Compound system for 28 Multi-InDel markers based on eight-color fluorescence detection technology, detection primer and application

Abstract

The invention discloses a 28 Multi-InDel marker composite system based on an eight-color fluorescence detection technology, a detection primer and application, and belongs to the technical field of forensic medicine identification. According to the invention, the eight-color fluorescence technology is applied to Multi-InDel detection for the first time, 28 autosomal short segment site genetic marker systems are integrated, and the amount of detection information is significantly increased. The system has excellent adaptability to highly degraded DNA samples, the detection rate is high, the extremely low cumulative matching probability (2.3292 * 10 <-18 >) and the extremely high cumulative exclusion probability (0.9999669) are shown, and an efficient and reliable technical scheme is provided for forensic degraded sample identification.

Description

Technical Field

[0001] This invention relates to the field of forensic identification technology, and in particular to a 28-multi-InDel labeling complex system based on eight-color fluorescence detection technology, detection primers, and applications. Background Technology

[0002] In the field of forensic individual identification and paternity testing, capillary electrophoresis typing of short tandem repeats (STRs) is currently recognized as the gold standard. However, commonly used commercial STR kits typically amplify fragments larger than 150 bp. When analyzing highly degraded DNA samples (such as old bones and decaying tissue), severe DNA fragmentation often leads to the failure of large-fragment allele amplification, incomplete typing information, or even complete loss of information, severely limiting the effective identification of such difficult samples.

[0003] To overcome the limitations of STRs in degraded samples, researchers have gradually turned their attention to single nucleotide polymorphisms (SNPs) and insertion / deletion (InDel) markers, which have the advantage of short amplicon lengths. Among these, multi-InDel markers are particularly suitable for analyzing highly degraded DNA due to their low mutation rate, short amplified fragments, and almost absence of "shadow peak" interference. Currently, multi-InDel multiplex amplification systems based on five- or six-color fluorescence detection systems have been developed. These systems are compatible with existing forensic genetic analyzers, and their data analysis workflow is similar to that of STR testing, making them easy for technicians to master. However, due to the limitation of the number of fluorescence channels, existing systems can only accommodate a limited number of multi-InDel sites within short fragments below 130 bp, while the effective fragments of highly degraded DNA are concentrated precisely in this range. Therefore, the current systems still have a bottleneck in their ability to identify degraded samples, making it difficult to obtain sufficient genetic information within limited fragment lengths to meet high-confidence identification requirements.

[0004] Eight-color fluorescence detection technology has demonstrated significant advantages in STR multiplexing, effectively increasing the locus capacity and information content per unit of detection by adding fluorescence channels. However, to date, this technology has not been effectively applied in the field of Multi-InDel detection, lacking a mature eight-color fluorescence Multi-InDel detection system. Combining eight-color fluorescence with Multi-InDel markers could potentially integrate more genetic loci within the same amplicon length range (especially below 130 bp), thereby significantly improving the typing success rate and identification efficiency for highly degraded samples. However, developing such a system faces multiple technical challenges: First, it requires screening from massive genomic data for Multi-InDel markers that are closely spaced, have small length differences, good polymorphism, and are suitable for multiplexing amplification; second, it requires multi-channel primer design for the eight-color fluorescence platform to ensure that fragment lengths within each fluorescence channel do not overlap, amplification efficiency is balanced, and there are no non-specific products; third, it requires establishing a complete detection method and data analysis workflow, and verifying its applicability and reliability in actual samples, especially highly degraded samples. Currently, no publicly available eight-color fluorescence Multi-InDel composite detection system has been found that can simultaneously meet the above requirements. Summary of the Invention

[0005] The purpose of this invention is to provide a 28-inDel marker complex system based on eight-color fluorescence detection technology, detection primers, and applications to address the problems existing in the prior art. This invention is the first to apply eight-color fluorescence technology to Multi-InDel detection, integrating 28 short autosomal fragment loci, significantly increasing the amount of detection information. This system exhibits excellent adaptability to highly degraded DNA samples, a high detection rate, and demonstrates an extremely low cumulative matching probability (2.3292 × 10⁻⁶). -18 With its extremely high cumulative exclusion probability (0.9999669), it provides an efficient and reliable technical solution for the identification of forensic degradation samples.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a genetic marker system for typing highly degraded samples, the genetic marker system consisting of Multi-InDel sites located at GRCh37.p13 of the reference genome as shown in the table below:

[0007] The present invention also provides a primer set for amplifying the genetic marker system, which consists of primers with nucleotide sequences as shown in SEQ ID NO. 1-56.

[0008] The present invention also provides the application of the primer set described above in the preparation of a reagent kit for forensic identification.

[0009] The present invention also provides a reagent kit for forensic identification, the reagent kit comprising the aforementioned primer set.

[0010] Optionally, the kit may also include a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture.

[0011] Optionally, the DNA standard includes standard DNA 2800M.

[0012] The present invention also provides the application of the primer set or the kit described herein in the identification of highly degraded forensic specimens.

[0013] Optionally, the highly degradable specimen is selected from at least one of the following: biological specimens from large-scale catastrophic events, old bones, decaying samples, formalin-fixed paraffin-embedded tissue, and follicularless hair.

[0014] The present invention also provides the application of the primer set or the kit described herein in individual identification or kinship identification.

[0015] Optionally, the DNA of the biological sample to be tested is amplified by multiplex PCR using the primer set or the kit to obtain amplification products; the amplification products are detected by capillary electrophoresis; based on the capillary electrophoresis results, the genotype of the biological sample to be tested at the Multi-InDel site is analyzed; based on the genotype analysis results, the biological sample to be tested is used for individual identification or kinship determination.

[0016] The present invention discloses the following technical effects: This invention provides a 28-multi-InDel labeled composite system based on eight-color fluorescence detection technology and its application, which effectively solves the key technical bottlenecks in the current field of Multi-InDel detection.

[0017] This invention fills the gap in the application of eight-color fluorescence technology in Multi-InDel detection systems for the first time. Compared to existing five-color and six-color systems, which suffer from insufficient locus capacity due to limitations in fluorescence channels, this system successfully integrates 28 autosomal Multi-InDel loci using eight-color fluorescence technology, significantly increasing the amount of genetic information obtained in a single detection. Crucially, the amplified fragments at all loci are strictly controlled within a short fragment range, giving it a natural fit for highly degraded DNA samples. Even under severe degradation conditions, it maintains a high detection rate, fundamentally overcoming the limitations of traditional STR kits and existing Multi-InDel systems in analyzing degraded samples.

[0018] Experimental results demonstrate that this system exhibits excellent forensic performance indicators: in a sample of 306 Han Chinese individuals, the cumulative matching probability (CMP) reached 2.3292 × 10⁻⁶. -18 The cumulative exclusion probability (CPE) is 0.9999669. It also possesses high sensitivity, strong resistance to inhibition, and good species specificity. This system is fully compatible with existing capillary electrophoresis platforms, enabling stable detection without equipment upgrades. It provides a reliable, efficient, and easily scalable technical solution for individual identification and paternity testing of highly degraded samples in forensic practice. Attached Figure Description

[0019] 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.

[0020] Figure 1 This is a diagram showing the primer system layout. The top of the diagram represents the fragment size (in bp), the left side represents the selected fluorescent markers for the 7 fluorescent channels, and the middle represents the sites contained in the 7 channels. The system contains 28 Multi-InDel markers from autosomes, distributed across 7 fluorescent channels (the eighth fluorescent channel is used as an internal standard and is not shown in the diagram). The 7 selected fluorescent markers are 6-FAM, HEX, NED, NH598, NH618, NH635, and NH650. As can be seen from the diagram, the maximum fragment length of all selected fluorescent markers is less than 125 bp. Figure 2 The results of capillary electrophoresis detection of standard DNA 2800M are shown in the figure. The horizontal axis represents the size of the amplified fragment (in bp), the name of the Multi-InDel marker is above the peak, and the number below the peak is the allele name of the amplified peak from the sample. The vertical axis represents the fluorescence signal intensity (in RFU). As can be seen from the figure, the allele typing of two 8-locus sites from standard DNA 2800M was successfully obtained through amplification electrophoresis, proving that this system can simultaneously detect up to 28 Multi-InDel markers from the sample in a single reaction. Figure 3 The image shows the detection results of DNA samples incubated in a boiling water bath at 100°C for 100 min using the eight-color fluorescence detection system for 28 Multi-InDel labels of this invention. Figure 4The image shows the detection results of DNA samples incubated in a boiling water bath at 100°C for 100 min using the GlobalFiler commercial kit. Figure 5 To use the eight-color fluorescence detection system for 28 Multi-InDel labels of this invention to detect the genotyping spectrum of the father sample in a triplet sample; Figure 6 To use the eight-color fluorescence detection system for 28 Multi-InDel labels of this invention to detect the genotyping pattern of the mother's sample in a triplet sample; Figure 7 This invention was used to detect the genotyping profile of child samples in triplet samples using an eight-color fluorescence detection system for 28 Multi-InDel labels. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Screening of 28 Multi-InDels 1. Database Sources and Group Selection Human genome variation data were obtained from the 1000 Genomes Project public database (https: / / feb2014.archive.ensembl.org / Homo_sapiens / Info / Index). Genotyping data from the Southern Chinese Han population (CHS, 105 individuals) and the Northern Chinese Han population (CHB, 103 individuals) in this database were selected as the basis for screening.

[0027] 2. Site selection criteria (i) All insertion / deletion (InDel) sites are located in non-coding regions to avoid potential functional impacts; (ii) Within a Multi-InDel marker, the physical distance between the selected insertion / deletion sites should be less than 70 bp; (iii) In each Multi-InDel marker, the length difference between the shortest and longest alleles must be less than 12 bp.

[0028] Based on the above criteria, 28 eligible Multi-InDel markers were screened, each with three alleles. Specific information on the 28 Multi-InDel markers is shown in Table 1 (reference genome: GRCh37.p13). Each Multi-InDel marker consists of two insertion / deletion sites. The naming convention for each Multi-InDel marker is as follows: taking D2MIW1 as an example, D represents DNA, 2 represents chromosome 2, MI indicates Multi-InDel, and W1 represents the laboratory internal number. All markers are located on chromosomes 1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 15, 18, and 20.

[0029] Table 1. Insertion / deletion sites contained in the 28 Multi-InDel markers 3. Design of multiplex amplification primer system For the 28 Multi-InDel markers selected above, primers that meet the requirements are designed according to the following rules: (i) PCR product size: 60-125 base pairs; (ii) Annealing temperature: 52-60℃; (iii) GC content: 30-70%; (iv) No primer mismatch, primer dimer, or hairpin structure; (v) The length ranges of the amplified fragments containing the Multi-InDel label in each fluorescence channel do not overlap.

[0030] A total of 56 primers were designed. For each Multi-InDel label, there are upstream and downstream primers, with the upstream primer labeled with different fluorescent molecules, as shown in parentheses in Table 2. Seven different fluorescent molecules were selected in the primer system of this invention: 6-FAM, HEX, NED, NH598, NH618, NH635, and NH650. The eighth fluorescent channel is a molecular weight internal standard channel, labeled with the fluorescent molecule NH680. Sites labeled with the same fluorescent molecule are placed within the same fluorescent channel in the eight-color system in the primer arrangement diagram. See the primer system arrangement diagram below. Figure 1 .

[0031] Table 2. Nucleotide sequences of 56 amplification primers 4. Working principle For the degraded samples to be tested, the sample was mixed with a mixture of 28 Multi-InDel labeled primers and an amplification reaction mix. After PCR amplification and capillary electrophoresis, the typing results of highly degraded samples were obtained using GA-marker analysis software. Simultaneous amplification of standard DNA 2800M and enzyme-free water served as positive and negative controls, respectively. The reliability of the test results can be determined based on the accuracy of the typing results of the positive and negative controls.

[0032] Example 2: Detection of Standard DNA 2800M 1. Main instruments and reagents (1) GA118-24B genetic analyzer (First Research Institute of the Ministry of Public Security), used for capillary electrophoresis separation and fluorescence signal collection; (2) ProFlex PCR instrument (ThermoFisher), used for polymerase chain reaction; (3) High-speed centrifuge (ThermoFisher), used for rapid sample centrifugation; (4) Qubit 3.0 fluorescence quantitative instrument (ThermoFisher), used for precise quantification of template DNA; (5) 96-well plate centrifuge (Eppendorf), used for centrifugation of electrophoresis plates; (6) Pipette (Eppendorf), used for precise pipetting; (7) Ultrapure water (Changsha Yuanke Instrument Experimental Equipment Co., Ltd.) is used to prepare the reaction system; (8) Hi-Di formamide (ABI), used for denaturation of PCR products; (9) The molecular weight internal standard NH680V2 (Suzhou Xinhai Biotechnology Co., Ltd.) is used for precise calibration of fragment length in capillary electrophoresis. (10) QIAGEN MultiplexPCR mix (Qagen); (11) Standard DNA 2800M (Promega), used as a positive control and reference sample for system validation; (12) Specific primers corresponding to the 28 Multi-InDel markers identified in Example 1.

[0033] 2. Testing Mix the primers according to the concentrations shown in Table 2, and follow the steps below to complete the detection of standard DNA 2800M: (1) Mix the components required for multiplex amplification according to the proportions shown in Table 3.

[0034] Table 3. Multiplex amplification system (2) PCR amplification PCR amplification program: 95℃, 5 min; 95℃, 30 s, 59℃, 60 s, 72℃, 30 s, 29 cycles; then 72℃, 10 min; store at 4℃.

[0035] (3) Capillary electrophoresis The sample mixture was prepared in the following proportions: 1 μL PCR amplification product, 8.8 μL Hi-Di formamide, and 0.2 μL molecular weight internal standard NH680V2. Detection was performed using a GA118-24B genetic analyzer. Capillary electrophoresis conditions were as follows: 1.5 kV injection for 10 seconds, followed by electrophoretic separation at 15 kV and 60°C for 1700 seconds. Electrophoresis data were analyzed using GA-Marker software (First Research Institute of the Ministry of Public Security). Electrophoresis detection results for standard DNA 2800M are shown below. Figure 2 .

[0036] Example 3: Detection of Artificially Degraded DNA This embodiment aims to simulate the high degree of DNA degradation commonly encountered in forensic practice. By constructing an artificial DNA degradation model, it compares and evaluates the 28-fold Multi-InDel multiplex amplification system described in this invention with mainstream commercially available STR kits (GlobalFiler). TMThe detection efficiency of this system for degraded DNA was evaluated, verifying its advantages in detecting degraded samples.

[0037] The artificial degradation model was created by incubating 2 ng / µL DNA samples (human genome samples derived from volunteer saliva swabs, extracted using the Magen Universal DNA Extraction Kit) in a 100°C water bath for specific time periods (0 min, 20 min, 40 min, 60 min, 80 min, and 100 min). These degradation samples were amplified using the detection system described in Example 2 and the GlobalFiler kit, detected using GA118-24B, and analyzed using the GA-marker. The final detection results are as follows: Figure 3 and Figure 4 As shown.

[0038] Figure 3 This is a graph showing the detection results of samples incubated for 100 minutes using the system of Example 2. Figure 4 The image shows the detection results of samples incubated for 100 min using the GlobalFiler commercial kit. At an analysis threshold of 100 RFU (relative fluorescence units), the allele detection rate was 100% (47 / 47) using the system in Example 2; the detection rate was 26.2% (11 / 42) using the GlobalFiler commercial kit.

[0039] from Figure 3 and Figure 4 As can be seen, human genomic DNA undergoes severe degradation after a 60-minute boiling water bath, exhibiting a clear trend of high allele detection at the beginning and low detection at the end in the genotyping map. The GlobalFiler kit can no longer obtain large-fragment STR typing, and its typing results are unreliable. In contrast, the detection system in Example 2 maintains a high allele detection rate, demonstrating a significant advantage for detecting highly degraded samples.

[0040] Example 4: Sample Detection and Population Genetic Parameter Calculation of Han Chinese Population This embodiment aims to evaluate the application effect of the multiplex amplification system for 28 Multi-InDel markers constructed in this invention in a real population by testing samples from the Han Chinese population, and to calculate its population genetic parameters, thereby verifying its applicability and effectiveness as a tool for forensic individual identification and paternity testing.

[0041] 1. Sample collection Bloodstain samples were collected from 306 unrelated individuals of Han Chinese descent (including samples from Han Chinese in southern and northern China), and all donors signed informed consent forms.

[0042] 2. Testing process a. Extract DNA from population samples using a universal column-based DNA extraction kit, quantify the DNA using a Nano Drop One micro-volume spectrophotometer, and then dilute it to 1 ng / µL for later use.

[0043] b. Using the DNA from step a as a template, perform multiplex PCR amplification in the amplification system shown in Table 3 using the multiplex amplification primers in Table 2. The amplification procedure is the same as in Example 2.

[0044] c. Perform electrophoresis and analysis on the above population samples according to the capillary electrophoresis and analysis parameters in Example 2.

[0045] Software analysis was performed on the obtained genotyping data to obtain the allele frequencies (Table 4) and forensic parameters (Table 5) of the above 28 Multi-InDel markers, including matching probability (MP), polymorphic information content (PIC), exclusion probability (PE), typical paternity index (TPI), expected heterozygosity (He), observed heterozygosity (Ho), Hardy-Weinberg equilibrium p-value (HWE-p), and effective number of alleles (Ae).

[0046] Table 4. Allele frequencies at 28 multiple insertion / deletion sites Table 5. Forensic parameters related to 28 multiple insertion / deletion sites. As shown in Table 4, complete genotyping of all 28 loci was successfully obtained for all samples, indicating that the system has a high amplification success rate and stability. The allele frequencies are reasonably distributed, providing a reliable basis for subsequent analysis. All 28 loci exhibit good polymorphism. The expected heterozygosity (He) of individual loci ranges from 0.44 to 0.59, with an average He > 0.5. The effective allele count (Ae) is greater than 1.96 for all loci, with five loci having an Ae greater than 2.80. The overall average Ae value is 2.54 (Table 5), indicating that these loci have high genetic diversity.

[0047] The HWE test p-values ​​for the vast majority of loci were greater than 0.05, conforming to Hardy-Weinberg equilibrium. This indicates that the population sample is in genetic equilibrium at this locus, without significant population stratification, inbreeding, or genotyping errors, and the data can be used for forensic evaluation. The probability of matching (MP) and probability of exclusion (PE) values ​​for all loci were within acceptable forensic thresholds, with D12MIW1 having the lowest MP value and D4MIW1 having the highest PE value. The entire detection system demonstrated excellent forensic identification efficiency, with a cumulative probability of matching (CMP) of 2.3292 × 10⁻⁶. -18This means that in a random population, the probability of two unrelated individuals having completely identical genotypes at all 28 loci is extremely low, demonstrating the system's extremely high individual identification capability. The cumulative exclusion probability (CPE) is 0.9999669, indicating that the system has a very strong ability to exclude non-paternity issues in paternity testing.

[0048] Example 5: Detection of Triplets in Paternity Testing Samples This embodiment aims to demonstrate and verify the reliability and accuracy of the 28-fold Multi-InDel multiplex amplification primer system provided by this invention in practical applications of paternity testing through a typical triplet (father-mother-child) paternity testing case.

[0049] The client (child, biological mother) requests a paternity test with the father being tested. The client provides blood cards (FTA cards) corresponding to the three individuals as biological samples to be tested.

[0050] The primers listed in Example 1, the PCR reaction system and human capillary electrophoresis parameters in Example 2 were used to detect a pair of triplet paternity test samples (from a Han Chinese population in southern China; the DNA sample used in the experiment was extracted and the concentration was 1 ng / µL). The final results are shown in [link to results]. Figures 5-7 .in Figure 5 The test results for the father. Figure 6 The test results for the mother. Figure 7 For children's test charts.

[0051] Table 6 shows the paternity-mother-child typing results and paternity index (PI) of the triplets (calculated based on the allele frequencies in Table 4 of Example 4). The nucleotide sequences of the allele standards in the allele standard mixture are shown in Table 7. The results show that all multiple insertion / deletion loci in the triplets conform to Mendelian inheritance, with a cumulative paternity index (CPI) of 142629.3751, demonstrating that the primer system designed for 28 Multi-InDel markers can be applied to paternity testing of highly degraded samples.

[0052] Table 6. Paternity index of the triad at 28 multiple insertion / deletion loci. Table 7. Nucleotide sequences of allele standards in the allele standard mixture. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A genetic marker system for typing highly degraded samples, characterized in that, The genetic marker system consists of the Multi-InDel sites located at GRCh37.p13 of the reference genome, as shown in the table below: 。 2. A primer set for amplifying the genetic marker system of claim 1, characterized in that, It consists of primers with nucleotide sequences as shown in SEQ ID NO.1-56.

3. The use of the primer set according to claim 2 in the preparation of a reagent kit for forensic identification.

4. A reagent kit for forensic identification, characterized in that, The kit includes the primer set as described in claim 2.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture.

6. The reagent kit according to claim 5, characterized in that, The DNA standards shown include standard DNA 2800M.

7. The application of the primer set according to claim 2 or the kit according to any one of claims 4-6 in the identification of highly degradable forensic specimens.

8. The application according to claim 7, characterized in that, The highly degradable specimens are selected from at least one of the following: biological specimens from large-scale catastrophic events, old bones, decaying samples, formalin-fixed paraffin-embedded tissues, and hair without follicles.

9. The application of the primer set according to claim 2 or the kit according to any one of claims 4-6 in individual identification or kinship identification.

10. The application according to claim 9, characterized in that, The DNA of the biological sample to be tested is amplified by multiplex PCR using the primer set described in claim 2 or the kit described in any one of claims 4-6 to obtain amplification products; the amplification products are detected by capillary electrophoresis; based on the capillary electrophoresis results, the genotype of the biological sample to be tested at the Multi-InDel site described in claim 1 is analyzed; based on the genotype analysis results, the biological sample to be tested is used for individual identification or kinship determination.

Images