker DNA attached to any item that can be forged or stolen. Such items include, but are not limited to, printed matter, bank notes, financial instruments, coins, labels, logo graphics, identification cards, fabrics, textiles, or other merchandise, Microchips and other electronic products such as desktops, notebooks, tablets, and smart phones are not just examples.

Each of the references, patents, and published patent applications disclosed in the specification of the present disclosure is incorporated herein in its entirety.

Although one or more embodiments of the present invention have been described in the drawings, it will be understood by those of ordinary skill in the art In the following, various forms and details can be changed.

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Claims (15)

A method for detecting a distinctive mark on-site, comprising: providing a copy from an item of interest; analyzing the sample to detect the presence of a distinctive mark, wherein the analyzing is performed using an on-site detecting instrument, and wherein the field detecting instrument A microsystem is included that is configured to perform sample in-out analysis; and to detect the presence of the distinctive marker in the sample. The method for detecting a distinctive mark on-site as described in claim 1, wherein the sample provided from the article of interest further comprises one or more DNA markers in addition to the distinctive marker. Taggants, wherein the DNA tag encodes information related to the item of interest; and in addition to detecting the presence of the distinctive marker in the sample, the presence of at least one of the DNA markers in the sample is also detected. A method of detecting a distinctive mark on-site as described in claim 1 wherein the sample further comprises the same identifier. The method for detecting a distinctive mark on-site as described in claim 3, wherein the sample identifier comprises an optical reporter, a digital code, a QR code or a code. The method for detecting a distinctive mark on-site according to claim 4, wherein the photoconductive agent comprises an upconverting phosphor, a fluorophore, a coloring agent, a coloring agent, Or a phosphor. A method for detecting a distinctive marker on-site as described in claim 1, wherein the distinctive marker comprises a biomolecule or an organic non-biological molecule. The method for detecting a distinctive marker on-site as described in claim 6, wherein the biomolecule is selected from the group consisting of a monobasic acid, a peptide, a protein, a lipid, an isoprene, and a single A group consisting of sugar, a polysaccharide, a fatty acid, a vitamin, a nucleic acid, a protein-DNA complex, and a peptide nucleic acid (PNA). A method of detecting a distinctive marker on-site as described in claim 7, wherein the distinctive marker comprises one or more nucleic acid sequences that are replicable to produce a unique amplicon profile. The method for detecting a distinctive mark on-site as described in claim 1, wherein the on-site detecting instrument communicates with a server, and the server includes authenticity data of the item of interest. The method for detecting a distinctive mark on-site as described in claim 1, wherein the on-site detecting device is a stand-alone device configured to store and process authenticity data of the item of interest. A method for detecting a distinctive marker on-site as described in claim 10, wherein the sample is analyzed by PCR, isothermal amplification, nucleic acid hybridization, or nucleic acid sequencing. A method for detecting a distinctive mark on-site, comprising: providing a copy from an item of interest; analyzing the sample to detect the presence of the distinctive mark, wherein the analyzing is performed using an on-site detecting instrument, and wherein the field detecting instrument A microsystem is included that is configured to perform a sample in-out analysis; and to verify the authenticity of the item of interest by detecting the presence of the distinctive marker in the sample. A method of detecting a distinctive marker on-site as described in claim 12, wherein the sample further comprises the same identifier. A method of detecting a distinctive marker on-site as described in claim 13 wherein the biomolecule comprises DNA. A kit for collecting samples from an item of interest, comprising: an on-site inspection instrument comprising the same collection unit configured to collect the same, the sample comprising a distinctive marker suitable for analysis in the on-site inspection instrument, Wherein the kit comprises a buffer solution for extracting the distinctive marker from the item of interest, and wherein the field detecting instrument is configured to analyze the sample to determine the presence of the distinctive marker in the sample .