A kit for human autosomal miniSTR typing and application thereof

Abstract

The application relates to the technical field of gene detection, and particularly discloses a kit for human autosomal miniSTR typing and application thereof. The kit comprises 10 pairs of STR primer groups for amplifying 10 miniSTR loci, and the 10 miniSTR loci are D3S1358, D21S11, D18S51, D7S820, D15S131, D15S12, D15S146, D16S539, D5S818 and TPOX. The kit can successfully perform STR typing on highly degraded DNA samples, and can be used as a supplement of traditional STR reagents.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a kit for human autosomal miniSTR typing and its application. Background Technology

[0002] In forensic science and court identification, DNA typing technology has become a core tool for individual identification, paternity testing, and crime scene evidence analysis. Short tandem repeats (STRs) are an important class of genetic markers in DNA, composed of repeating units of 2-6 base pairs, exhibiting high polymorphism and stability. STR typing generates a unique DNA fingerprint by amplifying repeating sequences at specific loci, thereby achieving high-precision differentiation of individuals. In forensic practice, STR kits are widely used in the analysis of biological samples such as blood, saliva, and hair, providing crucial evidence for criminal case investigation and disaster victim identification.

[0003] However, traditional STR typing techniques have limitations when dealing with degraded DNA samples (such as old, high-temperature, or chemically damaged samples). Because STR amplification fragments are typically long, amplification efficiency decreases significantly when DNA degradation causes fragment breakage, easily leading to typing failures or errors. To address this, researchers have developed mini-short tandem repeat (miniSTR) technology. MiniSTRs are STR sites shortened to less than 100-180 bp by redesigning primers. Their core advantage lies in their ability to more effectively amplify degraded DNA, improving detection success rates and reliability. In kits, miniSTRs supplement traditional STRs and are suitable for low-quality samples commonly encountered in forensic practice, such as bones, teeth, and cigarette butts, thus expanding the application range of DNA typing.

[0004] While miniSTR technology has shown great potential in the field of forensic medicine, research on miniSTRs targeting human autosomes is relatively limited. Therefore, there is an urgent need to develop highly specific and accurate human autosomal miniSTR typing kits to overcome the shortcomings of traditional STR typing techniques and provide a more comprehensive and efficient solution for forensic DNA analysis. Summary of the Invention

[0005] This invention relates to a kit for human autosomal miniSTR typing and its application. The kit redesigns miniSTRs for STR loci on human chromosomes 3, 5, 7, 15, 16, 18, and 21 as a supplement to traditional STRs. It not only maintains consistency with traditional STR typing results but also amplifies highly degraded DNA samples, thus overcoming the shortcomings of traditional STR typing technology.

[0006] On one hand, the present invention provides primers for human autosomal miniSTR typing, including 10 pairs of STR primers for amplifying 10 miniSTR loci, the 10 miniSTR loci being: D3S1358, D21S11, D18S51, D7S820, D15S131, D15S12, D15S146, D16S539, D5S818, and TPOX.

[0007] Of the 10 miniSTR loci mentioned above, D3S1358, D7S820, D16S539, D18S51, D21S11, D5S818, and TPOX are core loci in the CODIS system. This invention retains 7 CODIS system loci and adds 3 non-CODIS system loci. These 3 non-CODIS system loci meet relevant requirements for forensic medicine, such as gene frequency distribution conforming to Hardy-Weinberg equilibrium, no linkage between loci, high amplification efficiency, low mutation rate, and absence of mutation hotspots in flanking sequences.

[0008] Polymorphism verification of D15S131, D15S12, and D15S146 was performed using samples from Han Chinese populations in South China, Southeast China, Southwest China, and East China. Following the requirements for STR loci in forensic DNA testing, statistical analysis was conducted on the following forensic parameters of the above loci: individual identifiability (DP), non-paternal exclusion rate (EPP), polymorphism information content (PIC), expected heterozygosity (HE), and observed heterozygosity (HO). The analysis revealed that D15S131, D15S12, and D15S146 exhibit high polymorphism in the Han Chinese population, along with very high individual identifiability and non-paternal exclusion rate.

[0009] D15S131, D15S12, and D15S146 are three independent non-allelic genes located on human chromosome 15. Developing them into miniSTRs and incorporating them into the detection system can fill the gap in genetic marker density on chromosome 15 and further improve the overall resolution of the system. Furthermore, the flanking sequences of the core repeat regions of D15S131, D15S12, and D15S146 are highly conserved, which is beneficial for using small miniSTR primers to achieve specific amplification of highly degraded DNA samples.

[0010] In some embodiments, the primers described above are used to simultaneously amplify the following 10 miniSTR loci: D3S1358, D21S11, D18S51, D7S820, D15S131, D15S12, D15S146, D16S539, D5S818, and TPOX.

[0011] In some embodiments, the STR primer set for the miniSTR locus D3S1358 includes an upstream primer D3S1358_mini_F and a downstream primer D3S1358_mini_R, wherein the upstream primer D3S1358_mini_F has the nucleotide sequence shown in SEQ ID No. 1 and the downstream primer D3S1358_mini_R has the nucleotide sequence shown in SEQ ID No. 2.

[0012] Upstream primer D3S1358_mini_F: 5'-CAGACAGGGCTGAGGGTGG-3' (SEQ ID No. 1);

[0013] Downstream primer D3S1358_mini_R: 5'-GGCTACAGTAGATTCTGCTC-3' (SEQ ID No. 2).

[0014] In some embodiments, the STR primer set of the miniSTR locus D21S11 includes an upstream primer D21S11_mini_F and a downstream primer D21S11_mini_R; the upstream primer D21S11_mini_F has a nucleotide sequence as shown in SEQ ID No. 3, and the downstream primer D21S11_mini_R has a nucleotide sequence as shown in SEQ ID No. 4.

[0015] Upstream primer D21S11_mini_F: 5'-TGTATTAGTCAATGTTCTCCAGAG-3' (SEQ ID No. 3);

[0016] Downstream primer D21S11_mini_R: 5'-CTATGTGATACATTATGTGATACGTG-3' (SEQ ID No. 4).

[0017] In some embodiments, the STR primer set for the miniSTR locus D18S51 includes an upstream primer D18S51_mini_F and a downstream primer D18S51_mini_R, wherein the upstream primer D18S51_mini_F has a nucleotide sequence as shown in SEQ ID No. 5, and the downstream primer D18S51_mini_R has a nucleotide sequence as shown in SEQ ID No. 6.

[0018] Upstream primer D18S51_mini_F: 5'-GCTGGTCACAGGTAGCACC-3' (SEQ ID No. 5);

[0019] Downstream primer D18S51_mini_R: 5'-GGACAGATGATAGATGGATGGATAG-3' (SEQ ID No. 6).

[0020] In some embodiments, the STR primer set for the miniSTR locus D7S820 includes an upstream primer D7S820_mini_F and a downstream primer D7S820_mini_R, wherein the upstream primer D7S820_mini_F has a nucleotide sequence as shown in SEQ ID No. 7, and the downstream primer D7S820_mini_R has a nucleotide sequence as shown in SEQ ID No. 8.

[0021] Upstream primer D7S820_mini_F: 5'-GCTGGTGGGGGAGTTATTTTGC-3' (SEQ ID No. 7);

[0022] Downstream primer D7S820_mini_R: 5'-CAGAGTCTCACCAAAGTTTAG-3' (SEQ ID No. 8).

[0023] In some embodiments, the STR primer set for the miniSTR locus D15S131 includes an upstream primer D15S131_mini_F and a downstream primer D15S131_mini_R, wherein the upstream primer D15S131_mini_F has a nucleotide sequence as shown in SEQ ID No. 9, and the downstream primer D15S131_mini_R has a nucleotide sequence as shown in SEQ ID No. 10.

[0024] Upstream primer D15S131_mini_F: 5'-GCTGTGAATCCATCTGGTCC-3' (SEQ ID No. 9);

[0025] Downstream primer D15S131_mini_R: 5'-GGAGTTTGCTCATGATTTGG-3' (SEQ ID No. 10).

[0026] In some embodiments, the STR primer set for the miniSTR locus D15S12 includes an upstream primer D15S12_mini_F and a downstream primer D15S12_mini_R, wherein the upstream primer D15S12_mini_F has a nucleotide sequence as shown in SEQ ID No. 11 and the downstream primer D15S12_mini_R has a nucleotide sequence as shown in SEQ ID No. 12.

[0027] Upstream primer D15S12_mini_F: 5'-GGTGGCCACAGGCCAGGT-3' (SEQ ID No. 11);

[0028] Downstream primer D15S12_mini_R: 5'-CAGAGGCCTACCCAGGCA-3' (SEQ ID No. 12).

[0029] In some embodiments, the STR primer set for the miniSTR locus D15S146 includes an upstream primer D15S146_mini_F and a downstream primer D15S146_mini_R, wherein the upstream primer D15S146_mini_F has a nucleotide sequence as shown in SEQ ID No. 13, and the downstream primer D15S146_mini_R has a nucleotide sequence as shown in SEQ ID No. 14.

[0030] Upstream primer D15S146_mini_F: 5'-CTACCAAGCCCTTAGGTATCATC-3' (SEQ ID No. 13);

[0031] Downstream primer D15S146_mini_R: 5'-CAGAGGCCTACCCAGGCA-3' (SEQ ID No. 14).

[0032] In some embodiments, the STR primer set for the miniSTR locus D16S539 includes an upstream primer D16S539_mini_F and a downstream primer D16S539_mini_R, wherein the upstream primer D16S539_mini_F has a nucleotide sequence as shown in SEQ ID No. 15, and the downstream primer D16S539_mini_R has a nucleotide sequence as shown in SEQ ID No. 16.

[0033] Upstream primer D16S539_mini_F: 5'-GATCCCAAGCTCTTCCTCTT-3' (SEQ ID No. 15);

[0034] Downstream primer D16S539_mini_R: 5'-ACGTTTGTGTGCATCTGTA-3' (SEQ ID No. 16).

[0035] In some embodiments, the STR primer set for the miniSTR locus D5S818 includes an upstream primer D5S818_mini_F and a downstream primer D5S818_mini_R, wherein the upstream primer D5S818_mini_F has a nucleotide sequence as shown in SEQ ID No. 17, and the downstream primer D5S818_mini_R has a nucleotide sequence as shown in SEQ ID No. 18.

[0036] Upstream primer D5S818_mini_F: 5'-TCCCCATTGGCCTGTTCTC-3' (SEQ ID No. 17);

[0037] Downstream primer D5S818_mini_R: 5'-CCTGAACTCTCCTATTATCTCCA-3' (SEQ ID No. 18).

[0038] In some embodiments, the STR primer set of the miniSTR locus TPOX includes an upstream primer TPOX_mini_F and a downstream primer TPOX_mini_R, wherein the upstream primer TPOX_mini_F has a nucleotide sequence as shown in SEQ ID No. 19 and the downstream primer TPOX_mini_R has a nucleotide sequence as shown in SEQ ID No. 20.

[0039] Upstream primer TPOX_mini_F: 5'-GGTGACTCTCAGATCCCTAAG-3' (SEQ ID No. 19);

[0040] Downstream primer TPOX_mini_R: 5'-ACTGGCACAGAACAGGCACTTAGG-3' (SEQ ID No. 20).

[0041] In some embodiments, the upstream primer D3S1358_mini_F, the downstream primer D3S1358_mini_R, the upstream primer D21S11_mini_F, the downstream primer D21S11_mini_R, the upstream primer D18S51_mini_F, the downstream primer D18S51_mini_R, the upstream primer D7S820_mini_F, the downstream primer D7S820_mini_R, the upstream primer D15S131_mini_F, the downstream primer D15S131_mini_R, and the upstream primer... The 5' ends of the following primers are independently connected to fluorescent markers of the same or different colors: D15S12_mini_F, D15S12_mini_R, D15S146_mini_F, D15S146_mini_R, D16S539_mini_F, D16S539_mini_R, D5S818_mini_F, D5S818_mini_R, TPOX_mini_F, and TPOX_mini_R.

[0042] In some embodiments, the fluorescent marker is selected from one or more of ROX, FAM, HEX, TAMRA, VIC, NED, PET, LIZ, and Cy5.

[0043] ROX, FAM, HEX, TAMRA, VIC, NED, PET, LIZ, and Cy5 are all common fluorescent labeling groups. ROX and LIZ emit red fluorescence, FAM emits blue fluorescence, HEX and VIC emit green fluorescence, NED, TAMRA, and PET emit yellow fluorescence, and Cy5 emits deep red fluorescence. Using these different fluorescent colors, the loci corresponding to the electrophoretic bands can be effectively distinguished.

[0044] On the other hand, the present invention provides a kit for human autosomal miniSTR typing, comprising all of the primers described above.

[0045] In some embodiments, the kit of the present invention further includes allele standards corresponding to each locus to determine the alleles of each locus in the sample.

[0046] In some embodiments, the kit of the present invention further includes fluorescent dye correctors composed of 70, 100, 125, 150, 175, 200, 225, 250, 275, and 300 bp amplification products labeled with fluorescent markers. These fluorescent dye correctors can serve as a measure of amplicon size to facilitate accurate identification of amplicon size.

[0047] In some embodiments, the kit of the present invention further includes DNA polymerase and its buffer solution and dNTPs.

[0048] The kit of the present invention uses the following PCR amplification procedure when amplifying miniSTR loci in DNA samples: denaturation at 95°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 60°C for 15-60 s, extension at 72°C for 1 min, for 26-30 cycles, and finally incubation at 60°C for 10 min, and storage at 4°C.

[0049] On the other hand, the present invention provides a miniSTR typing method for degraded DNA samples, which uses the aforementioned miniSTR typing kit to perform multiplex amplification on the degraded DNA samples.

[0050] In this invention, the degraded DNA samples include environmentally exposed samples, aged and historical samples, forensic special evidence samples, pathologically processed samples, trace and minor samples, and artificially processed samples. Environmentally exposed samples refer to samples degraded due to long-term exposure to the natural environment, including but not limited to bloodstains, saliva stains, hair, bones, teeth, and skin tissue fragments aged by sunlight, rain, high temperature, high humidity, and microbial action. Aged and historical samples refer to biological samples preserved for more than one year, or originating from archaeological excavations, ancient biological remains, museum specimens, or historical relics. Forensic special evidence samples refer to trace DNA samples extracted after high-temperature carbonization, chemical corrosion, acid and alkali treatment, or immersion in water or soil. Pathologically processed samples refer to tissue sections fixed in formalin and embedded in paraffin, as well as samples with severely broken DNA due to uneven fixation solution penetration or excessively long fixation time. Trace and micro-samples refer to samples containing extremely low amounts of DNA template and usually accompanied by severe degradation, including but not limited to contact DNA samples, fingerprint DNA residues, hair shafts, small amounts of bone powder, and single hair follicles. Artificially processed samples refer to DNA samples artificially prepared to achieve degradation through physical or chemical methods (such as ultrasonic disruption, nuclease treatment, and oxidant treatment).

[0051] Therefore, the kit of the present invention can be used for DNA sample detection in a variety of scenarios, including but not limited to highly degraded DNA samples extracted from human bones, saliva, blood, hair, semen stains and old bloodstains.

[0052] On the other hand, the present invention provides applications of the above primers or kits in case investigation, individual identification, identification of the source of death, paternity testing, population genetic analysis and / or construction of human DNA databases.

[0053] The present invention has the following advantages and effects:

[0054] 1. Fill the gap in genetic marker density on chromosome 15, further improving the overall resolution of the system.

[0055] 2. When amplifying 10 loci simultaneously in a single tube, competition between primers and the formation of heterodimers are avoided, significantly improving the amplification efficiency of each locus. Detailed Implementation

[0056] To make the objectives, features, and advantages of this invention more apparent and understandable, the invention will be further described in detail below with reference to embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of the appended claims.

[0057] Unless otherwise specified, all experimental reagents and materials used in this invention are commercially available.

[0058] The kit used in this invention specifically includes 5% Chelex-100 solution, miniSTR primer set solution, reaction buffer (10mM DMSO, 50mM KCl, 10mM Tris-HCl (pH 8.3, 25℃), 2.0mM MgCl2, 0.1mg / ml BSA and a mixture of 0.2mM dNTPs), and hot-start Taq DNA polymerase solution. The kit is used as an example as follows:

[0059] - Add 5% Chelex-100 to the sample (such as blood, bone tissue, skin, etc.), incubate at 56℃ for 2 hours with constant temperature shaking, then take out the sample and shake for 2 minutes, boil for 8-10 minutes, centrifuge at 13000 rpm for 3 minutes, and take the supernatant.

[0060] - Perform amplification in a PCR instrument according to the following procedure: denature at 95℃ for 5 min, then denature at 94℃ for 30 s, anneal at 60℃ for 15-60 s, extend at 72℃ for 1 min, perform 26-30 cycles, and finally incubate at 60℃ for 10 min and store at 4℃.

[0061] - After the amplification reaction is complete, remove the reaction tube and perform electrophoresis and detection using an ABI 3500 genetic analyzer.

[0062] Example 1 Individual-Specific Test

[0063] Blood DNA samples from 10 unrelated Han Chinese individuals were randomly selected. All samples were genotyped using the kit of this invention and GlobalFiler (purchased from Thermo Fisher Scientific). The individual specificity of the kit was verified by comparing the genotyping results of the kit with those of GlobalFiler. The results showed that the genotyping concordance rate between the kit and GlobalFiler for all samples was 100%, with an average heterozygosity greater than 0.75, an average individual identification ability (DP) greater than 0.85, and a cumulative individual identification ability (CDP) greater than 0.99 for all miniSTR loci. These findings demonstrate that the kit of this invention possesses excellent individual-specific identification capabilities.

[0064] Example 2 Sensitivity Test

[0065] 2800 (male) DNA (10 ng / μL) (purchased from Promega) was used as a positive control and diluted to 15.625 pg, 31.25 pg, and 62.5 pg, respectively. Each diluted template DNA was subjected to PCR amplification using the kit of this invention. The amplification results were repeated 10 times to verify the sensitivity of the kit. The results showed that the minimum DNA detection limit of the kit was 31.25 pg.

[0066] Example 3: Organizational Identity Test

[0067] Five 50g samples each of muscle tissue, bone tissue, brain tissue, heart, kidney, and skin from three fresh male cadavers were collected. Bloodstains (over 3cm) were extracted using medical sterile gauze or medium-speed filter paper. The kit of this invention was used to perform PCR amplification on the muscle tissue, bone tissue, brain tissue, heart, kidney, skin, and bloodstains. The amplification results were repeated 10 times to verify the tissue identity of the kit. The results showed that the muscle tissue, bone tissue, brain tissue, heart, kidney, skin, and bloodstain samples from the three male cadavers amplified well and showed consistent typing, proving that the kit of this invention has good tissue identity.

[0068] Example 4 Species-specific test

[0069] Blood samples were collected from pigs, dogs, sheep, cattle, rabbits, chickens, ducks, and carp. DNA was extracted from the animal blood samples using the Chelex-100 method. A 2800 (male) DNA sample was used as a positive control, and deionized water as a negative control. PCR amplification was performed on all the above samples using the kit of this invention. The amplification results were repeated 10 times to verify the species specificity of the kit. The results showed that no amplification product peaks appeared in the eight common animal species within the genotyping region, demonstrating that the kit of this invention has good species specificity.

[0070] Example 5: Artificial DNA Degradation Model Test

[0071] DNA degradation model was created using DNase I (NEB, Ipswich, MA): 3 μg of whole blood DNA was digested with 0.01 U / μL DNase I at 37°C for 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, and 60 min, respectively. The samples were then amplified using the kit of this invention and GlobalFiler. The results are shown in Table 1.

[0072] Table 1. Genotyping results of the artificially degraded DNA model

[0073]

[0074] Note: This indicates that the peak height of a successfully genotyped locus exceeds 100 RFU; for example, 21 / 24 means that the number of identifiable loci is 24, and the number of successfully genotyped loci is 21.

[0075] The results showed that, for degraded DNA samples, the miniSTR kit of the present invention has a higher success rate in DNA sample typing compared to GlobalFiler, and can more accurately and effectively amplify small fragments and degraded DNA samples.

[0076] Example 6: Testing Naturally Degraded DNA Samples

[0077] Twelve old bone samples (5-10 years old), ten old teeth samples (5-10 years old), eight burned tissue fragments, and ten water-soaked skin samples (soaking time > 1 month) were collected and amplified using the kit of this invention and GlobalFiler. The results are shown in Table 2.

[0078] Table 2 Comparison of Genotyping Success Rates of Naturally Degraded DNA Samples

[0079]

[0080] Note: Genotyping success rate refers to the proportion of samples that obtain complete genotyping of at least 10 loci.

[0081] The results showed that GlobalFiler had a low typing success rate for naturally degraded DNA samples, making accurate typing difficult. The miniSTR kit of this invention had a significantly higher typing success rate than GlobalFiler and could be used as a supplement to GlobalFiler and other STR kits for accurate typing of naturally degraded DNA samples.

[0082] In this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0083] Although specific embodiments of the invention have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of the invention. All such modifications or alterations should fall within the scope of the appended claims.

Claims

1. A primer set for human autosomal miniSTR typing, characterized in that, It consists of 10 pairs of STR primers for simultaneous multiplexing amplification of 10 miniSTR loci, wherein the 10 miniSTR loci are: D3S1358, D21S11, D18S51, D7S820, D15S131, D15S12, D15S146, D16S539, D5S818, and TPOX; The STR primers for amplifying D3S1358 consist of an upstream primer D3S1358_mini_F and a downstream primer D3S1358_mini_R; the STR primers for amplifying D21S11 consist of an upstream primer D21S11_mini_F and a downstream primer D21S11_mini_R; the STR primers for amplifying D18S51 consist of an upstream primer D18S51_mini_F and a downstream primer D18S51_mini_R; the STR primers for amplifying D7S820 consist of an upstream primer D7S820_mini_F and a downstream primer D7S820_mini_R; and the STR primers for amplifying D15S131 consist of an upstream primer D15S131_mini_F and a downstream primer D15S131_mini_R. The STR primers for amplifying D15S12 consist of an upstream primer D15S12_mini_F and a downstream primer D15S12_mini_R; the STR primers for amplifying D15S146 consist of an upstream primer D15S146_mini_F and a downstream primer D15S146_mini_R; the STR primers for amplifying D16S539 consist of an upstream primer D16S539_mini_F and a downstream primer D16S539_mini_R; the STR primers for amplifying D5S818 consist of an upstream primer D5S818_mini_F and a downstream primer D5S818_mini_R; and the STR primers for amplifying TPOX consist of an upstream primer TPOX_mini_F and a downstream primer TPOX_mini_R. The D3S1358_mini_F is shown in SEQ ID No. 1; The D3S1358_mini_R is shown in SEQ ID No. 2; The D21S11_mini_F is shown in SEQ ID No. 3; The D21S11_mini_R is shown in SEQ ID No. 4; The D18S51_mini_F is shown in SEQ ID No. 5; The D18S51_mini_R is shown in SEQ ID No. 6; The D7S820_mini_F is shown in SEQ ID No. 7; The D7S820_mini_R is shown in SEQ ID No. 8; The D15S131_mini_F is shown in SEQ ID No. 9; The D15S131_mini_R is shown in SEQ ID No. 10; The D15S12_mini_F is shown in SEQ ID No. 11; The D15S12_mini_R is shown in SEQ ID No. 12; The D15S146_mini_F is shown in SEQ ID No. 13; The D15S146_mini_R is shown in SEQ ID No. 14; The D16S539_mini_F is shown in SEQ ID No. 15; The D16S539_mini_R is shown in SEQ ID No. 16; The D5S818_mini_F is shown in SEQ ID No. 17; The D5S818_mini_R is shown in SEQ ID No. 18; The TPOX_mini_F is shown in SEQ ID No. 19; The TPOX_mini_R is shown in SEQ ID No.

20.

2. The primer set according to claim 1, characterized in that, The D3S1358_mini_F, the D3S1358_mini_R, the D21S11_mini_F, the D21S11_mini_R, the D18S51_mini_F, the D18S51_mini_R, the D7S820_mini_F, the D7S820_mini_R, the D15S131_mini_F, the D15S131_mini_R, and the D15S12_mini The 5' ends of ni_F, D15S12_mini_R, D15S146_mini_F, D15S146_mini_R, D16S539_mini_F, D16S539_mini_R, D5S818_mini_F, D5S818_mini_R, TPOX_mini_F, and TPOX_mini_R are each independently connected to fluorescent markers of the same or different colors.

3. The primer set according to claim 2, characterized in that, The fluorescent marker is selected from one or more of ROX, FAM, HEX, TAMRA, VIC, NED, PET, LIZ, and Cy5.

4. A kit for human autosomal miniSTR typing, characterized in that, Includes the primer set as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, It also includes fluorescent dye correctors, which consist of 70, 100, 125, 150, 175, 200, 225, 250, 275, and 300 bp amplification products labeled with fluorescent markers.

6. A method for miniSTR typing of degraded DNA samples, characterized in that, The degraded DNA sample is amplified using the kit described in claim 4 or 5; the method is for non-therapeutic and non-diagnostic purposes.

7. The method according to claim 6, characterized in that, The degraded DNA samples include environmentally exposed samples, old and historical samples, forensic special evidence samples, pathologically processed samples, and trace and micro-samples.

8. Use of the primer set according to any one of claims 1-3, or the kit according to claim 4 or 5, in individual identification, paternity testing, population genetic analysis, and / or construction of a human DNA database.

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