STR typing technology for canine individual identification and parentage testing

Abstract

The present application relates to the field of gene detection technology, and specifically to a STR typing technology for dog individual identification and paternity testing. The present application can achieve complete STR typing detection of trace canine DNA as low as 0.1ng, and the target amplification peak bias of the 21 target STR loci in the system is less than 0.2nt; at the same time, it does not cross-reference common poultry and livestock sources such as humans, cattle, sheep, pigs, and chickens, and has high specificity; in addition, the present application is easy to compare results between different laboratories, ensuring the stability, repeatability, and authority of the typing results.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a STR typing method, a kit and an application thereof for dog individual identification and paternity determination. Background Art

[0002] There are more than 50 million pet dogs in my country. As working animals or companion animals, dogs are inseparable from human life. Because of this, dogs are inevitably closely related to the occurrence of certain crimes. For example, shed dog hair may be transferred between different individuals involved in the case, which can be used to link suspects and crime scenes, suspects and victims, or victims and crime scenes. Due to the increase in the number of dog owners, dog-injury cases are also common. The accurate individual identification and paternity identification of dogs is conducive to restoring the original appearance of the case, reconstructing the scene of the case, determining the person responsible, and providing key evidence for the trial of such cases. In addition, dogs, as companion animals, bring us a lot of companionship and joy in different families. At the same time, they also occupy an important position in the pet market. Some of these precious dog breeds need DNA identification during the transaction process to clarify their pedigree.

[0003] Short tandem repeat (STR), also known as microsatellite DNA, is a type of repetitive sequence on the genome. According to the number of bases in the repeating unit, it can be divided into dinucleotide sequences and tri-, tetra-, penta- and hexanucleotide sequences. STR is the most widely used type of genetic marker in forensic DNA analysis and plays an important role in individual identification and parentage testing. Among them, STR detection based on polymerase chain reaction (PCR) and capillary electrophoresis (CE) platforms has high sensitivity and accurate results, and is the current gold standard for STR analysis. STR analysis based on PCR-CE technology is also the main technical method for canine DNA detection. In order to manage dogs and investigate dog-related cases more accurately and efficiently, countries such as Australia, the United States, Hungary, and the United Kingdom have carried out the construction of dog-related STR databases.

[0004] At present, a series of dog individual identification typing detection systems have been developed for 10, 11, 15, 17, and 24 dog-specific STRs, which mainly detect STRs of dinucleotide and tetranucleotide repeating units. At the same time, the International Society for Animal Genetics (ISAG) recommended 21 STR loci for paternity testing of domestic dogs in order to standardize dog breeds and pedigree tracking. In the latest study, researchers developed a composite amplification typing detection system containing 24 STRs, with a cumulative exclusion probability of 0.9999 for the dog population. However, the loci included in this system are quite different from those in other international studies, which is not conducive to the comparison of results between laboratories and the promotion and large-scale application of related technical methods. In addition, there are limited STR typing technology systems in my country that can be used for dog individual identification and paternity testing. In order to make up for the technical deficiencies in dog individual identification and paternity testing, it is urgent to develop and construct a new STR typing technology system for dogs. Summary of the invention

[0005] In order to solve the aforementioned technical problems, the applicant has developed a STR typing technology for canine individual identification / parentage determination with high sensitivity, good specificity and excellent stability.

[0006] The first purpose of this application is to seek a new STR typing product or kit for canine individual identification and paternity determination;

[0007] The second objective of the present application is to seek a STR typing detection method for canine individual identification and paternity determination.

[0008] Specifically, this application adopts the following technical solutions:

[0009] The present application first provides a primer composition for dog individual identification and paternity typing, wherein the primers are directed to 21 dog-specific STR loci;

[0010] Further, the locus and size include the following:

[0011] FH2848(131-170nt), AHTh130(188-222nt), REN64E19(224-248nt), REN169O18(252-294nt), INU055(300-355nt), AHT 137(75-120nt), INU030(124-157nt), REN247M23(164-190nt), AHTh260(195-230nt), INRA21(250-294nt), REN105L03( 300-340nt), REN162C04(82-122nt), AHTk253(141-181nt), REN169D01(190-236nt), REN54P11(246-282nt), FH2054(29 3-353nt), CXX279(82-130nt), INU005(140-175nt), AHTk211(180-225nt), AHTh171(230-268nt), AHT121(279-339nt).

[0012] Preferably, the primer combination is directed against the sex identification gene locus Amelogenin (90-129nt).

[0013] Furthermore, the specific sequences of the primers are shown in SEQ ID NO.1-44.

[0014] Furthermore, the primers are divided into 4 groups and are fluorescently labeled;

[0015] Preferably, the fluorescent labels are FAM, HEX, TAMRA and ROX labels respectively:

[0016] Group 1: FAM blue fluorescent labeling: targeting loci Amelogenin (90-129nt), FH2848 (131-170nt), AHTh130 (188-222nt), REN64E19 (224-248nt), REN169O18 (252-294nt), INU055 (300-355nt);

[0017] Group 2: HEX green fluorescent labeling: targeting loci AHT137 (75-120nt), INU030 (124-157nt), REN247M23 (164-190nt), AHTh260 (195-230nt), INRA21 (250-294nt), REN105L03 (300-340nt);

[0018] Group 3: TAMRA yellow fluorescent labeling: targeting loci REN162C04 (82-122nt), AHTk253 (141-181nt), REN169D01 (190-236nt), REN54P11 (246-282nt), FH2054 (293-353nt);

[0019] Group 4: ROX red fluorescent labeling: targeting loci CXX279 (82-130nt), INU005 (140-175nt), AHTk211 (180-225nt), AHTh171 (230-268nt), AHT121 (279-339nt).

[0020] The present application also provides a kit for canine individual identification and paternity testing typing, characterized in that it comprises any of the primer combinations described above.

[0021] Furthermore, the kit further comprises: hot-start Taq DNA polymerase, PCR amplification buffer, quality control products and internal standards.

[0022] The present application also provides the use of any of the aforementioned primer combinations in canine individual identification and paternity testing typing, or in the preparation of a canine individual identification and paternity testing typing kit.

[0023] Further preferably, in the identification and typing process, the annealing temperature of the PCR amplification system DE is 57±2°C, the annealing time is 80-90s, the extension time is 5-15min, and the number of cycles is 29-30.

[0024] The present application also provides a method for dog individual identification and paternity testing typing, which includes constructing a composite PCR amplification system containing 22 dog-specific loci, using a method of fluorescent PCR amplification combined with capillary electrophoresis detection, detecting 21 dog-specific STR loci and AMEL loci for gender determination, and combining the fragment separation technology of capillary electrophoresis to achieve typing of all target loci in one experiment, which may specifically include the following steps:

[0025] A step of amplifying using the aforementioned primer combination, or a step of detecting using any of the aforementioned kits; further, the method further comprises the steps of capillary electrophoresis and data analysis and interpretation, and finally determining the genotype of the sample by the fragment size and the product peak height ratio of the locus.

[0026] Compared with the prior art, this application has at least the following advantages:

[0027] 1) The method of the present application has extremely high detection sensitivity, and the detection system can achieve complete STR typing detection of trace canine DNA as low as 0.1ng;

[0028] 2) The typing results of this application are more accurate, and the bias of the target amplification peaks of the 21 target STR loci in the system is less than 0.2nt;

[0029] 3) The species specificity of the detection in this application is high. All loci in the system have high canine species specificity. There is no product peak for non-canine DNA such as cat, mouse, pigeon, horse, cattle, Escherichia coli, fish and MDCK cell line in the detection.

[0030] 4) The application has strong applicability for test materials. The system can obtain accurate STR typing results for a variety of different types of test materials and template DNA obtained by different extraction methods, and the test materials are highly applicable. Moreover, compared with previous methods, the application is more stable. The system performs amplification reactions on a variety of different types of thermal cyclers under different primer concentrations, buffer concentrations, annealing temperatures, annealing times, and final extension times, and the results are accurate and stable. In summary, the repeatability and authority of this application are significantly better than previous technologies during the application process.

[0031] 5) The system of this application can achieve accurate amplification and detection of all loci in the system within 3 hours, with high speed, and related reagents and consumables can be mass-produced at low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 , 21 Initial arrangement of the dog-specific STR loci and the Amelogenin locus on five different color fluorescence channels;

[0034] Figure 2 , 21 The final optimized arrangement of the dog-specific STR loci and the Amelogenin locus on five different color fluorescence channels;

[0035] Figure 3 , Initially, the composite amplification pattern of the canine STR typing technology system using canine positive standards as amplification template;

[0036] Figure 4, typing patterns of the blue fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures;

[0037] Figure 5 , typing patterns of the green fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures;

[0038] Figure 6 , typing patterns of the yellow fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures;

[0039] Figure 7 , typing patterns of the red fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures;

[0040] Figure 8 , typing patterns of the purple fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures;

[0041] Fig. 9 , after multiple rounds of primer sequence and amplification conditions optimization, the canine STR typing technology system composite amplification map was performed using canine positive standard samples as amplification template;

[0042] Fig.10 , typing patterns of species-specific DNA samples (cat, mouse, pigeon, horse, cattle, E. coli, fish, and MDCK cell lines);

[0043] Fig.11 , typing patterns of quality control DNA at different DNA input amounts;

[0044] Fig.12 , Four The canine STR typing detection system correctly detects allele ratios at different concentrations of common inhibitors;

[0045] Fig.13 , typing patterns of quality control DNA at different primer concentrations;

[0046] Fig.14 , typing patterns of peripheral blood DNA samples at different primer concentrations;

[0047] Fig.15 , typing patterns of quality control DNA samples at different buffer concentrations;

[0048] Fig.16 , typing patterns of peripheral blood DNA samples at different buffer concentrations;

[0049] Fig.17 , typing patterns of quality control DNA samples at different annealing temperatures;

[0050] Fig.18, typing patterns of peripheral blood DNA samples at different annealing temperatures;

[0051] Fig.19 , typing patterns of quality control DNA samples at different annealing times;

[0052] Fig. 20 , typing patterns of peripheral blood DNA samples at different annealing times;

[0053] Fig.21 , typing patterns of quality control DNA samples at different terminal extension times;

[0054] Fig. 22 , typing patterns of peripheral blood DNA samples at different terminal extension times;

[0055] Fig.23 , typing patterns of quality control DNA samples at different cycle numbers;

[0056] Fig.24 , typing patterns of peripheral blood DNA samples at different cycle numbers;

[0057] Fig.25 , typing patterns of quality control DNA samples on different thermal cyclers and 3730XL genetic analyzer;

[0058] Fig.26 , the typing patterns of the quality control DNA samples on different thermal cyclers and the 3500XL Genetic Analyzer;

[0059] Fig. 27 , the typing patterns of the same peripheral blood DNA sample on the same thermal cycler instrument, 3500XL and 3730XL genetic analyzers, respectively. DETAILED DESCRIPTION

[0060] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0061] Definition of some terms

[0062] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0063] As used in this application, an indefinite or definite article used when referring to a singular noun eg "a" or "an", "the" includes a plural of that noun.

[0064] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0065] The term "approximately" in this application indicates an accuracy range that can be understood by those skilled in the art and still ensures the technical effect of the characteristic in question. The term generally indicates ±10%, preferably ±5%, of the indicated value.

[0066] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0067] The following are specific embodiments

[0068] Experimental Example 1: Locus grouping, primer design and optimization

[0069] 1. Screening of Canine STR Loci

[0070] In order to improve the comparability of STR typing results between different laboratories in the construction of the detection system, the STR loci in this application select the 21 STR loci recommended by ISAG. In addition, a gender determination locus Amelogenin is added for gender determination.

[0071] The arrangement of each locus in the detection system is determined based on the sequence characteristics and the fluorescent marker color of the final system. In the initial design, the 21 canine-specific STR loci can only be distributed on 5 different color fluorescent channels. Figure 1 .

[0072] The applicant then adjusted the size of the locus, combined with the adjustment of primer sequences and amplification procedures, and finally screened and established a new size locus, and arranged these canine-specific STR loci in 4 channels (partial adjustment can be found in the primer design and screening section below). For specific locus size and arrangement, see Figure 2 or as follows:

[0073] Channel 1: Amelogenin (90-129nt), FH2848 (131-170nt), AHTh130 (188-222nt), REN64E19 (224-248nt), REN169O18 (252-294nt), INU055 (300-355nt);

[0074] Channel 2: AHT137 (75-120nt), INU030 (124-157nt), REN247M23 (164-190nt), AHTh260 (195-230nt), INRA21 (250-294nt), REN105L03 (300-340nt);

[0075] Channel 3: REN162C04 (82-122nt), AHTk253 (141-181nt), REN169D01 (190-236nt), REN54P11 (246-282nt), FH2054 (293-353nt),

[0076] Channel 4: CXX279 (82-130nt), INU005 (140-175nt), AHTk211 (180-225nt), AHTh171 (230-268nt), AHT121 (279-339nt).

[0077] After optimization, the proteins can be evenly arranged on four different color fluorescent labeling channels (FAM, HEX, TAMRA and ROX), with high amplification efficiency and good balance.

[0078] 2. Primer Design and Screening

[0079] The present application aims to detect all target loci in one experiment, and fully considers multiple aspects such as the species specificity of the detection system to dogs, the amplification efficiency of the loci, the internal balance of alleles, the balance between loci, etc. The following examples illustrate the loci and preliminary primer screening test process of the present application from different angles.

[0080] 1. Single locus testing

[0081] For the target STR locus, the template sequence was downloaded from the public database, and the sex determination locus Amelogenin was used as an example: this application selected CM000039.4:7784625-7785836 in GENBANK, Canis lupusfamiliaris isolate Tasha breed boxer chromosome X, whole genome shotgun sequence. In order to ensure the balance of locus amplification and explore the most effective primer sequence, the applicant first designed multiple sets of primer pairs for the Amelogenin locus, specifically including 1 upstream primer and 7 downstream primers (see Table 1).

[0082] Table 1

[0083]

[0084]

[0085] Subsequently, the amplification specificity of the single primers of different primer pairs was tested respectively, and the primer pair with the best specificity and the highest amplification efficiency was selected for subsequent experiments. The first primer pair was screened as the preferred primer sequence.

[0086] Taking the AHTh130 locus as an example, two pairs of primers were designed in the early stage (Table 2 below). Both pairs of primers could obtain a single specific band in the single primer test. However, in the initial composite amplification system, the efficiency of primer pair 1 was low and no specific amplification peak was observed, so it was abandoned in the experiment (see the typing map for details). Figure 3 ); The primer pair 2 (bold sequence in the table below) designed by subsequent optimization showed good specificity and extension efficiency in both single primer test and adjusted composite system debugging. Fig. 9 result.

[0087] Table 2

[0088]

[0089] 2. Composite system optimization

[0090] A composite test of 21 canine-specific STR loci and the Amelogenin locus showed nonspecific amplification in the initial arrangement and overlap in the size ranges of amplified fragments at different loci (e.g. Figure 1 , the initial arrangement of 22 loci); the applicant adjusted the size and primer sequence of CXX279, INU005, AHth130, AMEL, REN105L03, and REN162C04 loci. After the adjustment, the loci can be correctly arranged in different color fluorescent labeling channels. However, as mentioned above Figure 3As shown in the figure, the REN64E19 and AHTh130 loci were missing. Then, in view of the missing REN64E19 and AHTh130 loci, a single color fluorescent marker was used to mark the loci, the annealing temperature was changed, and the strategy of adjusting the primer sequence in a small range was combined to perform compound amplification debugging to improve the detection performance of the system. For example, in the early design, the amplification efficiency of each color fluorescent marker channel under different temperature gradients can be seen. Figure 4-8 : Figure 4 This is the typing spectrum of the blue fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures. It can be seen that there is a non-specific peak at 276nt in the blue channel, and the non-specific peak gradually decreases with the increase of annealing temperature and disappears at 61-62°C; in addition, the peak value of the locus AHT121 is relatively low. Figure 5 This is the typing spectrum of the green fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures, and the effect is good. Figure 6 This is the typing spectrum of the yellow fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures, in which REN64E19 and AHTh130 have allele deletions. Figure 7 This is the typing spectrum of the red fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures, in which AHTh171 increases with the increase of annealing temperature. Figure 8 This is the typing spectrum of the purple fluorescent labeling channel of the canine STR typing detection system at different annealing temperatures. Based on the problems in the aforementioned channels, the corresponding loci were optimized and adjusted through multiple rounds of primer sequences and reaction conditions, and finally a clear typing spectrum was obtained. Fig. 9 This is the 22 loci typing map of the optimized positive control sample. It can be seen that clear and accurate typing results can be obtained for 21 dog-specific STR loci and dog sex determination sites, including the aforementioned REN64E19 and AHTh130 loci.

[0091] 3. Species-specific testing of the system

[0092] To test the species specificity of the canine STR typing system, DNA samples from cat, mouse, pigeon, horse, cattle, E. coli, fish, and MDCK cell lines were used for species specificity testing. For each species, 1 ng of DNA was added to the PCR amplification system. Fig.10The following are typing maps of DNA of different species. As can be seen from the figure, the canine STR typing technology system observed non-specific amplification peaks at the REN162C04 site of cat DNA samples, the INU055 site and all HEX marker sites of E. coli DNA samples, and the AHTh130 and AHTk211 sites of cattle DNA samples, which did not affect the allele typing reading of dogs. In general, the species specificity of the canine STR typing technology system is relatively good, and the allele typing of the target species can be accurately obtained.

[0093] Through the above-mentioned design, screening and optimization tests, the applicant finally determined the detection locus, repeat region structure, molecular weight and primer sequence of the present application, as shown in Table 3 for details.

[0094] Table 3

[0095]

[0096]

[0097] Example 2: Reaction system evaluation and testing

[0098] The canine STR typing technology system of the present application was constructed based on the determined primer sequences and locus sizes. In order to test the sensitivity (DNA template input amount) of the typing technology system, the stability of the amplification system to the primer concentration, buffer concentration, the amplification program to the annealing temperature, annealing time, final extension time, cycle number and other parameters and the accuracy of the results, a series of gradient experiments were designed, and the specific methods and results are as follows.

[0099] 1. The basic operation steps are as follows:

[0100] 1. PCR amplification reaction of target dog-specific STR loci

[0101] a) Determine the number of reactions N. The quality control sample and the no-template control (nuclease-free water) should be tested simultaneously in each PCR amplification reaction. N = number of samples to be tested + quality control sample control (1) + no-template control (1) + 1.

[0102] b) Place all components of the PCR amplification reaction at room temperature until they are completely dissolved. Vortex and mix the melted components for 10 seconds, and centrifuge for 10 seconds to concentrate the liquid at the bottom of the tube.

[0103] c) Prepare the PCR amplification reaction system except the template according to the table below. After adding each component, vortex and mix for 10 seconds, centrifuge for 10 seconds, and take 9 μL into 0.2 mL PCR tubes.

[0104]

[0105]

[0106] d) Add 1 μL of DNA sample to be tested, quality control product, or nuclease-free pure water to each tube of the amplification reaction system. Record the number sequence, vortex and mix for 10 seconds, and centrifuge for 10 seconds to concentrate the liquid at the bottom of the tube.

[0107] e) Place each reaction tube in the reaction tank of the PCR instrument and run the PCR program. The PCR amplification reaction program is shown in the table below.

[0108]

[0109] Note: After the PCR amplification program is completed, the PCR product can be tested immediately; or stored at 2-8℃ and tested within 24 hours; or stored at -20℃ and tested within three days. Please try to avoid repeated freezing and thawing, and the number of freezing and thawing should not exceed 3 times.

[0110] 2. PCR amplification product typing detection and result analysis

[0111] a) The PCR amplification product was centrifuged at 3500 rpm for 2 min in a benchtop or plate centrifuge.

[0112] b) Prepare a mixture of deionized formamide (Hi-Di) and molecular weight internal standard QD550 according to the table below. Mix the mixture using a vortex oscillator for 10 seconds, centrifuge it using a handheld centrifuge for 10 seconds, and dispense 9 μL into a 96-well plate. Add 1 μL of PCR amplification product (stock solution or dilution) to each well.

[0113] Components Volume per reaction (μL) N reaction volume (μL) Hi-Di 8.8 8.8N QD550 0.2 0.2N total 9 9N

[0114] c) Cover with a rubber pad and centrifuge briefly using a benchtop or plate centrifuge.

[0115] d) Denature at 95℃ for 5 min, then immediately place on ice at -20℃ for 5 min, and test within 24 hours. Centrifuge briefly before testing using a benchtop or plate centrifuge.

[0116] e) Use a gene analyzer to perform capillary electrophoresis detection. Select the "Fragment" electrophoresis method. For details, refer to the instruction manual of the gene analyzer.

[0117] f) Use IDX analysis software was used to analyze the capillary electrophoresis data.

[0118] Note: The molecular weight internal standard contains 22 fragments of different sizes, namely 70nt, 80nt, 100nt, 120nt, 140nt, 160nt, 180nt, 200nt, 225nt, 250nt, 275nt, 300nt, 325nt, 350nt, 375nt, 400nt, 425nt, 450nt, 475nt, 500nt, 525nt, and 550nt.

[0119] 2. Reaction System Evaluation and Testing

[0120] (I) System DNA template input (sensitivity) test

[0121] In order to test the sensitivity of the canine STR typing technology system, the DNA template input (sensitivity) of the system was tested by the gradient experiment method of peripheral blood sample DNA and quality control product DNA. The DNA template input gradient was 2ng, 1ng, 0.5ng, 0.2ng, and 0.1ng respectively.

[0122] The results are as follows Fig.11 As shown in the figure, when the amount of template DNA added was 0.2ng and 0.1ng, some loci were lost during the amplification of peripheral blood DNA samples. When the input amount was 2ng, 1ng, and 0.5ng, the peripheral blood DNA samples could be accurately typed. When the amount of template DNA added was 2ng, 1ng, 0.5ng, 0.2ng, and 0.1ng, the quality control DNA samples could be accurately typed. In order to consider the accuracy of typing, it is recommended that the amount of template DNA added be between 0.5-1ng when using this typing technology system.

[0123] (II) System anti-inhibition ability test

[0124] In order to test the anti-inhibition ability of the canine STR typing technology system, four common inhibitors were selected to test the anti-inhibition ability of the system. The gradients of the four inhibitors were set as follows: the eight concentration gradients of tannic acid were 25, 50, 75, 100, 120, 150, 200, and 250 ng / μL; the eight concentration gradients of humic acid were 75, 100, 150, 200, 250, 300, 450, and 500 ng / μL; the eight concentration gradients of hemoglobin were 800, 1000, 1200, 1500, 1800, 2000, 2500, and 5000 μM; the eight concentration gradients of indigo were 8, 10, 15, 20, 25, 30, 35, and 40 mM. The typing detection results of the four different inhibitors at different concentrations were statistically analyzed, and the results are shown in Figure 2. Fig.12. As can be seen from the figure, when the tannic acid concentration is 75ng / μL, the correct detection rate of the allele reaches 100%. When the humic acid concentration is 75ng / μL, 100ng / μL, 150ng / μL, 200ng / μL, 250ng / μL, 450ng / μL and 500ng / μL, the correct detection rate of the allele also reaches 100%. For hemoglobin, the correctly detected allele ratio reaches 100% at concentrations of 800μM, 1200μM, 1800μM and 2000μM. Similarly, when the indigo concentration is 8mM, 10mM, 20mM and 25mM, the correctly detected allele ratio reaches 100%. The results confirm that the canine STR typing technology system has a strong anti-inhibition ability.

[0125] (III) Primer concentration stability test of PCR amplification system

[0126] In order to test the stability of primers for PCR amplification of canine STR typing technology system, samples with 1 ng template DNA added and quality control DNA samples were amplified under the conditions of 80%, 90%, 100%, 110%, and 120% of the recommended primer concentrations.

[0127] The experimental results showed that when the primer addition amount of the typing technology system was 80%, 90%, 100%, 110%, and 120% of the recommended amount, the quality control product and peripheral blood DNA samples could detect all typings with good specificity and correct typing. The typing patterns of the quality control product and peripheral blood DNA samples at different primer concentrations were shown in Figure 2. Figure 13-14 ; The average peak height of the quality control DNA samples was not much different at 80%, 90%, 100%, 110%, and 120% of the recommended amount; the amplification efficiency of each locus (locus peak height / sample average peak height) was not much different, indicating that the primer amount had little effect on the canine STR system and could tolerate a difference of ±20% in primer amount. The peak heights of peripheral blood DNA and quality control DNA samples were balanced at different primer concentrations, the results were accurate, and the stability was good.

[0128] (IV) PCR amplification system buffer concentration stability test

[0129] In order to test the stability of PCR amplification of canine STR typing technology system to buffer, samples with 1 ng template DNA added and quality control DNA samples were amplified at 80%, 90%, 100%, 110%, and 120% of the recommended buffer concentration.

[0130] When the amount of amplification buffer added was 80%, 90%, 100%, 110%, and 120% of the recommended amount, the peripheral blood DNA and quality control DNA samples were accurately typed. The typing patterns of the quality control and peripheral blood DNA samples at different buffer concentrations are shown in Figure 15-16 . For peripheral blood DNA samples, when the amount of amplification buffer added was 80%, 90%, 100%, 110%, and 120% of the recommended amount, all samples could be amplified normally, and the difference in the average peak height of the samples increased first and then decreased with the amount of buffer added. The amplification efficiency was best at 100% of the recommended amount. For quality control DNA samples, when the amount of amplification buffer added was 80%, 90%, 100%, 110%, and 120% of the recommended amount, all samples could be amplified normally, and the difference in the average peak height of the samples increased first and then decreased with the amount of buffer added. At 100% of the recommended amount, the average peak height of the locus was low, and the amplification efficiency of each locus was relatively stable with little difference. This shows that the amplification system is stable when the amount of buffer added is within the range of ±20%. The peak heights of peripheral blood DNA and quality control DNA samples are balanced at different buffer concentrations, the results are accurate, and the stability is good.

[0131] (V) PCR amplification program annealing temperature test

[0132] In order to explore the effect of annealing temperature in PCR amplification procedure on canine STR typing technology system, samples with 1 ng template DNA added and quality control DNA samples were amplified at annealing temperatures of 55, 56, 57, 58, and 59°C, respectively.

[0133] When the annealing temperatures were 55, 56, 57, 58, and 59°C, all the DNA samples of peripheral blood and quality control products could be typed with good specificity and correct typing. The typing patterns of the quality control products and peripheral blood DNA samples at different annealing temperatures are shown in Figure 17-18 . For peripheral blood DNA samples, the annealing temperatures were 55, 56, 57, 58, and 59°C, respectively. The average peak height of the locus was the highest at 57°C, and the locus amplification efficiency (locus peak height / sample average peak height) did not differ much. There was little difference in the balance between sample loci. For quality control DNA samples, the annealing temperatures were 55, 56, 57, 58, and 59°C, respectively. The average peak height of the locus did not differ much, and the difference in the locus amplification efficiency (locus peak height / sample average peak height) did not change much, and the balance between loci was good. Taking all factors into consideration, the recommended annealing temperature for the canine STR typing technology system is 57°C. The system has good stability and can tolerate an annealing temperature difference of ±2°C.

[0134] (VI) PCR amplification program annealing time test

[0135] In order to explore the effect of annealing time in PCR amplification procedure on canine STR typing technology system, peripheral blood DNA samples with 1 ng template DNA added and quality control DNA samples were amplified at annealing temperatures of 60, 70, 80, 90, 100, and 110 seconds, respectively.

[0136] When the annealing time was 60, 70, 80, 90, 100, and 110 seconds, all samples could be typed with good specificity and correct typing. The typing patterns of the quality control products and peripheral blood DNA samples at different annealing times are shown in Figure 2. Figure 19-20 . For peripheral blood DNA samples, the annealing time was 60, 70, 80, 90, 100, and 110 seconds, respectively. The average peak height of the locus was generally higher at 80 or 90 seconds, and the locus amplification efficiency (locus peak height / sample average peak height) was not much different, except for one peripheral blood sample whose average locus peak height was reduced at 80 seconds. The average peak height of other samples and the balance between loci were good at 80 or 90 seconds. For quality control DNA samples, the annealing time was 60, 70, 80, 90, 100, and 110 seconds, respectively. The average peak height did not differ much, and the locus amplification efficiency (locus peak height / sample average peak height) did not change much, indicating that the annealing time had no significant effect on the amplification efficiency of quality control DNA samples. Considering the system amplification effect and program amplification time, the recommended annealing time is 80 seconds or 90 seconds.

[0137] (VII) PCR amplification program final extension time test

[0138] In order to explore the effect of the terminal extension time in the PCR amplification procedure on the canine STR typing technology system, peripheral blood DNA samples with 1 ng template DNA added and quality control DNA samples were amplified under the conditions of terminal extension time of 5 min, 7.5 min, and 10 min, respectively.

[0139] The results showed that when the final extension time was 5min, 7.5min, 10min, 12.5min, and 15min, all samples could be typed, with good specificity, correct typing, and no incomplete addition of A. The typing patterns of the quality control products and peripheral blood DNA samples at different annealing times were shown in Figure 2. Figure 21-22 For blood DNA and quality control samples, when the final extension time was 5min, 7.5min, 10min, 12.5min, and 15min, the average peak height and amplification efficiency were not much different, and the balance was good. This shows that the final extension time of 5-15min has a good effect of adding A, and 10min is the best.

[0140] (VIII) PCR amplification program cycle number test

[0141] In order to explore the effect of the cycle number in the PCR amplification program on the canine STR typing technology system, peripheral blood DNA samples with 1 ng template DNA added and quality control DNA samples were amplified at cycle numbers of 27, 28, 29, 30, and 31, respectively.

[0142] The results showed that when the cycle numbers were 27, 28, 29, 30, and 31, all samples could be typed with good specificity and correct typing. The typing patterns of the quality control products and peripheral blood DNA samples at different cycle numbers were shown in Figure 2. Figure 23-24 For peripheral blood DNA and quality control DNA samples, when the cycle numbers were 27, 28, 29, 30, and 31, the average peak height of the locus gradually increased, the amplification efficiency of the locus (locus peak height / average sample peak height) was not much different, and the balance between loci was not much different. Comprehensively considering the recommended cycle number of 29 or 30, the amplification effect is better.

[0143] (IX) PCR thermal cycler test

[0144] In order to explore the effects of different thermal cycling instruments on canine STR typing technology system, peripheral blood DNA samples with 1 ng template DNA added and quality control DNA samples were amplified using ABI 3500XL dx (pop 4), ABI 3500XL dx (pop7), and ABI3730XL, respectively.

[0145] The canine STR system can be applied to three PCR instruments, ABI 9700, ABI ProFlex, and ABI Veriti, and two detection instruments, ABI 3500XLdx (pop4) and ABI 3730XL. All of them can detect all the typing of samples with good specificity and correct typing. The typing patterns of quality control products on 3730 and 3500XL genetic analyzers and peripheral blood DNA samples on 3730 and 3500XL genetic analyzers are shown in Figure 25-27 .

[0146] The above data show that the typing technology system of the present application has high tolerance to primer concentration, buffer concentration, annealing temperature, annealing time, final extension time, cycle number changes and equipment, and has high detection sensitivity, good specificity and more accurate typing results.

[0147] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. A primer combination for canine individual identification and paternity testing typing, characterized in that: The primer combination is directed to dog-specific STR loci; the dog-specific STR loci include the following 21: FH2848(131-170nt), AHTh130(188-222nt), REN64E19(224-248nt), REN169O18(252-294nt), INU055(300-355nt), AHT137(75-120nt) , INU030(124-157nt), REN247M23(164-190nt), AHTh260(195-230nt), INRA21(250-294nt), REN105L03(300-340nt), REN162C04(82-1 22nt), AHTk253 (141-181nt), REN169D01 (190-236nt), REN54P11 (246-282nt), FH2054 (293-353nt), CXX279 (82-130nt), INU005 (140-175nt), AHTk211 (180-225nt), AHTh171 (230-268nt) and AHT121 (279-339nt); the primer combination also targets the gene locus Amelogenin (90-129nt) for sex identification; the sequences of the primers are shown in SEQ ID NO.1-44.

2. The primer composition according to claim 1, characterized in that The primers are fluorescently labeled.

3. The primer composition according to claim 2, characterized in that The fluorescent labels are divided into 4 groups, which are labeled with FAM, HEX, TAMRA and ROX respectively: Group 1: FAM blue fluorescent labeling: targeting loci Amelogenin (90-129nt), FH2848 (131-170nt), AHTh130 (188-222nt), REN64E19 (224-248nt), REN169O18 (252-294nt), INU055 (300-355nt); Group 2: HEX green fluorescent labeling: targeting loci AHT137 (75-120nt), INU030 (124-157nt), REN247M23 (164-190nt), AHTh260 (195-230nt), INRA21 (250-294nt), REN105L03 (300-340nt); Group 3: TAMRA yellow fluorescent labeling: targeting loci REN162C04 (82-122nt), AHTk253 (141-181nt), REN169D01 (190-236nt), REN54P11 (246-282nt), FH2054 (293-353nt); Group 4: ROX red fluorescent labeling: targeting loci CXX279 (82-130nt), INU005 (140-175nt), AHTk211 (180-225nt), AHTh171 (230-268nt), AHT121 (279-339nt).

4. A kit for dog individual identification and paternity testing, characterized in that: The invention comprises the primer composition described in any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit further comprises: hot start Taq DNA polymerase, PCR amplification buffer, quality control products and internal standards.

6. Use of the primer combination described in any one of claims 1 to 3 in preparing a typing kit for dog individual identification and paternity determination.

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