Amplification kit for 31 STR (short tandem repeat) gene loci marked by six-color fluorescence and application of amplification kit

Abstract

The invention relates to the technical field of biology, in particular to an amplification kit for 31 STR (short tandem repeat) gene loci marked by six-color fluorescence and application of the amplification kit. The STR typing primer disclosed by the invention comprises a primer pair for specifically amplifying 31 STR gene loci. The primer and the kit provided by the invention have the advantages of six-color fluorescence labeling, strong inhibition resistance and extremely high sensitivity, a complete genotyping map can be detected and obtained when low-concentration DNA is used as a template, the number of sites of the adopted site combination is more than that of sites of a conventional five-color fluorescence labeling amplification system, more genetic information can be provided, and the primer and the kit have the advantages of high sensitivity, high sensitivity and the like. Therefore, the individual identification capability is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an amplification kit for 31 STR loci with six-color fluorescence labeling and its application. Background Art

[0002] Short tandem repeats (STRs) are caused by slippage during DNA replication, or base mismatching between the slipped strand and the complementary strand during replication and repair, resulting in the deletion or insertion of one or several repeat units. STR inheritance conforms to Mendel's law of inheritance. Due to different repeat units and repeat numbers, the distribution of microsatellite DNA varies greatly among different races and populations, constituting STR genetic polymorphism. Moreover, the number of repeats at a homologous STR locus is also different among different individuals. Therefore, STR amplification detection technology has been widely used in forensic individual identification, paternity testing, population genetics analysis, and the construction of human DNA databases, etc.

[0003] Under normal circumstances, each STR locus is shared by approximately 5 - 20% of people, and the advantage of STR detection lies in the ability to simultaneously identify multiple STR loci.

[0004] STR detection has become the most commonly used and most mature technology in forensic science identification, and has been widely used in forensic identification and criminal case inspections by public security departments. STR data has become the main component of forensic science databases in various countries for comparing and screening criminal suspects. Theoretically, the individual identification rate can reach 0.999999999998 when 16 STR loci are jointly applied. However, in some identifications, more loci are required to provide more information to avoid possible misjudgments in some cases. Most of the case kits currently on the market select 13 CODIS (Combined DNA Index System: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D81179, D13S317, D16S539, D18S51, D21S11) loci, plus 8 - 15 preferred or alternative loci.

[0005] However, the common detection methods in the prior art are all five-color fluorescence labeling. For example, the Chinese invention patent CN116024357 A, a multiplex amplification system based on human Y-STR loci and its application, uses a five-color fluorescence labeling amplification system. The locus combination adopted by this invention is a five-color fluorescence labeling amplification system, which results in the problem of limited number of STRs detected synchronously in practical applications. The design of the kits on the market is adapted to genetic analyzers. Currently, commonly used genetic analyzers use capillary electrophoresis and five-color fluorescence excitation to convert optical signals into electrical signals for detection purposes. All the selected primer fluorescence labels are mainly five-color fluorescence labeling detection systems. However, with the innovation of corresponding industrial and software technologies, the combination of six-color fluorescence excitation acquisition system and capillary electrophoresis has given rise to the upgrading of corresponding STR typing detection kits. At the same time, due to the increase in fluorescence labeling in the same detection system, the number of STRs that can be detected synchronously has increased, which makes up for the defect that the number of STR loci in the five-color fluorescence labeling system cannot meet the requirements. For example, the Chinese invention patent CN115029450 A discloses a fluorescence multiplex amplification system for 32 short tandem repeat sequences and its application. The fluorescence multiplex amplification system of this invention includes 30 autosomal loci, one sex identification and one 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, thus improving the individual identification ability. Although its kit has a certain anti-inhibition ability and can detect samples with a concentration of 0.25 ng, its sensitivity cannot meet the high-standard requirements of the current market demand.

[0006] To achieve the above object, the present application provides a multiplex amplification system and a kit for 31 STR sequences. The locus combination of the six-color fluorescence labeling amplification system adopted has more loci than the five-color fluorescence labeling amplification system in the prior art, and these loci have extremely high individual recognition rates and non-paternity exclusion rates. It can be used for forensic individual identification, paternity testing, and population genetics analysis, and has the characteristics of more loci, high accuracy, strong anti-inhibition ability, and better sensitivity. Summary of the Invention

[0007] In view of this, the present invention provides an amplification kit and its application for 31 STR loci with six-color fluorescence labeling. The primers have strong anti-inhibition ability and extremely high sensitivity, and complete genotyping maps can be detected when using low-concentration DNA as a template.

[0008] To achieve the above invention object, the present invention provides the following technical solutions:

[0009] STR typing primers, including primer pairs that specifically amplify at least one of the following STR gene loci: D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659, D10S1435, D3S1358, D13S317, D7S820, D2S1338, D18S51, D3S3045, D19S433, D22S1045, D8S1179, vWA, TPOX, D8S1132, D10S1248, D12S391, CSF1PO, FGA, D6S477, Penta D, D6S1043, TH01, Penta E, D19S253, Amelogenin, and Y-indel.

[0010] In some embodiments, the STR typing primers include any one of the following primer pairs:

[0011] A specific primer pair for amplifying D2S441 as shown in SEQ ID NOs. 1-2;

[0012] A specific primer pair for amplifying D5S818 as shown in SEQ ID NOs. 3-4;

[0013] A specific primer pair for amplifying D21S11 as shown in SEQ ID NOs. 5-6;

[0014] A specific primer pair for amplifying D16S539 as shown in SEQ ID NOs. 7-8;

[0015] A specific primer pair for amplifying D1S1656 as shown in SEQ ID NOs. 9-10;

[0016] A specific primer pair for amplifying D15S659 as shown in SEQ ID NOs. 11-12;

[0017] A specific primer pair for amplifying D10S1435 as shown in SEQ ID NOs. 13-14;

[0018] A specific primer pair for amplifying Amelogenin as shown in SEQ ID NOs. 15-16;

[0019] A specific primer pair for amplifying D3S1358 as shown in SEQ ID NOs. 17-18;

[0020] A specific primer pair for amplifying D13S317 as shown in SEQ ID NOs. 19-20;

[0021] A specific primer pair for amplifying D7S820 as shown in SEQ ID NOs. 21-22;

[0022] Specific primer pairs for amplifying D2S1338 as shown in SEQ ID NO. 23 - 24;

[0023] Specific primer pairs for amplifying D18S51 as shown in SEQ ID NO. 25 - 26;

[0024] Specific primer pairs for amplifying D3S3045 as shown in SEQ ID NO. 27 - 28;

[0025] Specific primer pairs for amplifying Y - indel as shown in SEQ ID NO. 29 - 30;

[0026] Specific primer pairs for amplifying D19S433 as shown in SEQ ID NO. 31 - 32;

[0027] Specific primer pairs for amplifying D22S1045 as shown in SEQ ID NO. 33 - 34;

[0028] Specific primer pairs for amplifying D8S1179 as shown in SEQ ID NO. 35 - 36;

[0029] Specific primer pairs for amplifying vWA as shown in SEQ ID NO. 37 - 38;

[0030] Specific primer pairs for amplifying TPOX as shown in SEQ ID NO. 39 - 40;

[0031] Specific primer pairs for amplifying D8S1132 as shown in SEQ ID NO. 41 - 42;

[0032] Specific primer pairs for amplifying D10S1248 as shown in SEQ ID NO. 43 - 44;

[0033] Specific primer pairs for amplifying D12S391 as shown in SEQ ID NO. 45 - 46;

[0034] Specific primer pairs for amplifying CSF1PO as shown in SEQ ID NO. 47 - 48;

[0035] Specific primer pairs for amplifying FGA as shown in SEQ ID NO. 49 - 50;

[0036] Specific primer pairs for amplifying D6S477 as shown in SEQ ID NO. 51 - 52;

[0037] Specific primer pairs for amplifying PentaD as shown in SEQ ID NO. 53 - 54;

[0038] Specific primer pairs for amplifying D6S1043 as shown in SEQ ID NO. 55 - 56;

[0039] Specific primer pairs for amplifying TH01 as shown in SEQ ID NO. 57 - 58;

[0040] Specific primer pairs for amplifying Penta E as shown in SEQ ID NO. 59 - 60;

[0041] Specific primer pairs for amplifying D19S253 as shown in SEQ ID NO. 61 - 62.

[0042] The present invention divides the STR gene loci into five groups, and labels them with five different fluorescent dyes in sequence. In some specific embodiments, the five groups of STR gene loci are as follows:

[0043] The first group includes D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659, and D10S1435;

[0044] The second group includes Amelogenin, D3S1358, D13S317, D7S820, D2S1338, D18S51, and D3S3045;

[0045] The third group includes Y - indel, D19S433, D22S1045, D8S1179, vWA, TPOX, and D8S1132;

[0046] The fourth group includes D10S1248, D12S391, CSF1PO, FGA, and D6S477;

[0047] The fifth group includes Penta D, D6S1043, TH01, Penta E, and D19S253.

[0048] In some embodiments, the fluorescent dyes for labeling the amplification primers of the first to fifth groups of STR gene loci are selected from blue fluorescent dyes, green fluorescent dyes, yellow fluorescent dyes, red fluorescent dyes, or purple fluorescent dyes. In some specific embodiments, the primers of the first to fifth groups are labeled with FAM, HEX, TAMAR, ROX, and PURP in sequence.

[0049] The present invention also provides an STR genotyping kit, which includes the STR genotyping primers of the present invention.

[0050] In some embodiments, the kit further includes a sixth group of components for electrophoresis detection: an orange - fluorescent - labeled internal standard.

[0051] In some embodiments, the kit further includes PCR amplification reaction reagents; the PCR amplification reagents include PCR Mix and template DNA.

[0052] The present invention also provides an STR genotyping method, which uses the STR genotyping primers or the kit of the present invention to perform PCR amplification on a DNA sample to be tested, and obtains an STR genotyping map and data according to the amplification result.

[0053] In some specific embodiments, after the PCR amplification, the obtained amplification product is subjected to fragment analysis by capillary electrophoresis, and detected by an ABI series genetic analyzer. The detection result is analyzed on data analysis software such as GeneMapper TM ID-X, etc., to obtain the corresponding STR genotyping map and data.

[0054] The present invention also provides the application of the STR genotyping primers or kit in individual identification, paternity testing or population genetics analysis.

[0055] In the genotyping method of the present invention, the detection procedure includes denaturation - thermal cycling - final extension - incubation;

[0056] The DNA sample to be tested is derived from blood (stain), blood stain, semen, seminal stain, saliva (stain), body fluid, hair, muscle, tissue or nail.

[0057] Using the primer system and kit of 31 STR sequences provided by the present invention, it has six-color fluorescence labeling, strong anti-inhibition ability, extremely high sensitivity, and can detect and obtain a complete gene typing map even when using low-concentration DNA as a template. The locus combination adopted has more locus numbers than the conventional five-color fluorescence labeling amplification system, can provide more genetic information, and thus improves the individual identification ability; moreover, these loci have extremely high individual identification rates and non-paternity exclusion rates, and can be used for forensic individual identification, paternity testing and population genetics analysis. They have many loci, high accuracy, strong anti-inhibition ability, and better sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Querying the core locus of the gene in STRbase for Example 1 of the present invention;

[0059] Figure 2 Selecting the core sequence of the gene locus for Example 1 of the present invention;

[0060] Figure 3 Selecting forward and reverse primers according to the fragment size of the core sequence of the gene locus for Example 1 of the present invention;

[0061] Figure 4 Arrangement of gene loci for Example 1 of the present invention;

[0062] Figure 5 This is the 0.25 ng / μL 9948 standard DNA map in Example 2 of the present invention;

[0063] Figure 6 This is the genotyping locus map of the sensitivity (from high to low concentration) of the multiplex amplification system in Example 2 of the present invention;

[0064] Figure 7 This is the genotyping map of the multiplex amplification system kit under the influence of humic acid in Example 3 of the present invention;

[0065] Figure 8 This is the VeriFiler TM Plus kit genotyping map under the influence of humic acid in Example 3 of the present invention;

[0066] Figure 9 This is the genotyping map of individual identification of saliva (stain) samples using the multiplex amplification system kit in Example 4 of the present invention;

[0067] Figure 10 This is the genotyping map of individual identification of blood (stain) samples using the multiplex amplification system kit in Example 4 of the present invention;

[0068] Figure 11 This is the genotyping map of individual identification of hair follicle (root) samples using the multiplex amplification system kit in Example 4 of the present invention;

[0069] Figure 12 This is the genotyping map of individual identification of exfoliated cell (tissue DNA) samples using the multiplex amplification system kit in Example 4 of the present invention. Detailed implementation manners

[0070] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are a part rather than all of the embodiments of the present application. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0071] The multiplex amplification system provided by the present invention includes 31 pairs of amplification primers, which can simultaneously amplify 31 STR gene loci; the gene loci include D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659, D10S1435, D3S1358, D13S317, D7S820, D2S1338, D18S51, D3S3045, D19S433, D22S1045, D8S1179, vWA, TPOX, D8S1132, D10S1248, D12S391, CSF1PO, FGA, D6S477, Penta D, D6S1043, TH01, Penta E, D19S253, Amelogenin and Y-indel.

[0072] In the multiplex amplification system provided by the present invention, the amplified loci are respectively fluorescently labeled with species colors, and the same fluorescent label is regarded as the same group. The five groups of combinations are as follows:

[0073] The first group: D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659, D10S1435;

[0074] The second group: Amelogenin, D3S1358, D13S317, D7S820, D2S1338, D18S51, D3S3045;

[0075] The third group: Y-indel, D19S433, D22S1045, D8S1179, vWA, TPOX, D8S1132;

[0076] The fourth group: D10S1248, D12S391, CSF1PO, FGA, D6S477;

[0077] The fifth group: Penta D, D6S1043, TH01, Penta E, D19S253.

[0078] The six fluorescent labels are FAM, HEX, TAMAR, ROX, PURP and ORG respectively.

[0079] The first group is labeled with FAM, the second group is labeled with HEX, the third group is labeled with TAMAR, the fourth group is labeled with ROX, and the fifth group is labeled with PURP. The multiplex amplification system also includes a sixth component: internal standard; the internal standard is labeled with ORG.

[0080] Furthermore, the amplification system also includes PCR Mix and template DNA.

[0081] The PCR Mix was purchased from Multiplex PCR Mix.

[0082] The PCR amplification program is as follows: The first step is denaturation: 95°C for 1 minute; thermal cycling: 95°C for 10 seconds; 60°C for 90 seconds, for a total of 29 cycles; final extension at 60°C for 20 minutes; incubation at 4°C. The amplification products of the present invention need to be subjected to capillary electrophoresis for fragment analysis.

[0083] The amplification system or kit can be applied to forensic individual identification, paternity testing, and population genetics research.

[0084] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0085] The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

[0086] The technical solutions of the present invention will be further described in detail below through specific examples.

[0087] Example 1

[0088] 1. Primer sequence design and gene locus arrangement in the multiplex amplicon of 31 STRs

[0089] The primer design specifically includes searching for the core repeat sequence of the locus in NCBI or STRbase; selecting 500 - 700 bp before and after the core repeat sequence of the locus; selecting the upstream and downstream primers according to the required fragment size, and performing Blast alignment on the upstream and downstream primers, with the TM value between 56 - 62°C and ΔT ± 3°C; using Oligo 7 software to evaluate the upstream and downstream primers, without 3'-terminal dimers; the amplification efficiency of each locus should not vary too much.

[0090] In this example, 31 pairs of STR locus primers were designed according to the above conditions. The specific operation steps include:

[0091] (1) Search for the core repeat sequence of the locus in NCBI or STRbase (https: / / strbase.nist.gov / ); refer to Figure 1 , which is the query for the core sequence of the locus in STRbase.

[0092] (2) Select 700 bp before and after the core repeat sequence of the locus. The reason for selecting 700 bp is that the sequence size can be moved at any time when designing primers and large fragment sequences can be fully included; refer to Figure 2 .

[0093] (3) Select forward and reverse primers according to the required fragment size. When designing the primers, we fully consider the core sequence size, TM value, and GC content of the locus. Perform Blast alignment on the forward and reverse primers, and evaluate that the TM values of the forward and reverse sequences are between 56 - 62 °C, with ΔT ± 3 °C; refer to Figure 3 。

[0094] (4) Use Oligo 7 software to evaluate the upstream and downstream primers, and there are no 3'-terminal dimers;

[0095] Design 31 pairs of STR locus primers according to the conditions in step (3), refer to Table 1, and arrange them according to the difficulty of locus primer design, and arrange the loci according to the TM value in the primer Blast and the number of repeats of the locus core sequence. Refer to Figure 1 。

[0096] Table 1 Primer sequences of 31 STR loci

[0097]

[0098]

[0099]

[0100] Label the 5'-end of at least one primer in each primer pair with a fluorescent dye. Group and label the above primers as follows. Primers with the same fluorescent label are regarded as the same group. The five groups of combinations are:

[0101] Group 1: D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659, D10S1435;

[0102] Group 2: Amelogenin, D3S1358, D13S317, D7S820, D2S1338, D18S51, D3S3045;

[0103] Group 3: Y-indel, D19S433, D22S1045, D8S1179, vWA, TPOX, D8S1132;

[0104] Group 4: D10S1248, D12S391, CSF1PO, FGA, D6S477;

[0105] Group 5: PentaD, D6S1043, TH01, Penta E, D19S253;

[0106] Group 6 is the internal standard.

[0107] The six groups of fluorescent labels are FAM, HEX, TAMAR, ROX, PURP, and ORG respectively.

[0108] The label of the first group is the FAM label, the label of the second group is the HEX label, the label of the third group is the TAMAR label, the label of the fourth group is the ROX label, and the label of the fifth group is the PURP label. The internal standard used in the detection of this multiplex amplification system is the ORG label, which is the sixth group of fluorescent labels.

[0109] 2. Preparation of the multiplex amplification detection system

[0110] (1) Mix 31 pairs of single-site primers in a 1:1 ratio, and add 1×Low TE to make up to 105 μL to prepare a multiplex primer; the specific ratio is shown in Table 2.

[0111] (2) Oscillate and mix each reaction component to prepare a PCR reaction solution. Specifically, the total volume of the amplification system is 10 μL, including 1 μL of primer mixture (10×Primer mix), 2 μL of reaction buffer (5×PCR mix), 1 μL of 0.25 ng / μL 9948 standard DNA, and 6 μL of 1×Low TE, to obtain a multiplex amplification detection system.

[0112] Table 2 Primer formulations for 31 STR loci

[0113]

[0114]

[0115] 3. Amplification

[0116] Amplify the amplification detection system in step 2 according to the reaction program. The amplification program of the multiplex amplification system is: the first step of denaturation: 95°C, 1 minute; thermal cycling: 95°C, 10 seconds; 60°C, 90 seconds, for a total of 29 cycles; final extension 60°C, 20 minutes; incubation 4°C. Amplify according to the above amplification program to obtain an amplification product.

[0117] 4. Fragment analysis by capillary electrophoresis

[0118] Detect the amplification product by capillary electrophoresis. Mix the internal standard and formamide in a volume ratio of 2:100, take 9 μL of the mixture and add it to a 96-well plate, then add 1 μL of the amplification product or allele standard, mix well, centrifuge, and place it on an ABI3500 genetic analyzer for preparation of detection.

[0119] 5. Data analysis

[0120] Use GeneMapper TMThe ID-X software analyzes the obtained data and generates a spectrogram. The data analysis method includes: importing the original data, selecting Add sample to project in the File menu on the main page, finding the sample file, selecting the folder, clicking add to list, and then clicking add. The sample file will be displayed in the Project window: Select the analysis parameters. Define analysismethod, panel, and size standard. Browse the original data of the sample electrophoresis, select the file name of a certain sample, and select Raw data in the "sample" menu. Move the tracking line to stop the cursor on the right side of the primer peak (before the first orange internal standard peak). Use the value displayed on the X-axis at the lower left corner of the window at this time as the starting point in the analysis parameters of the analysis method: Click the green analysis button. A save project dialog box will appear. After naming and saving, the software will start processing the data. After the analysis is completed, analysis completed will be displayed at the lower left corner.

[0121] Repeat steps 1-5 to make the average intra-color balance ≥ 0.5; the average inter-color balance ≥ 0.5; the average large fragment amplification index greater than 1, so as to obtain the primer concentration values of each locus. Refer to Table 3. The multiplex primers formulated according to the seventh formula in Table 3 have high sensitivity at this concentration.

[0122] Table 3 Primer Concentrations of 31 STR Loci

[0123]

[0124]

[0125]

[0126] Example 2

[0127] This example is a test example for detecting the sensitivity using the multiplex amplification system kit of 31 STRs in Example 1.

[0128] The specific steps include:

[0129] 1. Select positive DNA 9948 as the control sample. Refer to Figure 5 , which is the 9948 standard DNA spectrogram at 0.25 ng / μL.

[0130] 2. Six concentration gradients of 500 pg, 250 pg, 125 pg, 62.5 pg, 50 pg, and 31.25 pg were selected respectively. Generally, the DNA content of conventional forensic samples is above 62.5 pg, but the DNA content of trace forensic samples in cases is below 62.5 pg. Currently, existing kits can generally detect conventional samples (above 62.5 pg). Therefore, in this example, two concentrations of 50 pg and 31.25 pg below 62.5 pg were selected for sensitivity detection.

[0131] After mixing each reaction component by oscillation, the PCR reaction solution was prepared in the following manner. The total volume of the amplification system was 10 μL, including 1 μL of primer mixture (10×Primer mix), 2 μL of reaction buffer (5×PCR mix), 1 μL of genomic DNA, and 6 μL of 1×Low TE. The amplification procedure for the above amplification detection system according to the reaction program of the multiplex amplification system was: the first step of denaturation: 95°C, 1 minute; thermal cycling: 95°C, 10 seconds; 60°C, 90 seconds, for a total of 29 cycles; final extension 60°C, 20 minutes; incubation 4°C. Amplification was carried out according to the above amplification procedure to obtain the amplification product.

[0132] 3. Capillary electrophoresis detection was performed for fragment analysis. The internal standard and formamide were mixed at a ratio of 2:100. 9 μL of the mixture was added to a 96-well plate, and then 1 μL of the amplification product or allele standard was added. After mixing and centrifugation, it was placed on an ABI3500 genetic analyzer for preparation of detection.

[0133] 4. Finally, data analysis was performed, and the data obtained were analyzed using GeneMapper TM ID-X software and generate a map. The analysis steps were the same as in Example 1.

[0134] Refer to Figure 6 , for the sensitivity genotyping locus map. As can be seen from the figure, the map contains the common allele genotypes of each locus.

[0135] Furthermore, through Table 4 and Figure 6 sensitivity analysis was performed. The positive DNA 9948 control sample amount was as low as 62.5 pg, and the allele locus detection rate was 100%; the allele detection rate was >95% under the 31.25 pg DNA template. It can be seen from the results that the multiplex amplification system provided in this example has high detection efficiency and can meet the amplification of different concentration samples in daily life.

[0136] Table 4 Sensitivity analysis results

[0137]

[0138]

[0139] Example 3

[0140] This example is for anti-inhibition detection using the multiplex amplification system kit of 31 STRs provided in Example 1.

[0141] The specific steps are as follows:

[0142] 1. Preparation of DNA inhibitor

[0143] Take 25 μL of 1 ng / μL 9948 standard DNA and 15 μL of 2000 ng / μL humic acid and add them to 60 μL of 1×Low TE to prepare 100 μL of DNA inhibitor.

[0144] After shaking and mixing each reaction component, prepare the PCR reaction solution in the following manner. The total volume of the amplification system is 10 μL, including 1 μL of primer mixture (10×Primer mix), 2 μL of reaction buffer (5×PCR mix), 5 μL of DNA inhibitor, and 2 μL of 1×Low TE. The amplification program of the above amplification detection system according to the reaction program of the multiplex amplification system is: the first step of denaturation: 95°C, 1 minute; thermal cycling: 95°C, 10 seconds; 60°C, 90 seconds, for a total of 29 cycles; final extension 60°C, 20 minutes; incubation 4°C. Amplify according to the above amplification program to obtain the amplification product.

[0145] 2. The comparison product is VeriFiler TM Plus PCR Amplification Kit, and prepare the amplification according to the instructions.

[0146] 3. Perform capillary electrophoresis detection. Mix the internal standard and formamide in a ratio of 2:100. Take 9 μL of the mixture and add it to a 96-well plate, then add 1 μL of the amplification product or allele standard, mix well, centrifuge, and place it on an ABI 3500 genetic analyzer for preparation of detection.

[0147] 4. Finally, perform data analysis. Use GeneMapper TM ID-X software to analyze the obtained data and generate a map. The map of the influence of 150 ng / μL humic acid on the amplification system of the present invention is shown in Figure 7 , and the influence on the comparison product is shown in Figure 8 . From Figure 7 and Figure 8 the peak heights of the obtained maps (refer to Table 5) are compared, and it can be seen that the anti-humic acid inhibition ability of the amplification system provided in this example is stronger than that of the imported product VeriFiler TM Plus.

[0148] Table 5 Influence of the amplification system of this example and VeriFiler at the level of 150 ng / μL humic acidTM Plus Peak Height Comparison

[0149]

[0150]

[0151] Example 4

[0152] In this Example 4, the application of the multiplex amplification system kit of 31 STRs provided in Example 1 in individual identification was used to perform individual identification on the test samples respectively, and individual STR genotyping was obtained.

[0153] 1. Obtaining of Samples

[0154] They respectively include: Sample 1 human saliva (stain) sample; Sample 2 human blood (stain) sample; Sample 3 human hair sample with hair follicles (roots); Sample 4 human exfoliated cell (tissue DNA) sample.

[0155] 2. DNA Extraction

[0156] The Chelex-100 method was used to extract locus DNA. Blood stains of (1-3mm) * (2-5mm) were placed in 1.5 mL centrifuge tubes, and the Chelex solution was shaken and mixed well to make Chelex fully suspended. 195 μL of Chelex-100 (5%) solution and 5 μL of proteinase K (20 mg / mL) were added to each tube and shaken and mixed well. After incubation at 56 °C for 2 h, it was taken out and shaken for 2 min, heated at 95 °C for 10 min, centrifuged at 13000 rpm for 5 min, and the supernatant was transferred to a new centrifuge tube.

[0157] After shaking and mixing each reaction component, the PCR reaction solution was prepared in the following manner. The total volume of the amplification system was 10 μL, including 1 μL of primer mixture (10×Primer mix), 2 μL of reaction buffer (5×PCR mix), 1 μL of DNA sample, and 6 μL of 1×LowTE. The amplification program of the above amplification detection system according to the reaction procedure of the multiplex amplification system was: The first step of denaturation: 95 °C, 1 minute; Thermal cycling: 95 °C, 10 seconds; 60 °C, 90 seconds, for a total of 29 cycles; Final extension 60 °C, 20 minutes; Incubation at 4 °C. Amplification was carried out according to the above amplification program to obtain amplification products.

[0158] 3. Using GeneMapper TM ID-X software to analyze the obtained data and generate a map

[0159] The obtained individual STR genotyping is shown in Figures 9 - 12As shown by the results in the figure, complete STR genotyping was obtained for all samples 1-4 to be tested, and the peak patterns were sharp and clear, without background bands, with good balance, no Pull-up peaks, stutter bands, and no non-specific amplification products, fully meeting the requirements of STR testing.

[0160] According to Figures 9 - 12 Calculate the likelihood ratio, refer to Table 6.

[0161] Table 6 Calculation results of the likelihood ratio for each sample in individual identification

[0162]

[0163]

[0164] Among them, PM: random match probability; CPM: cumulative random match probability; TDP: cumulative individual identification ability; LR: likelihood ratio.

[0165] Example 5

[0166] In this example, a multiplex amplification system kit of 31 STRs provided in Example 1 was used for paternity testing.

[0167] Perform kinship identification on a group of triadic families. The genotyping of each individual in the triadic family obtained by using the multiplex amplification system of 31 STRs provided in this example is shown in Table 7. Identification basis: According to the "Technical Specification for Paternity Testing GB / T 37223—2018", 29 STR loci such as D2S441 are all genetic markers of humans, following Mendel's genetic law. The combined application can be used for paternity testing, and its cumulative probability of excluding non-fathers is greater than 0.999999. Based on the above result analysis, at each STR locus, F can provide the necessary alleles for D. After calculation, the cumulative paternity index is 1.355225×10^13 (Note: greater than 10000).

[0168] Among them, the test samples in paternity testing also include samples such as semen (stain), hair with follicles, amniotic fluid, tissue blocks, etc. It can be seen from the amplification and extraction of samples using the amplification system provided in this example that the sensitivity of this kit can meet the requirements of daily experiments, can be applied to the amplification of various different types of extracted samples, and has a wide practical application range.

[0169] Table 7 Individual genotyping of triadic families

[0170]

[0171] Among them, PI: paternity index; CPI: cumulative paternity index; RCP: paternity probability.

[0172] The above are only the preferred embodiments of the present invention, and thus do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any changes, modifications, substitutions, integrations, and parameter changes to these embodiments that are made by means of conventional substitutions or that can achieve the same functions without departing from the principle and spirit of the present invention fall within the protection scope of the present invention.

Claims

1. STR typing primers, characterized in that, A primer pair that specifically amplifies at least one of the following STR gene loci: D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659, D10S1435, D3S1358, D13S317, D7S820, D2S1338, D18S51, D3S3045, D19S433, D22S1045, D8S1179, vWA, TPOX, D8S1132, D10S1248, D12S391, CSF1PO, FGA, D6S477, Penta D, D6S1043, TH01, Penta E, D19S253, Amelogenin, and Y-indel.

2. The STR typing primer according to claim 1, characterized in that, It includes any one of the following primer pairs: A specific primer pair for amplifying D2S441 as shown in SEQ ID NO. 1-2; A specific primer pair for amplifying D5S818 as shown in SEQ ID NO. 3-4; A specific primer pair for amplifying D21S11 as shown in SEQ ID NO. 5-6; A specific primer pair for amplifying D16S539 as shown in SEQ ID NO. 7-8; A specific primer pair for amplifying D1S1656 as shown in SEQ ID NO. 9-10; A specific primer pair for amplifying D15S659 as shown in SEQ ID NO. 11-12; A specific primer pair for amplifying D10S1435 as shown in SEQ ID NO. 13-14; A specific primer pair for amplifying Amelogenin as shown in SEQ ID NO. 15-16; A specific primer pair for amplifying D3S1358 as shown in SEQ ID NO. 17-18; A specific primer pair for amplifying D13S317 as shown in SEQ ID NO. 19-20; A specific primer pair for amplifying D7S820 as shown in SEQ ID NO. 21-22; A specific primer pair for amplifying D2S1338 as shown in SEQ ID NO. 23-24; A specific primer pair for amplifying D18S51 as shown in SEQ ID NO. 25-26; A specific primer pair for amplifying D3S3045 as shown in SEQ ID NO. 27-28; A specific primer pair for amplifying Y-indel as shown in SEQ ID NO. 29-30; A specific primer pair for amplifying D19S433 as shown in SEQ ID NO. 31-32; A specific primer pair for amplifying D22S1045 as shown in SEQ ID NO. 33-34; A specific primer pair for amplifying D8S1179 as shown in SEQ ID NO. 35-36; A specific primer pair for amplifying vWA as shown in SEQ ID NO. 37-38; A specific primer pair for amplifying TPOX as shown in SEQ ID NO. 39-40; Specific primer pairs for amplifying D8S1132 as shown in SEQ ID NO. 41-42; Specific primer pairs for amplifying D10S1248 as shown in SEQ ID NO. 43-44; Specific primer pairs for amplifying D12S391 as shown in SEQ ID NO. 45-46; Specific primer pairs for amplifying CSF1PO as shown in SEQ ID NO. 47-48; Specific primer pairs for amplifying FGA as shown in SEQ ID NO. 49-50; Specific primer pairs for amplifying D6S477 as shown in SEQ ID NO. 51-52; Specific primer pairs for amplifying Penta D as shown in SEQ ID NO. 53-54; Specific primer pairs for amplifying D6S1043 as shown in SEQ ID NO. 55-56; Specific primer pairs for amplifying TH01 as shown in SEQ ID NO. 57-58; Specific primer pairs for amplifying Penta E as shown in SEQ ID NO. 59-60; Specific primer pairs for amplifying D19S253 as shown in SEQ ID NO. 61-62.

3. The STR genotyping primer according to claim 1 or 2, characterized in that, The primer pairs are divided into five groups, wherein the primer pairs between the five groups are respectively labeled with five different fluorescent dyes, and the primer pairs in the same group are labeled with the same fluorescent dye.

4. The STR typing primer according to claim 3, wherein The first group includes D2S441, D5S818, D21S11, D16S539, D1S1656, D15S659 and D10S1435; The second group includes Amelogenin, D3S1358, D13S317, D7S820, D2S1338, D18S51 and D3S3045; The third group includes Y-indel, D19S433, D22S1045, D8S1179, vWA, TPOX and D8S1132; The fourth group includes D10S1248, D12S391, CSF1PO, FGA and D6S477; The fifth group includes Penta D, D6S1043, TH01, Penta E and D19S253; The fluorescent dyes for labeling the amplification primers of the first to fifth groups are selected from any one of blue fluorescent dye, green fluorescent dye, yellow fluorescent dye, red fluorescent dye or purple fluorescent dye, and are different from each other.

5. STR genotyping kit, characterized in that, Including the STR typing primer according to claim 1 or 2.

6. The kit according to claim 5, characterized in that, It further includes a sixth group component: an orange fluorescently labeled internal standard.

7. The kit according to claim 5, characterized in that, It further includes PCR amplification reaction reagents; the PCR amplification reaction reagents include PCR Mix and template DNA.

8. Use of the STR typing primer according to any one of claims 1-4 or the kit according to any one of claims 5-7 in forensic individual identification, paternity testing or population genetics analysis.

9. STR typing method, characterized in that, Perform PCR detection on the DNA sample to be tested using the STR typing primer according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 7, and obtain the STR typing map and data based on the detection results.

10. The STR typing method according to claim 9, wherein the procedure of the detection includes denaturation - thermal cycling - final extension - incubation; the DNA sample to be tested is derived from blood (stain), bloodstain, semen, seminal stain, saliva (stain), body fluid, hair, muscle, tissue or nail.

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