Animal-derived product authentication based on DNA methylation

Abstract

The present invention relates to a method of authenticating a test animal-derived product sample, the method comprising the steps of: (a) determining a test methylation profile of one or more preselected methylation sites within a genomic material obtained from the test animal-derived product sample; and (b) comparing the test methylation profile obtained from (a) to a reference methylation profile obtained from a control animal of the same biometric population of the test animal from which the product sample is derived, where the control animal is not by a single transection of the throat, the control animal is slaughtered by severing the bilateral carotid artery, bilateral jugular vein, bilateral vagus nerve, trachea and / or esophagus, and / or the control animal is not lethal by bleeding, where a difference in the test methylation profile of (a) as compared to a reference methylation profile from the control animal indicates that the test animal has passed a transecting throat, and a difference in the test methylation profile of (b) as compared to a reference methylation profile from the control animal indicates that the test animal has passed a transecting throat. Cutting off the bilateral carotid artery, bilateral jugular vein, bilateral vagus nerve, trachea and / or esophagus for slaughtering, and / or testing that the animal is death due to bleeding, and testing the animal-derived product so authenticated; and wherein the preselected methylation site is a CpG site selected from gene or genomic DNA regions from control and test animals that exhibit the highest degree of methylation variation during training of the method.

Description

Field of the Invention

[0001] The present invention relates to a method for determining the authentication of unknown animal-derived product samples based on a specific gene panel, wherein the specific gene panel provides a source for generating a DNA methylation profile specific for a clear authentication. Specifically, DNA methylation profile analysis can be used to determine the clear authentication of animal-derived products. The clear authentication may include halal, kosher, and the like. Background of the Invention

[0003] Sustainable food production is currently seen as one of the most important societal needs worldwide. As value chains in sectors such as agriculture and aquaculture are very complex, certification has been established to strengthen consumer relations and trust. However, certification is based on audits at specific farms and can be easily tampered with by moving livestock from non-certified to certified farms. Furthermore, oversight of sustainable agricultural practices is imperfect as it is largely limited to audits. This leaves room for fraudulent activity in the industry and there is an urgent need for tamper-proof analysis and certification for meat.

[0004] Further, some people in the world observe dietary restrictions that are particularly relevant to the tradition or religion they believe in. For example, some Jews only eat foods that have been considered kosher according to Jewish law. Similarly, some Muslims may wish to only eat foods that have been considered halal according to Islamic law. Others may have other dietary restrictions related to other traditions, religions, moral codes, etc. Kosher certification (i.e., designating food as kosher) and halal certification (i.e., designating food as halal) for foods including meat both involve compliance with specific laws related to animal slaughter. For example, the Jewish slaughtering ritual (Shechita) and the Islamic slaughtering ritual (Zabihah) require a one-time clean crosscutting of the neck of the animal with a non-serrated blade to cut off the main blood vessels of the animal and drain the blood of the animal. Considerations of health, safety, and animal welfare require that the origin of animal products and particularly meat products should be traceable, so that quality assurance audits and monitoring procedures can be effectively and reliably performed. However, there is currently no means to accurately and reliably track the origin of meat. Specifically, there is no reliable, effective, and easy-to-use method for distinguishing halal meat from non-halal meat, and kosher meat from non-kosher meat, other than trusting the labels found on meat packaging and stores. That is, although meat may be certified as kosher (i.e., permitted according to Jewish tradition) or halal (i.e., permitted according to Islamic tradition), buyers may not be able to determine based on a visual inspection whether the meat was properly slaughtered. Furthermore, when dining at a restaurant, customers may not be able to view the inspection label affixed to the meat packaging before the meat is prepared. Furthermore, customers may not be able to determine based on a visual inspection the type of animal from which the meat was derived. This may result in people who adhere to dietary restrictions being reluctant to consume animal products.

[0005] The physical health and / or chemical condition of an animal may change based on the manner in which the animal has been slaughtered. For example, it is well known that the Cortisol levels of an animal that may have been slaughtered kosher or halal may be different from the Cortisol levels of an animal that has undergone another means of slaughter. In addition, different types of meat obtained from different animals may be associated with physical and / or chemical differences. For example, the protein from pork from a pig may be different from the protein from beef from a cow. There may also be differences related to different breeds of a particular type of animal, different cuts of meat from a particular animal, and the like. This may limit the effectiveness of comparing an unknown meat sample with a set of known reference meat samples.

[0006] Recent studies have shown that the DNA methylation patterns of animals contain important information about their rearing conditions. For example, comparison of genome-wide methylation patterns and variation at the DNA level revealed that a large portion of epigenetic variation can be associated with adaptive differences and rearing conditions (e.g., captive salmon) (Le Luyer J et al., 2017 PNAS Vol. 114, No. 49).

[0007] Koop et al. and Rhein et al. also demonstrated, respectively, that methylation patterns are maintained postmortem and are consistent regardless of the level of decomposition of the sample. One of the only aspects that still affects the methylation pattern of a sample is DNA integrity and the amount of DNA found in the sample (Koop et al., 2021 International Journal of Legal Medicine 135: 167-173 and Rhein et al., 2015 Frontiers in Genetics 6: 182).

[0008] In view of the above, there is an urgent need to provide means for identification and quality control of biological, particularly food and more particularly authentication of animal-derived materials to meet dietary restrictions related to traditions, religions, moral codes and the like that are strictly followed by people. SUMMARY OF THE INVENTION

[0010] The present invention attempts to solve the above-mentioned problems by providing a method that uses DNA methylation patterns to distinguish one class of animal-derived products from another class of animal-derived products, and is able to determine the killing or slaughtering method of the animal from which the animal-derived product is derived, and thereby accurately and reliably authenticate the product. The present invention is based on the following discovery: the means of slaughtering animals leads to stress in the internal environment of the animal, and this can permanently change the genome of the animal through epigenetics. In particular, the ability to adapt to the environment (i.e. the slaughtering method) and maintain an adapted biological pattern depends on epigenetic mechanisms, including DNA methylation. In particular, the present invention is based on the following discovery: the method of slaughtering animals for food consumption may also lead to changes in the epigenetic mechanisms of the animal, including DNA methylation patterns, and these patterns may be passed on to different products that may be derived from the animal.

[0011] The inventors have unexpectedly discovered that this property can be used to identify "epigenetic fingerprints" on the genome that are specific not only to the slaughter method of one animal, but may also be specific to all animals that undergo the same slaughter method. For example, these 'epigenetic fingerprints' may be specific not only to animals slaughtered in a particular way, but also to any product derived from that animal (individual). Based on these findings, the present invention provides a means for identifying specific slaughter methods for specific animals, such as farmed animals from which animal-derived products are derived, also known as livestock and poultry. In particular, the method according to any aspect of the present invention can be used to determine whether an animal-derived product is derived from an animal that has been slaughtered in a particular way. More particularly, the method according to any aspect of the present invention can be used to determine whether an animal-derived product is derived from an animal that has been slaughtered by cutting the throat with a single knife, cutting off the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, and / or whether the animal has bled to death. In this way, the method according to any aspect of the present invention can then be used to accurately and reliably determine the method of slaughter of an animal, and subsequently provide and / or confirm the authentication of any sample derived from said animal. Further, the method according to any aspect of the present invention can also be used to identify whether any animal-derived product is accurately and reliably authenticated, particularly when the authentication is based on the method of slaughter of the animal.

[0012] According to one aspect of the present invention, there is provided a method for authenticating and testing a sample of an animal-derived product, the method comprising the following steps:

[0013] (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and

[0014] (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic group as the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death,

[0015] wherein a difference in the test methylation profile of (a) compared to a reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, and the test animal-derived product is so authenticated; and

[0016] The preselected methylation sites are CpG sites selected from genes or genomic DNA regions from control and test animals that showed the highest degree of methylation variation during the training process of the method.

[0017] According to another aspect of the present invention, there is provided a method for authenticating and testing a sample of an animal-derived product, the method comprising the following steps:

[0018] (a) determining the methylation status of at least one CpG site within genomic material obtained from a sample of a product of animal origin,

[0019] (b) comparing the methylation status of the CpG sites from (a) with the methylation status of a control animal of the same taxonomic group as the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death,

[0020] wherein a difference in the test methylation status of (a) in the test animal compared to the CpG site in the control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, and the test animal-derived product is so authenticated.

[0021] According to another aspect of the present invention, there is provided a method for authenticating and testing a sample of an animal-derived product, the method comprising the following steps:

[0022] (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and

[0023] (b) comparing the test methylation profile determined in (a) to a panel of predetermined reference methylation profiles of the same taxonomic group of the test animal from which the product sample was derived, wherein each predetermined reference methylation profile is specific for a clearly authenticated reference animal that:

[0024] - has been slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus; and / or

[0025] - Bleeding to death;

[0026] wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, then the test animal-derived product sample has a clear authentication as being the same as the predetermined reference methylation profile, and is derived from a test animal that has been slaughtered and / or bled to death by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and the test animal-derived product is so authenticated.

[0027] As used herein, the term 'animal-derived product' refers to a product derived from an animal. In particular, the term 'test animal-derived product' refers to a sample or object discussed that will undergo a method according to any aspect of the present invention. These products from animals may include meat and meat products, and also include fat, meat, blood and little-known products such as fish glue and dry stomach membrane (rennet), poultry products (meat and eggs), dairy products (milk and cheese) and non-food products such as fiber (wool, mohair, cashmere, leather, etc.). Animal-derived products may also include products that can be prepared using animal products (such as fat), such as soap, cream, etc. In an example, animal-derived products are meat, eggs, blood, brain, sperm, milk and any other tissue or sample providing genomic DNA. In particular, animal-derived products are meat.

[0028] The term 'quality certification' refers to a certificate or confirmation issued by a designated certification body, which recognizes the quality of a specific animal-derived product, including food for human use and / or consumption. The term 'quality certification' is used interchangeably with the term 'certification'. These certifications are usually found on the packaging of animal-derived products including food to be consumed, and are printed by product manufacturers. Clear examples of food quality certifications can include 'feeding patterns (Haltungsform)', 'animal welfare (Tierwohl)', 'non-GMO certification (Ohne Gentechnik)', 'halal certification', 'kosher certification' and other safety labels, which confirm that the products sold have been prepared in accordance with specific religious or safety regulations. Specifically, the term 'food quality certification' refers to a certificate or confirmation issued by a designated certification body, which recognizes the quality, source or slaughter means of a specific food for human consumption. According to any aspect of the present invention, the quality of the certification can be a clearly certified food quality or a clear certification, and this can be a kosher certification, a non-kosher certification, a halal certification or a non-halal certification.

[0029] In one example, according to any aspect of the invention, the explicitly certified food quality or certification of the sample X can be kosher, non-kosher, halal or non-halal. More particularly, kosher or halal means that the sample X originates from an animal that was slaughtered by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus. Even more particularly, the animal was drained of blood.

[0030] The term 'kosher' used in combination with food according to any aspect of the present invention refers to food that complies with the Jewish dietary regulations of kashrut or food that can be eaten according to halakha (law). Kosher used in connection with meat particularly relates to the manner in which the animal is prepared for consumption. According to Jewish tradition, meat can be considered kosher when it comes from an animal that has been slaughtered according to Jewish law, wherein the animal is slaughtered by a single cut across the throat to a precise depth not higher than the epiglottis and not lower than the beginning of the cilia in the trachea, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and esophagus, causing the animal to bleed to death. Such slaughtering must be carried out using a sharp broadsword, which is checked before each slaughtering to ensure that it has no irregularities (e.g., scratches and dents). The slaughtering is also usually performed by a butcher or rabbi. Kosher meat generally refers to most meats other than pork. In particular, kosher meat can be selected from beef, chicken, lamb, mutton, goat and mixtures thereof. Kosher meat does not include shellfish, which is not permitted according to Jewish tradition. Although Jewish tradition permits the consumption of vertebrate fish, since no special method exists for slaughtering vertebrate fish, all vertebrate fish can be considered kosher. Any food or meat that does not fall within the definition of 'kosher' would then be considered 'non-kosher'.

[0031] The term 'halal' used in conjunction with food according to any aspect of the invention refers to food that complies with Islamic dietary laws, and in particular meat that has been processed and prepared in accordance with these requirements. Slaughtering is performed in which the animal is slaughtered using a sharp blade without serrations to make a single clean cross-section of the neck to create a wound that cuts the front of the throat, esophagus and jugular vein, but does not sever the spinal cord. In addition to the instructions, the permitted animals should be slaughtered after reciting the Islamic prayer Bismillah. The animal must also be drained of blood after slaughter. The slaughter must be performed by an adult Muslim. Halal meat generally refers to most meats except pork. In particular, Halal meat can be selected from beef, chicken, lamb, mutton, goat and mixtures thereof. Although Islamic tradition allows the consumption of shellfish and vertebrate fish, since there is no special method for preparing shellfish, all shellfish and vertebrate fish can be considered Halal. Any food or meat that does not meet the definition of 'Halal' will then be considered 'Non-Halal'. The definition of halal is further provided in https: / / www.smiic.org / en / project / 24 (Organisation of Islamic Cooperation (OIC) / Standards and Metrology Institute for the Islamic Countries (SMIIC), OIC / SMIIC 1:2019 General Requirements for Halal Food, accessed on 8 June 2022).

[0032] The term "test" used in conjunction with the term object and / or animal in the present disclosure refers to an entity subjected to the method according to any aspect of the present invention and is the basis for the analytical application of the present invention. Thus, a "(individual) test object", "(individual) test object group" or "test profile" or "test animal-derived product" is an (individual) object or group of objects to be tested according to the present invention or a profile obtained or generated in this context. Conversely, the term "reference" should mean an entity, mostly predetermined, for comparison with the test entity. For example, the term 'reference animal' refers to an animal for comparison or as a control for a reference to a 'test animal'. Similarly, the terms 'sample' and / or 'test animal-derived product sample' used in accordance with any aspect of the present invention refer to an entity that can be subjected to the method of the present invention. In particular, a sample can be any (test) animal-derived product that can be subjected to the method of the present invention to determine a clear authentication by first determining a DNA methylation profile and then comparing the test methylation profile with a control. Blockchain can also be used to make information easily accessible to consumers.

[0033] The method according to any aspect of the present invention can be used to identify unknown samples (i.e., animal-derived product samples) based on DNA methylation patterns. These DNA methylation patterns can then be compared with reference DNA methylation patterns to trace the animal-derived product samples back to the slaughterhouse or farm from which the samples originated, and then determine whether the unknown samples correspond to animals that have been slaughtered according to kosher, halal, non-kosher, or non-halal practices. In this way, a purchaser or consumer of meat can verify that meat sold or marketed as kosher or halal food is authentic.

[0034] As used herein, the term "comprising" should be interpreted as covering both "including" and "consisting of", both of which have clear intentions and are therefore aspects of the individual disclosures of the present invention. When used herein, "and / or" should be regarded as the specific disclosure of two specified features or components, each with or without the other. For example, "A and / or B" should be regarded as the specific disclosure of (i) A, (ii) B and (iii) A and B, respectively, as if each is individually set forth herein. In the context of the present invention, the terms "about" and "approximately" represent that those skilled in the art will understand that the accuracy interval of the technical effect of the feature in question is still ensured. The terms generally indicate deviations of ±20%, ±15%, ±10% and, for example, ±5% from the indicated numerical value. As will be understood by a person of ordinary skill, the specific deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have such deviations that are greater than artificial or engineering technical effects. When an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0035] In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation status" or "methylation state" are used herein to describe the status, situation or condition of methylation of a genomic sequence, and such terms refer to the properties of a DNA segment at a specific genomic locus that are associated with methylation. Such properties include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of methylated C residues, the percentage of methylated C at any specific residue stretch, and allelic differences in methylation due to, for example, differences in allelic origin.

[0036] The term "methylation state" refers to the state of a specific methylation site (i.e., methylation relative to non-methylation), which means that a residue or methylation site is methylated or unmethylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined. Accordingly, the term "methylation profile" or also referred to as "methylation pattern" refers to the relative concentration or absolute concentration of methylated C residues or unmethylated C residues at any specific residue segment in the genomic material of a biological sample. For example, if a cytosine (C) residue that is not usually methylated in a DNA sequence is methylated, it can be referred to as "hypermethylated"; and if a cytosine (C) residue that is usually methylated in a DNA sequence is not methylated, it can be referred to as "hypomethylated". Similarly, if a cytosine (C) residue in a DNA sequence (e.g., DNA from a sample nucleic acid of a test subject) is methylated compared to another sequence from a different region or from a different individual (e.g., relative to a standard nucleic acid of a normal nucleic acid or a reference sequence), the sequence is considered to be hypermethylated compared to another sequence. Alternatively, if the cytosine (C) residues within a DNA sequence are non-methylated compared to another sequence from a different region or from a different individual, the sequence is considered to be hypomethylated compared to the other sequence. These sequences are referred to as "differentially methylated". The measurement of differential methylation levels can be accomplished in a variety of ways known to those skilled in the art. As a non-limiting example, one method is to measure the methylation levels of the CpG sites of each query determined by a bisulfite sequencing method.

[0037] The term "hypermethylation" refers to the average methylation status corresponding to an increased presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides within a normal control DNA sample. In particular, the control refers to an animal-derived product that has not been slaughtered by slicing the throat with a single knife, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or has not been bled to death.

[0038] The term "hypomethylation" refers to the average methylation status corresponding to a decrease in the presence of 5-mCyt at one or more CpG dinucleotides within the DNA sequence of the test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides within a normal control DNA sample. In particular, the control refers to an animal-derived product that has not been slaughtered by slicing the throat with a single knife, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, and / or has not been bled to death.

[0039] As used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not generally present in a recognized typical nucleotide base. For example, cytosine in its usual form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine in its usual form may not be considered a methylated nucleotide, while 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring, however, for the purposes of this article, thymine may not be considered a methylated nucleotide when present in DNA. Typical nucleotide bases for DNA are thymine, adenine, cytosine, and guanine. Typical bases for RNA are uracil, adenine, cytosine, and guanine. Accordingly, a "methylation site" is a location in a target gene nucleic acid region where methylation is possible. For example, a location containing CpG is a methylation site where cytosine may be methylated or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group attached to a nucleotide position where methylation can occur. These methylated nucleotides are commonly found in nature, and to date, methylated cytosines occurring mainly in the context of the dinucleotide CpG, but also in the context of CpNpG- and CpNpN-sequences can be considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but they will not be considered for any aspect of the present invention.

[0040] As used herein, a "CpG site" or "methylation site" is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation, either by naturally occurring events in vivo or by events that chemically methylate the nucleotide in vitro.

[0041] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more nucleotides that are methylated.

[0042] As used herein, the term 'epigenetic changes' refers to chemical (e.g., methylation) changes or protein (e.g., histone) changes that occur to the body of a gene or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can affect genes that are passed from one generation to the next.

[0043] As used herein, the term "bisulfite" encompasses any suitable type of bisulfite, such as sodium bisulfite, or another chemical reagent, which can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosine, and can therefore be used to differentially modify DNA sequences based on the methylation state of DNA, such as U.S. Patent Publication US 2010 / 0112595 (Menchen et al.). As used herein, the reagents for "differentially modifying" methylated or unmethylated DNA encompass any reagents that modify methylated and / or unmethylated DNA in the process, by which the product can be distinguished to be produced in methylated and unmethylated DNA, thereby allowing the identification of DNA methylation state. Such processes may include, but are not limited to, chemical reactions (e.g., C to U conversion by bisulfite) and enzymatic treatments (e.g., by cleavage of methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one that is able to cleave or digest a DNA molecule with much greater efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly greater efficiency when the DNA is not methylated.

[0044] In the context of the present invention, any "non-bisulfite-based method" and "non-bisulfite-based quantitative method" are also included to test the methylation status at any given methylation site to be tested. Such terms refer to any method for quantifying methylated or non-methylated nucleic acids that does not require the use of bisulfite. These terms also refer to methods for preparing nucleic acids to be quantified that do not require bisulfite treatment. Examples of non-bisulfite-based methods include, but are not limited to, methods using one or more methylation-sensitive enzymes for digesting nucleic acids and methods using reagents that bind nucleic acids based on methylation status for separating nucleic acids. The terms "methyl-sensitive enzyme" and "methylation-sensitive restriction enzyme" are DNA restriction endonucleases that rely on the methylation status of their DNA recognition sites for activity. For example, there are methyl-sensitive enzymes that cut or digest their DNA recognition sequences only when their DNA recognition sequences are not methylated. Therefore, unmethylated DNA samples will be cut into smaller fragments than methylated DNA samples. Similarly, highly methylated DNA samples are not cut. In contrast, there are methyl-sensitive enzymes that cut their DNA recognition sequences only when their DNA recognition sequences are methylated. As used herein, the terms "cleavage," "shearing," and "digestion" are used interchangeably.

[0045] A "biological sample" in the context of the present invention may comprise any biological material containing genomic material obtained from a subject or group of subjects, and may be liquid, solid or both, may be tissue or bone, or body fluids such as blood, lymph, etc. In particular, a biological sample useful in the present invention may comprise a biological cell or a fragment thereof.

[0046] As used herein, the term "preselected methylation sites" refers to methylation sites selected from genes or regions that show the highest degree of methylation variation during the training process of the method, and meet certain quality criteria such as considering a minimum sequencing coverage of ≥5x and a total of ≥5 qualified CpG sites. In addition, due to their limited dynamic range, genes with an average methylation level of <0.1 or an average methylation level of >0.9 can be excluded. A "reference methylation profile" can be defined based on multiple training samples using multivariate statistical methods such as principal component analysis or multidimensional scaling analysis. In particular, according to any aspect of the present invention, in particular, the CpG sites in the preselected methylation sites are selected from the following Table A:

[0047]

[0048]

[0049] The term "predetermined reference profile" as used in the context of the present invention refers to a typical or standard methylation profile of genomic material of a class of reference animal-derived products that is confirmed to be correctly labeled or authenticated. In one example, the predetermined reference profile can be used in the context of a control animal, wherein the control animal has been correctly authenticated (i.e., wherein the control animal was neither slaughtered by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, nor bleeding to death). Therefore, the control animal may have been slaughtered using non-halal and non-kosher slaughtering means, which means can be referred to as normal slaughtering means. The experimental subject group of the predetermined reference profile of the control animal may include profiles from different samples, which have been obtained from different parts of the control animal (not slaughtered by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, nor bleeding to death). For example, a panel of predetermined reference profiles may include at least one profile for eggs, at least one profile for meat (muscle, tissue, organ, etc.), at least one profile for milk, etc. Each of these samples may have its own unique predetermined methylation reference profile, which also forms part of the panel of predetermined reference profiles.

[0050] In another example, a predetermined reference profile can be used in the context of a reference animal that may have been slaughtered and / or bled to death by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea, and / or esophagus. Panels of predetermined reference profiles can be prepared for different samples from animals that have been confirmed to have been slaughtered and / or bled to death by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea, and / or esophagus. Here again, there can be a panel of predetermined reference profiles for each product derived from an animal that has been slaughtered and / or bled to death by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea, and / or esophagus. For example, a panel of predetermined reference profiles may include at least one profile for eggs, at least one profile for meat (muscle, tissue, organ, etc.), at least one profile for milk, etc., from an animal that has been slaughtered by slicing the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea, and / or esophagus, and / or has bled to death, etc. Each of these samples may have its own unique predetermined methylation reference profile, which also forms part of the panel of predetermined reference profiles.

[0051] For example, the control can be a type of meat of the same species or the same animal taxonomic group as a sample of an animal-derived product or an unknown sample with a known certification. Alternatively, a predetermined reference profile can be obtained from different types of meat with a known certification. In one example, the control can be a chicken certified as 'halal'. Halal chicken can be a mixture of different parts of a chicken (i.e., breast meat, thigh meat, kidney, liver, etc.). In another example, halal chicken may come from a part of a chicken. From halal chicken, a methylation profile can be obtained as a predetermined reference profile (control), which can be used to determine whether the test sample (meat) is halal or non-halal by determining whether the methylation profile of the test sample is significantly similar to the predetermined reference profile. The methylation profiles of different types of halal meat from one animal species may be the same. The methylation profiles of different types of halal meat from different animal species may also be the same. In one example, the control can be a chicken certified as "kosher". Kosher chicken can be a mixture of different parts of a chicken (i.e., breast meat, thigh meat, kidney, liver, etc.). In another example, kosher chicken may be from one part of a chicken. From the kosher chicken, a methylation profile may be obtained as a predetermined reference profile (control), which may be used to determine whether a test sample (meat) is kosher or non-kosher by determining whether the methylation profile of the test sample is significantly similar to the predetermined reference profile. The methylation profiles of different types of kosher meat from one animal species may be the same. The methylation profiles of different types of kosher meat from different animal species may also be the same.

[0052] There may be a collection of several predetermined reference profiles, and comparing the methylation profile of the test sample to the predetermined reference profiles in the collection may enable the identification of a specific predetermined reference profile that is (significantly) similar to the methylation profile of the test sample, and the test sample may then be confirmed as being slaughtered by a single cut across the throat, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus and / or bleeding to death. In one example, the predetermined reference profile may include methylation profiles of meat from different parts (i.e., breast, thigh, kidney, liver, shoulder, ribs, intestine, etc.) of an animal (chicken, goat, cattle, lamb, sheep, etc.) that has been slaughtered by a single cut across the throat, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus and / or bleeding to death.

[0053] In particular, the panels of predetermined reference methylation profiles according to any aspect of the invention are specific for different test animal-derived products. That is, each predetermined reference methylation profile is specific for a single animal-derived product. Thus, the panels of predetermined reference methylation profiles may include many different predetermined reference methylation profiles from different parts of an animal or several animals of the same biological taxonomic group as the test animal. There will also be different panels of predetermined reference methylation profiles for different animal taxonomic groups, and the relevant panels of predetermined reference methylation profiles specific to an animal taxonomic group will depend on the animal taxonomic group of the test animal.

[0054] In the context of the present disclosure, and in particular in the context of methylation profile comparisons, such as comparisons between a test profile (from a test subject (i.e., sample X)) and a reference profile, the term "significantly similar" shall mean similarities observed by statistical tools (i.e., by using bioinformatics) and / or also by using observation by eye. For example, significant similarity is observed if, by multivariate statistical methods such as principal component analysis or multidimensional scaling analysis, a test profile overlaps with a reference profile defined by multiple training samples. In particular, a test profile is significantly similar to a predetermined reference profile if more than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% of the methylation pattern / profile overlaps with its reference profile. Similarity of a test profile to more than one, such as two, three or even all reference profiles, reduces the significance of the similarity. Of course, the similarity of a test methylation profile to a reference methylation profile takes into account experimental errors that occur in all methods.

[0055] Animal-derived product samples can be a single type of meat, different types of meat, a single part of a type of meat, different parts of a single type of meat or different parts of different types of meat. The sample can be from any biological entity with a DNA genome and DNA genome methylation. In particular, the methylation site is a CpG site. The biological entity can be any animal except pig. In particular, the animal can be selected from chicken, lamb, camel, cattle, goat, sheep, horse, donkey, turkey, duck, goose, quail, rabbit and mule. More particularly, the animal can be selected from cattle, sheep, goat, camel, chicken, goose, duck and turkey. Therefore, the term 'meat' can be understood in this article to include chicken, lamb, beef, mutton, goat, camel meat, chicken, goose, duck, turkey and mixtures thereof.

[0056] The one or more preselected methylation sites in (a) are methylation sites associated with tissue-specific gene expression, preferably wherein the preselected methylation sites are associated with gene expression in a defined tissue.

[0057] The tissue may be selected from

[0058] (i) metabolic tissue such as intestinal tissue, preferably the ileum or jejunum,

[0059] (ii) muscle tissue,

[0060] (iii) skin tissue, and

[0061] (iv) organ tissue, preferably liver and / or pancreatic tissue.

[0062] According to another aspect of the present invention, there is provided a method for verifying the authentication of a sample of a test animal-derived product that has been authenticated as being derived from a test animal, the test animal having been slaughtered and / or bled to death by cutting the throat with a single knife, severing bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, the method comprising the following steps:

[0063] (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and

[0064] (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic group as the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death

[0065] wherein a difference in the test methylation profile of (a) compared to a reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, and the test animal-derived product is properly authenticated, and

[0066] wherein the similarity in the test methylation profile of (a) compared to the reference methylation profile of a control animal indicates that the test animal was not slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal bled to death, and the product derived from the test animal was misauthenticated, and

[0067] The preselected methylation sites are CpG sites selected from genes or genomic DNA regions from control and test animals that showed the highest degree of methylation variation during the training process of the method.

[0068] According to a further aspect of the present invention, there is provided a method for confirming or rejecting a presumptive authentication of a sample of a product of animal origin, the method comprising:

[0069] - comparing a test methylation profile obtained from genomic material of a product sample of animal origin with a reference methylation profile obtained from a control animal of the same taxonomic group from which the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death,

[0070] wherein a difference in the test methylation profile compared to the reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death

[0071] wherein the presumptive certification is that the test animal-derived product sample is kosher or halal, wherein the test animal is slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus and / or the test animal is bled to death.

[0072] According to another aspect of the present invention, there is provided a method for determining whether a sample of a test animal-derived product is obtained from a test animal that has been bled to death or slaughtered by cutting the throat with a single knife, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, the method comprising:

[0073] - Comparing a test methylation profile obtained from genomic material of a test animal-derived product sample with a reference methylation profile obtained from a control animal of the same taxonomic group from which the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death.

[0074] In particular, the certification is kosher or halal when the animal is slaughtered by transecting the throat, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus. Additionally or alternatively, the certification is kosher or halal when the animal is drained of blood.

[0075] According to yet a further aspect of the present invention, there is provided a certification of a sample of a test animal-derived product, wherein the test animal is slaughtered by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus,

[0076] The method for determining that the test animal is slaughtered by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus comprises the following steps:

[0077] (a) determining a test methylation profile of one or more preselected methylation sites within genomic material contained in a test animal-derived product sample; and

[0078] (b) comparing the test methylation profile determined in (a) to a panel of predetermined reference methylation profiles from the same taxonomic group of the test animal from which the product sample was derived, wherein each predetermined reference methylation profile is from a different reference animal and / or a different supplier,

[0079] Wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, the test animal is slaughtered by cutting the throat with a single knife, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus.

[0080] Additionally or alternatively, the test animal is bled dry.

[0081] According to yet another aspect of the present invention, there is provided the use of DNA methylation profiling for determining the certification of an animal-derived product sample, in particular, the certification of the animal-derived product sample is kosher or halal when the test animal is slaughtered by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus and / or the test animal bleeds to death. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Results of principal component analysis (PCA) of 6,706,643 CpG sites with minimum coverage of 10 in all halal and non-halal samples.

[0084] Figure 2 Results of principal component analysis (PCA) of the 76,339 differentially methylated positions (DMPs) identified. Example

[0085] The foregoing describes preferred embodiments, which, as will be appreciated by those skilled in the art, may be subject to changes or modifications in design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be covered by the scope of the claims.

[0086] Example 1

[0087] Wet Lab Methods

[0088] Halal and non-halal chicken breasts were purchased from five different suppliers. Suppliers for halal meat included Pasar, ZAC Butchery, Hego premium, RedMart and Keesong, while non-halal meat was purchased from Pasar, Tegel, Farm fresh, Ryan and AW's in Singapore. Genomic DNA was purified from breast tissue samples using DNeasy Blood & Tissue Kit (Qiagen) and quantified using PicroGreen or NanoDropTM 2000.

[0089] Genomic DNA (500 ng) from breast tissue samples was used to prepare libraries for Whole Genome Bisulfite Sequencing (WGBS). Sequencing of the libraries was performed by a third party on the NovaSeq platform, which generated 125 GB of data / sample with 20X coverage.

[0090] Muslim Non-Halal brand brand Pasar Pasar ZAC Butchery Tegel Hego Premium Farm fresh RedMart Ryan Keesong AW's

[0091] Calculation method

[0092] deal with:

[0093] Sequencing reads were trimmed and aligned using the assembled version 5.0 of the chicken (Gallus gallus) genome as the reference sequence using BSMAP1 version 2.5. Methylation ratios were determined using a Python script (methratio.py) distributed with the BSMAP package after deduplication using picard2 (http: / / broadinstitute.github.io / picard / ). BSMAP is further explained in Xi, Y. et al., BMC Bioinform. 10, 232 (2009). For all further analyses, only CpGs covered by at least ten reads were considered, resulting in 6,706,643 CpG sites.

[0094] Differential methylation analysis

[0095] Differential methylation analysis was performed for halal and non-halal groups using MethylKit3 (version 1.12.0). MethylKit used logistic regression to calculate p-values, and p-values ​​were adjusted to q-values ​​using a sliding linear model method 4. The use of the linear model method is further described here: Wang HQ et al., Bioinformatics. 2011; 27: 225–231. CpG sites with an FDR below 0.05 and a methylation change greater than 25% between groups were considered as significant differentially methylated positions (DMPs), which resulted in 76,339 CpG sites. The precise locations of the 8,000 CpG sites are provided in Table 1 below.

[0096] MethylKit generates a "prcomp" object that can be used to extract and plot principal components. The use of MethylKit is disclosed in Akalin A et al., Genome Biol. 2012; 13(10): R87. Principal component analysis (PCA) is a dimensionality reduction method that can transform a large data set into a few principal components. The first few principal components generally retain most of the variation present in the data set and can be used to emphasize grouping structure in the data.

[0097] Principal component analysis (PCA) of CpG sites with minimum coverage of 10 in all samples: 6,706,643, such as Figure 1 as shown in .

[0098] The principal component analysis (PCA) of the 76,339 DMPs identified is shown in Figure 2 middle.

[0099] Plotting of the first two principal components of CpGs before and after differential methylation analysis revealed meaningful clustering of samples. Differentially methylated positions (DMPs) were able to effectively cluster halal and non-halal groups in a meaningful manner.

[0100] Table 1. 8000 CpG sites with 76339 significant differentially methylated positions (DMPs) are provided.

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

Claims

1. A method for authenticating and testing a sample of an animal-derived product, the method comprising the following steps: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic group as the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death, wherein a difference in the test methylation profile of (a) compared to a reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, and the test animal-derived product is so authenticated; and The preselected methylation sites are CpG sites selected from genes or genomic DNA regions from control and test animals that showed the highest degree of methylation variation during the training process of the method.

2. A method for authenticating and testing a sample of an animal-derived product, the method comprising the following steps: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and (b) comparing the test methylation profile determined in (a) to a panel of predetermined reference methylation profiles of the same taxonomic group of the test animal from which the product sample was derived, wherein each predetermined reference methylation profile is specific for a clearly authenticated reference animal, - has been slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus; and / or - Bleeding to death; wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, then the test animal-derived product sample has a positive authentication as being the same as the predetermined reference methylation profile and is derived from a test animal that has been slaughtered and / or bled to death by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and the test animal-derived product is so authenticated; and The preselected methylation sites are CpG sites selected from genes or genomic DNA regions from control and test animals that showed the highest degree of methylation variation during the training process of the method.

3. A method for authenticating and testing a sample of an animal-derived product, the method comprising the following steps: (a) determining the methylation status of at least one CpG site within genomic material obtained from a sample of a product of animal origin, (b) comparing the methylation status of the CpG sites from (a) with the methylation status of a control animal of the same taxonomic group as the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death, wherein a difference in the test methylation status of (a) in the test animal compared to the CpG site in the control animal indicates that the test animal has been slaughtered by cutting the throat with a single knife, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, and the test animal-derived product is so authenticated.

4. The method according to any one of the preceding claims, wherein the test animal-derived product sample is explicitly certified as kosher or halal, wherein the test animal is slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal is bled to death.

5. A method for verifying the authentication of a sample of a test animal-derived product that has been authenticated as being derived from a test animal that has been slaughtered and / or bled to death by cutting the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, the method comprising the following steps: (a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic group as the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death, wherein a difference in the test methylation profile of (a) compared to a reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, and the test animal-derived product is properly authenticated, and wherein a similarity in the test methylation profile of (a) compared to a reference methylation profile of a control animal indicates that the test animal was not slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal bled to death, and the test animal-derived product is misauthenticated; and The preselected methylation sites are CpG sites selected from genes or genomic DNA regions from control and test animals that showed the highest degree of methylation variation during the training process of the method.

6. The method according to any one of the preceding claims, wherein the CpG sites are selected from the list provided in Table A.

7. A method for confirming or rejecting the presumptive authentication of a sample of a product of animal origin, the method comprising: - comparing a test methylation profile obtained from genomic material of a product sample of animal origin with a reference methylation profile obtained from a control animal of the same taxonomic group from which the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death, wherein a difference in the test methylation profile compared to the reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death, wherein the presumptive certification is that the test animal-derived product sample is kosher or halal, wherein the test animal is slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus and / or the test animal is bled to death.

8. A method for determining whether a sample of a product derived from a test animal was obtained from a test animal that had been bled to death or slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, the method comprising: - Comparing a test methylation profile obtained from genomic material of a test animal-derived product sample with a reference methylation profile obtained from a control animal of the same taxonomic group from which the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death.

9. The method according to claim 8, wherein the method comprises the following steps: a) determining a test methylation profile of one or more preselected methylation sites within genomic material obtained from a test animal-derived product sample; and b) comparing the test methylation profile determined in (a) to a panel of predetermined reference methylation profiles of the same taxonomic group of the test animal from which the product sample was derived, wherein the reference methylation profile is obtained from a control animal of the same taxonomic group of the test animal from which the product sample was derived, wherein the control animal was not slaughtered by a single cut across the throat, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the control animal was not bled to death, Wherein a difference in the test methylation profile of (a) compared to the reference methylation profile from a control animal indicates that the test animal has been slaughtered by slicing the throat with a single cut, severing both carotid arteries, both jugular veins, both vagus nerves, trachea and / or esophagus, and / or the test animal has bled to death.

10. The method according to any one of the preceding claims, wherein the animal is selected from the group consisting of cattle, sheep, goats, camels, chickens, geese, ducks and turkeys.

11. The method according to any of the preceding claims, wherein the test animal-derived product sample is meat, eggs, milk and any other tissue or sample providing genomic DNA.

12. The method according to any one of the preceding claims, wherein the test animal-derived product sample is meat.

13. The method according to any one of the preceding claims, wherein the difference is hypomethylation or hypermethylation.

14. Use of DNA methylation profile analysis for authenticating samples of animal-derived products, The certification of the animal-derived product sample is Kosher or Halal, wherein the test animal is slaughtered by cutting the throat with a knife, severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus / or the test animal is bled to death.

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