Composite system and method for skin identification and application
By constructing an ARMS-PCR skin-specific DNA methylation detection system, the technical challenges in skin identification were solved, achieving highly sensitive and specific skin identification results.
Patent Information
- Application Number
- CN202511283819.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies are insufficient for effectively identifying skin samples, especially due to the low DNA content and susceptibility to environmental factors. Traditional methods are prone to producing false positive and false negative results and lack skin-specific identification capabilities.
A skin-specific DNA methylation detection system was constructed using ARMS-PCR technology. Skin samples were identified using specific primer combinations, and genotyping analysis was performed using a capillary electrophoresis platform.
It achieves highly sensitive and specific skin identification, accurately distinguishing skin from other body fluid samples under low DNA content conditions, thus reducing the false positive rate.
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Figure CN121023035A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of forensic identification, and particularly relates to a composite system for skin identification, a method and use. BACKGROUND
[0002] In forensic practice, accurate identification of biological samples is of great significance for solving cases and maintaining judicial justice. On the one hand, based on the STR-based capillary electrophoresis typing technology system and the rapid development of DNA database, individual identification has been highly standardized. On the other hand, the identification of the source of body fluid samples is crucial for scene reconstruction and even the determination of the nature of the case. Skin is the largest organ of the human body, and its exfoliated cells are widely distributed on the contact surface of the crime scene (such as clothes, weapons, and electronic devices), which is an important biological sample.
[0003] However, the identification of skin samples has long faced technical challenges. On the one hand, the DNA content of skin cells that can be extracted from a crime scene is extremely low and easily affected by environmental factors, leading to degradation. On the other hand, traditional body fluid identification methods lack specificity in identifying skin cells, making it difficult to distinguish between epithelial cells. For example, traditional enzymatic and chemical methods are commonly used for blood and semen identification, but these methods can produce false positives and false negatives, and the identification process can cause significant sample loss. In addition, there is no effective and feasible method for skin identification. DNA methylation markers, as an epigenetic marker, have strong tissue specificity and outstanding anti-degradation ability, providing a new way to break through this bottleneck.
[0004] In recent years, the development of high-throughput DNA methylation detection technology has injected new energy into forensic body fluid identification. Illumina Infinium MethylationEPIC BeadChip (850K chip) developed by Illumina can cover 853,307 CpG sites, adding 413,745 sites compared to Illumina HumanMethylation450BeadChip (450K chip), significantly improving the screening efficiency of differential methylation regions. Combined with bioinformatics tools such as R language ChAMP package and public methylation databases such as GEODataSets, researchers can systematically screen body fluid / tissue-specific methylation markers and construct a multiple detection system.
[0005] At present, the detection method of DNA methylation is developing rapidly. The amplification refractory mutation system is a detection technology with simple operation process and low cost. Its principle is to realize the specific amplification of alleles by designing two allele-specific forward / reverse primers and a universal reverse / forward primer. When designing ARMS primers for methylation sites, the 3' end of the forward primer needs to be complementary to the CpG site, and a mismatch is introduced at the second or third base at the 3' end to improve the amplification specificity of the primer. At the same time, a reverse / forward primer is shared to improve the detection efficiency. This technology can be used for specific detection of target methylation sites. At present, there is no research report on the construction of a skin identification system based on ARMS-PCR technology. The present application aims to establish a skin-specific ARMS-PCR methylation detection system compatible with the capillary electrophoresis platform, and to provide a new solution for the identification of skin sources in biological samples. SUMMARY
[0006] In view of the above shortcomings in the prior art, the present application provides a composite system, method and use for skin identification. The present application uses ARMS-PCR technology to identify and analyze body fluid samples, and constructs a DNA methylation composite system for detecting skin components.
[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application to solve its technical problems is: The present application aims to provide a composite system for skin identification, which comprises a primer combination for amplifying a detection site; the sequence of the primer combination is shown in SEQ ID NO. 1-30; the detection site comprises cg06833110, cg14542120, cg05942459, cg02745009, cg02807788, cg10959820, cg17320707, cg25855753, cg22226904 and cg20603548.
[0008] Another object of the present application is to provide a primer combination for skin identification, which comprises the primer sequence shown in SEQ ID NO. 1-30. The primer specific information is shown in Table 1.
[0009]
[0010] Another object of the present application is to provide a kit comprising the above-mentioned primer combination.
[0011] Another object of the present application is to provide the use of the above-mentioned primer combination or kit in the preparation of products for forensic detection and identification.
[0012] Another object of the present application is to provide use of the primer combination or kit as described above in the preparation of a product for skin detection and identification.
[0013] Another object of the present application is to provide a method for skin identification, comprising the following steps: (1) extracting DNA of a sample to be detected, and transforming with bisulfite; (2) then using the primer combination or kit as described above for amplification detection, and determining whether the sample is a skin sample according to the methylation value of the detection site.
[0014] Further, the amplification system comprises 5 μL Master Mix, 1 μL Primer Mix, and 4 μL of the transformed DNA.
[0015] Further, the amplification procedure is: 95℃ pre-denaturation for 15 min; 94℃ denaturation for 30 s, 58℃ annealing for 90 s, 72℃ extension for 60 s, for a total of 33 cycles; final extension at 60℃ for 30 min.
[0016] Advantages of the present application: DNA methylation has tissue specificity, and can be used for body fluid identification, but there is no ARMS-PCR complex system that can be used for skin identification. The present application uses ARMS-PCR technology to identify and analyze body fluid samples, and constructs a DNA methylation complex system that can be used for skin component detection, which has excellent sensitivity and specificity. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 is an electropherogram of the complex amplification results of a skin shed cell sample; wherein sites cg06833110, cg05942459, and cg17320707 present a high methylation state; and sites cg02745009, cg25855753, cg02807788, cg10959820, cg14542120, cg22226904, and cg20603548 present a low methylation state.
[0018] Figure 2 is an electropherogram of the complex amplification results of a peripheral blood sample; wherein sites cg06833110, cg05942459, and cg17320707 present a low methylation state; and sites cg02745009, cg25855753, cg02807788, cg10959820, cg14542120, cg22226904, and cg20603548 present a high methylation state.
[0019] Figure 3Electropherogram of saliva sample composite amplification result; wherein sites cg06833110, cg05942459, cg17320707 present hypomethylation state; sites cg02745009, cg25855753, cg02807788, cg10959820, cg14542120, cg22226904, cg20603548 present hypermethylation state.
[0020] Figure 4 Electropherogram of saliva sample composite amplification result; wherein sites cg06833110, cg05942459, cg17320707 present hypomethylation state; sites cg02745009, cg25855753, cg02807788, cg10959820, cg14542120, cg22226904, cg20603548 present hypermethylation state.
[0021] Figure 5 Electropherogram of saliva sample composite amplification result; wherein sites cg06833110, cg05942459, cg17320707 present hypomethylation state; sites cg02745009, cg25855753, cg02807788, cg10959820, cg14542120, cg22226904, cg20603548 present hypermethylation state.
[0022] Figure 6 Electropherogram of saliva sample composite amplification result; wherein sites cg06833110, cg05942459, cg17320707 present hypomethylation state; sites cg02745009, cg25855753, cg02807788, cg10959820, cg14542120, cg22226904, cg20603548 present hypermethylation state.
[0023] Figure 7 Electropherogram of 0.25 ng skin exfoliated cell sample DNA (* indicates non-specific peak); the result shows that when the input amount of genomic DNA is 250 pg (i.e. 0.25 ng), the established skin identification composite amplification system can obtain complete and accurate typing results. DETAILED DESCRIPTION
[0024] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0025] Example 1 Sample Extraction This study collected 60 skin exfoliated cell samples and semen samples from volunteers with their informed consent. Twenty samples each of oral swabs, peripheral blood, menstrual blood, and vaginal secretions were collected. DNA was then extracted from the samples using the phenol-chloroform organic extraction method.
[0026] Example 2 Site Screening The following public datasets were used: GSE188593 (containing 32 skin samples), GSE131433 (containing 233 blood samples), GSE185445 (containing 379 semen samples), GSE111631 (containing 168 saliva samples), and GSE182874 (containing 2 vaginal secretion samples). Tissue-specific methylation sites were screened using the ChAMP package in R with a P < 0.05 criterion. The absolute value of the average methylation difference (|Δβ|) between skin fluids and the other four bodily fluids was calculated, and the top 30 sites with the highest |Δβ| were included as candidate sites. Site information is shown in Table 2.
[0027] Table 2 Information on the top 30 candidate sites sorted by |Δβ|
[0028] Then, based on the sites obtained from Table 2, skin-specific sites with |Δβ|>0.5 were further screened, resulting in the following sites: cg06833110, cg14542120, cg05942459, cg02745009, cg02807788, cg10959820, cg17320707, cg25855753, cg22226904, and cg20603548.
[0029] Example 3 Primer Design Based on the skin-specific sites with |Δβ| > 0.5 obtained in Example 1, ARMS primers were designed as follows: The design principles of the amplification primers are: a. length 15-30 bp, Tm value 50-65°C, GC content <60%; b. avoid hairpin structure and primer dimer; c. try to avoid CpG sites in the primer sequence, if it cannot be avoided, replace it with degenerate base Y (C / T) or R (G / A), try to avoid it at the 3' end of the primer; d. avoid polymorphic sites; e. try to make the amplification fragment short (less than 300 bp). ARMS primers are designed using Primer3web (v4.1.0), and the specific information of the primers is shown in Table 3.
[0030] Table 3 Primer information of 10 skin-specific methylation sites
[0031] Example 4 Construction of composite system According to the length of the amplification product of each CpG site in Table 3 and the different fluorescent labels of each primer, a primer composite primer system (Primer Mix) is established, and the concentration ratio of the primers in the system is adjusted according to the electrophoretic typing diagram of the amplification product. Finally, two Panels are constructed, and the primer concentration of the Panel is shown in Table 4.
[0032] Table 4 Information of 10 skin-specific methylation sites in composite amplification system
[0033] 1. Bisulfite conversion: according to the instructions, use Epitect® Fast Bisulfite Conversion Kit for bisulfite conversion.
[0034] 2. PCR amplification: use QIAGEN Multiplex PCR Master Mix kit for composite amplification. The amplification system includes: 5 μL Master Mix, 1 μL Primer Mix, 4 μL converted DNA. The amplification program is: 95°C for 15 min; 94°C for 30 s, 58°C for 90 s, 72°C for 60 s, 33 cycles; final extension 60°C for 30 min. The specific reaction system and reaction program are shown in Tables 5 and 6.
[0035] Table 5 Composite PCR reaction system
[0036] Table 6 Composite PCR reaction program
[0037] 3. Capillary electrophoresis typing: Take 1 μL of product, 0.02 μL of intramolecular marker and 9 μL of formamide, mix well, and then use ABI3500 sequencer for capillary electrophoresis. Use Gene Mapper ID-X Software v1.5 software to analyze the results, and set the threshold value to 50 RFU. The capillary electrophoresis condition setting is shown in Table 7. In this study, the methylation value was calculated by the peak area of the 6-FAM labeled methylation product and the peak area of the HEX labeled non-methylation product, and the methylation state of the target site in different samples was quantitatively characterized. The methylation value calculation formula is: methylation value = methylation product peak area / (methylation product peak area + non-methylation product peak area).
[0038] Table 7 Main electrophoresis conditions of 3500 capillary electrophoresis
[0039] 4. The detection results of skin, peripheral blood, semen, saliva, vaginal secretion, menstrual blood samples are shown in Figures 1-6 .
[0040] Example 5 Sensitivity The initial DNA template amount was diluted to different input amounts using RNase / DNase-free water, and the methylation expression of 10 CpG sites in skin exfoliated cell samples was detected to evaluate the sensitivity of the complex system. When the input amount of genomic DNA was 10 ng, 5 ng, 1 ng, 500 pg and 250 pg, accurate typing results could be obtained; but when the input amount was reduced to 100 pg, 50 pg and 25 pg, some sites could not be accurately typed, so the sensitivity of the complex amplification system was 0.25 ng.
[0041] Example 6 Specificity By detecting six kinds of forensic common body fluids / tissue samples such as skin exfoliated cells, peripheral blood, menstrual blood, vaginal secretion, saliva and semen, the skin specificity of the complex amplification system was verified. The average value and standard deviation of the methylation value of 10 skin-specific methylation sites in different body fluids / tissue samples are shown in Table 8.
[0042] Table 8 Methylation pattern of 10 skin-specific CpG sites
[0043] Finally, it should be noted that the above detailed description is merely illustrative of the technical solutions of the present application and is not limiting, and although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and all should be covered in the scope of the claims of the present application.
Claims
1. A composite system for skin identification, characterized in that, It includes primer combinations for amplifying detection sites; the sequences of the primer combinations are shown in SEQ ID NO.1~30; the detection sites include cg06833110, cg14542120, cg05942459, cg02745009, cg02807788, cg10959820, cg17320707, cg25855753, cg22226904 and cg20603548.
2. A primer combination for skin identification, characterized in that, Includes primer sequences as shown in SEQ ID NO.1~30.
3. A reagent kit, characterized in that, Includes the primer combination described in claim 2.
4. The use of the primer combination of claim 2 or the kit of claim 3 in the preparation of products for forensic testing and identification.
5. Use of the primer combination of claim 2 or the kit of claim 3 in the preparation of products for skin testing and identification.
6. A method for skin identification, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested and convert it with bisulfite; (2) Then, amplification and detection are performed using the primer combination described in claim 2 or the kit described in claim 3, and the methylation value of the detection site is used to determine whether the sample is a skin sample.
7. The method according to claim 6, characterized in that, The amplification system consisted of 5 μL Master Mix, 1 μL Rimer Mix, and 4 μL of transformed DNA.
8. The method according to claim 6, characterized in that, The amplification program was as follows: 95℃ pre-denaturation for 15 min; 94℃ denaturation for 30 s, 58℃ annealing for 90 s, 72℃ extension for 60 s, for a total of 33 cycles; final extension at 60℃ for 30 min.