A multiplex amplification system of 32 STR loci and application thereof

Abstract

The application belongs to the technical field of biological gene detection, and particularly relates to a 32-STR-gene-locus composite amplification system and application thereof. The amplification system comprises a specific amplification primer combination for simultaneously amplifying 32 STR gene loci, and has a nucleotide sequence as shown in SEQ ID NO. 1-64. The combination of the 32 STR gene loci has high polymorphism and individual identification capability. The system solves the problem of low individual identification rate caused by insufficient sites of a traditional kit, optimizes a multi-gene-locus arrangement strategy, improves the detection rate of degraded samples, and improves the detection sensitivity and reliability.

Description

Technical Field

[0001] This invention belongs to the field of biological gene detection technology, specifically relating to a multiplex amplification system with 32 STR loci and its application. Background Technology

[0002] Short tandem repeat sequences ( short tandem repeat STRs are a class of DNA polymorphic loci that are widely present in the human genome. They are highly polymorphic due to differences in DNA fragment length or DNA sequence between individuals and are inherited in a Mendelian codominant manner during gene transmission.

[0003] Forensic STR testing has evolved from single-locus assays to high-throughput multiplex systems, driven by improvements in individual identification capabilities and database compatibility. In the 1990s, the UK's SGM kit (6 loci) and the US's Identifiler (15 loci) established the CODIS standard framework. After 2010, GlobalFiler™ (24 loci, including D2S1338, D19S433, D12S391, etc.) became the international mainstream. In recent years, the number of loci in kits has continued to increase, with PowerPlex® Fusion 6C (27 loci) and GlobalFiler™ Plus (28 loci) further expanding the detection range. Technological development focuses on overcoming the physical limits of single-tube multiplex amplification, achieving higher throughput and sensitivity through optimized primer design and fluorescent labeling systems.

[0004] Forensic STR kits have evolved towards higher sensitivity, greater compatibility, and greater adaptability in practical applications, with a core focus on the stable detection of low-quality samples. Industry technological innovation focuses on the precision of primer design—by shortening amplicon length and optimizing the thermostability of primer binding sites, the aim is to improve the capture efficiency of fragmented DNA; simultaneously, multiplex PCR systems achieve dynamic equilibrium, utilizing modified polymerases and intelligent buffer formulations to coordinate the amplification uniformity of dozens of STR sites in a single reaction, inhibiting allele loss caused by dominant amplification.

[0005] However, with the increasingly complex environments for collecting evidence at crime scenes (such as high temperature and humidity, exposure to sunlight, or chemical contamination), the ability of STR kits to detect degraded DNA and trace samples still faces severe challenges. Furthermore, high individual identification rate is the core objective of forensic STR technology, driving the evolution of kits towards high-information-content composite detection systems. This involves enhancing the uniqueness of allele combinations by covering more than 28 STR loci in a single amplification (such as GlobalFiler™ Plus), and relying on amplicon length optimization design (shortening key sites such as D18S51 to 280bp) and an anti-inhibitor PCR system (amplification deviation ±15%) to ensure typing accuracy to meet the matching needs of tens of millions of samples in international databases. However, the inherent defects of low-quality samples severely limit the improvement of recognition rates: DNA fragmentation (<200bp) in highly degraded bone results in a detection rate of less than 60% for large STR loci, causing allele deletions; picogram-level micro-samples exhibit increased stutter peak proportions (reaching the main peak of 30%) due to random amplification bias, coupled with the resolution defects of autosomal STRs in mixed patches, leading to a typing error rate exceeding 40%, significantly reducing the confidence in individual identification. These bottlenecks highlight the urgent need for synergistic breakthroughs in picogram-level detection technology and novel locus combination strategies.

[0006] Overcoming the bottleneck in detecting low-quality samples remains a key challenge that needs to be addressed in current technology optimization. Summary of the Invention

[0007] The purpose of this invention is to provide a multiplex amplification system for 32 STR loci. This invention provides 32 highly polymorphic STR loci, solving the problem of low individual recognition rate caused by insufficient loci in traditional kits. To achieve the above objectives, the present invention provides the following technical solution: This invention provides a multiplex amplification system for 32 STR loci, the multiplex amplification system comprising a specific amplification primer combination for simultaneously amplifying 32 STR loci; The 32 STR loci are Amel, D16S539, D7S820, D22S1045, D19S433, CSF1PO, D1S1656, D8S1179, D2S441, vWA, D12S391, TPOX, D2S1338, Penta D, RS771783753, D3S1358, TH01, D21S11, D18S51, D10S1248, D13S317, D15S659, D3S3045, D6S477, D6S1043, FGA, RS199815934, D5S818, D10S1435, D19S253, D8S1132, and Penta E.

[0008] Preferably, the sequences of the specific amplification primers for the 32 loci are as follows: The upstream and downstream primer sequences of the Amel are shown in SEQ ID NO. 1 and SEQ ID NO. 2; The upstream and downstream primer sequences of D16S539 are shown in SEQ ID NO. 3 and SEQ ID NO. 4; The upstream and downstream primer sequences of the D7S820 are shown in SEQ ID NO. 5 and SEQ ID NO. 6; The upstream and downstream primer sequences of D22S1045 are shown in SEQ ID NO. 7 and SEQ ID NO. 8; The upstream and downstream primer sequences of D19S433 are shown in SEQ ID NO. 9 and SEQ ID NO. 10; The upstream and downstream primer sequences of the CSF1PO are shown in SEQ ID NO. 11 and SEQ ID NO. 12; The upstream and downstream primer sequences of D1S1656 are shown in SEQ ID NO. 13 and SEQ ID NO. 14; The upstream and downstream primer sequences of D8S1179 are shown in SEQ ID NO. 15 and SEQ ID NO. 16; The upstream and downstream primer sequences of the D2S441 are shown in SEQ ID NO. 17 and SEQ ID NO. 18; The upstream and downstream primer sequences of the vWA are shown in SEQ ID NO. 19 and SEQ ID NO. 20; The upstream and downstream primer sequences of D12S391 are shown in SEQ ID NO. 21 and SEQ ID NO. 22; The upstream and downstream primer sequences of the TPOX are shown in SEQ ID NO. 23 and SEQ ID NO. 24; The upstream and downstream primer sequences of the D2S1338 are shown in SEQ ID NO. 25 and SEQ ID NO. 26; The upstream and downstream primer sequences of the Penta D are shown in SEQ ID NO. 27 and SEQ ID NO. 28; The upstream and downstream primer sequences of RS771783753 are shown in SEQ ID NO. 29 and SEQ ID NO. 30; The upstream and downstream primer sequences of the D3S1358 are shown in SEQ ID NO. 31 and SEQ ID NO. 32; The upstream and downstream primer sequences of TH01 are shown in SEQ ID NO. 33 and SEQ ID NO. 34; The upstream and downstream primer sequences of D21S11 are shown in SEQ ID NO. 35 and SEQ ID NO. 36; The upstream and downstream primer sequences of the D18S51 are shown in SEQ ID NO. 37 and SEQ ID NO. 38; The upstream and downstream primer sequences of D10S1248 are shown in SEQ ID NO. 39 and SEQ ID NO. 40; The upstream and downstream primer sequences of D13S317 are shown in SEQ ID NO. 41 and SEQ ID NO. 42; The upstream and downstream primer sequences of D15S659 are shown in SEQ ID NO. 43 and SEQ ID NO. 44; The upstream and downstream primer sequences of the D3S3045 are shown in SEQ ID NO. 45 and SEQ ID NO. 46; The upstream and downstream primer sequences of D6S477 are shown in SEQ ID NO. 47 and SEQ ID NO. 48; The upstream and downstream primer sequences of D6S1043 are shown in SEQ ID NO. 49 and SEQ ID NO. 50; The upstream and downstream primer sequences of the FGA are shown in SEQ ID NO. 51 and SEQ ID NO. 52; The upstream and downstream primer sequences of RS199815934 are shown in SEQ ID NO. 53 and SEQ ID NO. 54; The upstream and downstream primer sequences of the D5S818 are shown in SEQ ID NO. 55 and SEQ ID NO. 56; The upstream and downstream primer sequences of D10S1435 are shown in SEQ ID NO. 57 and SEQ ID NO. 58; The upstream and downstream primer sequences of D19S253 are shown in SEQ ID NO. 59 and SEQ ID NO. 60; The upstream and downstream primer sequences of the D8S1132 are shown in SEQ ID NO. 61 and SEQ ID NO. 62; The upstream and downstream primer sequences of the Penta E are shown in SEQ ID NO. 63 and SEQ ID NO. 64.

[0009] Preferably, the multiplex amplification system further includes one or more of the following: PCR buffer, PCR polymerase, DNA extraction reagent, sequencing library construction reagent, and nucleic acid purification reagent.

[0010] The present invention also provides the application of the multiplex amplification system described in any of the above claims in the preparation of products for human individual identification, paternity testing or genotyping.

[0011] The present invention also provides a reagent kit for forensic paternity testing, the reagent kit comprising the aforementioned multiplex amplification system.

[0012] This invention also provides a method for STR typing using the aforementioned multiplex amplification system or the aforementioned kit, comprising the following steps: (1) Extract DNA from the sample to be tested; (2) Using the primer mixture and PCR reaction premix in the multiplex amplification kit, prepare the amplification system and perform multiplex PCR amplification on the DNA extracted in step (1); (3) Capillary electrophoresis is performed on the amplification products obtained in step (2); (4) Collect electrophoresis data and perform genotyping using data analysis software to obtain the genotyping results of the sample to be tested at the 32 STR loci.

[0013] Preferably, the reaction procedure for multiplex PCR amplification in step (2) includes the following steps: first stage: hot start at 94℃ for 5 minutes, for 1 cycle; second stage: denaturation at 94℃ for 10 seconds, annealing at 64℃ for 1 minute, and extension at 72℃ for 1 minute, for 26-30 cycles; third stage: final extension at 60℃ for 10 minutes, for 1 cycle.

[0014] Preferably, the sample to be tested is a trace DNA sample or a degraded DNA sample.

[0015] Preferably, the internal standard used in the capillary electrophoresis detection in step (3) is SIZE-550.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention integrates 32 highly polymorphic STR loci, solving the problem of low individual identification rate caused by insufficient loci in traditional reagent kits; it optimizes the multi-locus arrangement strategy, improves the detection rate of degraded samples, and enhances detection sensitivity and reliability. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Electrophoretic pattern for detecting trace sample 1.

[0019] Figure 2 Electrophoretic pattern for detecting trace sample 2.

[0020] Figure 3 Electrophoretic patterns for sensitivity testing of 30 pg 9948 standard. Detailed Implementation

[0021] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0022] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0023] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0024] Example 1 1. This invention contains 32 STR loci, namely Amel, D16S539, D7S820, D22S1045, D19S433, CSF1PO, D1S1656, D8S1179, D2S441, vWA, D12S391, TPOX, D2S1338, PentaD, RS771783753, D3S1358, TH01, D21S11, D18S51, D10S1248, D13S317, D15S659, D3S3045, D6S477, D6S1043, FGA, RS199815934, D5S818, D10S1435, D19S253, D8S1132, and Penta E.

[0025] Table 1 Primer sequences for each locus

[0026] Among them, Amel, D16S539, D7S820, D22S1045, D19S433, CSF1PO, D1S1656, and D8S1179 were labeled with FAM fluorescence; D2S441, vWA, D12S391, TPOX, D2S1338, and Penta were labeled with FAM fluorescence. D was labeled with VIC fluorescence; RS771783753, D3S1358, TH01, D21S11, D18S51, D10S1248, and D13S317 were labeled with PFS570 fluorescence; D15S659, D3S3045, D6S477, D6S1043, and FGA were labeled with PFS590 fluorescence; RS199815934, D5S818, D10S1435, D19S253, D8S1132, and Penta E were labeled with PFS610 fluorescence; and the internal standard was labeled with PFS650 fluorescence.

[0027] 2. The multiplex PCR reaction system is shown in Figure 2: Table 2 Amplification System

[0028] 3. The amplification procedure is shown in Table 3: Table 3 Amplification Procedure

[0029] 4. Electrophoresis sample preparation is shown in Table 4:

[0030] Example 2: Detection effect of trace samples This embodiment is used to verify the detection capability of the 32 STR loci multiplex amplification system provided by this invention for trace samples. The specific steps are as follows: 2.1 Simulation of Trace Samples (1) Trace sample 1: Take a clean plastic bag, press or rub it firmly with your fingers for 5 seconds, cut the area, and then extract it. (2) Trace sample 2: Wipe the table clean, press firmly with your palm on the designated area of ​​the table for 5 seconds, wipe with a cotton swab using a two-step dry and wet method, and then proceed with the extraction.

[0031] 2.2 Extraction of DNA from Trace Samples Using the Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit, DNA was extracted according to the instructions to obtain a trace sample of DNA.

[0032] 2.3 Preparation of the amplification system 32 pairs of fluorescently labeled primers were added to ultrapure water and diluted to the final concentration in Table 1 of Example 1 and prepared to 1 μL / person.

[0033] The amplification system was prepared according to Table 2 in Example 1, wherein the DNA sample was DNA extracted from trace samples (each trace sample was tested 3 times).

[0034] 2.4 Amplification Amplification was performed according to the amplification procedure in Table 3 of Example 1, and the amplification products were retained.

[0035] 2.5 Preparation of the capillary electrophoresis system The amplification products were prepared according to the system in Table 4 of Example 1 and detected by capillary electrophoresis using an Applied Biosystems 3500xL genetic analyzer.

[0036] 2.6 Electrophoresis Data Analysis Import the raw data from capillary electrophoresis into GeneMapper ID-X software to generate electrophoretic maps.

[0037] The test results are as follows: Figure 1 (Trace sample 1) and Figure 2 (Trace sample 2) is shown. As can be seen from the figure, the kit of the present invention obtained clear, complete and balanced genotyping maps of all 32 STR loci in the two trace samples. The allele peak heights were balanced, and there was no obvious dominant amplification or allele loss, indicating that the kit has excellent detection ability for trace samples.

[0038] Example 3: Reagent Kit Sensitivity Test This embodiment is used to evaluate the detection sensitivity of the kit of the present invention.

[0039] 3.1 Preparation of the amplification system 32 pairs of fluorescently labeled primers were added to ultrapure water and diluted to the final concentration in Table 1 of Example 1 and prepared to 1 μL / person.

[0040] The amplification system was prepared according to Table 2 in Example 1, with the DNA sample being 30 pg of 9948 standard (20 repeated tests).

[0041] 3.2 Amplification Amplification was performed according to the amplification procedure in Table 3 of Example 1, and the amplification products were retained.

[0042] 3.3 Preparation of the capillary electrophoresis system The amplification products were prepared according to the system in Table 4 of Example 1 and detected by capillary electrophoresis using an Applied Biosystems 3500xL genetic analyzer.

[0043] 3.4 Electrophoresis Data Analysis Import the raw data from capillary electrophoresis into GeneMapper ID-X software to generate electrophoretic maps.

[0044] Test results as follows Figure 3 As shown, the kit of this invention successfully produced accurate, clear, and complete genotyping maps of all 32 STR loci in a 30 pg sample of 9948 standard samples, without any interference from other peaks, indicating that its detection sensitivity consistently reaches the 30 pg level. This high sensitivity ensures accurate identification of all loci even with extremely low DNA input, meeting the stringent reliability requirements for trace samples in forensic evidence science.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A multiplex amplification system for 32 STR loci, characterized in that, The composite amplification system includes a specific combination of amplification primers for simultaneously amplifying 32 STR loci; The 32 STR loci are Amel, D16S539, D7S820, D22S1045, D19S433, CSF1PO, D1S1656, D8S1179, D2S441, vWA, D12S391, TPOX, D2S1338, Penta D, RS771783753, D3S1358, TH01, D21S11, D18S51, D10S1248, D13S317, D15S659, D3S3045, D6S477, D6S1043, FGA, RS199815934, D5S818, D10S1435, D19S253, D8S1132, and Penta E.

2. The multiplex amplification system according to claim 1, characterized in that, The sequences of the specific amplification primers for the 32 loci are shown below: The upstream and downstream primer sequences of the Amel are shown in SEQ ID NO. 1 and SEQ ID NO. 2; The upstream and downstream primer sequences of D16S539 are shown in SEQ ID NO. 3 and SEQ ID NO. 4; The upstream and downstream primer sequences of the D7S820 are shown in SEQ ID NO. 5 and SEQ ID NO. 6; The upstream and downstream primer sequences of D22S1045 are shown in SEQ ID NO. 7 and SEQ ID NO. 8; The upstream and downstream primer sequences of D19S433 are shown in SEQ ID NO. 9 and SEQ ID NO. 10; The upstream and downstream primer sequences of the CSF1PO are shown in SEQ ID NO. 11 and SEQ ID NO. 12; The upstream and downstream primer sequences of D1S1656 are shown in SEQ ID NO. 13 and SEQ ID NO. 14; The upstream and downstream primer sequences of D8S1179 are shown in SEQ ID NO. 15 and SEQ ID NO. 16; The upstream and downstream primer sequences of the D2S441 are shown in SEQ ID NO. 17 and SEQ ID NO. 18; The upstream and downstream primer sequences of the vWA are shown in SEQ ID NO. 19 and SEQ ID NO. 20; The upstream and downstream primer sequences of D12S391 are shown in SEQ ID NO. 21 and SEQ ID NO. 22; The upstream and downstream primer sequences of the TPOX are shown in SEQ ID NO. 23 and SEQ ID NO. 24; The upstream and downstream primer sequences of the D2S1338 are shown in SEQ ID NO. 25 and SEQ ID NO. 26; The upstream and downstream primer sequences of the Penta D are shown in SEQ ID NO. 27 and SEQ ID NO. 28; The upstream and downstream primer sequences of RS771783753 are shown in SEQ ID NO. 29 and SEQ ID NO. 30; The upstream and downstream primer sequences of the D3S1358 are shown in SEQ ID NO. 31 and SEQ ID NO. 32; The upstream and downstream primer sequences of TH01 are shown in SEQ ID NO. 33 and SEQ ID NO. 34; The upstream and downstream primer sequences of D21S11 are shown in SEQ ID NO. 35 and SEQ ID NO. 36; The upstream and downstream primer sequences of the D18S51 are shown in SEQ ID NO. 37 and SEQ ID NO. 38; The upstream and downstream primer sequences of D10S1248 are shown in SEQ ID NO. 39 and SEQ ID NO. 40; The upstream and downstream primer sequences of D13S317 are shown in SEQ ID NO. 41 and SEQ ID NO. 42; The upstream and downstream primer sequences of D15S659 are shown in SEQ ID NO. 43 and SEQ ID NO. 44; The upstream and downstream primer sequences of the D3S3045 are shown in SEQ ID NO. 45 and SEQ ID NO. 46; The upstream and downstream primer sequences of D6S477 are shown in SEQ ID NO. 47 and SEQ ID NO. 48; The upstream and downstream primer sequences of D6S1043 are shown in SEQ ID NO. 49 and SEQ ID NO. 50; The upstream and downstream primer sequences of the FGA are shown in SEQ ID NO. 51 and SEQ ID NO. 52; The upstream and downstream primer sequences of RS199815934 are shown in SEQ ID NO. 53 and SEQ ID NO. 54; The upstream and downstream primer sequences of the D5S818 are shown in SEQ ID NO. 55 and SEQ ID NO. 56; The upstream and downstream primer sequences of D10S1435 are shown in SEQ ID NO. 57 and SEQ ID NO. 58; The upstream and downstream primer sequences of D19S253 are shown in SEQ ID NO. 59 and SEQ ID NO. 60; The upstream and downstream primer sequences of the D8S1132 are shown in SEQ ID NO. 61 and SEQ ID NO. 62; The upstream and downstream primer sequences of the Penta E are shown in SEQ ID NO. 63 and SEQ ID NO.

64.

3. The multiplex amplification system according to claim 1, characterized in that, The multiplex amplification system also includes one or more of the following: PCR buffer, PCR polymerase, DNA extraction reagent, sequencing library construction reagent, and nucleic acid purification reagent.

4. The use of the multiplex amplification system according to any one of claims 1-3 in the preparation of products for human individual identification, paternity testing or genotyping.

5. A reagent kit for forensic paternity testing, characterized in that, The kit comprises the multiplex amplification system according to any one of claims 1-3.

6. A method for STR typing using the multiplex amplification system according to any one of claims 1-3 or the kit according to claim 5, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the primer mixture and PCR reaction premix in the multiplex amplification kit, prepare the amplification system and perform multiplex PCR amplification on the DNA extracted in step (1); (3) Capillary electrophoresis is performed on the amplification products obtained in step (2); (4) Collect electrophoresis data and perform genotyping using data analysis software to obtain the genotyping results of the sample to be tested at the 32 STR loci.

7. The method according to claim 6, characterized in that, The reaction procedure for multiplex PCR amplification described in step (2) The process includes the following steps: First stage: 94℃ hot start for 5 minutes, one cycle; Second stage: 94℃ denaturation for 10 seconds, 64℃ annealing for 1 minute, and 72℃ extension for 1 minute, 26-30 cycles; Third stage: 60℃ final extension for 10 minutes, one cycle.

8. The method according to claim 6, characterized in that, The sample to be tested is a trace DNA sample or a degraded DNA sample.

9. The method according to claim 6, characterized in that, The internal standard used in the capillary electrophoresis detection in step (3) is SIZE-550.

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