A method for paternity testing of cynomolgus monkeys and rhesus monkeys using microsatellite markers

Abstract

The present invention relates to the technical field of molecular biology, and particularly relates to a method for paternity testing of cynomolgus monkeys and rhesus monkeys using microsatellite markers. The present invention provides a specific primer set based on microsatellite markers and its uses. The inventors of the present invention found that only 12 pairs of specific primer sets based on microsatellite markers can be used to simultaneously perform paternity testing on cynomolgus monkeys and rhesus monkeys, expanding the scope of application, reducing the complexity and cost of simultaneously performing paternity testing on cynomolgus monkeys and rhesus monkeys, and improving the detection efficiency at the same time. The present invention has created an integrated paternity testing technical process applicable to cynomolgus monkeys and rhesus monkeys, eliminating the need to prepare different schemes for different monkey species, and improving work efficiency and convenience.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a method for paternity testing of cynomolgus monkeys and rhesus monkeys using microsatellite markers. Background Art

[0002] Microsatellites, also known as Simple Sequence Repeats (SSRs), are simple repetitive sequences evenly distributed in the eukaryotic genome, consisting of tandem repeat fragments of 2 - 6 nucleotides. Microsatellites follow Mendel's genetic laws and have characteristics such as a large quantity, co-dominant inheritance, rich polymorphism, and easy detection. They are widely used in aspects such as population genetic diversity analysis of animals and plants, paternity testing, and construction of genetic linkage maps.

[0003] The cynomolgus monkey, also known as the long-tailed macaque or crab-eating macaque, is a species native to tropical South Asia and Southeast Asia. Cynomolgus monkeys can obtain social behaviors homologous to human behaviors through artificial feeding and training. It is precisely because cynomolgus monkeys exhibit characteristics similar to humans that they are used as important biological models in biological research, such as drug safety evaluation. Clinical research often requires mutual verification among multiple experimental animals, and the moderate body size, anatomical structure, and physiological characteristics similar to humans of cynomolgus monkeys play an extremely important role in scientific research and clinical research.

[0004] The rhesus monkey (Macaca mulatta), as a non-human primate experimental animal, is commonly used in research fields such as physiology, ethology, AIDS, reproductive science, diabetes, and pathology, and is the most widely used and in-demand experimental animal in the world.

[0005] In this context, the demand for cynomolgus monkeys and rhesus monkeys in scientific research and drug research has increased. To obtain experimental animals with clear genetic backgrounds, paternity testing is indispensable, especially important in the research of gene-edited monkeys. Therefore, research on paternity testing and population polymorphism has also developed accordingly. However, the existing technology lacks an effective method for paternity testing of both cynomolgus monkeys and rhesus monkeys, and is not suitable for research or management scenarios involving two types of experimental monkeys. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems in the related art to some extent. For this purpose, an object of the present invention is to provide a set of specific primers based on microsatellite markers and their uses. The inventors of the present invention have found that only 12 pairs of specific primers based on microsatellite markers can be used to simultaneously perform paternity testing on cynomolgus monkeys and rhesus monkeys, expanding the scope of application, reducing the complexity and cost of simultaneously performing paternity testing on cynomolgus monkeys and rhesus monkeys, and improving the detection efficiency at the same time. The present invention has created an integrated paternity testing technical process applicable to cynomolgus monkeys and rhesus monkeys, eliminating the need to prepare different schemes for different monkey species, and improving work efficiency and convenience.

[0007] For this purpose, in the first aspect of the present invention, a set of specific primers based on microsatellite markers is provided. According to the embodiments of the present invention, the primer set includes 12 pairs of primers, which are respectively:

[0008] The first pair of primers, as shown in SEQ ID NO:1 and SEQ ID NO:2;

[0009] The second pair of primers, as shown in SEQ ID NO:3 and SEQ ID NO:4;

[0010] The third pair of primers, as shown in SEQ ID NO:5 and SEQ ID NO:6;

[0011] The fourth pair of primers, as shown in SEQ ID NO:7 and SEQ ID NO:8;

[0012] The fifth pair of primers, as shown in SEQ ID NO:9 and SEQ ID NO:10;

[0013] The sixth pair of primers, as shown in SEQ ID NO:11 and SEQ ID NO:12;

[0014] The seventh pair of primers, as shown in SEQ ID NO:13 and SEQ ID NO:14;

[0015] The eighth pair of primers, as shown in SEQ ID NO:15 and SEQ ID NO:16;

[0016] The ninth pair of primers, as shown in SEQ ID NO:17 and SEQ ID NO:18;

[0017] The tenth pair of primers, as shown in SEQ ID NO:19 and SEQ ID NO:20;

[0018] The eleventh pair of primers, as shown in SEQ ID NO:21 and SEQ ID NO:22;

[0019] The twelfth pair of primers, as shown in SEQ ID NO:23 and SEQ ID NO:24.

[0020] The following Table 1 shows the specific sequences of SEQ ID NO:1~24:

[0021] Table 1

[0022]

[0023] The inventors screened and obtained 12 pairs of primers for specific microsatellite markers, which are microsatellite markers that can perform well in both cynomolgus monkeys and rhesus monkeys, improving the targeting and effectiveness of the markers; created an integrated paternity testing technical process applicable to cynomolgus monkeys and rhesus monkeys, providing new ideas and methods for the research and management of multiple monkey species; and achieved a good balance of cost-effectiveness by reducing the number of markers and optimizing the process while ensuring the accuracy of paternity testing.

[0024] In the second aspect of the present invention, there is provided the use of the specific primer set based on microsatellite markers described in the first aspect in the preparation of a paternity testing kit for cynomolgus monkeys and / or rhesus monkeys.

[0025] The specific primer set provided by the present invention can be used in the preparation of a paternity testing kit for cynomolgus monkeys and rhesus monkeys.

[0026] In the third aspect of the present invention, there is provided a paternity testing kit for cynomolgus monkeys and / or rhesus monkeys. According to the embodiments of the present invention, the paternity testing kit includes the specific primer set based on microsatellite markers described in the first aspect.

[0027] In the fourth aspect of the present invention, there is provided a paternity testing method for cynomolgus monkeys or rhesus monkeys based on microsatellite markers. According to the embodiments of the present invention, the paternity testing method includes:

[0028] 1) Extract the genomic DNA of the sample to be tested;

[0029] 2) Perform a PCR amplification reaction on the genomic DNA using the specific primer set based on microsatellite markers described in the first aspect to obtain an amplification product;

[0030] 3) Determine the genotypes of 12 microsatellite markers of the sample to be tested based on the amplification product to determine the paternity of the cynomolgus monkey or rhesus monkey to be tested.

[0031] The prior art uses 16 microsatellite markers for paternity testing of cynomolgus monkeys, which cannot meet the needs of paternity testing of rhesus monkeys and is insufficient for the applicability in research or management scenarios involving two species of experimental monkeys. At the same time, this technology uses 16 microsatellite markers, which may increase the complexity and cost of operations, and may also consume more time and resources during the detection and analysis process. In contrast, the present invention can simultaneously complete paternity testing of cynomolgus monkeys and rhesus monkeys using only 12 special markers, reducing the number of markers, lowering the operation complexity and cost, and improving the detection efficiency. Moreover, the present invention can be used on both cynomolgus monkeys and rhesus monkeys, expanding the application scope. Meanwhile, the present invention creates an integrated paternity testing technical process applicable to cynomolgus monkeys and rhesus monkeys, eliminating the need to prepare different schemes for different monkey species and improving work efficiency and convenience.

[0032] In some embodiments of the present invention, the sample to be tested is blood, tissue, or hair from a suspected parent or suspected offspring of a cynomolgus monkey or a rhesus monkey.

[0033] In some embodiments of the present invention, the paternity testing method further includes:

[0034] Comparing the genotypes of 12 microsatellite markers of the offspring and the suspected parent respectively. If the genotypes of all 12 microsatellite markers can find the same genotypes in the suspected parent, it is determined that the suspected parent is the parent of the offspring.

[0035] In some embodiments of the present invention, the paternity testing method further includes:

[0036] Comparing the genotypes of 12 microsatellite markers of the parent and the suspected offspring respectively. If the genotypes of all 12 microsatellite markers can find the same genotypes in the suspected offspring, it is determined that the suspected offspring is the offspring of the parent.

[0037] In some embodiments of the present invention, the paternity testing method further includes:

[0038] Comparing the genotypes of 12 microsatellite markers of multiple suspected parents and multiple suspected offspring respectively. If the genotypes of all 12 microsatellite markers of suspected parent A can find the same genotypes in a certain suspected offspring B, it is determined that there is a parent-child relationship between the suspected parent A and the suspected offspring B.

[0039] The present invention has the following beneficial effects:

[0040] (1) In terms of scientific research: It provides a more efficient and accurate paternity testing method for the genetic research of cynomolgus monkeys and rhesus monkeys, which helps to deeply understand the genetic characteristics and evolutionary relationships of the two monkey species;

[0041] (2) In terms of breeding management: During the breeding process of monkeys, the ability to accurately identify parent-offspring relationships helps optimize breeding plans, improve the quality of the population and management level;

[0042] (3) In terms of conservation work: It provides strong technical support for the conservation of cynomolgus monkeys and rhesus monkeys, helps formulate more scientific conservation strategies, and protects these two precious primate resources.

[0043] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0045] Figure 1 Shows the genotypes of a certain family (suspected father, suspected mother, and offspring) detected by the method in Embodiment 2 of the present invention at a certain microsatellite marker locus. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention.

[0047] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0048] The endpoints and any values disclosed in this document are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0049] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0050] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0051] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent described event or condition can but does not necessarily occur, and this description includes the case where the event or condition occurs, and the case where the event or condition does not occur.

[0052] According to a specific embodiment of the present invention, the present invention provides a set of specific primers based on microsatellite markers, including 12 pairs of primers, namely:

[0053] The first pair of primers, as shown in SEQ ID NO:1 and SEQ ID NO:2;

[0054] The second pair of primers, as shown in SEQ ID NO:3 and SEQ ID NO:4;

[0055] The third pair of primers, as shown in SEQ ID NO:5 and SEQ ID NO:6;

[0056] The fourth pair of primers, as shown in SEQ ID NO:7 and SEQ ID NO:8;

[0057] The fifth pair of primers, as shown in SEQ ID NO:9 and SEQ ID NO:10;

[0058] The sixth pair of primers, as shown in SEQ ID NO:11 and SEQ ID NO:12;

[0059] The seventh pair of primers, as shown in SEQ ID NO:13 and SEQ ID NO:14;

[0060] The eighth pair of primers, as shown in SEQ ID NO:15 and SEQ ID NO:16;

[0061] The ninth pair of primers, as shown in SEQ ID NO:17 and SEQ ID NO:18;

[0062] The tenth pair of primers, as shown in SEQ ID NO:19 and SEQ ID NO:20;

[0063] The eleventh pair of primers, as shown in SEQ ID NO:21 and SEQ ID NO:22;

[0064] The twelfth pair of primers, as shown in SEQ ID NO:23 and SEQ ID NO:24.

[0065] The inventors screened and obtained 12 pairs of primers for specific microsatellite markers, which are microsatellite markers that perform well in both cynomolgus monkeys and rhesus monkeys, improving the targeting and effectiveness of the markers; established an integrated paternity testing technical process applicable to cynomolgus monkeys and rhesus monkeys, providing new ideas and methods for the research and management of multiple monkey species; and achieved a good balance of cost-effectiveness by reducing the number of markers and optimizing the process while ensuring the accuracy of paternity testing.

[0066] According to a specific embodiment of the present invention, the specific primer set based on microsatellite markers provided by the present invention can be used to prepare a paternity testing kit for cynomolgus monkeys and rhesus monkeys.

[0067] According to a specific embodiment of the present invention, the kit contains at least 12 pairs of primers for specific microsatellite markers. This kit can be used for paternity testing of cynomolgus monkeys and also for paternity testing of rhesus monkeys, breaking the limitation of the prior art which is only applicable to cynomolgus monkeys, meeting the need for paternity testing of different monkey species, so as to obtain experimental animals with clear genetic backgrounds. This kit can achieve accurate paternity testing by using only 12 microsatellite markers, reducing the operation complexity, cost and improving the efficiency.

[0068] According to a specific embodiment of the present invention, the present invention provides a paternity testing kit for cynomolgus monkeys and / or rhesus monkeys, including the specific primer set based on microsatellite markers described above.

[0069] It should be noted that in addition to the specific primer set, the kit provided by the present invention may also contain other reagents for PCR reactions, such as enzymes, buffers, ddH2O or some metal ion solutions, or alternatively, these reagents may also be included in the kit in the form of a mixture.

[0070] Compared with the prior art, using the kit of the present invention for paternity testing is not only applicable to cynomolgus monkeys but also to rhesus monkeys, providing convenience for research and management involving different monkey species; this kit only needs to detect 12 microsatellite markers, reducing the operation complexity and cost compared with more markers in the prior art, and also improving the detection efficiency; highly efficient and convenient: one set of technical process is applicable to two monkey species, without the need to prepare different schemes for different monkey species, saving time and resources and improving work efficiency.

[0071] According to a specific embodiment of the present invention, the present invention provides a paternity testing method for cynomolgus monkeys or rhesus monkeys based on microsatellite markers, including:

[0072] 1) Extract the genomic DNA of the sample to be tested;

[0073] 2) Using the specific primer set based on microsatellite markers described in the first aspect, perform a PCR amplification reaction on the genomic DNA to obtain an amplification product;

[0074] 3) Based on the amplification product, determine the genotypes of 12 microsatellite markers of the sample to be detected, so as to judge the parent - offspring relationship of the cynomolgus macaque or rhesus macaque to be detected.

[0075] The present invention can simultaneously complete the parent - child identification of cynomolgus macaques and rhesus macaques only by using 12 special markers, reducing the number of markers, lowering the operation complexity and cost, while improving the detection efficiency. Moreover, the present invention can be used on both cynomolgus macaques and rhesus macaques, expanding the application scope. At the same time, the present invention has created an integrated parent - child identification technical process applicable to cynomolgus macaques and rhesus macaques, without the need to prepare different schemes for different monkey species, improving the work efficiency and convenience.

[0076] According to a specific embodiment of the present invention, the sample to be detected is blood, tissue or hair from a suspected parent or suspected offspring of a cynomolgus macaque or rhesus macaque. It should be noted that the sample to be detected is not limited to blood, tissue or hair with hair follicles from a suspected parent or suspected offspring of a cynomolgus macaque or rhesus macaque. In fact, all parts containing genomic DNA from a suspected parent or suspected offspring of a cynomolgus macaque or rhesus macaque can be used as the sample to be detected and are covered by the protection scope of the present invention.

[0077] According to a specific embodiment of the present invention, the parent - child identification method further includes:

[0078] Compare the genotypes of 12 microsatellite markers of the offspring and the suspected parent respectively. If the genotypes of all 12 microsatellite markers can find the same genotypes in the suspected parent, it is determined that the suspected parent is the parent of the offspring.

[0079] According to a specific embodiment of the present invention, the parent - child identification method further includes:

[0080] Compare the genotypes of 12 microsatellite markers of the parent and the suspected offspring respectively. If the genotypes of all 12 microsatellite markers can find the same genotypes in the suspected offspring, it is determined that the suspected offspring is the offspring of the parent.

[0081] According to a specific embodiment of the present invention, the parent - child identification method further includes:

[0082] Compare the genotypes of 12 microsatellite markers of multiple suspected parents and multiple suspected offspring respectively. If the genotypes of all 12 microsatellite markers of the suspected parent A can find the same genotypes in a certain suspected offspring B, it is determined that there is a parent - offspring relationship between the suspected parent A and the suspected offspring B.

[0083] The present invention provides a paternity testing method that can be used in both cynomolgus monkeys and rhesus monkeys, breaking through the limitation of the prior art which is only applicable to cynomolgus monkeys and meeting the need for paternity testing of different monkey species. Through careful screening and optimization, accurate paternity testing can be achieved using only 12 microsatellite markers, reducing the operation complexity, cost and improving the efficiency. Moreover, the inventor has developed an integrated technical process applicable to cynomolgus monkeys and rhesus monkeys, so that different technical solutions do not need to be prepared for different monkey species during paternity testing, improving the convenience and versatility of the work.

[0084] The present invention is applicable to paternity testing of two non-human primate experimental animals, cynomolgus monkeys and rhesus monkeys. It can determine the parent-child relationship of cynomolgus monkeys and rhesus monkeys of different ages and genders. The applicable scenario of the present invention is during the breeding process of monkeys. Accurately identifying the parent-child relationship helps to optimize the breeding plan, improve the population quality and management level. For example, inbreeding can be avoided and excellent breeding monkeys can be selected for breeding. In addition, it can also provide technical support for the protection of cynomolgus monkeys and rhesus monkeys. Through paternity testing, the genetic relationship of the population can be determined, and more scientific protection strategies can be formulated to protect these two precious primate resources.

[0085] The solutions of the present disclosure will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those not specifying the specific technology or conditions in the embodiments, the technology or conditions described in the literature in the art or according to the product instructions are followed. For the reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0086] Example 1 Screening process of microsatellite markers

[0087] The MISA software was used to screen microsatellite markers from the cynomolgus monkey reference genome (Macaca_fascicularis_6.0) from the Ensembl database. The parameters were set as: definition(unit_size,min_repeats): 4-5, that is, microsatellite markers with 4-base repeats and the number of repeats above 5. After the screening was completed, the software Primer3 was used to design SSR primers. Finally, 300 microsatellite markers and primers with 4-base repeats were obtained in this study.

[0088] Subsequently, primers were designed for these 300 microsatellite markers. Five cynomolgus monkeys without blood relationship within three generations were selected, and blood samples were collected to extract DNA. The above 300 microsatellite markers were amplified and capillary electrophoresis was performed. Twelve microsatellite markers containing 4 or more different alleles, with a polymorphic information content greater than 0.6 and an expected heterozygosity exceeding 0.7 were screened out.

[0089] In order to identify the polymorphisms of the above 12 microsatellite markers (see Table 2 in Example 2) in a large population, in this study, 74, 35, and 29 cynomolgus monkeys were randomly selected from our unit, a certain biological company in Suzhou, and a certain biological company in Guangzhou, respectively, with a total of 138 cynomolgus monkeys for large-population verification. The 12 markers were amplified by designing specific primers. The Cervus software was used to calculate the genetic diversity of the 12 markers, the probability of excluding an incorrect father for each marker, and the cumulative probability of exclusion for all marker combinations. After calculation, the cumulative probability of exclusion for the 12-marker combination reached over 99.99%, indicating that the 12 selected loci had high polymorphisms and could meet the needs of paternity testing for cynomolgus monkeys.

[0090] Considering that cynomolgus monkeys and rhesus monkeys belong to the same genus Macaca and have a relatively high genomic similarity, in this study, blood samples from a total of 52 rhesus monkeys were collected from our unit and a certain biological company in Guangzhou. After DNA extraction, the genotypes of these 12 markers in the rhesus monkeys were detected. The Cervus software was used to calculate the genetic diversity of the 12 markers, the probability of excluding an incorrect father for each marker, and the cumulative probability of exclusion for all marker combinations. After calculation, the cumulative probability of exclusion for the 12-marker combination reached over 99.99%, indicating that the 12 selected loci had high polymorphisms and could also meet the needs of paternity testing for rhesus monkeys.

[0091] Example 2 Paternity testing process for cynomolgus monkeys and rhesus monkeys

[0092] The following method was used for paternity testing of cynomolgus monkeys and rhesus monkeys:

[0093] (1) DNA extraction:

[0094] Genomic DNA was extracted from the blood samples of cynomolgus monkeys and rhesus monkeys using the Tiangen genomic DNA extraction kit.

[0095] (2) PCR amplification: Specific primers targeting 12 microsatellite markers (primer sequences shown in Table 1) were designed for PCR amplification. The detailed information of the 12 microsatellite markers in cynomolgus monkeys is shown in Table 2 below.

[0096] Table 2 Detailed information of 12 microsatellite markers in cynomolgus monkeys

[0097]

[0098] The amplification system conditions for the PCR reaction are shown in Table 3 below:

[0099] Table 3 PCR amplification system

[0100]

[0101] The above amplification system was amplified according to the procedure in Table 4 below:

[0102] Table 4 PCR reaction procedure

[0103]

[0104] (3)Detection and analysis

[0105] The PCR amplification products were detected and analyzed by capillary electrophoresis, and the Genemapper software was used to determine the genotypes of each individual at 12 microsatellite marker loci. Only the genotypes of a certain family (alleged father, alleged mother, and offspring) at a certain microsatellite marker locus were shown exemplarily. Figure 1 Only the genotypes of a certain family (alleged father, alleged mother, and offspring) at a certain microsatellite marker locus were shown exemplarily.

[0106] (4)Paternity determination

[0107] The genotypes of the 12 microsatellite markers of the alleged parents were compared with those of the offspring monkeys. If the same genotype could be found in the alleged parents at all 12 loci, they were identified as the parents of the offspring monkey; otherwise, they were not. This algorithm is applicable to cynomolgus monkeys and rhesus monkeys and can accurately determine paternity.

[0108] To verify the accuracy of the 12 microsatellite marker combinations, in this study, 16 cynomolgus monkey families with known paternity and 11 rhesus monkey families with known paternity were randomly selected from our unit, a certain biological company in Suzhou, and a certain biological company in Guangzhou for kinship verification. The genotypes of the 12 microsatellite marker loci of 16 offspring cynomolgus monkeys and 11 offspring rhesus monkeys could all be traced back to the father and mother and were consistent with the known pedigree records. As shown in Table 5, in cynomolgus monkeys, the 12 microsatellite loci of the offspring monkeys matched successfully with the alleged father and alleged mother, and they were identified as the parents of the offspring monkey, while only 8 and 9 loci of the other fathers and other mothers matched successfully, respectively, and they were identified as not being the parents of the offspring monkey. Similarly, in rhesus monkeys (Table 6), the 12 microsatellite loci of the offspring monkeys matched successfully with the alleged father and alleged mother, and they were identified as the parents of the offspring monkey. While only 4 and 6 loci of the other fathers and other mothers matched successfully, respectively, and they were identified as not being the parents of the offspring monkey.

[0109] Table 5 Matching results of offspring cynomolgus monkeys with alleged parents and other parents

[0110]

[0111] Table 6 Matching results of offspring rhesus monkeys with alleged parents and other parents

[0112]

[0113] The above results indicate that the primer set provided by the present invention can be used to detect 12 microsatellite markers of cynomolgus monkeys or rhesus monkeys, enabling accurate paternity testing.

[0114] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", "some implementation manners" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0115] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A specific primer set for identifying cynomolgus monkeys and rhesus monkeys based on microsatellite markers, characterized in that: The primer set includes 12 pairs of primers, which are: A first pair of primers, as shown in SEQ ID NO: 1 and SEQ ID NO: 2; A second pair of primers, as shown in SEQ ID NO:3 and SEQ ID NO:4; a third pair of primers, as shown in SEQ ID NO:5 and SEQ ID NO:6; a fourth pair of primers, as shown in SEQ ID NO:7 and SEQ ID NO:8; a fifth pair of primers, as shown in SEQ ID NO:9 and SEQ ID NO:10; a sixth pair of primers, as shown in SEQ ID NO:11 and SEQ ID NO:12; a seventh pair of primers, as shown in SEQ ID NO:13 and SEQ ID NO:14; an eighth pair of primers, as shown in SEQ ID NO: 15 and SEQ ID NO: 16; a ninth pair of primers, as shown in SEQ ID NO: 17 and SEQ ID NO: 18; a tenth pair of primers, as shown in SEQ ID NO: 19 and SEQ ID NO: 20; an eleventh pair of primers, as shown in SEQ ID NO:21 and SEQ ID NO:22; The twelfth pair of primers is shown as SEQ ID NO:23 and SEQ ID NO:

24.

2. Use of the specific primer set according to claim 1 in preparing a paternity test kit for cynomolgus monkeys and / or rhesus monkeys.

3. A paternity test kit for cynomolgus monkeys and / or rhesus monkeys, characterized in that: The specific primer set according to claim 1 is included.

4. A method for parentage identification of cynomolgus monkeys or rhesus monkeys based on microsatellite markers, characterized in that: The paternity testing method comprises: 1) Extract genomic DNA from the sample to be tested; 2) performing a PCR amplification reaction on the genomic DNA using the specific primer set according to claim 1 to obtain an amplified product; 3) Determine the genotype of 12 microsatellite markers of the sample to be tested based on the amplified product, so as to determine the parent-offspring relationship of the cynomolgus monkey or rhesus monkey to be tested.

5. The paternity testing method according to claim 4, characterized in that: The sample to be tested is blood, tissue or hair from a suspected parent or suspected offspring of cynomolgus monkeys or rhesus monkeys.

6. The paternity testing method according to claim 4, characterized in that: The paternity testing method further comprises: The genotypes of the 12 microsatellite markers of the offspring and the suspected parent are compared respectively. If the genotypes of the 12 microsatellite markers can all be found to be the same genotypes in the suspected parent, the suspected parent is determined to be the parent of the offspring.

7. The paternity testing method according to claim 4, characterized in that: The paternity testing method further comprises: The genotypes of the 12 microsatellite markers of the parents and the suspected offspring are compared respectively. If the genotypes of the 12 microsatellite markers can all be found to be the same genotype in the suspected offspring, the suspected offspring is determined to be the offspring of the parents.

8. The paternity testing method according to claim 4, characterized in that: The paternity testing method further comprises: The genotypes of the 12 microsatellite markers of multiple suspected parents and multiple suspected offspring are compared respectively. If the genotypes of the 12 microsatellite markers of the suspected parent A can all find the same genotype in a suspected offspring B, it is determined that the suspected parent A and the suspected offspring B have a parent-child relationship.

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