Multiplex amplification kit for simultaneously detecting 21 X-STR gene loci and application of multiplex amplification kit

Abstract

The invention belongs to the technical field of biological detection, and relates to a composite amplification kit for detecting X-STR gene loci, in particular to a composite amplification kit for simultaneously detecting 21 X-STR gene loci and application of the composite amplification kit in individual recognition and genetic relationship identification. Seven linkage groups are used in the detection kit, and the defect that in the prior art, the linkage relation of gene loci is not very clear, or few linkage groups are used, and the identification efficiency is low due to the fact that few linkage groups are recognized on X chromosomes is overcome. The detection kit provided by the invention can improve the identification efficiency and accuracy, is suitable for forensic individual identification and special genetic relationship identification, and has a good forensic application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a multiplex amplification kit for detecting X-STR loci. Specifically, it relates to a multiplex amplification kit for simultaneously detecting 21 X-STR loci and its application in individual identification and kinship determination. Background Art

[0002] The prior art records that in the field of forensic physical evidence, individual identification and paternity testing are mainly achieved by detecting genetic markers of biological samples, and short tandem repeat (STR) genetic markers are mainly used for detection and analysis.

[0003] According to the literature, STR genetic markers are scattered throughout the human genome. They have high polymorphism, moderate length and are easy to detect, and are currently the most widely used genetic markers in forensic applications. After years of development, the STR genetic markers of autosomes and the Y chromosome are very rich in both quantity and applicable detection kits. In contrast, the research on STR genetic markers of the X chromosome (X-STR) is less.

[0004] The research literature also records that one of the X chromosomes of a female can be randomly inactivated to form a Barr body. Although gene expression is silenced, it can undergo homologous recombination with the non-inactivated X chromosome during mitosis and meiosis. The X-STR of male individuals is transmitted to their daughters in the form of a haplotype. In females, the two X chromosomes undergo homologous recombination during meiosis and randomly transmit one of the two alleles to their offspring. On the same chromosome, two adjacent X-STRs are more likely to be inherited downward in the form of a haplotype, resulting in linkage disequilibrium between the two genetic markers and forming a relatively tight linkage group. Therefore, due to the same chromosome localization and special genetic laws of X-STR, its application in judicial practice is less.

[0005] Currently, in the X-STR detection kits used in practice, the linkage relationships of some loci are not very clear, or the number of linkage groups used is small. There are only 4 generally recognized linkage groups on the X chromosome, which also results in low efficiency in identification. Therefore, the industry believes that using more linkage groups will be an important means to improve the detection and analysis efficiency of X-STR.

[0006] Based on the basis and current situation of the prior art, the inventors of the present application intend to provide a new multiplex amplification kit for detecting X-STR loci. Specifically, it relates to a multiplex amplification kit for simultaneously detecting 21 X-STR loci and its application in individual identification and kinship determination. The detection kit of the present invention can improve the identification efficiency and accuracy, is suitable for forensic individual identification and special kinship determination, and has good forensic application prospects. Summary of the Invention

[0007] Based on the basis and current situation of the prior art, the object of the present invention is to provide a new multiplex amplification kit for detecting X-STR loci, specifically relating to a multiplex amplification kit for simultaneously detecting 21 X-STR loci and its application in individual identification and kinship identification.

[0008] The present invention provides a multiplex amplification detection system that can detect 21 X-STR genetic markers in 7 linkage groups, which is applicable to forensic individual identification and kinship identification involving females.

[0009] The present invention screened X-STR genetic markers on 3 new linkage groups. Based on the genetic markers on the existing 4 linkage groups, through the analysis of genetic markers, 21 X-STR loci were selected to establish a detection kit, which has a high identification efficiency and is named FX-21Rplex.

[0010] Specifically, from the existing 7 linkage groups, according to the polymorphism of genetic markers, the tightness of genetic linkage, etc., 21 X-STRs were screened, including DXS10135, DXS8378, DXF02, DXF07, DXF08, DXF09, DXS10079, DXF13, DXS10074, DXS10075, DXF15, DXS6803, DXF18, DXF28, DXF29, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134, DXS7423. The specific information is shown in Table 1 below:

[0011] Table 1 X-STR names, linkage groups, chromosomal locations, and physical locations

[0012]

[0013]

[0014] As a further preferred scheme, the primers and usage concentrations of 21 X-STRs are as follows:

[0015] Table 2 Primer pair sequences and final amplification reaction concentrations of 21 X-STR loci

[0016]

[0017]

[0018] As a further preferred scheme, 21 X-STRs are divided into 4 groups, and one of each pair of primers is labeled with a corresponding color fluorescent dye, specifically as follows:

[0019] Table 3 Distribution of Four Groups of X-STR Fluorescent Labels

[0020] Fluorescent label X-STR grouping 5-FAM DXS10103, DXF02, DXF15, DXF13, DXF28 and DXF29 HEX DXF09, DXS10146, DXF18 and DXS10134 TRAMA DXS6803, DXF07, DXS10075, DXS10074, HPRTB and DXS8378 ROX DXS7423, DXS10079, DXS10101, DXF08 and DXS10135

[0021] As a further preferred solution, the conditions of the amplification system are shown in Table 4 and Table 5.

[0022] Table 4 Recommended Components of the Multiplex Amplification System

[0023] Component Volume 2×Multiplex PCR Master Mix 12.5 μL 5× Primer mixture 5 μL Template DNA 0.1 - 5 ng 4 mg / ml BSA 1 μL ddH2O Make up to 25 μL

[0024] Table 5 Recommended PCR Amplification Conditions of the Multiplex Amplification System

[0025]

[0026]

[0027] In the present invention, the sources of human genomic DNA include blood / bloodstains, semen / semen stains, saliva stains, sweat, tissues, hairs, bones, and other tissues and body fluids of humans.

[0028] Furthermore, the present invention provides an application in individual identification.

[0029] Furthermore, the present invention provides an application in kinship determination.

[0030] In the present invention, the detection system has good amplification specificity, and the peak heights of each locus are balanced; the results of the species-specificity test verification show that the detection system has high specificity.

[0031] In the present invention, DNA detection and comparison research was carried out:

[0032] Using DNA standard 9947A as a template, X-STR genotyping was performed using Argus X-12 and FX-21Rplex respectively. The results show that for the overlapping loci of the two kits, the genotyping is consistent;

[0033] Using blood cards and saliva swabs from the same individual source, cutting and extracting DNA using Chelex 100 as a template for X-STR genotyping. The results show that the genotyping results of the two types of specimens are consistent, indicating good genotyping effects for conventional biological specimens.

[0034] In the present invention, kinship analysis was carried out:

[0035] Using blood cards of a mother and son, extracting DNA using Chelex 100 and performing X-STR genotyping respectively. The results show that there is one identical allele at each X-STR locus between the mother and son.

[0036] The present invention provides a multiplex amplification kit for simultaneously detecting 21 X-STR loci. Seven linkage groups are used in the detection kit of the present invention, which overcomes the defects in the prior art that the linkage relationship of loci is not very clear, or the number of linkage groups used is small, and the recognized linkage groups on the X chromosome are few, resulting in low efficiency in identification. The detection kit of the present invention can improve the identification efficiency and accuracy, is applicable to forensic individual identification and special kinship identification, and has good forensic application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 1. Figure 1 It is a detection result diagram of DNA standard 2800M by the FX-21Rplex multiplex amplification kit.

[0038] 2. Figure 2 It is a detection result diagram of other species DNA samples by the FX-21Rplex multiplex amplification kit. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] For those skilled in the art to understand more clearly, the technical content of the present invention will be further clarified below in conjunction with specific embodiments.

[0040] Embodiment 1

[0041] Design of the kit:

[0042] 1. Selection of X-STR loci: According to the existing information and the data tested in this laboratory, 21 X-STR loci, namely DXS10135, DXS8378, DXF02, DXF07, DXF08, DXF09, DXS10079, DXF13, DXS10074, DXS10075, DXF15, DXS6803, DXF18, DXF28, DXF29, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134, DXS7423, are selected. The specific information is as follows:

[0043] Table 6 X-STR names, linkage groups, chromosomal locations, and physical locations

[0044]

[0045] 2. Primer design: Primer Premier 3 is used for primer design, and Autodimer is used for primer dimer analysis. The designed primers are repeatedly tested to ensure that each pair of primers has specific amplification, similar Tm values, and no primer dimers are formed between primers. The specific primer sequences are as follows:

[0046] Table 7 Primer pair sequences of 21 X-STR loci

[0047]

[0048]

[0049] 3. Primer Fluorescent Labeling: According to the amplicon size, the 21 X-STRs are divided into four groups, and one of each pair of primers is labeled with the corresponding fluorescent dye respectively:

[0050] Table 8 Distribution of Fluorescent Labels for Four Groups of X-STRs

[0051] Fluorescent label X-STR 5-FAM DXS10103, DXF02, DXF15, DXF13, DXF28 and DXF29 HEX DXF09, DXS10146, DXF18 and DXS10134 TRAMA DXS6803, DXF07, DXS10075, DXS10074, HPRTB and DXS8378 ROX DXS7423, DXS10079, DXS10101, DXF08 and DXS10135

[0052] 4. Optimization of Primer Usage Concentration: The primers are mixed at equal concentrations, and the primer concentration is adjusted according to the peak height of each locus until the peak height of each locus is balanced. The specific primer concentrations obtained are as follows:

[0053] Table 9 Final Concentrations of Primer Amplification Reactions for 21 X-STR Loci

[0054]

[0055]

[0056] 5. Optimization of Amplification Conditions: Different annealing temperatures, annealing times, extension times, cycle numbers, etc. are set to obtain optimized amplification conditions. The specific results are as follows:

[0057] Table 10 Recommended PCR Amplification Conditions for Multiplex Amplification System

[0058]

[0059] As Figure 1 shown, this detection system has good amplification specificity, and the peak height of each locus is balanced.

[0060] 6. Verification of Species Specificity: Using DNA from chicken, fish, cattle, duck, and pig, with 1 ng of DNA as the template, multiplex amplification is carried out according to the above primer concentration and amplification conditions, and the results obtained are as Figure 2 shown. The results indicate that this detection system has high specificity.

[0061] Example 2

[0062] DNA Detection and Comparison:

[0063] 1. Using DNA standard 9947A as the template, X-STR genotyping is performed using ArgusX-12 and FX-21Rplex respectively (Table 11). The results show that for the overlapping loci of the two kits, the genotyping is consistent.

[0064] Table 11 Genotyping Results of DNA Standard 9947A by Argus X-12 and FX-21Rplex

[0065]

[0066]

[0067] 2. Using blood cards and saliva swabs from the same individual, a small amount was cut and DNA was extracted using Chelex 100. 1 μL was used as a template for X-STR genotyping respectively. The results are shown in Table 12. The genotyping results of the two types of biological samples are consistent, indicating good genotyping effects for conventional biological samples.

[0068] Table 12 Genotyping Results of Blood Cards and Saliva Swabs from the Same Individual by FX-21Rplex Kit

[0069] X-STR locus Blood card genotyping Saliva swab genotyping DXS10135 20 / 23 20 / 23 DXS8378 10 10 DXF02 14.3 / 15 14.3 / 15 DXF07 9 / 10 9 / 10 DXF08 9 9 DXF09 14 14 DXS10079 19 / 20 19 / 20 DXF13 12 / 13 12 / 13 DXS10074 18 / 19 18 / 19 DXS10075 17 / 18 17 / 18 DXF15 9 / 11 9 / 11 DXS6803 12 / 12.3 12 / 12.3 DXF18 10.2 / 12.2 10.2 / 12.2 DXF28 17 / 18 17 / 18 DXF29 9 / 10 9 / 10 DXS10103 18 / 19 18 / 19 HPRTB 13 13 DXS10101 29 / 31 29 / 31 DXS10146 24 / 29 24 / 29 DXS10134 35 / 36 35 / 36 DXS7423 14 / 15 14 / 15

[0070] Example 3

[0071] Analysis of Kinship

[0072] Using blood cards of a mother and son, a small amount was cut and DNA was extracted using Chelex 100. 1 μL was used as a template for X-STR genotyping respectively. The results are shown in Table 13. The results indicate that there is one identical allele at each X-STR locus between the mother and son.

[0073] Table 13 Genotyping Results of Mother-Son Samples by FX-21Rplex Kit

[0074] X-STR locus Mother genotyping Son genotyping DXS10135 27 / 31 31 DXS8378 10 / 12 12 DXF02 13.3 / 17.3 13.3 DXF07 9 9 DXF08 9 / 10 9 DXF09 14 14 DXS10079 19 / 21 21 DXF13 12 / 14 14 DXS10074 14.2 / 18.2 14.2 DXS10075 17 / 18 18 DXF15 9 / 11 11 DXS6803 12 / 12.3 12.3 DXF18 9 9 DXF28 15 / 20 15 DXF29 9 / 10 9 DXS10103 17 / 20 17 HPRTB 13 13 DXS10101 29.1 / 31.1 31.1 DXS10146 28 / 33.2 33.2 DXS10134 35 35 DXS7423 14 / 16 16

[0075] The above embodiments are not intended to limit the protection scope of the present invention, but are intended to disclose the technical content of the present invention in more detail. If any modification and / or change is obvious to those skilled in the art, it will also be included in the concept of the present invention.

Claims

1. A multiplex amplification kit for simultaneously detecting 21 X-STR loci, characterized in that, The kit contains specific primers for detecting the following X-STR loci: DXS10135; DXS8378; DXF02; DXF07; DXF08; DXF09; DXS10079; DXF13; DXS10074; DXS10075; DXF15; DXS6803; DXF18; DXF28; DXF29; DXS10103; HPRTB; DXS10101; DXS10146; DXS10134; DXS7423.

2. The kit according to claim 1, wherein The primer sequences of the said loci are: DXS10135, as shown in SEQ ID NO: 1, 2; DXS8378, as shown in SEQ ID NO: 3, 4; DXF02, as shown in SEQ ID NO: 5, 6; DXF07, as shown in SEQ ID NO: 7, 8; DXF08, as shown in SEQ ID NO: 9, 10; DXF09, as shown in SEQ ID NO: 11, 12; DXS10079, as shown in SEQ ID NO: 13, 14; DXF13, as shown in SEQ ID NO: 15, 16; DXS10074, as shown in SEQ ID NO: 17, 18; DXS10075, as shown in SEQ ID NO: 19, 20; DXF15, as shown in SEQ ID NO: 21, 22; DXS6803, as shown in SEQ ID NO: 23, 24; DXF18, as shown in SEQ ID NO: 25, 26; DXF28, as shown in SEQ ID NO: 27, 28; DXF29, as shown in SEQ ID NO: 29, 30; DXS10103, as shown in SEQ ID NO: 31, 32; HPRTB, as shown in SEQ ID NO: 33, 34; DXS10101, as shown in SEQ ID NO: 35, 36; DXS10146, as shown in SEQ ID NO: 37, 38; DXS10134, as shown in SEQ ID NO: 39, 40; DXS7423, as shown in SEQ ID NO: 41, 42.

3. The kit according to claim 1, wherein DXS10103, DXF02, DXF15, DXF13, DXF28 and DXF29 are in the first group; DXF09, DXS10146, DXF18 and DXS10134 are in the second group; DXS6803, DXF07, DXS10075, DXS10074, HPRTB and DXS8378 are in the third group; DXS7423, DXS10079, DXS10101, DXF08 and DXS10135 are in the fourth group.

4. The kit according to claim 3, wherein One of the 5'-ends of each pair of primers is labeled with a fluorescent dye. Four sets of primers are labeled with blue, green, yellow, and red fluorescein respectively, and different fluorescent labels are used for each set. The primers are labeled using the following method: For blue labeling, 5-FAM (5-carboxyfluorescein), 6-FAM (6-carboxyfluorescein), or a fluorescein molecule with a similar spectrum is used for labeling; For green labeling, HEX (hexachloro-6-methylfluorescein), JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein succinimidyl ester), or a fluorescein molecule with a similar spectrum is used for labeling; For yellow, TAMRA (carboxytetramethylrhodamine) or a fluorescein molecule with a similar spectrum is used for labeling; for red, ROX (carboxy-X-rhodamine) or a fluorescein molecule with a similar spectrum is used for labeling.

5. The kit according to any one of claims 1-4, characterized in that The sources of human genomic DNA include blood / bloodstains, semen / seminal stains, saliva stains, sweat, tissues, hair, bones, and other tissues and body fluids of humans.

6. The kit according to claim 5, wherein Among them, The PCR reaction conditions are 95°C / 5 minutes → 30 cycles, and the reaction conditions for each cycle are 95°C / 30 seconds, 58.5°C / 90 seconds, 72°C / 30 seconds, → 68°C / 30 minutes → hold at 4°C.

7. Use of the kit according to claim 1 in individual identification and kinship determination.

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