Kit for rapidly detecting 21 Y-STR gene loci of micro-degraded sample and application of kit

Abstract

The invention discloses a kit for rapidly detecting 21 Y-STR gene loci of a micro-degraded sample and application of the kit. The kit comprises 19 pairs of specific amplification primer sequences for amplifying the 21 Y-STR gene loci. Compared with the prior art, the kit has the advantages that (1) the kit is combined with an eight-color fluorescence detection technology, the kit and a detection platform are deeply integrated, the detection process is optimized, and the detection speed and accuracy are improved; (2) according to the kit, the number of detectable sites in a 350bp range is greatly increased, so that the site detection number of detection materials such as conventional samples, old decayed samples, trace samples and degraded samples is greatly increased, and the purposes of forensic identification, genetic identification, family investigation and the like are achieved; and (3) in the forensic medicine application process, the kit has the advantages of high precision, high efficiency and high flux, so that the detection efficiency and accuracy of public security first-line cases are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic medicine and molecular biology, and relates to a scheme for detecting trace or trace amounts of test materials by using molecular marker primers and chemical fluorescence methods, and specifically is a 21 Y-STR gene loci kit for rapid detection of trace amounts of degraded test materials and its application. Background Art

[0002] Y-STR (short tandem repeats on the Y chromosome) is a genetic marker located on the male Y chromosome, with a core sequence consisting of 2 to 6 base pairs arranged in tandem repeats. This technology is widely used in many fields such as forensic evidence collection, paternity testing, and genealogical gene testing. As a male-specific chromosome, the Y chromosome has the characteristic of unidirectional inheritance from the paternal line, which makes Y-STR unique in forensic and genetic research.

[0003] At present, the amplified fragment size of mainstream Y-STR kits on the market is 550bp-600bp. Although multiple loci can be amplified at the same time, the fragment length cannot be taken into account, especially in trace and degraded samples, large fragments after 350bp cannot be detected, so complete and accurate genotyping cannot be obtained. In addition, the sequence of amplified site fragments of kits of various manufacturers is mostly arranged from small to large according to the core locus < preferred locus < alternative locus. It is difficult to increase the number of loci obtained by changing products and adding test reactions in the detection of trace and degraded samples, especially in major and difficult cases, it is difficult to achieve the purpose of forensic identification, kinship identification and family investigation. Summary of the invention

[0004] Technical problems to be solved: In order to overcome the shortcomings of the existing technology, the police frontline needs for high precision, high efficiency and high throughput forensic DNA detection reagents, and improve the efficiency and accuracy of case detection; at the same time, combined with the eight-color fluorescence detection technology, the kit is deeply integrated with the detection platform to optimize the detection process and improve the detection speed and accuracy. In addition, in order to significantly increase the number of detectable sites within the range of 350bp, thereby significantly increasing the number of sites detected for conventional samples, old and corrupt samples, trace samples, degraded samples and other samples, so as to achieve the purposes of forensic identification, kinship identification and family investigation. In view of this, the present invention provides a 21 Y-STR locus kit and application for rapid detection of trace degraded samples.

[0005] Technical solution: A kit for rapid detection of 21 Y-STR loci for trace degraded samples, the kit comprising 19 pairs of specific amplification primer sequences for amplifying 21 Y-STR loci, wherein the Y-STR loci are: DYS392, DYS389I / II, DYS447, DYS391, DYS456, DYS19, DYS437, DYS481, DYS533, DYS390, DYS393, GATA_H4, DYS439, DYS576, DYS458, DYS635, DYS448, DYS438, DYS385a / b.

[0006] Preferably, the sequences of the 19 pairs of specific amplification primers for amplifying 21 Y-STR loci are: DYS392, SEQ ID NOs: 1-2; DYS389I / II, SEQ ID NOs: 3-5; DYS447, SEQ ID NOs: 6-7; DYS391, SEQ ID NOs: 8-9; DYS456, SEQ ID NOs: 10-11; DYS19, SEQ ID NOs: 12-13; DYS437, SEQ ID NOs: 14-15; DYS481, SEQ ID NOs: 16-17; DYS533, SEQ ID NOs: 18-19; DYS390, SEQ ID NOs: 20-21; DYS393, SEQ ID NOs: 22-23; GATA_H4, SEQ ID NOs: 24-25; DYS439, SEQ ID NOs: 26-27; DYS576, SEQ ID NOs: ID NO: 28~29; DYS458, SEQ ID NO: 30~31; DYS635, SEQ ID NO: 32~33; DYS448, SEQ ID NO: 34~35; DYS438, SEQ ID NO: 36~37; DYS385a / b, SEQ ID NO: 38~39.

[0007] Preferably, the concentrations of the 19 pairs of specific amplification primers are: DYS392, 0.24 μM; DYS389I / II, 0.22 μM; DYS447, 0.14 μM; DYS391, 0.19 μM; DYS456, 0.21 μM; DYS19, 0.34 μM; DYS437, 0.18 μM; DYS481, 0.14 μM; DYS533, 0.33 μM; DYS390, 0.27μM; DYS393, 0.45μM; GATA_H4, 0.37μM; DYS439, 0.14μM; DYS576, 0.23μM ; DYS458, 0.24μM; DYS635, 0.19μM; DYS448, 0.12μM; DYS438, 0.25μM; DYS385a / b, 0.27μM.

[0008] The primers in the kit and their sequences and concentrations are shown in the following table:

[0009] Table 1 Specific amplification primer information of the kit of the present invention

[0010]

[0011]

[0012] Preferably, the locus and its 19 pairs of specific amplification primers are divided into 7 groups, specifically: DYS392, DYS389I / II, DYS447 are the first group; DYS391, DYS456, DYS19 are the second group; DYS437, DYS481, DYS533, DYS390 are the third group; DYS393, GATA_H4, DYS439 are the fourth group; DYS576, DYS458 are the fifth group; DYS635, DYS448 are the sixth group; DYS438, DYS385a / b are the seventh group.

[0013] Preferably, 7 groups of specific amplification primers are labeled with fluorescent dyes, and the 5' end of at least one primer in each pair of primers is labeled; the fluorescent dyes are 6-FAM, HEX, T13, SUM, LYN, PUR or H13, and each group uses a different fluorescent marker, and the internal standard is orange fluorescent SIZ.

[0014] Preferably, the fluorescent dyes used in each group of primers are: 6-FAM in the first group, HEX in the second group, T13 in the third group, SUM in the fourth group, LYN in the fifth group, PUR in the sixth group, and H13 in the seventh group.

[0015] Preferably, the kit comprises: a PCR reaction mixture, sdH 2O, genotyping standard AllelicLadder and fluorescent molecular weight internal standard; wherein, the composition of the PCR reaction mixture is: MgCl 2 8.0mM, Tris-HCl 145mM, KCl 135mM, dNTPs 8.5mM, BSA 4g / L, Hot Start Taq enzyme 2U / μL.

[0016] Preferably, the conditions for the amplification reaction of the kit are: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 10 seconds, annealing at 60°C for 60 seconds, extension at 68°C for 60 seconds, 29 cycles; final extension at 60°C for 15 minutes; and insulation at 4°C.

[0017] Application of any of the above-mentioned 21 Y-STR loci rapid detection kits for trace degraded samples in forensic identification, kinship identification and family screening analysis.

[0018] Preferably, the samples targeted by the forensic identification, kinship identification and family investigation include: conventional samples, old and corrupt samples, trace samples and degraded samples.

[0019] Beneficial effects: (1) The kit of the present invention combines the eight-color fluorescence detection technology, deeply integrates the kit with the detection platform, optimizes the detection process, and improves the detection speed and accuracy; (2) The kit greatly increases the number of detectable sites within the range of 350 bp, thereby greatly improving the number of sites detected for conventional samples, old and corrupt samples, trace samples, degraded samples and other samples, thereby achieving the purposes of forensic identification, kinship identification and family investigation; (3) In the process of forensic medicine application, because the kit has the advantages of high precision, high efficiency and high throughput, it greatly improves the efficiency and accuracy of front-line case detection in public security. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the arrangement of gene loci in the kit of the present invention;

[0021] Figure 2 The figure is a graph showing the amplification results of the kit of the present invention on a standard product;

[0022] Figure 3 This is a diagram showing the amplification results of the kit of the present invention on a degraded sample;

[0023] Figure 4 This is the amplification result of the same type of six-color kit for degraded samples. DETAILED DESCRIPTION

[0024] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0025] Example 1 Construction of the composite amplification kit of the present invention

[0026] The construction process includes the following stages: site screening, primer design, primer testing, primer optimization, and primer adjustment for multiple amplification, specifically:

[0027] First, through the study and optimization of the sequence composition characteristics of the conserved regions at both ends of the core repeat sequence of the Y-STR locus, 21 Y-STR loci were selected for system construction and the following were determined: Figure 1 The site arrangement scheme shown, in which the Y-STR loci are: DYS392, DYS389I / II, DYS447, DYS391, DYS456, DYS19, DYS437, DYS481, DYS533, DYS390, DYS393, GATA_H4, DYS439, DYS576, DYS458, DYS635, DYS448, DYS438, DYS385a / b.

[0028] Secondly, single gene primers are designed and synthesized based on the selected 21 Y-STR loci and site arrangement scheme, and the amplification conditions of a single STR locus are obtained through amplification experiments. The effect of the amplified products is observed by electrophoresis. If there is non-specific amplification or low amplification efficiency, the primers need to be redesigned until the single locus is successfully amplified.

[0029] Subsequently, the single primers need to be mixed and tested for composite amplification. As with the single-site amplification step, primers that produce nonspecificity and low amplification efficiency need to be excluded.

[0030] Finally, after the composite amplification primers for each locus are preliminarily determined, the primer concentration is adjusted. In the initial composite amplification, the same number of moles of primers should be added to the reaction system, and then the concentration ratio of each pair of primers is adjusted according to the test results until the amplification of the entire system is balanced.

[0031] Through the above process, the sequences of 19 pairs of specific amplification primers for amplifying 21 Y-STR loci of the kit of the present invention are determined as follows: DYS392, SEQ ID NO: 1-2; DYS389I / II, SEQ ID NO: 3-5; DYS447, SEQ ID NO: 6-7; DYS391, SEQ ID NO: 8-9; DYS456, SEQ ID NO: 10-11; DYS19, SEQ ID NO: 12-13; DYS437, SEQ ID NO: 14-15; DYS481, SEQ ID NO: 16-17; DYS533, SEQ ID NO: 18-19; DYS390, SEQ ID NO: 20-21; DYS393, SEQ ID NO: 22-23; GATA_H4, SEQ ID NO: 24-25; DYS439, SEQ ID NO: 26-27; NO: 26~27; DYS576, SEQ ID NO: 28~29; DYS458, SEQ ID NO: 30~31; DYS635, SEQ ID NO: 32~33; DYS448, SEQ ID NO: 34~35; DYS438, SEQ ID NO: 36~37; DYS385a / b, SEQ ID NO: 38~39.

[0032] The concentrations of the 19 pairs of specific amplification primers are: DYS392, 0.24 μM; DYS389I / II, 0.22 μM; DYS447, 0.14 μM; DYS391, 0.19 μM; DYS456, 0.21 μM; DYS19, 0.34 μM; DYS437, 0.18 μM; DYS481, 0.14 μM; DYS533, 0.33 μM ; DYS390, 0.27μM; DYS393, 0.45μM; GATA_H4, 0.37μM; DYS439, 0.14μM; DYS576, 0.23μM; DYS458, 0.24μM; DYS635, 0.19μM; DYS448, 0.12μM; DYS438, 0.25μM; DYS385a / b, 0.27μM.

[0033] The primers in the kit and their sequences and concentrations are shown in the following table:

[0034] Table 1 Specific amplification primer information of the kit of the present invention

[0035]

[0036]

[0037] The locus and its 19 pairs of specific amplification primers are divided into 7 groups, specifically: DYS392, DYS389I / II, DYS447 are the first group; DYS391, DYS456, DYS19 are the second group; DYS437, DYS481, DYS533, DYS390 are the third group; DYS393, GATA_H4, DYS439 are the fourth group; DYS576, DYS458 are the fifth group; DYS635, DYS448 are the sixth group; DYS438, DYS385a / b are the seventh group.

[0038] Seven groups of specific amplification primers were labeled with fluorescent dyes, and the 5' end of at least one primer in each pair of primers was labeled; the fluorescent dyes were 6-FAM, HEX, T13, SUM, LYN, PUR or H13, and each group used different fluorescent labels, and the internal standard used orange fluorescent SIZ. The fluorescent dyes used in each group of primers were: 6-FAM in the first group, HEX in the second group, T13 in the third group, SUM in the fourth group, LYN in the fifth group, PUR in the sixth group, and H13 in the seventh group.

[0039] The kit includes: PCR reaction mixture, sdH 2 O, genotyping standard Allelic Ladder and fluorescent molecular weight internal standard; wherein, the composition of the PCR reaction mixture is: MgCl 2 8.0mM, Tris-HCl 145mM, KCl 135mM, dNTPs 8.5mM, BSA 4g / L, Hot Start Taq enzyme 2U / μL.

[0040] Example 2 Establishment of PCR reaction system and procedure

[0041] Based on the composite amplification kit constructed in Example 1, the PCR reaction mixture is optimized, and after a large number of optimization tests of components and concentrations in the PCR reaction mixture, the influence of different components in the PCR reaction mixture on the system amplification performance is explored, thereby obtaining the optimal PCR reaction mixture. Further, the combined application test of each component in the PCR reaction system is carried out, the matching cycle parameters are optimized, and the PCR reaction system and program are finally established. The optimal PCR reaction system is shown in Table 2, and the optimal reaction program is shown in Table 3.

[0042] Table 2 PCR reaction system

[0043] Components Volume (10 μL) PCR reaction mixture 4μL Primer mix 2μL DNA template 1~4μL Deionized water Add to 10 μL

[0044] Table 3 PCR reaction program

[0045] Initial denaturation Thermal Cycle Final extension Insulation 95℃5min 29 cycles: 94°C for 10 seconds, 60°C for 60 seconds, 68°C for 60 seconds 60℃, 15min Maintain at 4℃

[0046] Example 3 The kit of the present invention is used to amplify highly degraded samples

[0047] The kit constructed in Example 1 and the amplification procedure and conditions established in Example 2 were used for comparison, and the commercially available six-color AGCU Y43CS fluorescence detection kit was used for comparison, and highly degraded samples were selected as the test materials. The specific process is as follows:

[0048] (1) Preparation of highly degraded samples

[0049] 0.5ng male genomic DNA with known DNA typing was placed in a metal bath at 95℃ for 2h to simulate sample degradation.

[0050] (2) PCR amplification

[0051] The PCR amplification system in Table 2 and the PCR reaction program in Table 3 were used.

[0052] (3) Detection of PCR products by capillary electrophoresis

[0053] The amplified product was centrifuged at 3000rpm for 5 minutes, 1μL of the product or Allelic Ladder was mixed with 0.3μL Marker SIZ-500 and 9.7μL deionized formamide, denatured at 95℃ for 3 minutes, and then placed in an ice bath for electrophoresis detection. Note: The amount of Allelic Ladder and Marker SIZ-500 can be adjusted according to the actual electrophoresis conditions.

[0054] (4) Data analysis

[0055] Analyze the data collected by the Genetic Analyzer assay using fragment analysis software.

[0056] (5) Test conclusion

[0057] The above-mentioned degraded samples were amplified using the composite amplification kit described in the present invention, and the six-color Y43CS fluorescence detection kit (Wuxi Sino-German Meilian Biotechnology Co., Ltd.) was used to amplify and analyze the same degraded and non-degraded samples (for obtaining complete and correct DNA typing results). The analysis data comparison results are shown in Table 4.

[0058] The test results show that for this degraded sample, the composite amplification kit and its eight-color fluorescence detection platform can obtain complete Y-STR typing results, and the peak type is normal, there is no missed detection, and the typing result is correct. Although the detection rate of this composite amplification kit is the same as that of the six-color product, both are full detection, but the overall amplification peak height of this composite amplification kit is about 4000RFU, the overall site peak height is close, and the balance is good, while the peak height of the six-color Y43CS fluorescence detection kit product amplification is not only high at the beginning and low at the end, but also the peak height of most sites is less than 1000RFU, and the peak height of some sites is even less than 500RFU, which is as low as the analysis threshold, which is not conducive to the deterministic interpretation of typing. If this composite amplification kit and the six-color Y43CS fluorescence detection kit simultaneously detect highly degraded samples with a lower degree of degradation, then the peak height of the large fragment of the six-color Y43CS fluorescence detection kit may be further reduced, or even most sites may be missed, and the overall peak height of this composite amplification kit may be reduced due to severe template degradation, but it will still be higher than the six-color Y43CS fluorescence detection kit. It can be seen from this embodiment that in terms of degradation sample detection, the composite amplification kit has obvious advantages over the existing six-color fluorescence detection kit of the same type in terms of overall amplification balance and amplification peak height, and can meet the needs of forensic identification and family investigation.

[0059] Table 4 Data comparison results

[0060]

Claims

1. A kit for rapid detection of 21 Y-STR loci for trace degraded samples, characterized in that: The kit includes 19 pairs of specific amplification primer sequences for amplifying 21 Y-STR loci, wherein the Y-STR loci are: DYS392, DYS389I / II, DYS447, DYS391, DYS456, DYS19, DYS437, DYS481, DYS533, DYS390, DYS393, GATA_H4, DYS439, DYS576, DYS458, DYS635, DYS448, DYS438, DYS385a / b.

2. The 21 Y-STR loci kit for rapid detection of trace degraded samples according to claim 1, characterized in that: The sequences of the 19 pairs of specific amplification primers for amplifying 21 Y-STR loci are: DYS392, SEQ ID NOs: 1-2; DYS389I / II, SEQ ID NOs: 3-5; DYS447, SEQ ID NOs: 6-7; DYS391, SEQ ID NOs: 8-9; DYS456, SEQ ID NOs: 10-11; DYS19, SEQ ID NOs: 12-13; DYS437, SEQ ID NOs: 14-15; DYS481, SEQ ID NOs: 16-17; DYS533, SEQ ID NOs: 18-19; DYS390, SEQ ID NOs: 20-21; DYS393, SEQ ID NOs: 22-23; GATA_H4, SEQ ID NOs: 24-25; DYS439, SEQ ID NOs: 26-27; DYS576, SEQ ID NOs: 28-29; NO: 28~29; DYS458, SEQ ID NO: 30~31; DYS635, SEQ ID NO: 32~33; DYS448, SEQ ID NO: 34~35; DYS438, SEQ ID NO: 36~37; DYS385a / b, SEQ ID NO: 38~39.

3. The 21 Y-STR loci kit for rapid detection of trace degraded samples according to claim 1, characterized in that: The concentrations of the 19 pairs of specific amplification primers are: DYS392, 0.24 μM; DYS389I / II, 0.22 μM; DYS447, 0.14 μM; DYS391, 0.19 μM; DYS456, 0.21 μM; DYS19, 0.34 μM; DYS437, 0.18 μM; DYS481, 0.14 μM; DYS533, 0.33 μM; DYS390, 0.27 μM; DYS393, 0.45 μM; GATA_H4, 0.37 μM; DYS439, 0.14 μM; DYS576, 0.23 μM; DYS458, 0.24 μM; DYS635, 0.19 μM; DYS448, 0.12μM; DYS438, 0.25μM; DYS385a / b, 0.27μM.

4. The 21 Y-STR loci kit for rapid detection of trace degraded samples according to claim 1, characterized in that: The locus and its 19 pairs of specific amplification primers are divided into 7 groups, specifically: DYS392, DYS389I / II, DYS447 are the first group; DYS391, DYS456, DYS19 are the second group; DYS437, DYS481, DYS533, DYS390 are the third group; DYS393, GATA_H4, DYS439 are the fourth group; DYS576, DYS458 are the fifth group; DYS635, DYS448 are the sixth group; DYS438, DYS385a / b are the seventh group.

5. The 21 Y-STR loci kit for rapid detection of trace degraded samples according to claim 4, characterized in that: Seven groups of specific amplification primers were labeled with fluorescent dyes, and the 5' end of at least one primer in each pair of primers was labeled; the fluorescent dyes were 6-FAM, HEX, T13, SUM, LYN, PUR or H13, and each group used a different fluorescent label, and the internal standard was orange fluorescent SIZ.

6. The 21 Y-STR loci kit for rapid detection of trace degraded samples according to claim 5, characterized in that: The fluorescent dyes used in each group of primers are: 6-FAM in the first group, HEX in the second group, T13 in the third group, SUM in the fourth group, LYN in the fifth group, PUR in the sixth group, and H13 in the seventh group.

7. The kit for rapid detection of 21 Y-STR loci of trace degraded samples according to claim 1, characterized in that: The kit comprises: a PCR reaction mixture, sdH2O, a genotyping standard Allelic Ladder and a fluorescent molecular weight internal standard; wherein the PCR reaction mixture comprises: MgCl2 8.0 mM, Tris-HCl 145 mM, KCl 135 mM, dNTPs 8.5 mM, BSA 4 g / L, and hot start Taq enzyme 2 U / μL.

8. The kit for rapid detection of 21 Y-STR loci of trace degraded samples according to claim 7, characterized in that: The conditions of the amplification reaction of the kit are: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 10 seconds, annealing at 60°C for 60 seconds, extension at 68°C for 60 seconds, 29 cycles; final extension at 60°C for 15 minutes; and insulation at 4°C.

9. Use of the 21 Y-STR loci kit for rapid detection of trace degraded samples according to any one of claims 1 to 8 in forensic identification, kinship identification and family screening analysis.

10. The use according to claim 9, characterized in that: The samples targeted by the forensic identification, kinship identification and family investigation include: routine samples, old and corrupt samples, trace samples and degraded samples.

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