CN112852972A

CN112852972A - Primer group and kit for simultaneously amplifying 34 STR loci of human and application of primer group and kit

Info

Publication number: CN112852972A
Application number: CN202011627296.1A
Authority: CN
Inventors: 冉凌飞; 蒿杰; 刘甲乾; 路瑶; 赵亚楠
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 and a kit for simultaneously amplifying 34 STR loci of human and application thereof, belonging to the technical field of molecular genetics. In the invention, the 34 STR loci comprise 29 autosomal STR loci, 1Y chromosome STR locus and 4 individual identification STR loci; the invention can realize that 34 STR loci are amplified simultaneously in one reaction, comprises 20 core loci and 10 preferred loci specified by the ministry of public security, comprises all loci of a mainstream kit in the market at present, and also comprises 5 individual identification loci, thereby effectively preventing the risk of sex identification error caused by Y chromosome deletion. The 34 STR loci are combined together and have the characteristics of high individual recognition power and high non-father exclusion rate.

Description

Primer group and kit for simultaneously amplifying 34 STR loci of human 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 34 STR loci of human 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 DNA in amounts insufficient for Southern blot analysis. In view of the above characteristics, STR typing techniques have been widely applied to forensic identification.

The forensic DNA database integrates the elements of modern DNA inspection technology, information technology, network technology, scientific management and the like. The core of the forensic DNA database lies in the recorded STR data information, and a perfect forensic DNA database should contain an autosomal STR database and a Y chromosome STR database. The Y chromosome STR data provides investigation clues for cases, reduces investigation range, can predict regions, ethnicities or even surnames where criminals may exist, and the autosome STR data is used for screening and confirming final criminal individuals.

The currently published 20 autosomal STR core loci (20 loci, including Amelogenin, CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, D2S1338, D19S433, D6S1043, D12S391, Penta D, Penta E) and 10 autosomal STR preferred loci (10 loci, including D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253, D6S477, D10S1435 for expanded use) correspond to DNA therapeutics identifying the number of loci, specificity and the higher sensitivity of the kit. At present, autosomal STR amplification kits are mainly used for case detection and genetic relationship identification processes, various kits are abundant in the market, and although the kits all contain 13 CODIS core loci, other loci are different. The public security is very inconvenient in the using process, and in order to use more gene loci to judge criminal suspects, a plurality of kits are usually needed to be used in combination, and more time is also needed to be consumed.

Disclosure of Invention

The invention aims to provide a primer group and a kit for amplifying 34 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 amplifying 34 STR loci of human simultaneously, wherein the 34 STR loci comprise 29 autosomal STR loci, 1Y chromosome STR locus and 4 individual identification loci; the 29 autosomal STR loci are respectively: D3S1358, TH01, D21S11, D18S51, D2S1338, D15S659, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta E, D6S1043, D22S1045, D19S433, D1S1656, D12S391, D10S1248, D2S441, vWA, D8S1179, TPOX, FGA, D3S3045, D10S1435, D19S253, D6S477, D8S1132 and Penta D; the 1Y chromosome STR locus is as follows: DYS 391; the 4 individual identification loci are respectively: RS759551978, RS2032678, RS771783753 and Amelogenin;

the nucleotide sequence of the upstream primer for amplifying RS759551978 is shown in 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 the D3S1358 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 TH01 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 the D21S11 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 the D18S51 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 the D2S1338 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 the D15S659 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 RS771783753 is shown in 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 the Amelogenin 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 the D5S818 is shown as 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 the D13S317 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 the D7S820 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 the D16S539 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 CSF1PO 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 Penta E 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 the D6S1043 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 the D22S1045 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 the D19S433 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 the D1S1656 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 DYS391 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 the D12S391 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 the D10S1248 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 the D2S441 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 vWA 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 the D8S1179 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 TPOX 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 FGA 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 the D3S3045 is shown in 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 the D10S1435 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 the D19S253 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 the D6S477 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 the D8S1132 is shown in 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 Penta D is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 68.

Preferably, the 34 STR loci are divided into five groups, wherein,

the first group includes: RS759551978, RS2032678, D3S1358, TH01, D21S11, D18S51, D2S1338 and D15S 659; the second group includes: RS771783753, Amelogenin, D5S818, D13S317, D7S820, D16S539, CSF1PO and Penta E; the third group includes: D6S1043, D22S1045, D19S433, D1S1656, DYS391, D12S391 and D10S 1248; the fourth group includes: D2S441, vWA, D8S1179, TPOX and FGA; the fifth group includes: D3S3045, D10S1435, D19S253, D6S477, D8S1132 and PentaD; the upstream primer and/or the downstream primer of the first group to the fifth group STR loci respectively adopt different fluorescent dyes.

The invention also provides a kit comprising 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 0.3-0.4 mu M;

SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.12-0.18 mu M;

SEQ ID NO: 5 and SEQ ID NO: 6 is 0.08-0.13 mu M independently;

SEQ ID NO: 7 and SEQ ID NO: 8 is 0.1-0.15 mu M independently;

SEQ ID NO: 9 and SEQ ID NO: 10 is independently 0.2-0.25 mu M;

SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.2-0.25 mu M;

SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is 0.35-0.45 mu M independently;

SEQ ID NO: 15 and SEQ ID NO: 16 is 0.2-0.3 mu M independently;

SEQ ID NO: 17 and SEQ ID NO: 18 is 0.08-0.13 mu M independently;

SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.07-0.11 mu M;

SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.07-0.11 mu M;

SEQ ID NO: 23 and SEQ ID NO: the concentration of the primers shown in 24 is 0.1-0.15 mu M independently;

SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.4-0.5 mu M;

SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.1-0.15 mu M;

SEQ ID NO: 29 and SEQ ID NO: the concentration of the primers shown in 30 is 0.1-0.15 mu M independently;

SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.4-0.5 mu M;

SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.3-0.4 mu M;

SEQ ID NO: 35 and SEQ ID NO: the concentration of the primers shown in 36 is 0.12-0.18 mu M independently;

SEQ ID NO: 37 and SEQ ID NO: 38 is 0.3-0.4 mu M independently;

SEQ ID NO: 39 and SEQ ID NO: 40 is independently 0.15-0.2 mu M;

SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.2-0.3 mu M;

SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.3-0.4 mu M;

SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.3-0.4 mu M;

SEQ ID NO: 47 and SEQ ID NO: 48 is 0.12-0.18 mu M independently;

SEQ ID NO: 49 and SEQ ID NO: the concentration of the primers shown by 50 is 0.12-0.18 mu M independently;

SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.25-0.35 mu M;

SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.4-0.5 mu M;

SEQ ID NO: 55 and SEQ ID NO: 56 is 0.2-0.3 mu M independently;

SEQ ID NO: 57 and SEQ ID NO: the concentration of the primer shown as 58 is 0.08-0.13 mu M independently;

SEQ ID NO: 59 and SEQ ID NO: the concentration of the primers shown as 60 is 0.06-0.11 mu M independently;

SEQ ID NO: 61 and SEQ ID NO: the concentration of the primers shown in 62 is 0.1-0.15 mu M independently;

SEQ ID NO: 63 and SEQ ID NO: the concentration of the primers shown by 64 is 0.06-0.11 mu M independently;

SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.1-0.15 mu M;

SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.1-0.15. mu.M.

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 corresponding relationship between the allelic ladder and the STR locus is:

in the first group: RS759551978 corresponds to 1, 2; RS 2032678: 1, 2; D3S1358 corresponds to 10,12,13,14,15,16,17,18,19, 20; TH01 corresponds to 5,6,8,9,9.3, 10; D21S11 corresponds to 26,27,28,28.2,29,29.2,30,31,31.2,32,32.2,33,33.2,34,34.2,35.2,36.2, 38; D18S51 corresponds to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,26, 27; D2S1338 corresponds to 16,17,18,20,21,23,24,25, 26; D15S659 corresponds to 10,12,13,14,15,16,17,18,19, 20;

in the second group: rs771783753 corresponds to 1, 2; amelogenin corresponds to X, Y; D5S818 corresponds to 7,8,9,10,11,12,13,14, 15; D13S317 corresponds to 7,8,9,10,11,12,13, 14; D7S820 corresponds to 7,8,9,10,11,12,13,14, 15; D16S539 corresponds to 5,6,8,9,10,11,12, 13; CSF1PO corresponds to 6,7,8,9,10,11,12,13,14,15, 16; penta E corresponds to 5,6,7,8,9,10,11,12,13,14,15,16,17,19,20,21,22,23,24, 26;

in the third group: D6S1043 corresponds to 9,10,11,12,13,14,15,16,17,18,19,20, 21; D22S1045 corresponds to 11,12,14,15,16,17,18, 19; D19S433 corresponds to 9,10,11,11.2,12,13,13.2,14,14.2,15,15.2,16,16.2,17, 18.2; D1S1656 corresponds to 10,11,12,13,14,15,16,17, 18; DYS391 corresponds to 6,7,8,9,10,11,12, 13; D12S391 corresponds to 15,16,17,18,20,21,23,24,25,26, 27; D10S1248 corresponds to 10,11,12,13,14,15,16,17, 18;

in the fourth group: D2S441 corresponds to 8.1,9,10,11,12,13,14, 15; vWA correspond to 12,13,14,15,16,17,18,19,20, 21; D8S1179 corresponds to 7,8,9,10,11,12,13,14,15,16,17, 18; TPOX corresponds to 5,6,7,8,9,10,11,12,13, 14; FGA corresponds to 16,17,18,19,20,21,23,24,25,26,27,29,30,31.2,43.2,44.2,45.2, 46.2;

in the fifth group: D3S3045 corresponds to 9,12,13,13.2,14, 15; D10S1435 corresponds to 7,8,9,10,11,12,13,14, 15; D19S253 corresponds to 7,9,11,12,13,14, 15; D6S477 corresponds to 11,14,15,16,18,19, 20; D8S1132 corresponds to 16,17,18,19,20,21,22,23,24, 25; penta D corresponds to 5,6,7,8,9,10,11,12,13,14,15, 16.

The invention also provides application of the primer group or the kit in the scheme in forensic individual identification, forensic DNA database construction, suspect family investigation or judicial 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, 72 ℃,30 sec, 29 cycles; 60 deg.C, 30 min.

The invention has the beneficial effects that: the invention provides a primer group for amplifying 34 STR loci of human simultaneously, wherein the 34 STR loci comprise 29 autosomal STR loci, 1Y chromosome STR locus and 4 individual identification STR loci. The 34 amplified STR loci comprise 20 core loci and 10 preferred loci specified by the ministry of public security, comprise all loci of mainstream kits in the market at present, and also comprise 5 individual identification loci, so that the risk of sex identification errors caused by Y chromosome deletion can be effectively prevented. The 34 STR loci are combined together and have the characteristics of high individual recognition power and high non-father exclusion rate. 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 the primers is between 18bp and 32bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 34 pairs of primers have no interaction, so that the primers corresponding to 34 loci can be compatible with one single tube. The length of the overall amplification product is between 66 and 500 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 the current forensic identity identification, the construction of a forensic DNA database and the identification of judicial genetic relationship.

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 invention provides a primer group for amplifying 34 STR loci of people simultaneously, wherein the 34 STR loci comprise 29 autosomal STR loci, 1Y chromosome STR locus and 4 individual identification STR loci; the 29 autosomal STR loci are respectively: D3S1358, TH01, D21S11, D18S51, D2S1338, D15S659, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta E, D6S1043, D22S1045, D19S433, D1S1656, D12S391, D10S1248, D2S441, vWA, D8S1179, TPOX, FGA, D3S3045, D10S1435, D19S253, D6S477, D8S1132 and Penta D; the 1Y chromosome STR locus is as follows: DYS 391; the 4 individual identification STR loci are respectively as follows: RS759551978, RS2032678, RS771783753 and Amelogenin;

The 34 loci examined by the present invention comprise: 29 autosomal STR loci, each: D3S1358, TH01, D21S11, D18S51, D2S1338, D15S659, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta E, D6S1043, D22S1045, D19S433, D1S1656, D12S391, D10S1248, D2S441, vWA, D8S1179, TPOX, FGA, D3S3045, D10S1435, D19S253, D6S477, D8S1132, Penta D; 1Y chromosome STR locus DYS 391; 4 individuality identification of loci RS759551978, RS2032678, RS771783753 and Amelogenin. The primer set of the present invention comprises 34 pairs of primers, each corresponding to 34 loci involved in the present invention. Aiming at 34 loci, 34 pairs of primers are designed, the length of the primers is 18-30 bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 34 pairs of primers have no interaction, so that the primers corresponding to the 34 loci can be compatible in a single tube. The length of the overall amplification product is between 66 and 500 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. In the invention, each pair of primers in the primer group can be stably compatible and do not react with each other in one tube, and the specificity, the secondary structure, the amplification efficiency, the stability and the like are greatly improved. The 34 STR loci of the present invention comprise 20 core loci defined by the Ministry of public Security, and 10 preferred loci, including all loci of the currently marketed mainstream kits. Meanwhile, the sex identification gene locus also comprises 5 individual identification gene loci, so that the risk of sex identification errors caused by Y chromosome deletion can be effectively prevented. The 34 gene loci are combined together and have the characteristics of high individual recognition power and high non-paternal exclusion rate. The STR loci in the primer sets of the invention are compared with the loci of the mainstream kits on the market in Table 1.

Table 1 STR loci in primer sets of the invention are compared to commercially available mainstream kit sites, see table 1.

In the invention, the amplification balance of the primer group comprehensively exceeds the standard (more than or equal to 70% in a gene locus, 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 34 STR loci are divided into five groups, wherein the first group comprises: RS759551978, RS2032678, D3S1358, TH01, D21S11, D18S51, D2S1338 and D15S 659; the second group includes: RS771783753, Amelogenin, D5S818, D13S317, D7S820, D16S539, CSF1PO and Penta E; the third group includes: D6S1043, D22S1045, D19S433, D1S1656, DYS391, D12S391 and D10S 1248; the fourth group includes: D2S441, vWA, D8S1179, TPOX and FGA; the fifth group includes: D3S3045, D10S1435, D19S253, D6S477, D8S1132 and Penta D; the upstream primer and/or the downstream primer of the first group to the fifth group STR loci respectively adopt different fluorescent dyes.

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 34 STR loci simultaneously in one reaction, and fully meets the compatibility of the current public security DNA database comparison.

In the present invention, the 5' end of the upstream primer and/or the downstream primer of each of the 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 0.3-0.4 mu M, and more preferably 0.361 mu M; SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.12-0.18. mu.M, and more preferably 0.166. mu.M; SEQ ID NO: 5 and SEQ ID NO: 6 is 0.08-0.13 muM independently, and more preferably 0.099 muM; SEQ ID NO: 7 and SEQ ID NO: 8 is independently 0.1-0.15. mu.M, more preferably 0.130. mu.M; SEQ ID NO: 9 and SEQ ID NO: 10 is independently 0.2 to 0.25. mu.M, and more preferably 0.227. mu.M; SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.2 to 0.25. mu.M, more preferably 0.224. mu.M; SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is 0.35-0.45 mu M independently, and the more preferable concentration is 0.428 mu M; SEQ ID NO: 15 and SEQ ID NO: 16 is independently 0.2-0.3. mu.M, more preferably 0.277. mu.M; SEQ ID NO: 17 and SEQ ID NO: 18 is 0.08 to 0.13. mu.M, and preferably 0.110. mu.M; SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.07-0.11. mu.M, more preferably 0.08. mu.M; SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.07-0.11. mu.M, more preferably 0.083. mu.M; SEQ ID NO: 23 and SEQ ID NO: 24 is independently 0.1-0.15. mu.M, more preferably 0.121. mu.M; SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.4-0.5. mu.M, more preferably 0.448. mu.M; SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.1-0.15. mu.M, more preferably 0.129. mu.M; SEQ ID NO: 29 and SEQ ID NO: 30 is independently 0.1-0.15. mu.M, more preferably 0.124. mu.M; SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.4-0.5. mu.M, and more preferably 0.449. mu.M; SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.3-0.4. mu.M, and more preferably 0.358. mu.M; SEQ ID NO: 35 and SEQ ID NO: 36 is independently 0.12-0.18. mu.M, more preferably 0.149. mu.M; SEQ ID NO: 37 and SEQ ID NO: 38 is 0.3-0.4. mu.M, and preferably 0.332. mu.M; SEQ ID NO: 39 and SEQ ID NO: 40 is independently 0.15-0.2. mu.M, more preferably 0.174. mu.M; SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.2 to 0.3. mu.M, more preferably 0.224. mu.M; SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.3-0.4. mu.M, and more preferably 0.361. mu.M; SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.3-0.4. mu.M, more preferably 0.333. mu.M; SEQ ID NO: 47 and SEQ ID NO: 48 is independently 0.12-0.18. mu.M, more preferably 0.150. mu.M; SEQ ID NO: 49 and SEQ ID NO: 50 is 0.12-0.18. mu.M, preferably 0.159. mu.M; SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.25-0.35. mu.M, more preferably 0.284. mu.M; SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.4-0.5. mu.M, more preferably 0.421. mu.M; SEQ ID NO: 55 and SEQ ID NO: 56 is independently 0.2 to 0.3. mu.M, more preferably 0.240. mu.M; SEQ ID NO: 57 and SEQ ID NO: 58 is independently 0.08 to 0.13. mu.M, and more preferably 0.090. mu.M; SEQ ID NO: 59 and SEQ ID NO: 60 is independently 0.06-0.11 mu M, and more preferably 0.085 mu M; SEQ ID NO: 61 and SEQ ID NO: 62 is independently 0.1-0.15. mu.M, more preferably 0.123. mu.M; SEQ ID NO: 63 and SEQ ID NO: 64 is independently 0.06-0.11 mu M, and more preferably 0.078 mu M; SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.1-0.15. mu.M, more preferably 0.111. mu.M; SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.1 to 0.15. mu.M, more preferably 0.126. mu.M.

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

The allele ladder 34A of the invention covers all the most common alleles and most rare alleles of each locus, and is more convenient for comparing the 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 forensic individual identification, forensic DNA database construction, suspect family investigation or judicial 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-500 and PCR amplification product or allele ladder 34A are mixed to obtain a mixture; the molecular weight internal standard BTY-5000.5 μ l, PCR amplification product or allele ladder 34A 1 μ l calculated by HiDi formamide volume of 8.5 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 12sec, 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 34 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, 72 ℃,30 sec, 29 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-500+ 1. mu.l of PCR product/1. mu.l of allele step 34A; 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 12sec, 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 34 loci genotypes, the invention can find that the 34 loci genetic information of the suspected father and son conforms to the genetic rule, and can judge the father-son relationship.

Example 2: application of kit of the invention in forensic medicine

The invention is mainly used for the construction of DNA database of criminals and the identification of the identity of criminal suspects in the field of forensic science. The national forensic DNA database is a national DNA database of illegal criminals established by the ministry of public security in China, breaks through 5000 thousands of data in the database at present, and is a main tool for case detection of a public security system. The database is usually built by adopting a method of directly amplifying and detecting blood samples, the detection material in the identification work of the identity of the criminal suspect is usually a complex detection material, and the amplification detection method is adopted for detection after DNA is extracted. The invention can take two different inspection materials into consideration, and is convenient for the construction of DNA databases in China and the identification of the identity of the criminal suspect. The detailed part of the operation is as follows:

firstly, it is known that the material to be detected by the criminal is usually blood card, which 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 criminals are generally case field inspection materials which are complex, and the detection analysis of data is generally carried out by 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, 72 ℃,30 sec, 29 cycles; 60 ℃ for 30 min; storing at constant temperature of 10 ℃.

Table 7: reaction System of this example

Fourthly, detecting by a genetic analyzer and analyzing data

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-500+ 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 12sec, after which electrophoresis was started. 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 national DNA database. FIG. 6 is a blood card sample typing chart, and FIG. 7 is a DNA extraction sample typing chart. 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 of the forensic.

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 34 STR loci of human and application thereof

<160> 68

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ccggaggtaa aggtgtctta aagt 24

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gaaagaattc tcttatttgg gttatt 26

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caatagtgtg caaggatggg t 21

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accctgtcct agccttctta tagc 24

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tgaggctgca aaaagctata attgta 26

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ccaggaaaga gagaaaccat gtgat 25

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accaaattgt gttcatgagt atagtttc 28

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caaacgtgag gttgactcta ctg 23

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ggctgcaggg cataacatta 20

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agaagcagag agaaagggag a 21

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tccatagatt agatagatca tagacagaca 30

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cttctgtcac agggctgatg a 21

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ttacctctgt tggtcattga gggttat 27

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gcctggaagg tcgaagctga 20

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attttttata taaaataact caccaaagga 30

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ataccttttt ttctactgat acctttgt 28

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agatccctcc tgtgggctga aaag 24

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atttaaatat gtgagtcaat tccccaag 28

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actaccagca acaacacaaa taaac 25

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atttgtgtgg agtggaggtg cctaaagac 29

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ataatttaaa cataagtacc catatgtgg 29

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attactagtg actgttctca aggt 24

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agagcttaaa ctgggaagct g 21

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ggtgattttc ctctttggta tcc 23

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attacagaag tctgggatgt ggagga 26

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agcttgaatt ataacgattc cacatttatc 30

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gggggtctaa gagcttgtaa aaag 24

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aaggtgcaca cttggacagc at 22

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attaccaaca tgaaagggta ccaata 26

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acttcccata ataaatccta tc 22

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aatgagacaa tatatgtaaa gtgctctc 28

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gatttcaata ggtttttaag gaac 24

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gaagaaaatc cccatataag ttcaag 26

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actagggaca tttaaggctg tatgg 25

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aatatatctc tcctggagct gcgacacatt 30

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atttccagtc caactagatc ctgtg 25

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ataagtggct gtggtgttat g 21

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aagccctagt ggatgataag aataatc 27

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attgcaactt atatgtattt ttgtatttca tg 32

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aaccgtcgac tggcacagaa ca 22

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gatatgctgt actttttcta tgact 25

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acatctgcat gggaaatcaa tatc 24

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acaaaaataa agagatagac agatagcc 28

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aacatgctgt tcattgcaga gg 22

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agagtctcta cctgtgctag tcc 23

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acatctctct ctccctctct ctttcg 26

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ataagaattc tttaatctgg acacaag 27

Claims

1. A primer group for amplifying 34 STR loci of human simultaneously, wherein the 34 STR loci comprise 29 autosomal STR loci, 1Y chromosome STR locus and 4 individual identification loci; the 29 autosomal STR loci are respectively: D3S1358, TH01, D21S11, D18S51, D2S1338, D15S659, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta E, D6S1043, D22S1045, D19S433, D1S1656, D12S391, D10S1248, D2S441, vWA, D8S1179, TPOX, FGA, D3S3045, D10S1435, D19S253, D6S477, D8S1132 and PentaD; the 1Y chromosome STR locus is as follows: DYS 391; the 4 individual identification loci are respectively: RS759551978, RS2032678, RS771783753 and Amelogenin;

the nucleotide sequence of the upstream primer for amplifying RS759551978 is shown in 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 the D3S1358 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 TH01 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 the D21S11 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 the D18S51 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 the D2S1338 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 the D15S659 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 RS771783753 is shown in 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 the Amelogenin 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 the D5S818 is shown as 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 the D13S317 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 the D7S820 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 the D16S539 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 CSF1PO 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 Penta E 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 the D6S1043 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 the D22S1045 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 the D19S433 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 the D1S1656 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 DYS391 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 the D12S391 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 the D10S1248 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 the D2S441 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 vWA 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 the D8S1179 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 TPOX 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 FGA 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 the D3S3045 is shown in 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 the D10S1435 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 the D19S253 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 the D6S477 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 the D8S1132 is shown in 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 Penta D is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 68.

2. The primer set of claim 1, wherein the 34 STR loci are grouped into five groups, wherein,

the first group includes: RS759551978, RS2032678, D3S1358, TH01, D21S11, D18S51, D2S1338 and D15S 659;

the second group includes: RS771783753, Amelogenin, D5S818, D13S317, D7S820, D16S539, CSF1PO and Penta E;

the third group includes: D6S1043, D22S1045, D19S433, D1S1656, DYS391, D12S391 and D10S 1248;

the fourth group includes: D2S441, vWA, D8S1179, TPOX and FGA;

the fifth group includes: D3S3045, D10S1435, D19S253, D6S477, D8S1132 and Penta D;

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

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 0.3-0.4 mu M;

SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.12-0.18 mu M;

SEQ ID NO: 5 and SEQ ID NO: 6 is 0.08-0.13 mu M independently;

SEQ ID NO: 7 and SEQ ID NO: 8 is 0.1-0.15 mu M independently;

SEQ ID NO: 9 and SEQ ID NO: 10 is independently 0.2-0.25 mu M;

SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.2-0.25 mu M;

SEQ ID NO: 15 and SEQ ID NO: 16 is 0.2-0.3 mu M independently;

SEQ ID NO: 17 and SEQ ID NO: 18 is 0.08-0.13 mu M independently;

SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.07-0.11 mu M;

SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.07-0.11 mu M;

SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.4-0.5 mu M;

SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.1-0.15 mu M;

SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.4-0.5 mu M;

SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.3-0.4 mu M;

SEQ ID NO: 37 and SEQ ID NO: 38 is 0.3-0.4 mu M independently;

SEQ ID NO: 39 and SEQ ID NO: 40 is independently 0.15-0.2 mu M;

SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.2-0.3 mu M;

SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.3-0.4 mu M;

SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.3-0.4 mu M;

SEQ ID NO: 47 and SEQ ID NO: 48 is 0.12-0.18 mu M independently;

SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.25-0.35 mu M;

SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.4-0.5 mu M;

SEQ ID NO: 55 and SEQ ID NO: 56 is 0.2-0.3 mu M independently;

SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.1-0.15 mu M;

SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.1-0.15. 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 STR locus correspond in relationship to one another as:

9. Use of the primer set of claim 1 or 2 and the kit of any one of claims 3 to 8 for forensic individual identification, forensic DNA database construction, suspect pedigree screening or forensic 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;