CN111378759A - Genetic loci for canine genotyping and uses thereof - Google Patents
Genetic loci for canine genotyping and uses thereof Download PDFInfo
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Abstract
The invention relates to a locus for canine genotyping and applications thereof. The invention provides a group of loci, including the following STR loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055 and REN54P 11. Primer sets, kits, methods of genotyping, and methods and systems for canine individual identification and genetic identification are thus provided. The STR locus provided by the invention can be accurately used for canine genotyping.
Description
Technical Field
The invention relates to the field of genetic determination, in particular to a composition for canine genotyping and application thereof, and particularly relates to a locus for canine genotyping, a primer set for amplifying each STR locus, a kit, a genotyping method, a genotyping system, a method and equipment for canine individual identification and genetic identification.
Background
Human genome STR (short tandem repeat) is a genetic marker which is commonly applied at present, and is a DNA genetic marker formed by tandem repeat with a few bases as a core unit. The length of the probe is between dozens and hundreds of bp. The DNA sequence formed by the tandem repeat can generate hundreds of millions of genotype combinations, and the occurrence frequency of each combination in the population is very low, so that STR typing is the most important technology for individual identification and paternity test of forensic individuals at home and abroad nowadays.
Dogs are the first domesticated livestock of humans and are also the pets with the highest feeding rates. According to statistics, the number of the pet dogs in 2017 reaches about 4990 thousands, and the proportion of the pet raising families in pet raising users is the highest, and reaches 60%, so that the pet raising dogs are the main pet category. Dogs, one of the most intimate partners for humans, have many specialized abilities, such as olfactory acuity, assisted hunting, and battle. With the problems of pet dog loss, police dog file management, genetic breeding and the like, establishing a DNA file database and a genetic identification technology for a love dog is particularly important.
However, the kit for typing dogs has many disadvantages in terms of individual recognition and genetic polymorphism. Typing and individual identification for dogs has yet to be further improved.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. To this end, it is an object of the present invention to provide a set of loci and kits for genotyping dogs, and methods and systems for genotyping dogs. The group of loci and the kit provided by the invention can realize the genotyping of the dogs, so that the loci and the kit can be used for individual identification and genetic identification of the dogs.
The invention is realized based on the following findings of the inventor: the genome STR fragment is always an effective tool for individual identification of biological characteristics, has the characteristics of smaller fragment, accurate typing, high amplification efficiency and the like, and is widely applied to the fields of forensic individual identification, paternity identification and the like. The fluorescent labeling multiplex amplification detection technology can detect a plurality of STR loci simultaneously, can distinguish the loci according to different fluorescence colors carried by allele fragments, and can distinguish the alleles of different loci by adopting different fluorescent dye labels even when the loci with the length of the allele fragments overlapped appear. The kit aiming at the canine typing has the main problems that the number of STR loci is small, and certain defects exist in individual identification power and genetic polymorphism. Therefore, it is necessary to develop a canine typing kit with a large number of loci, good genetic polymorphism, strong individual recognition ability and high accuracy.
However, aiming at all STR loci in dogs, all loci, some loci or some loci can be selected to be quickly and conveniently used for genotyping of dogs comprehensively, and creative analysis is needed. Through research, the inventor finds that comprehensive STR typing results can be obtained by combining as few STR loci as possible, so that the STR typing results are used for individual identification and genetic identification of dogs. The loci are suitable for canine groups in a plurality of countries and regions around the world, have the advantages of strong sensitivity, good stability, high accuracy and the like, and can be widely applied to canine individual identification, genetic breeding and the like.
Therefore, the invention provides the following technical scheme:
according to a first aspect of the invention, the invention provides a set of loci comprising the following autosomal STR loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055 and REN54P 11. Optionally, the set of loci can further include a sex chromosome locus DAMEL. The details of each site are shown in Table 1. Wherein the DAMEL is used as sex chromosome locus for dog sex identification. By utilizing each locus in the group of loci, the method can be conveniently and quickly used for genotyping of the dog so as to realize individual identification, genetic identification, establishment of a DNA archive database and the like of the dog, and has important value in the field of forensic medicine and commerce.
TABLE 1 information on each locus
According to a second aspect of the invention, there is provided a primer set suitable for specifically amplifying a nucleic acid sequence comprising the following loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055 and REN54P 11. By specifically amplifying the nucleic acid sequences of all loci in the composition, the gene can be quickly and conveniently used for genotyping of dogs, and the individual identification of the dogs is realized.
In some embodiments of the present invention, the primer set described above may further include the following technical features:
in some embodiments, the locus further comprises DAMEL. DMAEL as a locus on a sex chromosome can be used for sex determination of canines.
In some embodiments, the primer set is a nucleotide sequence shown in SEQ ID NO. 1-12, and SEQ ID NO. 15-40 or derivatives thereof. In some embodiments, the primer set further comprises the nucleotide sequences shown in SEQ ID NO. 13 to SEQ ID NO. 14 or derivatives thereof. In performing multiplex PCR reactions, great care should be taken in the selection of primer sequences. Because improper selection of primers may produce undesirable results, such as lack of amplification, amplification at one or more sites outside the intended target locus, formation of primer dimers, undesired interactions between primers at different loci, and the like. The invention designs specific primers aiming at 20 sites at the flanks of the repeated sequences of the sites. The annealing temperature of each pair of primers is about 60 ℃, primer dimer or non-specific amplification product can not be generated, and the length of the final amplification product is below 400 bp. Through tests, non-specific amplification and primer dimer are not generated, and other interaction or cross reaction is not generated. Therefore, the method can simultaneously realize the co-amplification of all STR loci, and can be conveniently and quickly used for genotyping and identifying the dogs at one time. In some embodiments, the derivative is a nucleic acid molecule that is obtained by substituting and/or deleting and/or adding one or more nucleotides to a nucleotide sequence and has the same function as the nucleotide sequence.
In some embodiments, the final concentration of the primer sequences used for specific amplification of AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, and REN54P11 loci in the amplification reaction is: (2.15. + -. 0.1) μ M, (0.45. + -. 0.1) μ M, (0.84. + -. 0.1) μ M, (0.60. + -. 0.1) μ M, (0.75. + -. 0.1) μ M, (1.25. + -. 0.1) μ M, (0.30. + -. 0.1) μ M, (0.38. + -. 0.1) μ M, (0.92. + -. 0.1) μ M, (0.35. + -. 0.1) μ M, (0.40. + -. 0.1) μ M, (2.00. + -. 0.1) μ M, (0.65. + -. 0.1) μ M, (0.50. + -. 0.1) μ M, (1.15. + -. 0.1) μ M, (0.30. mu.M) μ M, (1.25. + -. 0.1) μ M, (2.00. + -. 0.1) μ M and (0.63. + -. 0.1) μ M.
In some embodiments, the primer sequences for DAMEL locus-specific amplification are present at a final concentration of (0.65 ± 0.1) μ M in the amplification reaction system.
In some embodiments, at least one primer of each pair of primers of the primer set has a detectable label attached thereto.
In some embodiments, at least one primer of each pair of primers of the primer set has a detectable label attached to its 5' end.
In some embodiments, the primer sets comprise a first primer set, a second primer set, a third primer set, and a fourth primer set, each primer set having a distinguishable detectable label attached thereto; wherein the first primer set comprises primers for specific amplification of AHTk211, CXX279, INU030, REN169D01, REN247M23 locus, the second primer set comprises primers for specific amplification of INRA21, DAMEL, REN169O18, REN105L03, FH2848, AHTk253 locus, the third primer set comprises primers for specific amplification of AHT121, AHT137, REN162C04, AHTh171, FH2054 locus, and the fourth primer set comprises primers for specific amplification of INU005, REN64E19, INU055, REN54P11 locus. The primer groups are divided into different groups, namely a first primer group, a second primer group, a third primer group and a fourth primer group, wherein the primers in each primer group are provided with detectable labels, and the detectable labels of the primers in the different primer groups are different, so that the grouping is realized. And then combining the fragment size of the product obtained by amplification of each primer in each group of primer groups to determine which site is obtained, thereby realizing gene typing.
In some embodiments, the detectable label comprises a fluorescent label.
In some embodiments, the at least one selected from the group consisting of FAM fluorescent label, HEX fluorescent label, TAMRA fluorescent label, ROX fluorescent label, Cy5 fluorescent label. Discrimination of product fragments of the same or similar size amplified by different primers can be achieved by using FAM (6' -carboxyfluorescein) blue fluorescent label, HEX (hexachloro-6-methylfluorescein) green fluorescent label, TAMRA (4-methyl-6-carboxy-rhodamine) yellow fluorescent label, ROX (carboxy-X-rhodamine) red fluorescent label, Cy5(Indodicarbocyanine) purple fluorescent label, and the like.
According to a third aspect of the invention, there is provided a kit comprising a primer set capable of specifically amplifying the following loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11, and DAMEL. The kit selects a site with good stability, high amplification efficiency and strong sensitivity for detection. The method can be widely applied to individual identification and genetic identification of dogs, establishment of DNA archive databases and the like in forensic medicine and commerce.
In some embodiments of the present invention, the kit described above may further comprise the following technical features:
in some embodiments, the reagent comprises a primer set according to the second aspect of the invention.
In some embodiments, the kit further comprises a molecular internal standard that is a mixture containing a plurality of DNA fragments of known size. The size of the amplification product obtained can be indicated by molecular internal standards. The molecular internal standard may be labeled with a detectable label that is different from the detectable label carried on the primer.
In some embodiments, the kit further comprises at least one of: PCR buffer, DNA template, Taq DNA polymerase and dNTP mixture.
In some embodiments, the PCR buffer comprises 10mM DMSO, 50mM KCl, 10mM Tris-HCl (pH8.3), 20mM MgCl2And 0.1mg/ml BSA.
According to a fourth aspect of the invention there is provided the use of a kit for individual identification and/or relatedness of a dog, said kit being as described in relation to the third aspect of the invention.
According to a fifth aspect of the present invention, there is provided a method of genotyping comprising: amplifying STR loci of a sample to be detected to obtain allele amplification products, wherein the STR loci comprise AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055 and REN54P 11; analyzing the allele amplification product to obtain a genotyping result.
According to an embodiment of the present invention, the above genotyping method may further comprise the following technical features:
in some embodiments, the method further comprises amplifying the sex chromosome locus DAMEL of the test sample.
In some embodiments, the amplification is performed using a multiplex amplification reaction to obtain an allele amplification product. In the multiplex amplification reaction, primers are mixed in various ways in the same reaction to simultaneously amplify a plurality of genes, so that the convenient and rapid amplification of all STR loci can be realized.
In some embodiments, the test sample is from canine DNA.
In some embodiments, the test sample is derived from one or more of blood, blood stain, semen stain, bone, vaginal cells, hair, saliva stain, urine, sweat, amniotic fluid.
In some embodiments, prior to performing the analysis, further comprising separating the amplified alleles.
In some embodiments, the allele amplification products are separated by capillary gel electrophoresis.
In some embodiments, the nucleic acid sequence comprising the STR locus is specifically amplified using a primer set according to the second aspect of the invention.
According to a sixth aspect of the present invention, there is provided a genotyping system, the system comprising: an amplification unit for amplifying the following loci in a sample to be tested to obtain allele amplification products: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, and REN54P 11; and the allele determining unit is connected with the amplification unit and is used for analyzing the allele amplification products to obtain a genotyping result.
According to an embodiment of the present invention, the genotyping system described above may further comprise the following technical features:
in some embodiments, the locus further comprises DAMEL.
In some embodiments, the amplification unit performs the amplification using a multiplex amplification reaction to obtain an allele amplification product.
In some embodiments, the test sample is from canine DNA.
In some embodiments, the test sample is derived from one or more of blood, blood stain, semen stain, bone, vaginal cells, hair, saliva stain, urine, sweat, amniotic fluid.
In some embodiments, the system further comprises a separation unit, the separation unit being connected to the amplification unit and the allele determination unit, respectively, and the separation unit being configured to separate the allele amplification product.
In some embodiments, the separation unit separates the allele amplification products by capillary gel electrophoresis.
In some embodiments, the amplification unit specifically amplifies nucleic acid sequences containing the STR locus using a primer set according to the second aspect of the invention.
According to a seventh aspect of the present invention, there is provided a method for canine individual identification, comprising: obtaining DNA from a dog to be detected to obtain a sample containing the DNA of the dog; obtaining a genotyping result for said DNA based on a sample containing canine DNA, said genotyping result being obtained according to the method of the fifth aspect of the invention; and according to the genotyping result, carrying out individual identification on the dog. In some embodiments, the method further comprises further performing genetic identification on the dog based on the genotyping results.
According to an eighth aspect of the present invention, there is provided an apparatus for canine individual identification, comprising: the DNA acquisition system is used for acquiring DNA from a dog to be detected to obtain a sample containing the DNA of the dog; a genotyping system, connected to the DNA acquisition system, that obtains genotyping results for the DNA based on a sample containing dog DNA, the genotyping system being according to the sixth aspect of the invention; and the analysis system is connected with the genotyping system and is used for carrying out individual identification on the dog according to the genotyping result. In some embodiments, the assay system may further identify the dog based on genotyping results.
The beneficial effects obtained by the invention are as follows: the invention can simultaneously analyze 20 STR loci of dogs, the loci are all selected from the canine genotyping loci recommended by the International animal genetics society, and the invention is applicable to canine groups of various countries and regions in the world. One of the loci is a sex identification locus, and the sex of the dog can be accurately identified. Moreover, the primers provided by the invention can amplify a plurality of STR loci and sex determination loci AMEL in a single tube at one time, can complete PCR amplification within 90 minutes, and can obtain a genotyping map within 120 minutes, thereby greatly saving material and time costs. And the accuracy is high, and the method has important value.
Drawings
Fig. 1 is a schematic diagram of STR locus layout provided according to an embodiment of the present invention.
FIG. 2 is a graph of genotyping random dog 1 provided according to one embodiment of the invention.
FIG. 3 is a schematic diagram of a genotyping system provided in accordance with an embodiment of the invention.
FIG. 4 is a schematic diagram of a genotyping system provided in accordance with an embodiment of the invention.
Fig. 5 is a schematic diagram of an apparatus for canine individual identification and affinity identification provided in accordance with an embodiment of the present invention.
Figure 6 is a graph of genotyping random dog 2 provided in accordance with an embodiment of the invention.
FIG. 7 is a graph of genotyping random dog 1 provided in accordance with an embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Certain terms appearing in the present invention are explained and illustrated below to provide a better understanding of the invention. It should be noted that these explanations and illustrations are only intended to facilitate the understanding of the present invention, and should not be construed as limiting the present invention.
Herein, "DNA" refers to deoxyribonucleic acid in its various forms, such as genomic DNA, cDNA, isolated nucleic acid molecules, vector DNA, and chromosomal DNA, as is commonly understood in the art. "nucleic acid" refers to any form of DNA or RNA (ribonucleic acid).
Herein, a dog refers to a canine of the family canidae of the order carnivora of the class mammalia, and may be, for example, a domestic dog.
In this context, a locus or locus refers to the position that a gene occupies on a chromosome, and on a molecular level, a locus refers to a DNA sequence that has a genetic effect. A locus may be a gene, a portion of a gene, or a DNA sequence with some regulatory effect. In a chromosome, DNA encoding at the same locus is called an allele.
As used herein, the term "STR locus" or STR locus in "STR locus" or "STR loci" refers to a DNA genetic marker formed by the tandem repeat of 2-6 bases as core units. These expressions refer to the same concept.
The group of STR loci provided by the invention comprises the following STR loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11, and DAMEL. The group of STR loci provided by the invention refers to the combined application of the STR loci, and the combined application can be combined application in any form. For example, different primers are used to simultaneously amplify these STR loci to achieve canine genotyping. As another example, nucleic acid sequences comprising these STR loci can be used as control samples, and the like. Any form of information that simultaneously utilizes these STR loci is within the scope of the present invention.
In accordance with the STR loci described above, in at least some embodiments, the present invention provides a composite system that is capable of amplifying the following 20 loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11 and a sex-identifying locus DAMEL. The "complex system" can also be expressed as a "complex system" and means that the 20 loci can be amplified simultaneously in the same environment. The composite system provided by the invention is used for individual identification of dogs, the stability is good, the amplification efficiency is high, the sensitivity is high, and the 20 gene loci are specifically selected on the basis of the research results of dog groups in a plurality of countries and regions in the world, so that the composite system can be widely applied to individual identification, genetic identification, DNA archive database establishment and the like of dogs in the forensic and commercial fields.
In at least one embodiment, the composite system may further comprise: a first, second, third and fourth composite system, the first composite system for differentiating AHTk211, CXX279, INU030, REN169D01, REN247M 23; the second composite system is used for distinguishing INRA21, DAMEL, REN169O18, REN105L03, FH2848 and AHTk 253; the third composite system is used for distinguishing AHT121, AHT137, REN162C04, AHTh171 and FH 2054; the fourth composite system is used for distinguishing INU005, REN64E19, INU055 and REN54P 11; the internal standard is orange fluorescent label, and the fluorescent label is Atto 633. The first complex system, the second complex system, the third complex system and the fourth complex system are respectively provided with different fluorescent labels so as to realize the distinction of each complex system. For example, FAM (6' -carboxyfluorescein) blue fluorescent label, HEX (hexachloro-6-methylfluorescein) green fluorescent label, TAMRA (4-methyl-6-carboxy-rhodamine) yellow fluorescent label, ROX (carboxy-X-rhodamine) red fluorescent label, and Cy5 (indoxacyanine) violet fluorescent label can be used. In one embodiment of the present invention, the first complex system is labeled with FAM blue fluorescent label; the second complex system is marked with a HEX green fluorescent marker; the third composite system is marked with TAMRA yellow fluorescent marker; the fourth complex system is labeled with a ROX red fluorescent label. Amplification products at each site in each composite system are separated according to length difference, and two adjacent STR sites cannot overlap, so that all sites can be distinguished in one total composite system and used for genotyping.
The composite system of the invention can amplify all STR loci in a single tube at one time, and can complete PCR amplification within 90 minutes and obtain a genotyping map within 120 minutes by means of fluorescence labeling, thereby greatly saving material and time costs.
In at least one embodiment, the composite system further comprises: primer sequences SEQ ID NO 1-SEQ ID NO 40, and specific nucleotide sequences of the primer sequences and corresponding amplification sites are shown in Table 2. The length of each product is within 400bp by using the primers for amplification.
TABLE 2 primer information for amplification of each STR locus
And the primers are mixed, a composite amplification test is carried out to verify, after the conditions of non-specific amplification phenomenon, cross reaction and the like are determined to be absent, the concentration of each pair of primers is adjusted, and the peak value balance of each fragment in the group can reach more than 40%. To this end, according to the embodiment of the present invention, the primers are in a pair from small to large according to the numbers SEQ ID NO. 1 to SEQ ID NO. 40, and the final concentrations of the primers in the amplification reaction system are as follows according to the table 2: (2.15. + -. 0.1) μ M, (0.45. + -. 0.1) μ M, (0.84. + -. 0.1) μ M, (0.60. + -. 0.1) μ M, (0.75. + -. 0.1) μ M, (1.25. + -. 0.1) μ M, (0.30. + -. 0.1) μ M, (0.38. + -. 0.1) μ M, (0.92. + -. 0.1) μ M, (0.35. + -. 0.1) μ M, (0.40. + -. 0.1) μ M, (2.00. + -. 0.1) μ M, (0.65. + -. 0.1) μ M, (0.50. + -. 0.1) μ M, (1.15. + -. 0.1) μ M, (0.30. mu.M) μ M, (1.25. + -. 0.1) μ M, (2.00. + -. 0.1) μ M and (0.63. + -. 0.1) μ M. . Whereby co-amplification of all sites can be achieved.
According to an embodiment of the present invention, a kit may be prepared using the above-described complex system. What is needed isThe kit may comprise the above-described composite system. In addition, PCR buffer, DNA template, 1U to 2U Taq DNA polymerase and 0.2mM each dNTP mixture may be included in the kit. The buffer solution for PCR amplification reaction of the present invention comprises: 10mM DMSO, 50mM KCl, 10mM Tris-HCl (pH8.3, 25 ℃), 2.0mM MgCl20.1mg/ml BSA (bovine serum albumin). The dNTP mixture is an equimolar mixture of four deoxyribonucleotides (dATP, dTTP, dCTP, dGTP). In addition, the kit may further comprise: and (3) marking the molecular weight internal standard by using fluorescent marks different from the primers in the composite system. For example, it can be labeled orange with the fluorescent label Atto 633. The molecular weight internal standard is a DNA fragment mixture of a plurality of fluorescence labeled known fragment sizes, the fragment size of the composite amplification product can be calculated by taking the molecular weight internal standard as a reference, and the fragment size is compared with the allele ladder, so that the genotype of each site of the detected sample is analyzed and judged.
According to an embodiment of the present invention, there is also provided a genotyping method, comprising: amplifying loci in a sample to be detected to obtain amplified alleles, wherein the STR loci comprise AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11 and DAMEL; analyzing the amplified alleles to obtain a genotyping result.
According to an embodiment of the present invention, the sample to be tested is a sample containing canine DNA. In at least one embodiment, the DNA is extracted from canine blood (blood spots), tissue, saliva, hair, etc. by the Chelex-100 method. The amount of DNA may be in the range of 0.2 to 5ng, as required.
According to embodiments of the present invention, the amplification is performed using various reaction thermal cyclers (e.g., ABI 9700, ABI Veriti, Bio-RadmCycler, etc.). In at least one embodiment, amplification is performed using the following conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10 seconds, annealing at 58 ℃ for 1 minute, extension at 70 ℃ for 20 seconds, this step being repeated for 28 cycles; continuing the extension for 30 minutes at 60 ℃; storing at 4-12 deg.c. The detection method of the products after the composite amplification adopts a single-channel or multi-channel capillary electrophoresis genetic analyzer (3500, 3130) for detection.
According to the embodiment of the invention, the amplification product is mixed and denatured with formamide and an internal standard of molecular weight at a ratio of 30:1, and then separated by capillary electrophoresis. In the capillary electrophoresis technique, the fluorescence labeling products are separated due to different fragment sizes, and can emit recognizable optical signals under laser excitation, and can be successfully received by a genetic analyzer (ABI 3130, 3100, 3500 and the like) to accurately read the fragment sizes. The molecular weight internal standard is a DNA fragment mixture of a plurality of fluorescence labeled known fragment sizes, the fragment size of the composite amplification product can be calculated by taking the molecular weight internal standard as a reference, and the fragment size is compared with the allele ladder, so that the genotype of each site of the detected sample is analyzed and judged. The data after electrophoresis can be in
And (3) converting and analyzing the data on data analysis software such as IDx and GeneMarker to obtain accurate STR genotyping information and visual maps.
The present invention also provides a genotyping system, as shown in fig. 3, comprising: the kit comprises an amplification unit and an allele determining unit, wherein the amplification unit is used for amplifying the following loci in a sample to be detected to obtain allele amplification products, and the genes comprise: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11, and DAMEL; the allele determining unit is connected with the amplification unit and is used for analyzing the allele amplification products to obtain a genotyping result. According to an embodiment of the present invention, the genotyping system may further comprise a separation unit, as shown in FIG. 4, connected to the amplification unit, and an allele determination unit connected to the separation unit, the separation unit being adapted to separate the allele amplification products. The allele amplification products may be separated, for example, by capillary gel electrophoresis.
In addition, the present invention also provides an apparatus for identification of individual dogs, which comprises a DNA acquisition system, a genotyping system connected to the DNA acquisition system, and an analysis system connected to the genotyping system, as shown in fig. 5. The DNA acquisition system is used for acquiring DNA from a dog to be detected to obtain a sample containing the dog DNA; the genotyping system uses a sample containing canine DNA to obtain a genotyping result for the DNA; and the analysis system identifies the dog according to the genotyping result. The analysis system can also identify the relative of the dog according to the genotyping result.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1STR site determination
The 19 STR loci (AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11) and DAMEL, which are recommended by the international animal genetics society for genetics and identification loci, can simultaneously obtain 20 locus information.
When selecting STR loci for canine genotyping, a plurality of alternative loci are selected, and are combined and applied to as many loci as possible, so that on one hand, workload in genotyping is increased, and on the other hand, when the number of loci is more, results are interfered with each other in detection, and accuracy of genotyping is influenced. When the selected sites are insufficient or improper, the obtained typing results for two different samples are extremely similar, so that accurate distinguishing can not be realized. The inventors of the present invention finally determined the above-mentioned sites by comparatively analyzing the positions of the sites, the conditions of the repeating units, and the results of the overlay and cross-influence on the genome when different sites are used in combination. And the accurate typing result is obtained by typing at least 20 random dogs.
The 20 loci selected by the invention can be applied to the genotyping of the dogs, and the identification and the genetic identification of the individual dogs can be rapidly and comprehensively realized.
Example 2 genotyping results for random dogs 1
Example 2 provides results of genotyping using the STR loci provided in example 1, using random canine 1 samples as an example.
Randomly selecting a body hair sample of the dog 1, extracting DNA in the body hair sample, designing a primer to amplify the DNA so as to obtain a nucleic acid sequence containing 20 sites of the embodiment 1, and carrying out electrophoresis detection and analysis to obtain a genotyping map of the dog. Wherein the DNA is extracted by chelex-100 method (refer to Forensic DNA Protocol, Humana Press, 1998), the extracted DNA sample is amplified, the amplification reaction is carried out on a thermal cycler (such as ABI 9700, ABI Veriti, Bio-Radmycycler, etc.), the electrophoresis and detection are carried out on an ABI3500 genetic analyzer, and the data analysis adopts
IDx software. The reagents and materials used in the present invention such as formamide, internal standards, etc., are conventional materials commonly used by those skilled in the art.
1. Dilution of DNA concentration in Canine samples
DNA was extracted by the Chelex-100 method, and the obtained DNA was diluted to 1 ng/. mu.L at a concentration of 10.15 ng/. mu.L for the experiment.
2. Fluorescent labeling primer mixture
Primers were designed for each of the 20 sites in example 1, and the sequences of the primers are shown in Table 2. Then, different fluorophores are connected to each primer. Wherein the primers used for amplifying AHTk211, CXX279, INU030, REN169D01, REN247M23 are labeled with blue fluorescent FAM; primers for amplifying INRA21, DAMEL, REN169O18, REN105L03, FH2848, AHTk253, labeled with green fluorescent HEX; primers for amplification of AHT121, AHT137, REN162C04, AHTh171, FH2054, labeled with yellow fluorescent TAMRA; primers for amplification of INU005, REN64E19, INU055, REN54P11, labeled with red fluorescent ROX. The primers are used for amplification, the schematic size diagram of the amplified DNA fragment is shown in FIG. 1, and 100-350 in FIG. 1 is the size of the DNA fragment, and the unit is bp.
All primers were mixed at the concentrations given in table 3 to prepare a primer mixture. The final primer concentration shown in table 3 represents the sum of the forward primer and the reverse primer corresponding to each site, and the forward primer and the reverse primer have the same content and are 1: 1.
TABLE 3 primer information of each STR locus in the multiplex amplification system of the present invention
| Site name | Primer sequence numbering | Final concentration of primer in amplification System (μ M) |
| AHTk211 | |
2.15 |
| CXX279 | 3 and 4 of SEQ ID NO | 0.45 |
| INU030 | SEQ ID NO 5 and SEQ ID NO 6 | 0.84 |
| REN169D01 | SEQ ID NO 7 and SEQ ID NO 8 | 0.60 |
| REN247M23 | SEQ ID NO 9 and |
0.75 |
| INRA21 | 11 and 12 SEQ ID NO | 1.25 |
| DAMEL | 13 and 14 SEQ ID NO | 0.30 |
| REN169O18 | 15 and 16 SEQ ID NO | 0.38 |
| REN105L03 | 17 and 18 SEQ ID NO | 0.92 |
| FH2848 | |
0.35 |
| AHTk253 | 21 and 22 of SEQ ID NO | 0.40 |
| AHT121 | 23 and 24 SEQ ID NO | 2.00 |
| AHT137 | SEQ ID NO 25 and SEQ ID NO 26 | 0.65 |
| REN162C04 | SEQ ID NO 27 and SEQ ID NO 28 | 0.50 |
| AHTh171 | 29 and 30 of SEQ ID NO | 1.15 |
| FH2054 | 31 and 32 of SEQ ID NO | 0.30 |
| INU005 | 33 and 34 of SEQ ID NO | 1.25 |
| REN64E19 | 35 and 36 of SEQ ID NO | 2.00 |
| INU055 | 37 and 38 SEQ ID NO | 0.63 |
| REN54P11 | 39 and 40 of SEQ ID NO | 0.65 |
3. Polymerase Chain Reaction (PCR) amplification
1) Taking buffer solution, a primer mixture (the final concentration of the primer is shown in the table 3) and Taq enzyme, preparing a mixed solution according to the table 4, shaking, uniformly mixing, subpackaging into PCR reaction tubes with 25 mu L of each tube, and adding template DNA.
TABLE 4 multiplex amplification reaction System
| Components | Volume (μ L) |
| Primer mixture (5 × PrimerSets) | 5 |
| Buffer (2.5 × PCRMasterMix) | 10 |
| Hot start Taq enzyme | 0.4(2U) |
| DNA | 0.2ng-5ng |
| Nuclease-free water | Make up to 25 μ L |
2) A thermal cycler (G1000) was set up according to the reaction conditions shown in Table 5, and the PCR reaction tube was placed in the apparatus to perform PCR amplification.
TABLE 5 composite amplification thermal cycling conditions
3) After the amplification reaction is finished, the reaction tube is taken out, and electrophoresis and detection are carried out by using an ABI3500 genetic analyzer. For electrophoresis results
The IDx software was analyzed and the results of the typing of the randomized canine samples are detailed in figure 2.
The results of FIG. 2 show that PCR multiplex amplification and DNA typing detection have stable results and clear and complete maps, and the peak heights of the amplification products of 19 STR loci and 1 DAMEL sex gene are kept balanced, so that the STR loci and the typing method provided by the invention can be used for canine genotyping, and the results are accurate and reliable.
Example 3 genotyping results for random dog 2
Example 3 provides results of genotyping using the STR loci provided in example 1, exemplified by random canine 2 samples.
Randomly selecting a body hair sample of the dog 2, extracting DNA in the sample, designing a primer to amplify the DNA so as to obtain a nucleic acid sequence containing 20 sites of the embodiment 1, and carrying out electrophoresis detection and analysis to obtain a genotyping map of the dog. The specific implementation procedure is the same as in example 2.
The results of FIG. 6 show that PCR multiplex amplification and DNA typing detection have stable results and clear and complete maps, and the peak heights of the amplification products of 19 STR loci and 1 DAMEL sex gene are kept balanced, so that the STR loci and the typing method provided by the invention can be used for canine genotyping, and the results are accurate and reliable.
Example 4 genotyping of random Canine 1 Using different loci from the invention
Selecting 21 STR loci: AHTk211, INU055, INU030, REN105L03, REN169D01, AHTh260, REN247M23, INRA21, AHTh130, REN169O18, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, CXX279, and REN54P11, and 1 personalized authentication site DAMEL.
Among them, different STR loci from example 1 are AHTh260 and AHTh 130. And even the same sites, sites using different primer sequences from those used in example 2 were REN169D01, DAMEL, REN105L03, FH2848, INU005, INU055, REN54P 11. Specifically, the primer sequences used for these sites are as follows:
the primers used for amplifying REN169D01 site were:
AGTGGGTTGCAAGTGGAAC(SEQ ID NO:41)
AATAGCACATCTTCCCCACG(SEQ ID NO:42)
the primers used to amplify the DAMEL locus were:
GTGCCAGCTCAGCAGCCCGTGGT(SEQ ID NO:43)
TCGGAGGCAGAGGTGGCTGTGGC(SEQ ID NO:44)
the primers used for amplifying REN105L03 site were:
GGACAATGCTAAGGCTCACCTACC(SEQ ID NO:45)
CTTCTGGTGCCTGACAAGATGGAAT(SEQ ID NO:46)
the primers used for amplifying the FH2848 site were:
CAAAACCAACCCATTCACTC(SEQ ID NO:47)
GTCACAAGGACTTTTCTCCTG(SEQ ID NO:48)
the primers used to amplify the INU005 site were:
CTTTCTACCAGCAAGGTTAC(SEQ ID NO:49)
TTCCCATTTAATTGCCTCT(SEQ ID NO:50)
the primers used to amplify the INU055 site were:
CCAGGCGTCCCTATCCATCT(SEQ ID NO:51)
GCACCACTTTGGGCTCCTTC(SEQ ID NO:52)
the primers used for amplifying the REN54P11 site were:
GGGGGAATTAACAAAGCCTGAG(SEQ ID NO:53)
TGCAAATTCTGAGCCCCACTG(SEQ ID NO:54)
the primers used for amplifying the AHTh260 site were:
CGCTATACCCACACCAGGAC(SEQ ID NO:55)
CCACAGAGGAAGGGATGC(SEQ ID NO:56)
the primers used for amplifying the AHTH130 site were:
GTTTCTCTCCCTTCGGGTTC(SEQ ID NO:57)
GACGTGTGTTCACGCCAG(SEQ ID NO:58)
the primer sequences for the other STR sites were the same as in example 2.
Wherein, primers for amplifying AHTk211, INU005, INU030, REN105L03, REN169D01, AHTh260, REN247M23 are marked by blue fluorescence FAM; primers for amplifying INRA21, AHTH130, REN169O18, DAMEL, FH2848, AHTk253, labeled with green fluorescent HEX; primers for amplification of AHT121, AHT137, REN162C04, AHTh171, FH2054, labeled with yellow LTAM; primers for amplification of CXX279, REN64E19, INU055, REN54P11, labeled with red fluorescent ROX. Wherein the final concentration of each primer is as follows:
TABLE 6 primer concentrations
| Site name | Final concentration of primer in amplification System (μ M) |
| AHTk211 | 1.96 |
| INU005 | 0.98 |
| INU030 | 1.00 |
| REN105L03 | 0.65 |
| REN169D01 | 0.65 |
| AHTh260 | 0.20 |
| REN247M23 | 0.50 |
| INRA21 | 0.60 |
| AHTH130 | 0.50 |
| REN169O18 | 0.50 |
| DAMEL | 0.52 |
| FH2848 | 0.40 |
| AHTk253 | 1.88 |
| AHT121 | 0.50 |
| AHT137 | 0.50 |
| REN162C04 | 0.30 |
| AHTh171 | 0.30 |
| FH2054 | 0.50 |
| CXX279 | 0.50 |
| REN64E19 | 0.25 |
| INU055 | 0.50 |
| REN54P11 | 0.50 |
Referring to the method steps of example 2, the above sites were multiplexed amplified. After the amplification reaction is finished, the amplification product is subjected to electrophoresis and detection by using an ABI3500 genetic analyzer. For electrophoresis results
The IDx software was analyzed and the typing results are shown in FIG. 7.
The results showed that the typing effect was not good when the SNP sites of this example were used for gene analysis. The main points are as follows: REN169D01, DAMEL, REN105L03, FH2848, INU005, INU055, REN54P11, AHTh260 and AHTH130 loci, and the phenomena of low efficiency of multiplex amplification, poor typing effect, existence of a hetero-peak, cross-overlapping of allele positions of different loci, and the like appear.
On the basis of the above results, the inventors redesigned the primers for amplifying the above sites, and found that the typing effect of AHTh260 and AHTH130 is still poor after many experiments.
Experimental results show that the SNP locus provided by the embodiment 1 of the invention is used for genotyping of the dog, and the results are accurate and reliable. The SNP loci are applied, primers are designed, and the method can be used for dog individual identification, genetic identification, DNA archive database establishment and the like, and plays an important role in the forensic medicine and commercial fields.
In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; may be mechanically coupled, may be electrically coupled or may be in communication with each other; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Huada medical science Co Ltd
<120> locus for canine genotyping and use thereof
<130>PIDC3185391
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<213> Artificial sequence
<220>
<223> primer sequences
<400>58
gacgtgtgtt cacgccag 18
Claims (9)
1. A set of loci characterized by: including the following autosomal STR loci:
AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, and REN54P 11;
optionally, the locus further comprises the sex chromosome locus DAMEL.
2. A primer set adapted to specifically amplify a nucleic acid sequence comprising the following loci:
AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, and REN54P 11;
optionally, the locus further comprises DAMEL.
3. The primer group of claim 2, wherein the primer group has a nucleotide sequence shown in SEQ ID NO. 1-12, SEQ ID NO. 15-40 or a derivative thereof;
optionally, the primer group also comprises nucleotide sequences shown as SEQ ID NO 13-SEQ ID NO 14 or derivatives thereof;
optionally, the derivative is a nucleic acid molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to a nucleotide sequence and has the same function with the nucleotide sequence;
optionally, the final concentrations in the amplification reaction system of the primer sequences for specific amplification of AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055 and REN54P11 loci are: (2.15. + -. 0.1) μ M, (0.45. + -. 0.1) μ M, (0.84. + -. 0.1) μ M, (0.60. + -. 0.1) μ M, (0.75. + -. 0.1) μ M, (1.25. + -. 0.1) μ M, (0.30. + -. 0.1) μ M, (0.38. + -. 0.1) μ M, (0.92. + -. 0.1) μ M, (0.35. + -. 0.1) μ M, (0.40. + -. 0.1) μ M, (2.00. + -. 0.1) μ M, (0.65. + -. 0.1) μ M, (0.50. + -. 0.1) μ M, (1.15. + -. 0.1) μ M, (0.30. mu.M) μ M, (1.25. + -. 0.1) μ M, (2.00. + -. 0.1) μ M and (0.63. + -. 0.1) μ M;
optionally, the final concentration of the primer sequence for DAMEL locus-specific amplification in the amplification reaction system is (0.65 ± 0.1) μ M;
optionally, at least one primer of each pair of primers of the primer set has a detectable label attached thereto,
optionally, at least one primer of each pair of primers of the primer set has a detectable label attached to its 5' end;
optionally, the primer sets comprise a first primer set, a second primer set, a third primer set and a fourth primer set, wherein each primer set primer is connected with a distinguishable detectable label,
wherein the first primer set comprises primers for specific amplification of AHTk211, CXX279, INU030, REN169D01, REN247M23 locus,
the second primer set comprises primers for specific amplification of INRA21, DAMEL, REN169O18, REN105L03, FH2848, AHTk253 loci,
the third primer group comprises primers for specific amplification of AHT121, AHT137, REN162C04, AHTh171 and FH2054 loci,
the fourth primer set comprises primers for specific amplification of INU005, REN64E19, INU055, REN54P11 locus;
optionally, the detectable label comprises a fluorescent label;
optionally, the fluorescent label is at least one selected from the group consisting of FAM fluorescent label, HEX fluorescent label, TAMRA fluorescent label, ROX fluorescent label, Cy5 fluorescent label, Atto633 fluorescent label.
4. A kit comprising a primer set capable of specifically amplifying the following loci: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, REN54P11, and DAMEL;
optionally, the primer set is the primer set of claim 2 or 3;
optionally, the kit further comprises a molecular internal standard which is a mixture containing a plurality of DNA fragments with known sizes;
optionally, the kit further comprises at least one of: PCR buffer solution, DNA template, TaqDNA polymerase and dNTP mixture;
optionally, the PCR buffer comprises 10mM DMSO, 50mM KCl, 10mM Tris-HCl (pH8.3), 20mM MgCl2And 0.1mg/ml BSA.
5. Use of the kit of claim 4 for individual identification and/or genetic identification of a canine.
6. A method of genotyping, comprising:
amplifying STR loci of a sample to be detected to obtain allele amplification products, wherein the STR loci comprise AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055 and REN54P 11;
analyzing the allele amplification product to obtain a genotyping result;
optionally, further comprising amplifying the sex chromosome locus DAMEL of the test sample;
optionally, performing said amplification using a multiplex amplification reaction to obtain an allele amplification product;
optionally, the sample to be tested is from canine DNA;
optionally, the sample to be tested is from one or more of blood, blood spot, semen spot, bone, vaginal cell, hair, saliva spot, urine, sweat, amniotic fluid;
optionally, prior to performing the analysis, further comprising isolating the allele amplification product;
optionally, separating the allele amplification products by capillary gel electrophoresis;
optionally, specifically amplifying a nucleic acid sequence containing the STR locus using a primer set, the primer set being the primer set of claim 2 or 3.
7. A genotyping system, the system comprising:
an amplification unit, configured to amplify the following loci in a sample to be tested to obtain allele amplification products, where the STR loci include: AHTk211, CXX279, INU030, REN169D01, REN247M23, INRA21, REN169O18, REN105L03, FH2848, AHTk253, AHT121, AHT137, REN162C04, AHTh171, FH2054, INU005, REN64E19, INU055, and REN54P 11;
an allele determination unit connected to the amplification unit, the allele determination unit being configured to analyze the allele amplification product to obtain a genotyping result;
optionally, the locus further comprises DAMEL;
optionally, the amplification unit performs the amplification using a multiplex amplification reaction to obtain an allele amplification product;
optionally, the sample to be tested is from canine DNA;
optionally, the sample to be tested is from one or more of blood, blood spot, semen spot, bone, vaginal cell, hair, saliva spot, urine, sweat, amniotic fluid;
optionally, the system further comprises a separation unit, which is connected to the amplification unit and the allele determination unit, respectively, and which is used to separate the allele amplification product;
optionally, the separation unit separates the allele amplification products by capillary gel electrophoresis;
optionally, the amplification unit specifically amplifies nucleic acid sequences containing the STR locus using a primer set, the primer set being the primer set of claim 2 or 3.
8. A method for individual identification of a dog, comprising:
obtaining DNA from a dog to be detected to obtain a sample containing the DNA of the dog;
obtaining genotyping results for said DNA based on a sample containing canine DNA, said genotyping results obtained according to the method of claim 6;
according to the genotyping result, carrying out individual identification on the dog;
optionally, the canine is further subjected to genetic identification based on the genotyping results.
9. An apparatus for canine individual identification, comprising:
the DNA acquisition system is used for acquiring DNA from a dog to be detected to obtain a sample containing the DNA of the dog;
a genotyping system, said genotyping system being connected to said DNA acquisition system, said genotyping system obtaining genotyping results for said DNA based on a sample comprising canine DNA, said genotyping system being the genotyping system of claim 7;
an analysis system connected with the genotyping system, wherein the analysis system identifies the dog according to the genotyping result;
optionally, the analysis system further performs genetic identification of the dog based on the genotyping results.
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| CN201811612320.7A CN111378759A (en) | 2018-12-27 | 2018-12-27 | Genetic loci for canine genotyping and uses thereof |
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| CN102321752A (en) * | 2010-08-19 | 2012-01-18 | 无锡中德美联生物技术有限公司 | Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof |
| US20130071847A1 (en) * | 2011-09-19 | 2013-03-21 | BioPet Vet Lab | Methods and materials for determining the source of waste |
| WO2015063429A1 (en) * | 2013-10-31 | 2015-05-07 | Christian Doutremepuich | Method and kit for identifying a dog by analysing a biological sample |
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