STR (short tandem repeat) marker combination for dogs and application of STR marker combination

Abstract

The invention relates to an STR marker combination for dogs and application of the STR marker combination, and belongs to the technical field of molecular biology and forensic identification. The STR marker combination comprises a combination of any four STR gene loci in the following 20 gene loci: PEZ1, PEZ2, FH2010, PEZ5, PEZ12, FH2309, PEZ20, PEZ15, FH2079, PEZ8, FH3377, PEZ21, PEZ3, PEZ6, FH2004, FH2054, VWFX, FH2611, FH2132 and FH2328, and a combination of any four STR gene loci in the following 20 gene loci. The STR marker combination further comprises a gene locus Amel for identifying sex. Through reasonable gene locus and fragment combination and fluorescent combination, the problem of simultaneous PCR amplification of 21 fluorescent primer single tubes is solved. And secondly, the technical verification index of the 10 [mu] L reaction system completely meets the forensic medicine application requirements, and the cost is greatly reduced. And finally, through cooperation of multiple departments, an STR database of 1000 kinds of dogs in the Chengdu region based on the system is constructed for the first time, and the application basis for effectiveness calculation is solved.

Description

Technical Field

[0001] This invention relates to a combination of STR markers for dogs and its application, belonging to the fields of molecular biology and forensic identification technology. Background Technology

[0002] Dogs were the first animals domesticated by humans and are currently the most numerous and widely distributed companion animals in the world. According to statistics from the China Pet Industry White Paper and other related data, as of 2021, the number of dogs in urban households in my country had exceeded 50 million. Therefore, it is imperative to upgrade the standardized management and social governance and prevention system for dogs.

[0003] Information management methods for dogs include identification tags, electronic chips, and biometric identification. Currently, identification tags and electronic chips are the main methods used both domestically and internationally. However, with the continuous development and maturation of technology, and drawing on the experience and achievements of the Nanchang Police Dog Base's dog DNA database under the Ministry of Public Security and the national special population DNA database construction in recent years, biometric identification has become the trend and mainstream of future information management of dogs in the civilian sector. Canine biometric identification includes five technologies: DNA, nose print, facial recognition, iris recognition, and retinal recognition. Among these, DNA identification has the highest accuracy, unaffected by morphology, disease, or life cycle. Through the construction of DNA databases, law enforcement officers can detect, compare, and locate unknown biological traces at incident scenes, making it the most promising and advantageous method. Individual canine DNA identification mainly uses short tandem repeats (STRs) as genetic markers. Currently, there are few commercially available products, and those that exist are expensive and costly, hindering large-scale database construction and application. Furthermore, most STR loci are fewer than 20 (limiting the cumulative individual identification capacity). Representative examples include Thermo Fisher Scientific's "Thermo Scientific Canine Genotypes Panel 2.1" (USA), Wuxi Zhongde Meilian Biotechnology Co., Ltd.'s "Canine 17A STR Fluorescence Detection Kit" (China), and Yuewei Gene's "Canine STR Typing Kit." However, these kits suffer from the following problems: 1) Generally high prices: A kit for 100 canine samples from these three manufacturers costs around 20,000 RMB (meaning the testing cost for one canine biological sample is around 200 RMB), making large-scale library construction and application difficult; 2) Insufficient locus accumulation and individual identification capabilities: For example, the "Canine 17A STR Fluorescence Detection Kit" only has 17 loci, resulting in limited individual identification capabilities; 3) Loci do not meet the requirements of GA-T-1703-2019 "Laboratory Testing Standards for Canine DNA": For example, the "Thermo Scientific Canine Genotypes Panel 2.1" kit... 2.1” and “Canine STR Typing Kit” only have 6 and 1 loci respectively that meet the requirements of GA-T-1703-2019; 4) There is no effective and available canine STR genetic database: In some regions, there are currently no reported databases available for calculating the probability of matching (likelihood rate) and parentage index (cumulative exclusion probability) of individual dogs.

[0004] Given the above issues, it is particularly important to develop an efficient and cost-effective canine biological individual identification and detection technology based on STR genetic markers. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a combination of STR markers for dogs and its applications. This invention solves the problem of simultaneous PCR amplification using 21 fluorescent primers in a single tube through a rational combination of loci and fragments, and fluorescent combinations. Secondly, the technical verification indicators of the 10μL reaction system fully meet the requirements for forensic applications, significantly reducing costs. Finally, through multi-departmental collaboration, a database of STR markers from 1000 dogs of various species in the Chengdu area based on this system has been constructed for the first time, solving the application foundation for power calculation.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A combination of STR markers for dogs, wherein the combination of STR markers comprises any four STR loci from the following 20 loci: PEZ1, PEZ2, FH2010, PEZ5, PEZ12, FH2309, PEZ20, PEZ15, FH2079, PEZ8, FH3377, PEZ21, PEZ3, PEZ6, FH2004, FH2054, VWFX, FH2611, FH2132, and FH2328.

[0007] In a preferred embodiment of the STR marker combination described in this invention, the STR marker combination further includes a sex-identifying locus, wherein the sex-identifying locus is the Amel locus.

[0008] As a preferred embodiment of the STR marker combination described in this invention, the primer pair sequences for amplifying the STR loci are as follows: the primer pair sequence for amplifying PEZ1 is SEQ ID NO:1-2, the primer pair sequence for amplifying PEZ2 is SEQ ID NO:3-4, the primer pair sequence for amplifying FH2010 is SEQ ID NO:5-6, the primer pair sequence for amplifying PEZ5 is SEQ ID NO:7-8, the primer pair sequence for amplifying PEZ12 is SEQ ID NO:9-10, the primer pair sequence for amplifying FH2309 is SEQ ID NO:11-12, the primer pair sequence for amplifying PEZ20 is SEQ ID NO:13-14, the primer pair sequence for amplifying PEZ15 is SEQ ID NO:15-16, the primer pair sequence for amplifying FH2079 is SEQ ID NO:17-18, the primer pair sequence for amplifying PEZ8 is SEQ ID NO:19-20, and the primer pair sequence for amplifying FH3377 is SEQ ID NO:1-2. Primer pairs for amplifying PEZ21 (SEQ ID NO: 21-22), PEZ3 (SEQ ID NO: 27-28), PEZ6 (SEQ ID NO: 29-30), FH2004 (SEQ ID NO: 31-32), FH2054 (SEQ ID NO: 33-34), VWFX (SEQ ID NO: 35-36), FH2611 (SEQ ID NO: 37-38), FH2132 (SEQ ID NO: 39-40), and FH2328 (SEQ ID NO: 41-42) are also used.

[0009] More preferably, the primer pair sequence for amplifying the sex-identifying locus Amel is SEQ ID NO:23-24.

[0010] On the other hand, the present invention also provides a canine STR genotyping kit, which includes primer pairs of STR loci with STR marker combinations as described in the present invention, and primer pair sequences of the sex-identifying locus Amel.

[0011] Furthermore, the present invention also provides the application of the STR marker combination or the canine STR genotyping kit in canine individual identification or kinship determination.

[0012] Furthermore, the present invention also provides a canine STR genotyping method, wherein the canine STR genotyping method uses the aforementioned STR marker combination or the aforementioned canine STR genotyping kit, and includes the following steps: (1) Extract genomic DNA from canine samples; (2) Using the genomic DNA as a template, perform multiplex PCR amplification using the primer set; (3) Capillary electrophoresis was performed on the PCR amplification products; (4) Determine the alleles of each STR locus based on the electrophoresis results and complete the canine STR genotyping.

[0013] Preferably, the total volume of the multiplex PCR amplification reaction system in step (2) is 10 μL, and the amounts of each component are as follows: 4 μL of 2.5×Multiplex Mix, 0.25~5 ng of DNA template, 2 μL of primer mixture, and deionized water to make up to 10 μL.

[0014] Preferably, the procedure for multiplex PCR amplification in step (2) is as follows: 50℃ UNG enzyme digestion for 5 min, 95℃ pre-denaturation for 2 min, 95℃ denaturation for 10 s, 58℃ annealing for 60 s, 70℃ extension for 45 s, 35 cycles; 60℃ extension for 30 min; After the PCR reaction is completed, the sample is taken out, briefly centrifuged and then stored at 4℃.

[0015] Preferably, the canine sample in step (1) includes blood, saliva, hair, and tissue; the purity of the genomic DNA A260 / A280 is between 1.7 and 1.9, and the concentration is ≥0.25 ng / μL.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High site polymorphism and strong recognition ability: The STR marker combination provided by this invention for dogs is composed of 21 pairs of canine STR loci and sex loci primers in 4 colors. The cumulative individual recognition power (TDP) is >0.9999999999 and the cumulative paternity exclusion rate (CPE) is >0.9999. The system efficiency fully meets the practical needs and is far higher than the commonly used sites in the existing technology. It can meet the needs of high-precision individual identification and paternity testing.

[0017] (2) High detection efficiency, simple operation and low cost: The present invention adopts multiplex PCR technology, which can amplify 21 STR sites at the same time. Combined with capillary electrophoresis detection, the entire typing process can be completed within 4 hours. The kit is complete and the operation steps are simple. No complicated experimental equipment is required, which is convenient for use by grassroots laboratories and routine testing institutions. The present invention adopts multiplex PCR technology, and its reaction system is only 10μL, which significantly reduces the cost compared with the 20 or 25μL systems currently available on the market.

[0018] (3) High primer specificity and good amplification effect: The primer set of the present invention is designed for the core repeat sequence and flanking conserved sequence of each STR site, and optimizes the Tm value, GC content and secondary structure of the primers, effectively avoiding the formation of primer dimers and non-specific amplification; in multiplex PCR amplification, the amplification efficiency of each primer pair is balanced, ensuring the accuracy and stability of the detection results.

[0019] (4) The present invention also provides a canine STR genotyping method. The verification results show that the system has good species specificity, trace DNA level sensitivity, good degradation tolerance and tolerance to common interfering substances, and is suitable for the detection of routine canine biological samples (such as oral swabs, hair follicles, muscle tissue, etc.) and difficult biological samples (such as various exfoliated cells).

[0020] (5) Wide range of applications: This invention can be widely applied to areas such as individual dog identification, paternity testing, pet registration, and forensic evidence identification (e.g., tracing the source of evidence at the scene of dog-related cases). It has significant practical and economic value. The research results have been successfully applied to the investigation of dog-related cases, solving the problem of investigating sudden public health and safety incidents involving dogs, and providing a reference for the construction of a science and technology-driven policing and "smart law enforcement" innovation system. Moreover, the STR loci and primer set of this invention have been verified on more than 20 common dog breeds such as Golden Retrievers, Labrador Retrievers, German Shepherds, and Chinese Rural Dogs, and can achieve effective amplification and accurate typing, making it suitable for the detection of the vast majority of dog breeds. Attached Figure Description

[0021] Figure 1 A fluorescent labeling combination for the locus of a canine 4-color 21-fold STR multiplex amplification system.

[0022] Figure 2 This image shows the specificity verification results (capillary electrophoresis) of the canine 4-color 21-fold STR multiplex amplification system. From top to bottom, the images show the amplification results using genomic DNA from cats, fish, chickens, sheep, ducks, pigs, mice, rabbits, horses, cattle, humans, E. coli, and dogs as templates. The x-axis represents the PCR product fragment size (bp); the y-axis represents the fluorescence intensity (RFU).

[0023] Figure 3 Sensitivity test results (capillary electrophoresis) of the canine 4-color 21-fold STR multiplex amplification system. The results, from top to bottom, show the amplification results using canine DNA standards of 0.0625, 0.125, 0.25, 0.5, 1, 5, 10, and 20 ng as templates. The x-axis represents the PCR product fragment size (in bp), and the y-axis represents the fluorescence intensity (in RFU).

[0024] Figure 4The results of capillary electrophoresis were used to test the tolerance of the canine 4-color 21-fold STR multiplex amplification system. The x-axis represents the size of the PCR product fragment (in bp), and the y-axis represents the fluorescence intensity (in RFU).

[0025] Figure 5 The results of capillary electrophoresis were used to test the suitability of the canine 4-color 21-fold STR multiplex amplification system. The x-axis represents the size of the PCR product fragment (unit: bp), and the y-axis represents the fluorescence intensity (unit: RFU).

[0026] Figure 6 The capillary electrophoresis results of 4-color 21-fold STR multiplex amplification of canine pedigree 1 are shown. The x-axis represents the size of the PCR product fragment (in bp), and the y-axis represents the fluorescence intensity (in RFU). Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0028] To explain the technical solution of the present invention more clearly and in more detail, some embodiments are provided below for further illustration.

[0029] Example 1 1. Materials and Methods 1.1 STR locus selection According to literature reports, 20 canine autosomal STR loci with high individual identification ability and 1 sex identification locus were selected for primer design and combined into 4 groups of fluorescent channels (Table 1 and 2010). Figure 1 The average individual identifiability (DP) of the 20 STR loci is >0.9, the average heterozygosity (H) is >0.6, and there is no linkage between the loci. The cumulative individual identifiability (TDP) is >0.9999999999 (theoretically enough to distinguish all dogs on Earth), and the cumulative exclusion probability (CPE) is >0.9999.

[0030] Table 1. Locus and primer information for the canine 4-color 21-fold STR multiplex amplification system

[0031] 1.2 Sample Biological samples from cats, fish, chickens, sheep, ducks, pigs, mice, rabbits, horses, and cattle were collected from muscle tissue of each species. E. coli biological samples were collected from DH5α competent cells. Human genomic DNA standard 9947A was purchased from Basepoint Cognitive Technology (Beijing) Co., Ltd. Canine genomic DNA standard was purchased from Zyagen. Canine hair follicles, muscle tissue, and exfoliated cells were samples retained from routine cases at the Sichuan GeneGrid Forensic Identification Center. Canine oral swabs were collected from dogs managed by the Sichuan Provincial or Chengdu Municipal Public Security Bureau.

[0032] 1.3 Main Reagents and Equipment Blood / cell / tissue genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.), high-efficiency oral swab genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.), QIAamp® DNAInvestigator extraction kit (Kagem GmbH, Germany), Multiplex Mix (with UNG) (Shanghai Sangon Biotech), canine STR locus fluorescent amplification primers (Shanghai Sangon Biotech), Allelicladder 21plex (Shanghai Sangon Biotech), GeneScan 500LIZ molecular weight internal standard (Thermo Fisher Scientific, USA), spectral calibrators (Shanghai Sangon Biotech), formamide (Thermo Fisher Scientific, USA), POP7 gel (Thermo Fisher Scientific, USA), etc.

[0033] Veriti 96-well gene amplification instrument (Thermo Fisher Scientific, USA), Qubit 2.0 fluorescence spectrometer (Thermo Fisher Scientific, USA), Applied Biosystems™ 3730xl gene analyzer (Thermo Fisher Scientific, USA), GeneMapper™ ID-X V1.6 software (Thermo Fisher Scientific, USA), etc.

[0034] 1.4 DNA Extraction and Quantification Genomic DNA was extracted from biological samples of various species, DH5α competent cells, and canine oral swabs using a blood / cell / tissue genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.) and a high-efficiency oral swab genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The specific steps are as follows: 1) Take 0.1g tissue sample / 10 6 For each cell / oral swab sample, add 200 μL of buffer GA and 20 μL of proteinase K solution respectively, mix well, and incubate at 56°C for 1-2 hours for digestion. 2) Add 200 μl of buffer GB, mix thoroughly by inverting, and incubate at 70°C for 10 min; 3) Add 200 μl of anhydrous ethanol and shake thoroughly for 15 seconds; 4) Add the solution from the previous step to the adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube; 5) Add 500 μl of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and put the adsorption column CB3 into the collection tube; 6) Add 600 μl of washing buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube; 7) Repeat the previous step; 8) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for a few minutes to dry. 9) Transfer the adsorption column CB3 into a centrifuge tube, add 100 μl of elution buffer TE, incubate at room temperature for 5 min, and centrifuge at 12,000 rpm (~13,400×g) for 2 min.

[0035] Genomic DNA was extracted from canine hair follicles, muscle tissue, and exfoliated cells using the QIAamp® DNA Investigator extraction kit. The specific steps are as follows: 1) Place one hair follicle / 0.1 g muscle tissue / one exfoliated cell swab in a 2 ml centrifuge tube, add 20 μl proteinase K and 600 μl Buffer ATL, and vortex for 10 seconds; 2) After a brief centrifugation, place the 2ml tube in a constant temperature mixer and shake at 56°C and 900 rpm for at least 1 hour; 3) Add 600 μl of Buffer AL and vortex for 15 seconds; 4) Place the 2 ml tube in a thermostatic mixer and shake at 900 rpm for 10 minutes at 70°C; 5) After a brief centrifugation, add 300 μl of ethanol (96–100%) and vortex for 15 seconds; 6) After a brief centrifugation, carefully transfer the lysis buffer to a QIAamp MinElute column (placed in a 2 ml collection tube), and centrifuge at 6000×g (8000 rpm) for 1 minute. Carefully discard the eluent in the collection tube, and then return the column to the collection tube. 7) Add 500 μl of Buffer AW1 and centrifuge at 6000×g (8000 rpm) for 1 minute. Place the column in a clean 2 ml collection tube; 8) Add 700 μl of Buffer AW2 and centrifuge at 6000×g (8000 rpm) for 1 minute. Place the column in a clean 2 ml collection tube; 9) Add 700 μl of ethanol (96–100%), centrifuge at 6000 × g (8000 rpm) for 1 minute. Place the column in a clean 2 ml collection tube; 10) Centrifuge at full speed (20000×g; 14000 rpm) for 3 minutes to allow the membrane to dry completely; 11) Place the QIAamp MinElute column in a clean 1.5ml centrifuge tube, carefully open the column cap, and incubate at 56°C for 3 minutes; 12) Add 30µl of Buffer ATE to the center of the membrane, incubate at room temperature (15–25°C) for 5 minutes, and centrifuge at full speed (20000×g; 14000 rpm) for 1 minute.

[0036] After extraction, the DNA was analyzed using a Qubit 2.0 fluorescence spectrometer to determine the dsDNA concentration. The specific procedures are as follows: 1) Qubit dsDNA dye, buffer, and standards should be allowed to equilibrate at room temperature for 30 minutes before use; 2) Prepare the working solution according to the dye-buffer solution ratio of 1:200; 3) Take two tubes of 190 μL working solution and add 10 μL of standard 1 and standard 2 respectively. Take 1 μL of each DNA sample and add it to 199 μL of working solution. Vortex to mix. 4) Insert the incubated reaction tube into the sample slot of the Qubit 2.0 fluorometer, close the top cover, select dsDNA mode, first measure the standard to calculate the standard curve, and then measure the sample concentration.

[0037] 1.5 PCR Amplification PCR amplification was performed using the Veriti 96-well gene amplification instrument. The specific operating steps are as follows: 1) On an ice box, prepare each reaction solution according to the following amplification system: 4 μL of 2.5×Multiplex Mix (with UNG), 0.25~5 ng of DNA template, 2 μL of primer set, and add water to a total volume of 10 μL; 2) Place the prepared reaction solution on a PCR machine and amplify according to the following amplification program: 50℃ UNG enzyme digestion for 5 min; 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 58℃ annealing for 60 s, 70℃ extension for 45 s, 35 cycles; 60℃ extension for 30 min. 3) After the PCR reaction is complete, remove the sample, centrifuge briefly, and then store it at 4°C.

[0038] 1.6 Capillary electrophoresis and data analysis PCR products were subjected to capillary electrophoresis using an Applied Biosystems™ 3730xl genetic analyzer. The specific procedure was as follows: 1 μL of PCR product, 0.2 μL of LIZ 500 internal standard, and 9.8 μL of deionized formamide were mixed and denatured at 95°C for 3 minutes, followed by an immediate 3-minute ice bath. After centrifugation for 3 minutes, the 96-well plate was placed in the adapter and then into the 3730XL genetic analyzer. The injection voltage was 2.0 kV, and the injection time was 20 seconds. Electrophoresis was then initiated.

[0039] Fragment size and genotype analysis was performed using GeneMapper® IDX V1.6 software. The specific steps are as follows: After electrophoresis, analysis was performed in GeneMapper software. Panels and Bins were imported, an Analysis Method was established, the electrophoresis data was imported, and the appropriate Panel, Analysis Method, and Size Standard parameters were selected. The allele ladder for that Panel was selected, and the data was analyzed.

[0040] 2. Results 2.1 Species specificity Using genomic DNA from cats, fish, chickens, sheep, ducks, pigs, mice, rabbits, horses, cattle, humans, *E. coli*, and dogs as templates, the above-mentioned 4-color 21-fold STR complex amplification system was performed, followed by capillary electrophoresis analysis. The results showed that this complex PCR amplification system was specific; compared to dogs, no obvious target product was observed in non-dog species. Figure 2 As shown. Figure 2 Specificity verification of the canine 4-color 21-fold STR multiplex amplification system (capillary electrophoresis results). From top to bottom, the amplification results are shown using genomic DNA from cats, fish, chickens, sheep, ducks, pigs, mice, rabbits, horses, cattle, humans, E. coli, and dogs as templates. Horizontal axis: PCR product fragment size (unit: bp); Vertical axis: fluorescence intensity (unit: RFU).

[0041] 2.2 Sensitivity Canine DNA standards were used as templates at concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 5, 10, and 20 ng for 4-color 21-strand STR multiplex amplification. The results showed... Figure 3 As shown, Figure 3 Sensitivity testing (capillary electrophoresis results) of the canine 4-color 21-fold STR multiplex amplification system is shown. From top to bottom, the amplification results are obtained using canine DNA standards as templates at concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 5, 10, and 20 ng. The x-axis represents PCR product fragment size (bp); the y-axis represents fluorescence intensity (RFU). The results indicate that all input concentrations can detect all loci. At input concentrations of 10 and 20 ng, the fluorescence signal is too strong, leading to oversaturation. Even at a concentration as low as 0.0625 ng, although the signal is weaker, all loci can be accurately detected. Therefore, the optimal template DNA input range for this system is set to 0.25–5 ng.

[0042] 2.3 Tolerance The canine DNA standards were processed according to the methods in Table 2, including enzyme digestion and treatment with either indigo or heme. These standards were then simultaneously subjected to 4-color 21-fold STR multiplex amplification with normal samples to test the system's tolerance to degradation of samples and common interfering agents. The results are as follows: Figure 4 .

[0043] Table 2. Sample processing methods for tolerance testing

[0044] Capillary electrophoresis results showed that... Figure 4 , Figure 4 Tolerance test of the canine 4-color 21-fold STR multiplex amplification system (capillary electrophoresis results). Horizontal axis: PCR product fragment size (unit: bp); Vertical axis: fluorescence intensity (unit: RFU). Results show that compared to the normal control: large fragment regions in the enzyme-digested samples showed a significant signal decrease, but correct genotyping was still possible; the signals of samples treated with indigo and heme showed virtually no change.

[0045] 2.4 Applicability Common canine biological samples of various types were collected, including oral swabs, hair follicles, muscle tissue, and exfoliated cells. DNA was extracted and subjected to 4-color 21-strand STR multiplex amplification. Capillary electrophoresis results showed... Figure 5 The results show that all loci can be detected. Figure 5 Suitability test of the canine 4-color 21-fold STR multiplex amplification system (capillary electrophoresis results). Horizontal axis: PCR product fragment size (bp); Vertical axis: Fluorescence intensity (RFU).

[0046] 2.5 Database Construction and Family Verification Using the aforementioned 4-color 21-fold canine STR multiplex amplification system, DNA from oral swabs of over 1,000 dogs of various types collected from the Sichuan Provincial Public Security Department's police dog base and dog shelters under the jurisdiction of the Chengdu Municipal Public Security Bureau was analyzed, constructing the first canine STR genetic database in the Chengdu area. The test results of 35 dogs from 7 families showed that the system can accurately determine their parentage (e.g., ...). Figure 6 and family 1) shown in Table 3.

[0047] Figure 6 Capillary electrophoresis results of 4-color 21-fold STR multiplex amplification in canine pedigree 1. x-axis: PCR product fragment size (bp); y-axis: fluorescence intensity (RFU).

[0048] Table 3. Size of 4-color 21-fold STR multiplex amplification fragments from canine pedigree 1 (unit: bp)

[0049] Example 2: Specific application case of the STR marker pairing for dogs provided by the present invention Case 1: In 2024, a public security department in Sichuan Province investigated a case of a retired police dog being stolen. Samples including the stolen dog's remaining hair, used collar, and food bowl, along with muscle tissue samples from a suspected stolen dog, were sent to the author's identification center for testing. Using the four-color 21-fold canine STR multiplexing system constructed in this invention, canine STR typing results were detected in the DNA extracted from hair follicles in the hair, the collar, and the food bowl. These results matched the STR typing results of the suspected stolen dog's muscle tissue DNA, providing crucial evidence for the police investigation.

[0050] Case 2: In 2025, a public outcry over a dog-injury incident occurred in Chengdu. The police submitted the knife used to injure the dog and other suspected contact samples. Using the four-color 21-fold canine STR multiplex amplification system constructed in this invention, the police successfully performed STR detection and comparison on the canine DNA extracted from the knife and contact samples, providing crucial evidence for the police to quickly handle dog-related public safety incidents.

Claims

1. A STR marker combination for dogs, characterized in that The STR marker combination comprises a combination of any 4 STR loci in the following 20 loci: PEZ1, PEZ2, FH2010, PEZ5, PEZ12, FH2309, PEZ20, PEZ15, FH2079, PEZ8, FH3377, PEZ21, PEZ3, PEZ6, FH2004, FH2054, VWFX, FH2611, FH2132, FH2328.

2. The STR marker combination of claim 1, wherein, The STR marker combination further comprises a gender identification locus, which is the Amel locus.

3. The STR marker combination of claim 1, wherein, The primer pair sequences for amplifying the STR loci are as follows: the primer pair sequences for amplifying PEZ1 are SEQ ID NO: 1-2, the primer pair sequences for amplifying PEZ2 are SEQ ID NO: 3-4, the primer pair sequences for amplifying FH2010 are SEQ ID NO: 5-6, the primer pair sequences for amplifying PEZ5 are SEQ ID NO: 7-8, the primer pair sequences for amplifying PEZ12 are SEQ ID NO: 9-10, the primer pair sequences for amplifying FH2309 are SEQ ID NO: 11-12, the primer pair sequences for amplifying PEZ20 are SEQ ID NO: 13-14, the primer pair sequences for amplifying PEZ15 are SEQ ID NO: 15-16, the primer pair sequences for amplifying FH2079 are SEQ ID NO: 17-18, the primer pair sequences for amplifying PEZ8 are SEQ ID NO: 19-20, the primer pair sequences for amplifying FH3377 are SEQ ID NO: 21-22, the primer pair sequences for amplifying PEZ21 are SEQ ID NO: 25-26, the primer pair sequences for amplifying PEZ3 are SEQ ID NO: 27-28, the primer pair sequences for amplifying PEZ6 are SEQ ID NO: 29-30, the primer pair sequences for amplifying FH2004 are SEQ ID NO: 31-32, the primer pair sequences for amplifying FH2054 are SEQ ID NO: 33-34, the primer pair sequences for amplifying VWFX are SEQ ID NO: 35-36, the primer pair sequences for amplifying FH2611 are SEQ ID NO: 37-38, the primer pair sequences for amplifying FH2132 are SEQ ID NO: 39-40, the primer pair sequences for amplifying FH2328 are SEQ ID NO: 41-42.

4. The STR marker combination of claim 2, wherein, The primer pair sequences for amplifying the gender identification locus Amel are SEQ ID NO: 23-24.

5. A canine STR genotyping kit characterized in that, The canine STR genotyping kit comprises the primer pairs of the STR loci of the STR marker combination of any one of claims 1-3, and further comprises the primer pairs of the locus Amel of claim 4.

6. Use of the STR marker combination of any one of claims 1-4 or the canine STR genotyping kit of claim 5 in canine individual identification or kinship identification.

7. A method of canine STR genotyping, characterized in that, The canine STR genotyping method is performed by using the STR marker combination according to any one of claims 1-4 or the canine STR genotyping kit according to claim 5, and the canine STR genotyping method comprises the following steps: (1) extracting genomic DNA of a canine sample; (2) using the genomic DNA as a template, performing multiplex PCR amplification by using the primer group; (3) performing capillary electrophoresis detection on the PCR amplification product; (4) determining alleles of each STR site according to the electrophoresis result, and completing canine STR genotyping.

8. The canine STR genotyping method according to claim 7, characterized in that, The total volume of the reaction system of the multiplex PCR amplification in step (2) is 10 μL, and the dosages of the components are as follows: 2.5 × Multiplex Mix 4 μL, DNA template 0.25-5 ng, primer group mixture 2 μL, and deionized water to 10 μL.

9. The method for canine STR genotyping according to claim 7, characterized in that, The program of the multiplex PCR amplification in step (2) is as follows: 50 ℃ UNG enzyme digestion for 5 min, 95 ℃ pre-denaturation for 2 min, 95 ℃ denaturation for 10 s, 58 ℃ annealing for 60 s, 70 ℃ extension for 45 s, 35 cycles; 60 ℃ extension for 30 min; after the PCR reaction is completed, the sample is taken out, centrifuged briefly, and then placed at 4 ℃ for storage.

10. The canine STR genotyping method according to claim 7, characterized in that, The canine sample in step (1) includes blood, saliva, hair, tissue, and exfoliated cells; the purity of the genomic DNA is A260 / A280 between 1.7-1.9, and the concentration is ≥0.25 ng / μL.

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