CN112852971A

CN112852971A - Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit

Info

Publication number: CN112852971A
Application number: CN202011622301.XA
Authority: CN
Inventors: 冉凌飞; 蒿杰; 刘甲乾; 赵亚楠; 路瑶
Original assignee: Biotech Original Biotechnology Beijing Co ltd
Current assignee: Biotech Original Biotechnology Beijing Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-05-28

Abstract

The invention provides a primer group for simultaneously amplifying 44 human Y-STR loci, a kit and application thereof, belonging to the technical field of molecular genetics. In the invention, the 44Y-STR loci comprise 41Y chromosome STR loci and 3Y-indel loci, and comprise all loci of a mainstream kit in the market; the invention can realize the simultaneous amplification of 44 loci in one reaction, can reduce the amplification time, and improve the identification efficiency and the material detection adaptability of the kit.

Description

Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit

Technical Field

The invention relates to the technical field of molecular genetics, in particular to a primer group and a kit for simultaneously amplifying 44 human Y-STR loci and application thereof.

Background

STR (short tandem repeats), also known as microsatellite sequences, is a short tandem repeat that is present in large amounts in human genomic DNA and has a repeat unit of 2-6 nucleotides. Because of the high polymorphism and stability, and the much smaller length of amplified product (less than 500bp) compared to the AMP-FLP and VNTR isotyping methods, STR genotyping requires less template quality and allows analysis even with degraded DNA templates. In addition, STR typing is suitable for DNA purified by a variety of DNA purification methods, which often yield insufficient amounts of DNA for Southern blot analysis. In view of the above characteristics, STR typing techniques have been widely used for identification.

The currently published 20Y-STR core loci (20 loci, including DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATAH4, DYS481, DYS533, DYS576) and 15Y-STR preferred loci (15 loci, including DYS643, DYS460, DYS549, DYS387S1a/b, DYS449, DYS518, DYS570, DYS527 a/b, DYS447, DYS437, DYS557, DYS 596) have a higher demand for the number, specificity and sensitivity of the DNA identification to the loci of the kit. Most of the current kits on the market only contain 29 cores and preferred loci, the kits of each family on the preferred loci are different, the use process is very inconvenient, multiple kits are usually needed to be used in combination to obtain more locus information, and more time is also needed to be consumed.

Disclosure of Invention

The invention aims to provide a primer set and a kit for amplifying 44 human Y-STR loci simultaneously and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a primer group for simultaneously amplifying 44Y-STR loci of a human, wherein the 44Y-STR loci comprise 41Y chromosome STR loci and 3Y-indel loci; the 41Y chromosome STR loci are respectively: DYS576, DYS392, DYS481, DYS549, DYS596, DYS627, dysf 404S1a/b, DYS393, DYS391, DYS390, DYS448, DYS449, DYS570, DYS593, dysf 456, DYS19, Y _ GATA _ H4, DYS533, DYS527, DYS557, DYS437, DYS522, DYS438, DYS389I, DYS635, DYS389II, DYS518, DYS645 388, DYS460, DYS458, DYS437, DYS439, DYS385a/b, DYS387S1a/b and DYS 643; the 3Y-indel loci are respectively: rs759551978, rs2032678 and rs 771783753;

the nucleotide sequence of the upstream primer for amplifying rs759551978 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying rs2032678 is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS576 is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS392 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS481 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS549 is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS596 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS627 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYF404S1a/b is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying rs771783753 is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS393 is shown in SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22; the nucleotide sequence of the upstream primer for amplifying 3DYS391 is shown as SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24; the nucleotide sequence of the upstream primer for amplifying DYS390 is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 26 is shown; the nucleotide sequence of the upstream primer for amplifying DYS448 is shown as SEQ ID NO: 27, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 28 is shown; the nucleotide sequence of the upstream primer for amplifying DYS449 is shown as SEQ ID NO: 29, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 30 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS570 is shown as SEQ ID NO: 31, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 32 is shown; the nucleotide sequence of the upstream primer for amplifying DYS593 is shown as SEQ ID NO: 33, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 34; the nucleotide sequence of the upstream primer for amplifying DYS456 is shown as SEQ ID NO: 35, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 36 is shown; the nucleotide sequence of the upstream primer for amplifying DYS19 is shown as SEQ ID NO: 37, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 38; the nucleotide sequence of the upstream primer for amplifying Y _ GATA _ H4 is shown as SEQ ID NO: 39, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 40 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS533 is shown as SEQ ID NO: 41, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 42 is shown; the nucleotide sequence of the upstream primer for amplifying DYS527 is shown as SEQ ID NO: 43, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 44 is shown; the nucleotide sequence of the upstream primer for amplifying DYS557 is shown as SEQ ID NO: 45, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 46; the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 47, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 48 is shown; the nucleotide sequence of the upstream primer for amplifying DYS522 is shown as SEQ ID NO: 49, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 50 is shown; the nucleotide sequence of the upstream primer for amplifying DYS438 is shown as SEQ ID NO: 51, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 52; the nucleotide sequence of the upstream primer for amplifying DYS389I is shown as SEQ ID NO: 53, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 54 is shown; the nucleotide sequence of the upstream primer for amplifying DYS635 is shown as SEQ ID NO: 55, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 56 is shown; the nucleotide sequence of the upstream primer for amplifying 3DYS389II is shown as SEQ ID NO: 57, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 58; the nucleotide sequence of the upstream primer for amplifying DYS447 is shown as SEQ ID NO: 59, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 60 is shown; the nucleotide sequence of the upstream primer for amplifying DYS518 is shown as SEQ ID NO: 61, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 62; the nucleotide sequence of the upstream primer for amplifying DYS388 is shown as SEQ ID NO: 63, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 64 is shown; the nucleotide sequence of the upstream primer for amplifying DYS645 is shown as SEQ ID NO: 65, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 66 is shown; the nucleotide sequence of the upstream primer for amplifying DYS460 is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 68; the nucleotide sequence of the upstream primer for amplifying DYS458 is shown as SEQ ID NO: 69, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 70 is shown; the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 71, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 72 is shown; the nucleotide sequence of the upstream primer for amplifying DYS439 is shown as SEQ ID NO: 73, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 74 is shown; the nucleotide sequence of the upstream primer for amplifying DYS385a/b is shown as SEQ ID NO: 75, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 76; the nucleotide sequence of the upstream primer for amplifying DYS387S1a/b is shown as SEQ ID NO: 77, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 78, respectively; the nucleotide sequence of the upstream primer for amplifying DYS643 is shown as SEQ ID NO: 79, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 80.

Preferably, the 44Y-STR loci are divided into five groups, wherein,

the first group includes: rs759551978, rs2032678, DYS576, DYS392, DYS481, DYS549, DYS596, DYS627 and DYF404S1 a/b;

the second group includes: rs771783753, DYS393, DYS391, DYS390, DYS448, DYS449, DYS570 and DYS 593;

the third group includes: DYS456, DYS19, Y _ GATA _ H4, DYS533, DYS527, DYS557, DYS437, and DYS 522;

the fourth group includes: DYS438, DYS389I, DYS635, DYS389II, DYS447, DYS518, DYS388 and DYS 645;

the fifth group includes: DYS460, DYS458, DYS437, DYS439, DYS385a/b, DYF387S1a/b and DYS 643;

the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.

The invention also provides a kit of the primer group in the scheme.

Preferably, the kit further comprises a PCR reaction premix and deionized water.

Preferably, the kit further comprises an internal molecular weight standard and an allelic ladder.

Preferably, in the primer set,

SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.37-0.47 mu M;

SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.04-0.14 mu M;

SEQ ID NO: 5 and SEQ ID NO: 6 is 0.03-0.13 mu M independently;

SEQ ID NO: 7 and SEQ ID NO: the concentration of the primers shown in 8 is 0.25-0.35 mu M independently;

SEQ ID NO: 9 and SEQ ID NO: 10 is 0.08-0.19 mu M independently;

SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.33-0.43 mu M;

SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is 0.1-0.2 mu M independently;

SEQ ID NO: 15 and SEQ ID NO: 16 is independently 0.31-0.41 mu M;

SEQ ID NO: 17 and SEQ ID NO: 18 is independently 0.11-0.21 mu M;

SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.05-0.15 mu M;

SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.13-0.22 mu M;

SEQ ID NO: 23 and SEQ ID NO: the concentration of the primers shown in 24 is 0.22-0.32 mu M independently;

SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.12-0.21 mu M;

SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.05-0.13 mu M;

SEQ ID NO: 29 and SEQ ID NO: 30 is independently 0.26-0.36 mu M;

SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.23-0.32 mu M;

SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.33-0.42 mu M;

SEQ ID NO: 35 and SEQ ID NO: the concentration of the primers shown by 36 is 0.04-0.12 mu M independently;

SEQ ID NO: 37 and SEQ ID NO: 38 is 0.16-0.21 mu M independently;

SEQ ID NO: 39 and SEQ ID NO: 40 is independently 0.07-0.15 mu M;

SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.12-0.18 mu M;

SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.05-0.14 mu M;

SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.04-0.13 mu M;

SEQ ID NO: 47 and SEQ ID NO: 48 is 0.08-0.17 mu M independently;

SEQ ID NO: 49 and SEQ ID NO: the concentration of the primers shown by 50 is 0.1-0.2 mu M independently;

SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.33-0.41 mu M;

SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.267-0.32 mu M;

SEQ ID NO: 55 and SEQ ID NO: 56 is 0.04-0.14 mu M independently;

SEQ ID NO: 57 and SEQ ID NO: the concentration of the primer shown as 58 is 0.03-0.1 mu M independently;

SEQ ID NO: 59 and SEQ ID NO: the concentration of the primers shown as 60 is 0.38-0.48 mu M independently;

SEQ ID NO: 61 and SEQ ID NO: the concentration of the primers shown in 62 is 0.1-0.19 mu M independently;

SEQ ID NO: 63 and SEQ ID NO: the concentration of the primers shown by 64 is 0.34-0.42 mu M independently;

SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.35-0.45 mu M;

SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.18-0.25 μ M;

SEQ ID NO: 69 and SEQ ID NO: 70 is independently 0.16-0.25 mu M;

SEQ ID NO: 71 and SEQ ID NO: 72 is 0.04-0.08 mu M independently;

SEQ ID NO: 73 and SEQ ID NO: 74 is 0.07-0.12 mu M independently;

SEQ ID NO: 75 and SEQ ID NO: 76 is 0.09-0.18 mu M independently;

SEQ ID NO: 77 and SEQ ID NO: 78 is independently 0.03-0.12 mu M;

SEQ ID NO: 79 and SEQ ID NO: the concentration of the primer indicated by 80 is 0.14-0.23 mu M independently.

Preferably, the PCR reaction premix takes deionized water as a solvent and comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl₂50 to 100mM (NH)₄)₂SO₄0.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction.

Preferably, the allelic ladder and the Y chromosome STR locus correspond in relationship to one another:

in the first group: rs759551978 corresponds to 1, 2; rs2032678 corresponds to 1, 2; DYS576 corresponds to 11,12,13,14,15,16,17,18,19,20,21,22, 23; DYS392 corresponds to 7,9,10,11,12,13,14,15,16,17,18,19,20, 21; DYS481 corresponds to 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31, 32; DYS549 corresponds to 7,8,9,10,11,12,13,14,15,16, 17; DYS596 corresponds to 12,13,14,15,16,17, 18; DYS627 corresponds to 15,16,17,18,19,20,21,22,23,24,25, 26; DYF404S1a/b corresponds to 11,12,13,14,15,16, 17;

in the second group: rs771783753 corresponds to 1, 2; DYS393 corresponds to 8,9,10,11,12,13,14,15, 16; DYS391 corresponds to 5,6,7,8,9,10,11,12,13,1,15, 16; DYS390 corresponds to 17,18,19,20,21,22,23,24,25,26, 27; DYS448 corresponds to 16,17,18,19,20,21,22,23, 24; DYS449 corresponds to 22,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,40,41, 42; DYS570 corresponds to 10,11,12,13,14,15,16,17,18,20,21,23,24, 25; DYS593 corresponds to 14,15,16,17, 18;

in the third group: DYS456 corresponds to 13,14,15,16,17,18, 19; DYS19 corresponds to 10,11,12,13,14,15,16,17,18,19, 20; y _ GATA _ H4 corresponds to 8,9,10,11,12,13,14 corresponds to DYS 533: 7,8,9,10,11,12,13,14,15, 16; DYS527 corresponds to 13,14,15,16,17,19,20,21,22,23,24,25,26, 27; DYS557 corresponds to 12,13,14,15,16,17,18,19,20, 21; DYS437 corresponds to 10,11,12,13,14,15, 16; DYS522 corresponds to 9,10,11,12,13, 14.

In the fourth group: DYS438 corresponds to 9,10,11,12,13, 14; DYS389I corresponds to 10,11,12,13,14, 15; DYS635 corresponds to 17,18,19,20,21,22,23,24,25, 26; DYS389II corresponds to 24,25,26,27,28,29,30,31,32,33, 34; DYS447 corresponds to 22,23,24,25,26,27,28,29, 30; DYS518 corresponds to 32,33,34,35,36,37,38,39,40, 41; DYS388 corresponds to 9,10,11,12,13,14, 15; DYS645 corresponds to 7,8, 9;

in the fifth group: DYS460 corresponds to 8,9,10,11,12,13, 14; DYS458 corresponds to 12,14,15,16,17,18,18.2,19,20,21, 22; DYS437 corresponds to 13,14,15, 16; DYS439 corresponds to 9,10,11,12,13,14, 15; DYS385a/b corresponds to 9,10,11,12,13,14,15,16,17,19,20,21,22, 24; DYF387S1a/b corresponds to 33,34,35,36,37,38,39, 40; DYS643 corresponds to 6,8,9,10,11,12,13,14,16, 17.

The invention also provides application of the primer group or the kit in the scheme in individual identification, DNA database construction, family investigation or genetic relationship identification.

Preferably, the application comprises the following steps:

putting a sample to be detected into an amplification system containing the primer group in the scheme, performing PCR amplification to obtain an amplification product, and detecting the amplification product;

when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;

when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;

the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 90sec, 71 ℃, 50sec, 28 cycles; 60 deg.C, 30 min.

The invention has the beneficial effects that: the invention provides a primer group for simultaneously amplifying 44Y-STR loci of a human, wherein the 44Y-STR loci comprise 41Y chromosome STR loci and 3Y-indel loci; the 44Y-STR loci have certain compatibility with the loci of market mainstream kits, and can share and communicate with the existing DNA data. The 44Y-STR loci comprise 20Y-STR core loci, 15 preferred Y-STR loci and 9 alternative Y-STR loci, and comprise all locus loci of the mainstream kit in the market at present. Meanwhile, the invention also comprises 3Y-indel loci, so that the risk of sex identification error caused by Y chromosome deletion can be effectively prevented. The 44 gene loci are combined to provide more information, have the characteristics of high individual identification power and high non-paternity exclusion rate, and can be applied to the research fields of paternity test, individual identification, anthropology and the like. The primer group disclosed by the invention can realize rapid amplification, has strong amplification specificity and no non-specific amplification, and simultaneously, the primers do not interfere with each other and do not form primer dimers. The length of each primer is 18-32 bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 44 pairs of primers have no interaction, so that the primers corresponding to 44 loci can be compatible with one single tube. The length of the overall amplification product is between 70 and 530 bp. After amplification reaction, 1-2 specific amplification bands can be obtained from each locus, and non-specific amplification peaks, primer peaks and other miscellaneous peaks are avoided. The primer group also has the advantage of high sensitivity, can be suitable for the amplification detection of the autosomal STR of various test materials, and can meet the requirements of multifunctional STR identification of current identification, DNA database construction and genetic relationship identification.

Drawings

FIG. 1 is a positive control DNA9948 genotyping map;

FIG. 2 is a ladder allelic typing map;

FIG. 3 is a BTY-550 spectrum of a molecular weight internal standard;

FIG. 4 is a chart of suspected subgenomic typing;

FIG. 5 is a suspected father genotyping map;

FIG. 6 is a blood card test material genotyping chart;

FIG. 7 is a genotyping chart of the extracted DNA sample.

Detailed Description

The 44 loci examined by the present invention comprise: the 41Y chromosome STR loci are: DYS576, DYS392, DYS481, DYS549, DYS596, DYS627, dysf 404S1a/b, DYS393, DYS391, DYS390, DYS448, DYS449, DYS570, DYS593, DYS456, DYS19, Y _ GATA _ H4, DYS533, DYS527, DYS557, DYS444, DYS522, DYS438, DYS389I, DYS635, DYS389II, DYS447, DYS518, DYS388 645, DYS460, DYS458, DYS437, DYS439, DYS385a/b, DYS387S1a/b, DYS643, 3Y-indel gene loci rs 7551978, rs 2678, rs 7717853; the primer set of the present invention comprises 44 pairs of primers, each corresponding to 44 loci involved in the present invention. Aiming at 44 loci, 44 pairs of primers are designed, the length of the primers is between 18bp and 30bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 44 pairs of primers have no interaction, so that the primers corresponding to the 44 loci can be compatible in a single tube. The length of the total amplification product is between 70 and 520 bp. After amplification reaction, 1-2 specific amplification bands can be obtained from each locus, and non-specific amplification peaks, primer peaks and other miscellaneous peaks are avoided.

The 44Y-STR loci comprise 20 core loci and 15 preferred loci, and comprise all locus loci of the mainstream kit in the market at present. Meanwhile, the invention also comprises 1Y-indel locus and 9 alternative loci, and can effectively prevent the risk of sex identification error caused by Y chromosome deletion. The 44 gene loci are combined to provide more information, have the characteristics of high individual identification power and high non-paternity exclusion rate, and can be applied to the research fields of paternity test, individual identification, anthropology and the like. The Y-STR locus in the primer set of the invention is compared with the sites of the mainstream kit in the market, and the comparison is shown in Table 1.

Table 1 comparison of the Y-STR locus in the primer set of the invention with the commercial mainstream kit sites is shown in table 1.

In the invention, the amplification balance of the primer group comprehensively exceeds the standard (more than or equal to 50% in the same color group and more than or equal to 30% among different color groups) in GB/T37226-2018 court science human fluorescence labeling STR composite amplification detection reagent quality basic requirements.

In the present invention, the 44Y-STR loci are preferably divided into five groups, wherein the first group comprises: rs759551978, rs2032678, DYS576, DYS392, DYS481, DYS549, DYS596, DYS627 and DYF404S1 a/b; the second group includes: rs771783753, DYS393, DYS391, DYS390, DYS448, DYS449, DYS570 and DYS 593; the third group includes: DYS456, DYS19, Y _ GATA _ H4, DYS533, DYS527, DYS557, DYS437, and DYS 522; the fourth group includes: DYS438, DYS389I, DYS635, DYS389II, DYS447, DYS518, DYS388 and DYS 645; the fifth group includes: DYS460, DYS458, DYS437, DYS439, DYS385a/b, DYF387S1a/b and DYS 643; the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.

In the present invention, the fluorescent dye used in the first group is preferably FAM; the fluorescent dye adopted by the second group is preferably HEX; the fluorescent dye used in the third group is preferably TAMAR; the fluorescent dye adopted by the fourth group is preferably ROX; the fluorescent dye adopted in the fifth group is preferably PURPLE; ORG was used as internal standard. The invention adopts six-color fluorescent labeling systems, namely FAM, HEX, TAMAR, ROX, PURPLE and ORG. Wherein FAM represents blue, HEX represents green, TAMAR represents yellow, ROX represents red, PURPLE represents PURPLE, and ORG represents orange. The invention can amplify 44 gene loci simultaneously in one reaction, and fully satisfies the compatibility of the current DNA database comparison.

In the present invention, the 5' end of the upstream primer and/or the downstream primer of each of the Y-STR loci is labeled with a fluorescent dye. The invention uses the fluorescence labeling method to label a fluorescent dye at the 5' end of the primer, the PCR product can emit optical signals with specific wavelength under the laser excitation state, the optical signals can be collected by electrophoresis detection through a genetic analyzer (ABI 3130/ABI 3500/ABI 3730 series one by one), and the detection is carried out through the collected optical signals.

The invention also provides a kit comprising the primer group in the scheme.

In the present invention, the kit preferably further comprises a PCR reaction premix and deionized water. In the present invention, the kit preferably further comprises a positive control DNA 9948. In the invention, the primer group, the PCR reaction premixed solution and the deionized water form a reaction system of the kit.

In the present invention, in the primer set, SEQ ID NO: 1 and SEQ ID NO: 2 is independently preferably 0.37-0.47 mu M, and more preferably 0.417 mu M; SEQ ID NO: 3 and SEQ ID NO: 4, the concentration of the primer is preferably 0.04-0.14 mu M independently, and more preferably 0.092 mu M independently; SEQ ID NO: 5 and SEQ ID NO: 6 is independently preferably 0.03-0.13 mu M, and more preferably 0.079 mu M; SEQ ID NO: 7 and SEQ ID NO: 8, the concentration of the primer is preferably 0.25-0.35 mu M independently, and more preferably 0.296 mu M; SEQ ID NO: 9 and SEQ ID NO: 10 is preferably 0.08-0.19. mu.M, and more preferably 0.136. mu.M; SEQ ID NO: 11 and SEQ ID NO: 12 is independently preferably 0.33 to 0.43. mu.M, and more preferably 0.382. mu.M; SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is independently preferably 0.1-0.2 mu M, and further preferably 0.146 mu M; SEQ ID NO: 15 and SEQ ID NO: 16 is independently preferably 0.31-0.41 mu M, and more preferably 0.356 mu M; SEQ ID NO: 17 and SEQ ID NO: 18 is independently preferably 0.11-0.21. mu.M, and more preferably 0.159. mu.M; SEQ ID NO: 19 and SEQ ID NO: 20 is independently preferably 0.05-0.15 mu M, and more preferably 0.098 mu M; SEQ ID NO: 21 and SEQ ID NO: 22 is independently preferably 0.13-0.22 mu M, and more preferably 0.184 mu M; SEQ ID NO: 23 and SEQ ID NO: 24 is independently preferably 0.22-0.32. mu.M, and more preferably 0.268. mu.M; SEQ ID NO: 25 and SEQ ID NO: 26 is independently preferably 0.12-0.21 mu M, and further preferably 0.066 mu M; SEQ ID NO: 27 and SEQ ID NO: 28, the concentration of the primer is preferably 0.05-0.13 mu M independently, and more preferably 0.083 mu M; SEQ ID NO: 29 and SEQ ID NO: 30 is independently preferably 0.26-0.36 mu M, and more preferably 0.314 mu M; SEQ ID NO: 31 and SEQ ID NO: 32 is independently preferably 0.23-0.32 mu M, and more preferably 0.277 mu M; SEQ ID NO: 33 and SEQ ID NO: 34, the concentration of the primer is preferably 0.33-0.42 mu M independently, and more preferably 0.378 mu M; SEQ ID NO: 35 and SEQ ID NO: the concentration of the primer shown as 36 is preferably 0.04-0.12 mu M independently, and more preferably 0.08 mu M independently; SEQ ID NO: 37 and SEQ ID NO: 38 is independently preferably 0.16-0.21. mu.M, and more preferably 0.107. mu.M; SEQ ID NO: 39 and SEQ ID NO: 40 is independently preferably 0.07-0.15 mu M, and more preferably 0.103 mu M; SEQ ID NO: 41 and SEQ ID NO: 42 is independently preferably 0.12-0.18 mu M, and more preferably 0.166 mu M; SEQ ID NO: 43 and SEQ ID NO: 44 is independently preferably 0.05-0.14 mu M, and more preferably 0.082 mu M; SEQ ID NO: 45 and SEQ ID NO: 46 is independently preferably 0.04-0.13 mu M, and further preferably 0.086 mu M; SEQ ID NO: 47 and SEQ ID NO: 48 is preferably 0.08-0.17 mu M independently, and more preferably 0.128 mu M independently; SEQ ID NO: 49 and SEQ ID NO: the concentration of the primer shown by 50 is preferably 0.1-0.2 mu M independently, and more preferably 0.16 mu M; SEQ ID NO: 51 and SEQ ID NO: 52 is independently preferably 0.33-0.41 mu M, and more preferably 0.372 mu M; SEQ ID NO: 53 and SEQ ID NO: 54 is independently preferably 0.267-0.32 mu M, and more preferably 0.725 mu M; SEQ ID NO: 55 and SEQ ID NO: 56, the concentration of the primer is preferably 0.04-0.14 mu M independently, and more preferably 0.084 mu M independently; SEQ ID NO: 57 and SEQ ID NO: the concentration of the primer shown by 58 is preferably 0.03-0.1 mu M independently, and more preferably 0.046 mu M; SEQ ID NO: 59 and SEQ ID NO: the concentration of the primer shown as 60 is 0.38-0.48 mu M independently and preferably 0.426 mu M; SEQ ID NO: 61 and SEQ ID NO: 62 is independently preferably 0.1-0.19. mu.M, and more preferably 0.149. mu.M; SEQ ID NO: 63 and SEQ ID NO: 64 is independently preferably 0.34-0.42 mu M, and more preferably 0.388 mu M; SEQ ID NO: 65 and SEQ ID NO: 66 is independently preferably 0.35-0.45 mu M, and more preferably 0.402 mu M; SEQ ID NO: 67 and SEQ ID NO: 68 is independently preferably 0.18-0.25. mu.M, and more preferably 0.204. mu.M; SEQ ID NO: 69 and SEQ ID NO: 70 is preferably 0.16-0.25. mu.M, and more preferably 0.203. mu.M; SEQ ID NO: 71 and SEQ ID NO: 72 is preferably 0.04-0.08 mu M independently, and more preferably 0.054 mu M independently; SEQ ID NO: 73 and SEQ ID NO: 74 is independently preferably 0.07-0.12. mu.M, and more preferably 0.098. mu.M; SEQ ID NO: 75 and SEQ ID NO: 76 is preferably 0.09-0.18 mu M independently, and is further preferably 0.131 mu M independently; SEQ ID NO: 77 and SEQ ID NO: 78, the concentration of the primer is preferably 0.03-0.12 mu M independently, and more preferably 0.077 mu M; SEQ ID NO: 79 and SEQ ID NO: the concentration of the primer represented by 80 is preferably 0.14 to 0.23. mu.M, and more preferably 0.185. mu.M, independently.

In the present invention, the PCR reaction premix preferably includes hot-start DNA polymerase, dNTP, magnesium ions, potassium ions, Tris buffer and enhancer; the PCR reaction premix takes deionized water as a solvent, and preferably comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl₂50 to 100mM (NH)₄)₂SO₄0.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction. In one embodiment of the present invention, the recipe for the PCR reaction premix is shown in Table 2.

TABLE 2 PCR reaction premix formula of the inventive example

H in Table 2₂O is deionized water which is highly deionized water, the resistivity reaches 18.2M omega, and the interference of external ions on an amplification system is completely eliminated.

In the invention, the PCR reaction premix has the characteristics of high speed, high sensitivity, strong adaptability and the like, and can not freeze at the temperature of-20 ℃, thereby effectively preventing the influence of freeze thawing on the performance of the reagent. The sensitivity of the PCR reaction premix liquid is as high as 0.03ng DNA, the effective amplification time is reduced to be within 1.5h, the PCR reaction premix liquid has excellent amplification effect on DNA from different sources (blood, blood stains, semen, seminal stains, saliva stains, hair, tissues, nails, body fluid and the like), and meanwhile, the PCR reaction premix liquid also has very good amplification effect on special blood cards, saliva cards and FTA cards.

In the invention, the positive control DNA9948 is used as an amplification standard substance for testing the quality of an amplification system. In the preferred embodiment of the invention, the positive control can be correctly typed, and the sensitivity is as high as 0.03ng and is far lower than the standard 0.125 ng. The 9948 fractal pattern amplified by the present invention is shown in FIG. 1 and Table 3.

TABLE 39948 genotyping

In the present invention, the kit preferably further comprises an internal molecular weight standard and an allelic ladder. In the present invention, the molecular weight internal standard and the allele ladder are detection reagents of the kit. In the present invention, the correspondence between the allelic ladder and the Y chromosome STR locus is specifically shown in table 4.

TABLE 4 correspondence of allelic ladders and the Y chromosome STR loci

In the present invention, the allele ladder encompasses all the most common alleles and most rare alleles at each locus, which is very convenient for data analysis and comparison of genotyping results.

In the present invention, the molecular weight internal standard is BTY-550; the BTY-550 comprises the following DNA fragments: 65. 75, 100, 139, 150, 160, 200, 250, 300, 340, 400, 450, 490, 500, 540, and 550. In the present invention, the internal molecular weight standard is labeled with the fluorescent dye ORG, representing orange. The molecular weight internal standard can effectively distinguish the sizes of 65-550 bp DNA fragments.

In the specific implementation process of the invention, the kit comprises two parts, wherein the first part is a reaction system and comprises a primer group, a reaction premix, a positive control DNA9948 and deionized water; the second part is a detection reagent comprising a molecular weight internal standard and an allele ladder.

The invention also provides application of the primer group and the kit in the scheme in individual identification, DNA database construction, family investigation or genetic relationship identification.

In the present invention, the application preferably comprises the steps of:

In the present invention, the sample to be detected preferably includes human blood, blood stain, semen, seminal stain, saliva, salivary stain, hair, tissue, nail or body fluid.

In the present invention, the standard amplification system for PCR amplification is 25. mu.l, and comprises 5. mu.l of the mixture of the primer sets, 10. mu.l of the PCR reaction premix, 7.5. mu.l of deionized water, and 2.5. mu.l of the positive control DNA 9948. The amplification procedure for the standard amplification system is shown in Table 5.

TABLE 5 amplification procedure for the Standard amplification System

The method for detecting the amplification product of the present invention preferably includes: carrying out electrophoretic detection on the amplification product; the electrophoresis detection equipment is preferably ABI3500 XL; in the specific implementation process of the invention, HiDi formamide, molecular weight internal standard BTY-550 and PCR amplification product or allele ladder 44Y are mixed to obtain a mixture; taking the volume of HiDi formamide as 8.5 μ l, and measuring the molecular weight internal standard BTY-5500.5 μ l and PCR amplification product or allele step 44Y 1 μ l; after the mixture was dispensed into a 96-well plate, centrifugation was carried out to remove air bubbles, and the 96-well plate was placed in a sample application tray and then in a 3500Xl genetic analyzer at a sample application voltage of 1.8kVolts for 24sec, after which electrophoresis was started.

The present invention preferably further comprises analyzing the results of the electrophoresis on GeneMapper _ IDX software after said electrophoresis. The positive control 9948 typing map is shown in FIG. 1, and the allelic ladder map is shown in FIG. 2; molecular weight internal standard BTY-550 is shown in FIG. 3.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Two hairs from the suspected father and son were subjected to direct multiplex amplification, and 44 loci were detected. The amplification method adopts a direct amplification method, namely a section of hair with hair follicles is taken and directly put into an amplification system, so that the hair follicles are completely immersed in liquid, the standard amplification program is adopted in the amplification program, an ABI Proflex PCR instrument is adopted in an amplification instrument, a 3500xl genetic analyzer is adopted in a genetic analyzer, and GeneMapper-ID-X analysis software is adopted in analysis software. The operation steps of this embodiment are as follows:

first, 0.5cm above the hair follicles was cut short with scissors, and the cut hair with follicles was placed in a 200. mu.l PCR tube.

Preparing 10 mul amplification system according to the standard reaction system of the invention: mu.l of the primer mixture, 4. mu.l of the reaction premix, and 4. mu.l of deionized water were added to the PCR tube with cut hair. Performing PCR amplification on an ABI Proflex PCR instrument by the following amplification program: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 90sec, 71 ℃, 50sec, 28 cycles; 60 ℃ for 30 min; storing at constant temperature of 15 ℃.

Detecting and data analyzing by genetic analyzer

After PCR amplification is finished, the amplification product is detected on ABI3500 XL. 8.5. mu.l of HiDi formamide + 0.5. mu.l of molecular weight internal standard BTY-550+ 1. mu.l of PCR product/1. mu.l of allele step 44Y; after being divided into 96-well plates, the plates were centrifuged to remove air bubbles, and the 96-well plates were placed in a sample tray and then in a 3500Xl genetic analyzer at a sample injection voltage of 1.8kVolts for 24sec, after which electrophoresis was started.

Analysis was performed on GeneMapper IDX software after the end of electrophoresis, see fig. 4: a suspected gene fractal graph; FIG. 5: a suspected father genotyping map; the results of the suspected parent and child gene typing are shown in Table 6.

Table 6: suspected paternal genotyping

By analyzing and comparing the obtained genotypes of the 44 loci, the invention can find that the genetic information of the 44 loci of the suspected father and son conforms to the genetic rule, and at least can judge the same family relationship.

Example 2

The invention is mainly used for the construction and identity identification of DNA databases.

The detailed part of the operation is as follows:

the known human detection material is usually a blood card and can be directly amplified. Using a 1mm diameter punch, a 1mm diameter blood piece was punched directly from the dried blood card and placed in a 200. mu.l PCR tube.

Secondly, unknown personnel usually take case on-site examination materials which are complex, and the detection and analysis of data are usually carried out by adopting a method of firstly extracting DNA and then amplifying. DNA was extracted by the Chelex-100 and magnetic bead method. (the extraction method refers to DNA extraction of Zhengxiufen 'forensic DNA analysis' Chapter four).

Thirdly, the preparation and amplification of the reaction system, referring to the standard system of the invention, 10 mul of reaction system is prepared, as shown in Table 7 below. Performing PCR amplification on an ABI Proflex PCR instrument by the following amplification program: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 90sec, 71 ℃, 50sec, 28 cycles; 60 ℃ for 30 min; storing at constant temperature of 15 ℃.

Table 7: reaction System of this example

Fourthly, detecting by a genetic analyzer and analyzing data

And after the electrophoresis is finished, analyzing on GeneMapper _ IDX software, exporting the analyzed data to a CODIS format file after the analysis is finished, and uploading the exported data to a DNA database. FIG. 6 is a blood card sample typing chart, and FIG. 7 is a DNA extraction sample typing chart. As can be seen from FIGS. 6 and 7, the invention detects the direct amplification sample and the extracted DNA sample, has correct result and good graphic balance, and can completely meet the daily application.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Baite Yuan Biotechnology (Beijing) Ltd

<120> primer group and kit for simultaneously amplifying 44 human Y-STR loci and application thereof

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<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

aacgtggtgg taagtgcatt ttatcc 26

<210> 80

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

ataggaatga cacaataaac ttttgg 26

Claims

1. A primer group for simultaneously amplifying 44Y-STR loci of human, wherein the 44Y-STR loci comprise 41Y chromosome STR loci and 3Y-indel loci; the 41Y chromosome STR loci are respectively: DYS576, DYS392, DYS481, DYS549, DYS596, DYS627, dysf 404S1a/b, DYS393, DYS391, DYS390, DYS448, DYS449, DYS570, DYS593, dysf 456, DYS19, Y _ GATA _ H4, DYS533, DYS527, DYS557, DYS437, DYS522, DYS438, DYS389I, DYS635, DYS389II, DYS518, DYS645 388, DYS460, DYS458, DYS437, DYS439, DYS385a/b, DYS387S1a/b and DYS 643; the 3Y-indel loci are respectively: rs759551978, rs2032678 and rs 771783753;

the nucleotide sequence of the upstream primer for amplifying rs759551978 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the upstream primer for amplifying rs2032678 is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;

the nucleotide sequence of the upstream primer for amplifying DYS576 is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification;

the nucleotide sequence of the upstream primer for amplifying DYS392 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification;

the nucleotide sequence of the upstream primer for amplifying DYS481 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying DYS549 is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification;

the nucleotide sequence of the upstream primer for amplifying DYS596 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying DYS627 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying DYF404S1a/b is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying rs771783753 is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying DYS393 is shown in SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22;

the nucleotide sequence of the upstream primer for amplifying 3DYS391 is shown as SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24;

the nucleotide sequence of the upstream primer for amplifying DYS390 is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 26 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS448 is shown as SEQ ID NO: 27, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 28 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS449 is shown as SEQ ID NO: 29, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 30 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying DYS570 is shown as SEQ ID NO: 31, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 32 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS593 is shown as SEQ ID NO: 33, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 34;

the nucleotide sequence of the upstream primer for amplifying DYS456 is shown as SEQ ID NO: 35, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 36 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS19 is shown as SEQ ID NO: 37, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 38;

the nucleotide sequence of the upstream primer for amplifying Y _ GATA _ H4 is shown as SEQ ID NO: 39, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 40 is shown in the figure;

the nucleotide sequence of the upstream primer for amplifying DYS533 is shown as SEQ ID NO: 41, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 42 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS527 is shown as SEQ ID NO: 43, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 44 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS557 is shown as SEQ ID NO: 45, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 46;

the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 47, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 48 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS522 is shown as SEQ ID NO: 49, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 50 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS438 is shown as SEQ ID NO: 51, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 52;

the nucleotide sequence of the upstream primer for amplifying DYS389I is shown as SEQ ID NO: 53, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 54 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS635 is shown as SEQ ID NO: 55, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 56 is shown;

the nucleotide sequence of the upstream primer for amplifying 3DYS389II is shown as SEQ ID NO: 57, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 58;

the nucleotide sequence of the upstream primer for amplifying DYS447 is shown as SEQ ID NO: 59, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 60 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS518 is shown as SEQ ID NO: 61, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 62;

the nucleotide sequence of the upstream primer for amplifying DYS388 is shown as SEQ ID NO: 63, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 64 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS645 is shown as SEQ ID NO: 65, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 66 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS460 is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 68;

the nucleotide sequence of the upstream primer for amplifying DYS458 is shown as SEQ ID NO: 69, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 70 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 71, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 72 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS439 is shown as SEQ ID NO: 73, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 74 is shown;

the nucleotide sequence of the upstream primer for amplifying DYS385a/b is shown as SEQ ID NO: 75, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 76;

the nucleotide sequence of the upstream primer for amplifying DYS387S1a/b is shown as SEQ ID NO: 77, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 78, respectively;

the nucleotide sequence of the upstream primer for amplifying DYS643 is shown as SEQ ID NO: 79, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 80.

2. The primer set of claim 1, wherein said 44Y-STR loci are grouped into five groups, wherein,

3. A kit comprising the primer set of claim 1 or 2.

4. The kit of claim 3, further comprising a PCR reaction premix and deionized water.

5. The kit of claim 3 or 4, further comprising an internal molecular weight standard and an allelic ladder.

6. The kit according to claim 3, wherein in the primer set,

SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.37-0.47 mu M;

SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.04-0.14 mu M;

SEQ ID NO: 5 and SEQ ID NO: 6 is 0.03-0.13 mu M independently;

SEQ ID NO: 9 and SEQ ID NO: 10 is 0.08-0.19 mu M independently;

SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.33-0.43 mu M;

SEQ ID NO: 15 and SEQ ID NO: 16 is independently 0.31-0.41 mu M;

SEQ ID NO: 17 and SEQ ID NO: 18 is independently 0.11-0.21 mu M;

SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.05-0.15 mu M;

SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.13-0.22 mu M;

SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.12-0.21 mu M;

SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.05-0.13 mu M;

SEQ ID NO: 29 and SEQ ID NO: 30 is independently 0.26-0.36 mu M;

SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.23-0.32 mu M;

SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.33-0.42 mu M;

SEQ ID NO: 37 and SEQ ID NO: 38 is 0.16-0.21 mu M independently;

SEQ ID NO: 39 and SEQ ID NO: 40 is independently 0.07-0.15 mu M;

SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.12-0.18 mu M;

SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.05-0.14 mu M;

SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.04-0.13 mu M;

SEQ ID NO: 47 and SEQ ID NO: 48 is 0.08-0.17 mu M independently;

SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.33-0.41 mu M;

SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.267-0.32 mu M;

SEQ ID NO: 55 and SEQ ID NO: 56 is 0.04-0.14 mu M independently;

SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.35-0.45 mu M;

SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.18-0.25 μ M;

SEQ ID NO: 69 and SEQ ID NO: 70 is independently 0.16-0.25 mu M;

SEQ ID NO: 71 and SEQ ID NO: 72 is 0.04-0.08 mu M independently;

SEQ ID NO: 73 and SEQ ID NO: 74 is 0.07-0.12 mu M independently;

SEQ ID NO: 75 and SEQ ID NO: 76 is 0.09-0.18 mu M independently;

SEQ ID NO: 77 and SEQ ID NO: 78 is independently 0.03-0.12 mu M;

7. The kit according to claim 4, wherein the PCR reaction premix solution uses deionized water as a solvent and comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl₂50 to 100mM (NH)₄)₂SO₄0.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction.

8. The kit of claim 5, wherein the allelic ladder and the Y chromosome STR locus correspond in relationship to one another as follows:

9. Use of the primer set according to claim 1 or 2 and the kit according to any one of claims 3 to 8 for individual identification, DNA database construction, family investigation or genetic relationship identification.

10. The application according to claim 9, characterized in that it comprises the following steps:

placing a sample to be detected into an amplification system containing the primer group of claim 1 or 2, performing PCR amplification to obtain an amplification product, and detecting the amplification product;