A fluorescence multiplex amplification system for 32 short tandem repeats and its application

Abstract

The present invention belongs to the technical field of biological gene detection, and relates to a fluorescence multiplex amplification system for 32 short tandem repeats and its application. The fluorescence multiplex amplification system of the present invention includes 30 autosomal loci, one sex identification and a Yindel on the Y chromosome for auxiliary sex determination. The combination of 19 Class A loci, 10 Class B loci and 1 Class C locus can provide more genetic information, thereby improving the individual identification ability. The kit of the present invention can not only amplify various conventional extracted samples, but also directly amplify fresh and aged blood stains or saliva stains, and can use DNA testing technology to confirm the identities of abducted women and children. At the same time, it is also applicable to forensic genetics paternity testing and individual identification.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological gene detection, and relates to a fluorescence multiplex amplification system for 32 short tandem repeats and its application. Background Art

[0002] Short tandem repeats (STRs) are a class of DNA polymorphic loci widely present in the human genome. They are highly polymorphic due to differences in DNA fragment length or DNA sequence between individuals and follow the Mendelian codominant mode of inheritance during gene transmission. Autosomal STR loci are widely used in forensic individual identification and paternity testing due to their low mutation rate, stable inheritance, and high genetic polymorphism. In 1997, the United States officially designated 13 autosomal STR loci as the CODIS core STR loci and established a series of testing methods, evaluation criteria, and basic personnel information databases. In April 2009, the European Network of Forensic Science Institutes (ENFSI) unanimously decided to add another 5 STR loci (D1S1656, D2S441, D10S1248, D12S391, D22S1045) to the existing 7 STRs (D3S1358, D8S1179, D18S51, D21S11, FGA, TH01, vWA) of the European Standard Set (ESS).

[0003] The fluorescence detection typing technology for STR multiplex amplification comprehensively applies multiple multiplex amplification, capillary electrophoresis, and fluorescence detection technologies and has been widely used in various forensic DNA laboratories. It has the characteristics of being fast, sensitive, accurate, stable, and having good repeatability, and can achieve automation. In the initial stage in China, STR kits were monopolized by foreign countries. With the improvement of domestic scientific research capabilities, more and more self-developed kits have come to the market. Currently, domestic autosomal kits with relatively high sensitivity on the market can amplify extracted samples, but cannot simultaneously be compatible with the amplification of direct amplification samples because there are various PCR inhibitors in direct amplification samples. Summary of the Invention

[0004] The object of the present invention is to propose a fluorescence multiplex amplification system for 32 short tandem repeats with strong kinship recognition ability and high comprehensive detection efficiency in view of the above problems existing in the prior art.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] A fluorescence multiplex amplification system for 32 short tandem repeats, comprising 32 pairs of amplification primers, which are respectively used for simultaneously amplifying 30 autosomal STR loci, 1 C-class locus D4S2366, and 1 Y-Indel locus; the 30 autosomal STR loci are respectively: Amelogenin, D18S51, D21S11, D3S1358, FGA, D8S1179, vWA, CSF1PO, D16S539, D7S820, D13S317, D5S818, D2S1338, D19S433, TH01, TPOX, D6S1043, Penta D, Penta E, D12S391, D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253, D6S477, D10S1435.

[0007] In the above fluorescence multiplex amplification system for 32 short tandem repeats, the 32 pairs of amplification primers include the sequences shown below:

[0008] The upstream and downstream primer sequences of D3S1358 are shown as SEQ ID NO.1 and SEQ ID NO.2;

[0009] The upstream and downstream primer sequences of TH01 are shown as SEQ ID NO.3 and SEQ ID NO.4;

[0010] The upstream and downstream primer sequences of D21S11 are shown as SEQ ID NO.5 and SEQ ID NO.6;

[0011] The upstream and downstream primer sequences of D18S51 are shown as SEQ ID NO.7 and SEQ ID NO.8;

[0012] The upstream and downstream primer sequences of Penta E are shown as SEQ ID NO.9 and SEQ ID NO.10;

[0013] The upstream and downstream primer sequences of Y-indel are shown as SEQ ID NO.11 and SEQ ID NO.12;

[0014] The upstream and downstream primer sequences of D19S253 are shown as SEQ ID NO.13 and SEQ ID NO.14;

[0015] The upstream and downstream primer sequences of D12S391 are shown as SEQ ID NO.15 and SEQ ID NO.16;

[0016] The upstream and downstream primer sequences of D6S1043 are shown as SEQ ID NO.17 and SEQ ID NO.18;

[0017] The upstream and downstream primer sequences of D2S1338 are shown as SEQ ID NO.19 and SEQ ID NO.20;

[0018] The upstream and downstream primer sequences of D15S659 are shown as SEQ ID NO.21 and SEQ ID NO.22;

[0019] The upstream and downstream primer sequences of D6S477 are shown as SEQ ID NO.23 and SEQ ID NO.24;

[0020] The upstream and downstream primer sequences of Amelogenin are shown as SEQ ID NO.25 and SEQ ID NO. 26;

[0021] The upstream and downstream primer sequences of D5S818 are shown as SEQ ID NO.27 and SEQ ID NO.28;

[0022] The upstream and downstream primer sequences of D13S317 are shown as SEQ ID NO.29 and SEQ ID NO.30;

[0023] The primer pair sequence of D7S820 is that the upstream and downstream primer sequences are shown as SEQ ID NO.31 and SEQ ID NO.32;

[0024] The upstream and downstream primer sequences of D19S433 are shown as SEQ ID NO.33 and SEQ ID NO.34;

[0025] The upstream and downstream primer sequences of CSF1PO are shown as SEQ ID NO.35 and SEQ ID NO.36;

[0026] The upstream and downstream primer sequences of Penta D are shown as SEQ ID NO.37 and SEQ ID NO.38;

[0027] The upstream and downstream primer sequences of D2S441 are shown as SEQ ID NO.39 and SEQ ID NO.40;

[0028] The upstream and downstream primer sequences of VWA are shown as SEQ ID NO.41 and SEQ ID NO.42;

[0029] The upstream and downstream primer sequences of D8S1179 are shown as SEQ ID NO.43 and SEQ ID NO.44;

[0030] The upstream and downstream primer sequences of TPOX are shown in SEQ ID NO.45 and SEQ ID NO.46;

[0031] The upstream and downstream primer sequences of FGA are shown in SEQ ID NO.47 and SEQ ID NO.48;

[0032] The upstream and downstream primer sequences of D4S2366 are shown in SEQ ID NO.49 and SEQ ID NO.50;

[0033] The upstream and downstream primer sequences of D3S3045 are shown in SEQ ID NO.51 and SEQ ID NO.52;

[0034] The upstream and downstream primer sequences of D16S539 are shown in SEQ ID NO.53 and SEQ ID NO.54;

[0035] The upstream and downstream primer sequences of D22S1045 are shown in SEQ ID NO.55 and SEQ ID NO.56;

[0036] The upstream and downstream primer sequences of D8S1132 are shown in SEQ ID NO.57 and SEQ ID NO.58;

[0037] The upstream and downstream primer sequences of D1S1656 are shown in SEQ ID NO.59 and SEQ ID NO.60;

[0038] The upstream and downstream primer sequences of D10S1248 are shown in SEQ ID NO.61 and SEQ ID NO.62;

[0039] The upstream and downstream primer sequences of D10S1435 are shown in SEQ ID NO.63 and SEQ ID NO.64.

[0040] Preferably, the final concentrations of the primer pairs in the amplification volume are as follows:

[0041] D3S1358 0.08 μM;

[0042] TH01 0.06 μM;

[0043] D21S11 0.21 μM;

[0044] D18S51 0.1 μM;

[0045] Penta E 0.32 μM;

[0046] Y-indel 0.11 μM;

[0047] D19S253 0.18 μM;

[0048] D12S391 0.25 μM;

[0049] D6S1043 0.1 μM;

[0050] D2S1338 0.35 μM;

[0051] D15S659 0.39 μM;

[0052] D6S477 0.39 μM;

[0053] Amelogenin 0.09 μM;

[0054] D5S818 0.06 μM;

[0055] D13S317 0.15 μM;

[0056] D7S820 0.07 μM;

[0057] D19S433 0.12 μM;

[0058] CSF1PO 0.08 μM;

[0059] Penta D 0.12 μM;

[0060] D2S441 0.16 μM;

[0061] VWA 0.1 μM;

[0062] D8S1179 0.09 μM;

[0063] TPOX 0.12 μM;

[0064] FGA 0.13 μM;

[0065] D4S2366 0.13 μM;

[0066] D3S3045 0.33 μM;

[0067] D16S539 0.12 μM;

[0068] D22S1045 0.11 μM;

[0069] D8S1132 0.18 μM;

[0070] D1S1656 0.15 μM;

[0071] D10S1248 0.08 μM;

[0072] D10S1435 at 0.22 μM.

[0073] In the above fluorescence multiplex amplification system of 32 short tandem repeats, the 5' ends of the 32 pairs of primers in the fluorescence multiplex amplification system are respectively labeled with 5 different fluorescent dyes. The same fluorescent dye label is regarded as the same group. The five groups of combinations are as follows:

[0074] The first group: D3S1358, TH01, D21S11, D18S51, and Penta E;

[0075] The second group: Y-indel, D19S253, D12S391, D6S1043, D2S1338, D15S659, and D6S477;

[0076] The third group: Amelogenin, D5S818, D13S317, D7S820, D19S433, CSF1PO, and Penta D;

[0077] The fourth group: D2S441, VWA, D8S1179, TPOX, FGA, and D4S2366;

[0078] The fifth group: D3S3045, D16S539, D22S1045, D8S1132, D1S1656, D10S1248, and D10S1435.

[0079] Preferably, the fluorescent dye is any one of blue fluorescent dye, green fluorescent dye, yellow fluorescent dye, red fluorescent dye, and purple fluorescent dye.

[0080] In the above fluorescence multiplex amplification system of 32 short tandem repeats, the fluorescent dye is at least one of FAM, HEX, TAMRA, ROX, and AF549.

[0081] In the above fluorescence multiplex amplification system of 32 short tandem repeats, it further includes a set of fluorescent molecular weight internal standard SIZE-500. The fragment sizes of the molecular weight internal standard are 75, 87, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500.

[0082] In the above fluorescence multiplex amplification system of 32 short tandem repeats, the fluorescent dye label of the molecular weight internal standard uses AF633 dye.

[0083] In the above fluorescence multiplex amplification system of 32 short tandem repeats, it further includes an allelic typing standard Allelic Ladder.

[0084] The present invention also provides a kit, comprising the above-mentioned fluorescence multiplex amplification system.

[0085] The present invention also provides a method for using the above-mentioned kit, and the method comprises the following steps:

[0086] S1. Extract human genomic DNA by using the Chelex method, magnetic bead extraction method and silica bead extraction method, or obtain amplification templates by using extraction-free blood spot collection cards and oral cell collection cards;

[0087] S2. Amplify the obtained DNA;

[0088] S3. Mix the molecular weight internal standard and deionized formamide in the kit to prepare a loading mixture, and then perform electrophoresis detection on the loading mixture and the amplification product or the allelic typing standard of 32 loci in the kit by using a genetic analyzer;

[0089] S4. Analyze the results.

[0090] Application of the above-mentioned fluorescence multiplex amplification system and kit in the information system for searching abducted / missing children.

[0091] Compared with the prior art, the present invention has the following beneficial effects: The fluorescence multiplex amplification system of the present invention includes 30 autosomal loci, a sex identification and a Yindel on the Y chromosome for auxiliary sex determination. The combination of 19 Class A loci, 10 Class B loci and 1 Class C locus can provide more genetic information, thereby improving the individual identification ability. The kit of the present invention can not only amplify various conventional extracted samples, but also directly amplify fresh and old blood spots or saliva spots. The fluorescence multiplex amplification system of the present invention can confirm the identities of abducted women and children, and is also applicable to forensic genetics paternity testing and individual identification. Description of the Drawings

[0092] Figure 1 It is the genotyping diagram of 0.25 ng 9948 standard product of the kit in Example 1;

[0093] Figure 2 It is the allelic typing standard of the kit in Example 1: Allelic Ladder diagram;

[0094] Figure 3 It is the amplification result diagram of Bokun saliva card of the kit in Example 1;

[0095] Figure 4 It is the amplification result diagram of Whatman FTA blood card sample of the kit in Example 1;

[0096] Figure 5 Amplification result graph of hair Chelex extraction sample of the kit in Example 1.

[0097] Figure 6 Amplification result graph of Bokun automatic extraction sample of the kit in Example 1. Detailed implementation manners

[0098] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0099] Example 1:

[0100] The present invention contains a total of 32 gene loci, namely Amelogenin, D18S51, D21S11, D3S1358, FGA, D8S1179, vWA, CSF1PO, D16S539, D7S820, D13S317, D5S818, D2S1338, D19S433, TH01, TPOX, D6S1043, Penta D, Penta E, D12S391, D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253, D6S477, D10S1435 and Yindel. The sequences of the above primer pairs are shown in Table 1:

[0101] Table 1: Primer sequences of each gene locus

[0102]

[0103]

[0104] The 5'-end of at least one primer in each primer pair is labeled with a fluorescent dye. The above primers are grouped and labeled as follows:

[0105] The first group is labeled with the blue fluorescent dye 6-FAM: D3S1358, TH01, D21S11, D18S51 and Penta E;

[0106] The second group is labeled with the green fluorescent dye HEX: Y-indel, D19S253, D12S391, D6S1043, D2S1338, D15S659 and D6S477;

[0107] The third group is labeled with the yellow fluorescent TAMRA dye: Amelogenin, D5S818, D13S317, D7S820, D19S433, CSF1PO and Penta D;

[0108] The fourth group is labeled with the red fluorescent ROX dye: D2S441, VWA, D8S1179, TPOX, FGA, and D4S2366;

[0109] The fifth group is labeled with the purple fluorescent AF549 dye: D3S3045, D16S539, D22S1045, D8S1132, D1S1656, D10S1248, and D10S1435.

[0110] In some other embodiments of the present invention, any one of the blue fluorescent dye 6-FAM, green fluorescent dye HEX, yellow fluorescent dye TAMRA, red fluorescent dye-labeled ROX, and purple fluorescent dye AF549 can be used to label each group with a fluorescent dye, as long as the dyes between each group are different. The above primers constitute a specific amplification primer pair. Among them, the molecular weight internal standard is the orange fluorescent-labeled SIZE-500.

[0111] The primer concentrations are:

[0112] D3S1358 0.08 μM;

[0113] TH01 0.06 μM;

[0114] D21S11 0.21 μM;

[0115] D18S51 0.1 μM;

[0116] Penta E 0.32 μM;

[0117] Y-indel 0.11 μM;

[0118] D19S253 0.18 μM;

[0119] D12S391 0.25 μM;

[0120] D6S1043 0.1 μM;

[0121] D2S1338 0.35 μM;

[0122] D15S659 0.39 μM;

[0123] D6S477 0.39 μM;

[0124] Amelogenin 0.09 μM;

[0125] D5S818 0.06 μM;

[0126] D13S317 0.15 μM;

[0127] D7S820 0.07 μM;

[0128] D19S433 0.12 μM;

[0129] CSF1PO 0.08 μM;

[0130] Penta D 0.12 μM;

[0131] D2S441 0.16 μM;

[0132] VWA 0.1 μM;

[0133] D8S1179 0.09 μM;

[0134] TPOX 0.12 μM;

[0135] FGA 0.13 μM;

[0136] D4S2366 0.13 μM;

[0137] D3S3045 0.33 μM;

[0138] D16S539 0.12 μM;

[0139] D22S1045 0.11 μM;

[0140] D8S1132 0.18 μM;

[0141] D1S1656 0.15 μM;

[0142] D10S1248 0.08 μM;

[0143] D10S1435 0.22 μM.

[0144] 1. Kit operation steps

[0145] 1.1 System configuration: Configure the PCR reaction solution and the specific amplification primer pair according to the proportions in the instruction manual to form the reaction system. After vortex mixing, centrifuge with a centrifuge, and then aliquot by volume using a pipette. When the kit is used for amplification, the 25 μL amplification reaction system is shown in Table 2:

[0146] Table 2: Amplification reaction system

[0147]

[0148]

[0149] 1.2 Amplification program

[0150] The amplification program on the PCR instrument is shown in Table 3.

[0151] Table 3: Amplification system

[0152]

[0153] 1.3 Detection of amplification products on a genetic analyzer

[0154] The loading mixture is composed of deionized formamide and the molecular weight internal standard (Size-500) in the system {(1 μL Size-500 + 12 μL deionized formamide) × (number of injections)}. Mix 1 μL of the amplification product or the Allelic Ladder of the allelic analysis standard with 9 μL of the loading mixture, centrifuge to remove air bubbles and perform electrophoresis as soon as possible. Detect and analyze with an ABI 3500 genetic analyzer (purchased from ABI Company, USA). The specific analysis parameters are injection voltage: 1.2 kv, injection time: 15 s.

[0155] 1.4 Analysis of data

[0156] Use GeneMapper ID-X (Lifetech Company, USA) software for result analysis

[0157] Figure 1 This is the genotyping map of the 0.25 ng 9948 standard product of the kit; it can be seen from the figure that the kit has high efficiency and can meet the amplification of daily samples with different concentrations.

[0158] Figure 2 This is the result map of the allelic typing standard; it can be seen from the figure that the allelic typing standard of this kit contains the common allelic typings of each locus, corrects the results detected due to different instruments and conditions, and ensures the accuracy of the results.

[0159] Bokun saliva cards and Whatman FTA blood cards are the cards for direct amplification that are used more frequently in the market. They contain relatively high contents of components that inhibit PCR amplification and are somewhat representative. This kit amplifies Bokun saliva cards and Whatman FTA blood cards, and the amplification results are as Figure 3 、 4 shown. It can be seen from the figure that for these direct amplification samples with longer storage times, the amplification efficiency is high, and there is good balance. The results are accurate and reliable, indicating that this kit has a certain anti-inhibition ability and can be applied to the amplification of various different types of direct amplification samples, and can also obtain good experimental results for old direct amplification samples.

[0160] Example 2:

[0161] The specimens are hair and semen stain samples.

[0162] 1. Operating steps of the kit

[0163] 1.1 DNA extraction: For hair samples, extraction is carried out according to the polystyrene divinylbenzene resin method in the "Forensic DNA Laboratory Test Specification" GAT 383—2014 (May 9, 2014). Cut 5 mm - 10 mm of the hair root part of the hair sample, rinse it with absolute ethanol, water, and absolute ethanol respectively once, dry it, and then add Chelex-100, proteinase K, and DTT, and digest at 56 °C until completely dissolved. Put the dissolved sample into a thermostat at 95 °C for 10 min, centrifuge and store at 4 °C for later use. For semen stain samples, extraction is carried out according to the instruction manual of the Bokun magnetic bead automatic extractor.

[0164] 1.2 System configuration: Configure the PCR reaction solution and the specific amplification primer pair according to the ratio in the instruction manual to form the reaction system. After vortex mixing, centrifuge with a centrifuge, and then aliquot by volume with a pipette. When the kit is used for amplification, the 25 μL amplification reaction system is shown in Table 4:

[0165] Table 4: Amplification system

[0166] Component Volume PCR reaction solution 12.5 μL Specific amplification primer pair 6.25 μL Human locus DNA 2 μL of extracted sample Nuclease-free water Make up to 25 μL

[0167] 1.3 Amplification program

[0168] The amplification program on the PCR instrument is shown in Table 5:

[0169] Table 5: Amplification program

[0170]

[0171] 1.4 Detection of amplification products on a genetic analyzer

[0172] The loading mixture is composed of deionized formamide and the molecular weight internal standard (Size-500) in the system {(1 μL Size-500 + 12 μL deionized formamide) × (number of injections)}. Mix 9 μL of the loading mixture with 1 μL of the amplification product, avoid generating bubbles, and perform electrophoresis as soon as possible. Detect and analyze with an ABI 3500 genetic analyzer (purchased from ABI Company, USA). The specific analysis parameters are injection voltage: 1.2 kv, injection time: 15 s.

[0173] 1.5 Data analysis

[0174] Use GeneMapper ID-X (Lifetech Company, USA) software for result analysis

[0175] The detection samples in paternity testing also include samples such as semen (stain), hair with hair follicles, amniotic fluid, tissue blocks, etc. The amplification and extraction results of this kit are as Figure 5 、 6As shown, it can be seen from the figure that the sensitivity of this kit can meet the needs of daily experiments, and it can be applied to the amplification of various different types of extraction samples, with a wide range of practical applications.

[0176] For the points in the technical scope claimed by the present invention that are not exhausted in the embodiments herein and the new technical solutions formed by the equivalent replacement of single or multiple technical features in the technical solutions of the embodiments, they are also within the scope claimed by the present invention; at the same time, in all the exemplified or unexemplified embodiments of the present invention, the various parameters in the same embodiment only represent an example (i.e., a feasible solution) of its technical solution, and there is no strict coordination and limitation relationship between the various parameters. Among them, the various parameters can be replaced with each other without violating the axioms and the requirements of the present invention, unless otherwise specifically stated.

[0177] The technical means disclosed in the present invention are not limited to the technical means disclosed in the above technical means, but also include the technical solutions formed by any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches are also regarded as the protection scope of the present invention.

[0178] The specific embodiments described herein are only illustrative of the spirit of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims. Sequence Listing <110> Ningbo HaierShi Gene Technology Co., Ltd. <120> Fluorescent multiplex amplification system for 32 short tandem repeats and its application <141> 2022-06-23 <160> 64 <170> SIPOSequenceListing 1.0 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <400> 2 tctcttatac tcatgaaatc aacagaggct 30 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <400> 2 aatctgggtg acagagcaag acc 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gcccacacag tcccctgtac 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <400> 4 gcaaataggg ggcaaaattc aaagg 25 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 tatgggactt ttctcagtct ccataaatat 30 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <400> 6 ccagagacag actaatagga ggtagatag 29 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <400> 7 agttcttgag cccagaaggt taa 23 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <400> 8 tactttaaaa ataacaaacc cgactacc 28 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 ctggagtgca atggcgtgat 20 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <400> 10 ccttagtttt actactggtc tactttggg 29 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <400> 11 taataatact gactgtcttt gagcaagaaa 30 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 tgagaagaga agtgagaagg catgtg 26 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 cctgggaaat gtatttattt ctccagagta 30 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <400> 14 gattagatag atcatagaca gacagacgg 29 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <400> 15 ctccagagag aaagaatcaa caggatc 27 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <400> 16 cttgcagatg gactgtcatg agatttttca 30 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <400> 17 ccatgctact agattctgtg gttctcca 28 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <400> 18 tcctttcata cagaatggca ctcttattca 30 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <400> 19 ctcacccctt ttcctaccag aat 23 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <400> 20 tggagtggag gtgcctaaag a 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <400> 21 tattttccat cctgtgcctc cc 22 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <400> 22 ggcagtaatg gttagtggag aatatttaac 30 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <400> 23 taaaactcta aggaggcaac aaaagattta 30 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <400> 24 tagcagcagg actgtgagtt ctaa 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <400> 25 gtttgcgtta acaatgccct g 21 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <400> 26 ggaactgtaa aatcaggacc acttgaga 28 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <400> 27 caatcatagc cacagtttac aacatttg 28 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <400> 28 ttcctctttg gtatccttat gtaatatttt 30 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <400> 29 gttcatttct ttagtgggca tccgtga 27 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <400> 30 tcctccttca acttgggttg ag 22 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <400> 31 ctcagaggga atatatattc ttaagaatta 30 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <400> 32 aggttaatat atataaaggg tatgatagaa 30 <210> 33 <211> 29 <212> DNA <213> Artificial Sequence <400> 33 gtgttggtta catgaataag ttctttagc 29 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <400> 34 aggcatgttg gcacattcct gta 23 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <400> 35 ctgagccttc tcagatacta tctcctg 27 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <400> 36 aataactgca tcttaaccta ttgggaggtc 30 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <400> 37 ttgagcctgg aaggtcgaag 20 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <400> 38 attgatgtgt aagaattctt taatctggac 30 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <400> 39 tcatctatga aaacttctat ctatct 26 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <400> 40 aaattggagc taagtggctg tggtgt 26 <210> 41 <211> 28 <212> DNA <213> Artificial Sequence <400> 41 agatagagat aggacagatg ataaatac 28 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <400> 42 atgataagaa taatcagtat gtgacttgga 30 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <400> 43 ctttctgccc acacggcctg gcaact 26 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <400> 44 tatagtttca cgtagctata attagt 26 <210> 45 <211> 26 <212> DNA <213> Artificial Sequence <400> 45 tcaggggagg aactgggaac cccaca 26 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <400> 46 tttcagggct gtgatcacta gcaccc 26 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <400> 47 ctgcatatag tagacactca gtgcat 26 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <400> 48 tgggattact aattgctatt aggaca 26 <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <400> 49 tcaaagtttc atcatgaaaa atgatg 26 <210> 50 <211> 26 <212> DNA <213> Artificial Sequence <400> 50 tgttagtaca aacttgctag aaaatt 26 <210> 51 <211> 26 <212> DNA <213> Artificial Sequence <400> 51 agaagcagag agaaagggag agatag 26 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <400> 52 atctgcatgg gaaatcaata tcatct 26 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <400> 53 tgggtgagct gcttgccaag gagtgg 26 <210> 54 <211> 26 <212> DNA <213> Artificial Sequence <400> 54 gatttgtgca caacgagcat ctggca 26 <210> 55 <211> 26 <212> DNA <213> Artificial Sequence <400> 55 ccccgatgat agtagtctca ttatta 26 <210> 56 <211> 26 <212> DNA <213> Artificial Sequence <400> 56 ctcagctgta gaatggaaat agtgac 26 <210> 57 <211> 26 <212> DNA <213> Artificial Sequence <400> 57 ccagggcaac aggagcaaaa ctctgt 26 <210> 58 <211> 26 <212> DNA <213> Artificial Sequence <400> 58 ttagtggctt aatgtatttg tttttc 26 <210> 59 <211> 30 <212> DNA <213> Artificial Sequence <400> 59 caagccagga aagagagaaa ccatgtgatt 30 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <400> 60 gggtggtaga gatggaagaa aatccccata 30 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <400> 61 ttctgtgcca agcacttgac atatatcatt 30 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <400> 62 tattataatt aacacttatt ctgacagttc 30 <210> 63 <211> 30 <212> DNA <213> Artificial Sequence <400> 63 acaaaatagg tgattctcac aaaaataaag 30 <210> 64 <211> 30 <212> DNA <213> Artificial Sequence <400> 64 ggtgggagaa tgacctgagc ctgggaggtc 30

Claims

1. A fluorescence multiplex amplification system for 32 short tandem repeats, characterized in that, It includes 32 pairs of amplification primers, which are respectively used for simultaneously amplifying 30 autosomal STR loci, one C locus D4S2366, and one Y-Indel locus; the 30 autosomal STR loci are respectively: Amelogenin, D18S51, D21S11, D3S1358, FGA, D8S1179, vWA, CSF1PO, D16S539, D7S820, D13S317, D5S818, D2S1338, D19S433, TH01, TPOX, D6S1043, PentaD, Penta E, D12S391, D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253, D6S477, D10S1435; The 32 pairs of amplification primers include the sequences shown as follows: The upstream and downstream primer sequences of D3S1358 are shown as SEQ ID NO.1 and SEQ ID NO.2; The upstream and downstream primer sequences of TH01 are shown as SEQ ID NO.3 and SEQ ID NO.4; The upstream and downstream primer sequences of D21S11 are shown as SEQ ID NO.5 and SEQ ID NO. 6; The upstream and downstream primer sequences of D18S51 are shown as SEQ ID NO.7 and SEQ ID NO. 8; The upstream and downstream primer sequences of Penta E are shown as SEQ ID NO.9 and SEQ ID NO. 10; The upstream and downstream primer sequences of Y-indel are shown as SEQ ID NO.11 and SEQ ID NO. 12; The upstream and downstream primer sequences of D19S253 are shown as SEQ ID NO.13 and SEQ ID NO. 14; The upstream and downstream primer sequences of D12S391 are shown as SEQ ID NO.15 and SEQ ID NO. 16; The upstream and downstream primer sequences of D6S1043 are shown as SEQ ID NO.17 and SEQ ID NO. 18; The upstream and downstream primer sequences of D2S1338 are shown as SEQ ID NO.19 and SEQ ID NO. 20; The upstream and downstream primer sequences of D15S659 are shown as SEQ ID NO.21 and SEQ ID NO. 22; The upstream and downstream primer sequences of D6S477 are shown as SEQ ID NO.23 and SEQ ID NO.24; The upstream and downstream primer sequences of Amelogenin are shown as SEQ ID NO.25 and SEQ ID NO. 26; The upstream and downstream primer sequences of D5S818 are shown as SEQ ID NO.27 and SEQ ID NO. 28; The upstream and downstream primer sequences of D13S317 are shown as SEQ ID NO.29 and SEQ ID NO. 30; The primer pair sequences of D7S820 are shown as the upstream and downstream primer sequences in SEQ ID NO. 31 and SEQ ID NO. 32; The upstream and downstream primer sequences of D19S433 are shown in SEQ ID NO. 33 and SEQ ID NO. 34; The upstream and downstream primer sequences of CSF1PO are shown in SEQ ID NO. 35 and SEQ ID NO. 36; The upstream and downstream primer sequences of Penta D are shown in SEQ ID NO. 37 and SEQ ID NO. 38; The upstream and downstream primer sequences of D2S441 are shown in SEQ ID NO. 39 and SEQ ID NO. 40; The upstream and downstream primer sequences of VWA are shown in SEQ ID NO. 41 and SEQ ID NO. 42; The upstream and downstream primer sequences of D8S1179 are shown in SEQ ID NO. 43 and SEQ ID NO. 44; The upstream and downstream primer sequences of TPOX are shown in SEQ ID NO. 45 and SEQ ID NO. 46; The upstream and downstream primer sequences of FGA are shown in SEQ ID NO. 47 and SEQ ID NO. 48; The upstream and downstream primer sequences of D4S2366 are shown in SEQ ID NO. 49 and SEQ ID NO. 50; The upstream and downstream primer sequences of D3S3045 are shown in SEQ ID NO. 51 and SEQ ID NO. 52; The upstream and downstream primer sequences of D16S539 are shown in SEQ ID NO. 53 and SEQ ID NO. 54; The upstream and downstream primer sequences of D22S1045 are shown in SEQ ID NO. 55 and SEQ ID NO. 56; The upstream and downstream primer sequences of D8S1132 are shown in SEQ ID NO. 57 and SEQ ID NO. 58; The upstream and downstream primer sequences of D1S1656 are shown in SEQ ID NO. 59 and SEQ ID NO. 60; The upstream and downstream primer sequences of D10S1248 are shown in SEQ ID NO. 61 and SEQ ID NO. 62; The upstream and downstream primer sequences of D10S1435 are shown in SEQ ID NO. 63 and SEQ ID NO.

64.

2. The fluorescent multiplex amplification system for 32 short tandem repeat sequences according to claim 1, wherein The 5'-ends of the 32 pairs of primers in the fluorescence multiplex amplification system are respectively labeled with 5 different fluorescent dyes. The same fluorescent dye labeling is regarded as the same group. The five groups of combinations are as follows: The first group: D3S1358, TH01, D21S11, D18S51 and Penta E; The second group: Y-indel, D19S253, D12S391, D6S1043, D2S1338, D15S659 and D6S477; The third group: Amelogenin, D5S818, D13S317, D7S820, D19S433, CSF1PO and Penta D; The fourth group: D2S441, VWA, D8S1179, TPOX, FGA and D4S2366; The fifth group: D3S3045, D16S539, D22S1045, D8S1132, D1S1656, D10S1248 and D10S1435.

3. The fluorescent multiplex amplification system for 32 short tandem repeats according to claim 2, wherein The fluorophore is at least one of FAM, HEX, TAMRA, ROX and AF549.

4. A fluorescence multiplex amplification system for 32 short tandem repeat sequences according to claim 1, characterized in that, It also includes a set of fluorescent molecular weight internal standard SIZE-500, and the fragment sizes of the molecular weight internal standard are: 75, 87, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500.

5. A fluorescence multiplex amplification system for 32 short tandem repeat sequences according to claim 4, characterized in that, The fluorophore labeling of the molecular weight internal standard uses AF633 dye.

6. The fluorescence multiplex amplification system of 32 short tandem repeat sequences according to claim 1, wherein It also includes an Allelic Ladder as an allelic typing standard.

7. A kit, characterized in that, It includes the fluorescent multiplex amplification system according to any one of claims 1-6.

8. A method for using the kit according to claim 7, characterized in that, The method includes the following steps: S1. Obtain the amplification template by using human genomic DNA extracted by the Chelex method, magnetic bead extraction method and silica bead extraction method, or by using a blood spot collection card and an oral cell collection card without extraction; S2. Amplify the obtained DNA; S3. Mix the molecular weight internal standard in the kit and deionized formamide to prepare a loading mixture, and then perform electrophoresis detection on the loading mixture and the amplification product or the allelic typing standard of 32 loci in the kit by a genetic analyzer; S4. Analyze the results.

9. The application of the fluorescent multiplex amplification system according to any one of claims 1-6, or the kit according to claim 7 in the abducted / missing children information system.

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