CN112852973A - Primer group and kit for simultaneously amplifying 13 STR loci of human and application thereof - Google Patents
Primer group and kit for simultaneously amplifying 13 STR loci of human and application thereof Download PDFInfo
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Abstract
The invention provides a primer group and a kit for simultaneously amplifying 13 STR loci of human and application thereof, belonging to the technical field of molecular genetics. In the invention, the 13 STR loci comprise 12 autosomal STR loci and 1 individual identification STR locus; the invention can simultaneously amplify 13 STR loci with fragments less than 320bp in one reaction, comprises 8 core loci and 5 preferred loci specified by the ministry of public security, and also comprises 1 individual identification locus, thereby effectively preventing the problem of detection loss of the loci of trace and degraded test material large fragments. The 13 STR loci are combined with the mainstream kit in the market, so that more loci information can be obtained for trace and degraded DNA, and the individual identification capability is effectively improved.
Description
Technical Field
The invention relates to the technical field of molecular genetics, in particular to a primer group and a kit for simultaneously amplifying 13 STR loci and application thereof.
Background
STR (short tandem repeats), also known as microsatellite sequences, is a short tandem repeat that is present in large amounts in human genomic DNA and has a repeat unit of 2-6 nucleotides. Because of the high polymorphism and stability, and the much smaller length of amplified product (less than 500bp) compared to the AMP-FLP and VNTR isotyping methods, STR genotyping requires less template quality and allows analysis even with degraded DNA templates. In addition, STR typing is suitable for DNA purified by a variety of DNA purification methods, which often yield DNA in amounts insufficient for Southern blot analysis. In view of the above characteristics, STR typing techniques have been widely applied to forensic identification.
The forensic DNA database is an important scientific and technological apparatus integrating the elements of modern DNA inspection technology, information technology, network technology, scientific management and the like, realizing cross-space-time diversified application, accurately fighting crimes, protecting people and maintaining social stability. The core of the forensic DNA database lies in the recorded STR (short distance repeat) data information, and a perfect forensic DNA database should include an autosomal STR database and a Y chromosome STR database. The Y chromosome STR data provides investigation clues for cases, reduces investigation range, can predict regions, ethnicities or even surnames where criminals may exist, and the autosome STR data is used for screening and confirming final criminal individuals.
The currently published 20 autosomal STR core loci (20 loci, including Amelogenin, CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, D2S1338, D19S433, D6S1043, D12S391, Penta D, Penta E) and 10 autosomal STR preferred loci (10 loci, including D1S1656, D2S441, D22S1045, D10S1248, D8S1132, D15S659, D3S3045, D19S253, D6S477, D10S1435 for expanded use) correspond to DNA therapeutics identifying the number of loci, specificity and the higher sensitivity of the kit. However, in a complex and variable natural environment, especially, environmental factors such as humidity, high temperature, ultraviolet rays, exposure to sunlight, microorganisms, strong acid and the like can cause DNA damage in a biological test material, double strand break and the length of DNA molecules is shortened. When the conventional STR typing test is carried out on the degraded biological sample, the allele loss of large-fragment STR locus loci is easy to occur, and the typing failure is caused. The application of STR parting technology in trace and degraded biological detection materials is greatly limited, so that the value of some important biological detection materials in case detection cannot be fully embodied, and the rapid case detection is not facilitated.
In order to fundamentally meet the use requirements of public security users, improve the compatibility of the kit, reduce the amplification time, improve the material detection adaptability of the kit, improve the sensitivity of the kit and make up for the application defect of the conventional STR detection technology, on the basis of the original STR parting technology, the forward and reverse primers are redesigned to move to the core repetitive sequence as far as possible, the length of the amplified product fragment is reduced, and meanwhile, the genetic polymorphism of the original STR locus site and the original parting result are kept unchanged.
Disclosure of Invention
The invention aims to provide a primer group and a kit for amplifying 13 STR loci simultaneously and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer group for amplifying 13 STR loci of a person simultaneously, wherein the 13 STR loci comprise 12 autosomal STR loci and 1 individual identification STR locus; the 12 autosomal STR loci are respectively: D10S1248, Penta E, CSF1PO, D2S1338, D18S51, D1S1656, D12S391, D6S477, FGA, D15S659, D8S1132 and Penta D; the 1 individual identification STR loci are as follows: amelogenin;
the nucleotide sequence of the upstream primer for amplifying the D10S1248 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying Penta E is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying CSF1PO is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying the D2S1338 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying the D18S51 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the Amelogenin is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying the D1S1656 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the D12S391 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the D6S477 is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying FGA is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the D15S659 is shown as SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22; the nucleotide sequence of the upstream primer for amplifying the D8S1132 is shown in SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24; the nucleotide sequence of the upstream primer for amplifying Penta D is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 26.
Preferably, the 13 STR loci are divided into five groups, wherein,
the first group includes: D10S1248 and Penta E;
the second group includes: CSF1PO, D2S1338 and D18S 51;
the third group includes: amelogenin, D1S1656 and D12S 391;
the fourth group includes: D6S477 and FGA;
the fifth group includes: D15S659, D8S1132 and Penta D;
the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.
The invention also provides a kit comprising the primer group in the scheme.
Preferably, the kit further comprises a PCR reaction premix and deionized water.
Preferably, the kit further comprises an internal molecular weight standard and an allelic ladder.
Preferably, in the primer set,
SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.2-0.25 mu M;
SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.17-0.23 mu M;
SEQ ID NO: 5 and SEQ ID NO: 6 is 0.1-0.16 mu M independently;
SEQ ID NO: 7 and SEQ ID NO: the concentration of the primers shown in 8 is 0.13-0.18 mu M independently;
SEQ ID NO: 9 and SEQ ID NO: 10 is independently 0.11-0.15 mu M;
SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.08-0.12 mu M;
SEQ ID NO: 13 and SEQ ID NO: 14 is independently 0.11-0.15 mu M;
SEQ ID NO: 15 and SEQ ID NO: 16 is independently 0.13-0.18 mu M;
SEQ ID NO: 17 and SEQ ID NO: 18 is independently 0.11-0.18 mu M;
SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.2-0.25 mu M;
SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.45-0.52 mu M;
SEQ ID NO: 23 and SEQ ID NO: 24 is independently 0.14-0.19 μ M;
SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.08-0.13. mu.M.
Preferably, the PCR reaction premix takes deionized water as a solvent and comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl 250 to 100mM (NH)4)2SO40.5 to 1 μ M dNTP, 1000 to 1500mM betaine, volume fraction of0.19 to 0.38 percent of Triton, 2 to 3mg/ml of BSA, 5 to 15 percent of Tween and 2.5 to 7.5 percent of glycerol.
Preferably, the corresponding relationship between the allelic ladder and the STR locus is:
in the first group: D10S1248 corresponds to 10,11,12,13,14,15,16,17, 18; penta E corresponds to 5,6,7,8,9,10,11,12,13,14,15,16,17,19,20,21,22,23,24, 26; in the second group: CSF1PO corresponds to 6,7,8,9,10,11,12,13,14,15, 16; D2S 1338: 16,17,18,20,21,23,24,25, 26; D18S51 corresponds to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,26, 27; in the third group: amelogenin corresponds to X, Y; D1S1656 corresponds to 10,11,12,13,14,15,16,17, 18; D12S391 corresponds to 15,16,17,20,21,23,24,25,26, 27; in the fourth group: D6S477 corresponds to 11,14,15,16,18,19, 20; FGA corresponds to 16,17,18,19,20,21,23,24,25,26,27,29,30,31.2,43.2,44.2,45.2, 46.2; in the fifth group: D15S659 corresponds to 10,12,13,14,15,16,17,18,19, 20; D8S1132 corresponds to 16,17,18,19,20,21,22,23,24, 25; penta D corresponds to 5,6,7,8,9,10,11,12,13,14,15, 16.
The invention also provides application of the primer group or the kit in the scheme in forensic individual identification, forensic DNA database construction, suspect family investigation or judicial genetic relationship identification.
Preferably, the application comprises the following steps:
putting a sample to be detected into an amplification system containing the primer group in the scheme, performing PCR amplification to obtain an amplification product, and detecting the amplification product;
when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;
when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;
the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 60sec, 71 ℃,20 sec, 28 cycles; 60 deg.C for 10 min.
The invention has the beneficial effects that: the invention provides a primer group for amplifying 13 STR loci of human simultaneously, wherein the 13 STR loci comprise 12 autosomal STR loci and 1 individual identification STR locus. The invention can simultaneously amplify 13 STR loci with fragments less than 320bp in one reaction, comprises 8 core loci and 5 preferred loci specified by the ministry of public security, and also comprises 1 individual identification locus, thereby effectively preventing the problem of detection loss of the loci of trace and degraded test material large fragments. The 13 STR loci are combined with the mainstream kit in the market, so that more loci information can be obtained for trace and degraded DNA, and the individual identification capability is effectively improved. The primer group disclosed by the invention can realize rapid amplification, has strong amplification specificity and no non-specific amplification, and simultaneously, the primers do not interfere with each other and do not form primer dimers. The length of the primers is 18-32 bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 13 pairs of primers have no interaction, so that the primers corresponding to 13 STR loci can be compatible in a single tube. The length of the total amplification product is between 70 and 320 bp. After amplification reaction, 1-2 specific amplification bands can be obtained from each locus, and non-specific amplification peaks, primer peaks and other miscellaneous peaks are avoided. The primer group also has the advantage of high sensitivity, can be suitable for the amplification detection of the autosomal STR of various test materials, and can meet the requirements of multifunctional STR identification of the current forensic identity identification, the construction of a forensic DNA database and the identification of judicial genetic relationship.
Drawings
FIG. 1 is a positive control DNA9948 genotyping map;
FIG. 2 is a ladder allelic typing map;
FIG. 3 is a BTY-550 spectrum of a molecular weight internal standard;
FIG. 4 is a chart of suspected subgenomic typing;
FIG. 5 is a suspected father genotyping map;
FIG. 6 is a genotyping chart for DNA samples extracted;
FIG. 7 is a blood card test material genotyping chart.
Detailed Description
The invention provides a primer group for amplifying 13 STR loci of a person simultaneously, wherein the 13 STR loci comprise 12 autosomal STR loci and 1 individual identification STR locus; the 12 autosomal STR loci are respectively: D10S1248, Penta E, CSF1PO, D2S1338, D18S51, D1S1656, D12S391, D6S477, FGA, D15S659, D8S1132 and Penta D; the 1 individual identification STR loci are as follows: amelogenin;
the nucleotide sequence of the upstream primer for amplifying the D10S1248 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying Penta E is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying CSF1PO is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying the D2S1338 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying the D18S51 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the Amelogenin is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying the D1S1656 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the D12S391 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the D6S477 is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying FGA is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying the D15S659 is shown as SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22; the nucleotide sequence of the upstream primer for amplifying the D8S1132 is shown in SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24; the nucleotide sequence of the upstream primer for amplifying Penta D is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 26.
In the invention, each pair of primers in the primer group can be stably compatible and do not react with each other in one tube, and the specificity, the secondary structure, the amplification efficiency, the stability and the like are greatly improved.
The 13 STR loci of the present invention comprise 8 core loci defined by the Ministry of public Security, and 5 preferred loci, which comprise 1 individual identification locus. The 13 STR loci are combined with the conventional autosomal STR kit, so that the detection rate of trace and degraded detected materials can be improved, and the individual identification power can be improved. The STR locus sites in the primer set of the present invention are shown in Table 1.
TABLE 1 STR locus sites in the primer sets of the invention
In the invention, the amplification balance of the primer group comprehensively exceeds the standard (more than or equal to 70% in a gene locus, more than or equal to 50% in the same color group, and more than or equal to 30% among different color groups) in GB/T37226-2018 court science human fluorescence labeling STR composite amplification detection reagent quality basic requirements.
In the present invention, the 13 STR loci are divided into five groups, wherein,
a first group: D10S1248 and Penta E;
second group: CSF1PO, D2S1338 and D18S 51;
third group: amelogenin, D1S1656 and D12S 391;
and a fourth group: D6S477 and FGA;
and a fifth group: D15S659, D8S1132 and Penta D;
the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.
In the present invention, the fluorescent dye used in the first group is preferably FAM; the fluorescent dye adopted by the second group is preferably HEX; the fluorescent dye used in the third group is preferably TAMAR; the fluorescent dye adopted by the fourth group is preferably ROX; the fluorescent dye adopted in the fifth group is preferably PURPLE; ORG was used as internal standard. The invention adopts six-color fluorescent labeling systems, namely FAM, HEX, TAMAR, ROX, PURPLE and ORG. Wherein FAM represents blue, HEX represents green, TAMAR represents yellow, ROX represents red, PURPLE represents PURPLE, and ORG represents orange. The invention can simultaneously amplify 13 STR loci in one reaction, and fully meets the compatibility of the current public security DNA database comparison. In the present invention, the 5' end of the upstream primer and/or the downstream primer of each of the STR loci is labeled with a fluorescent dye. The invention uses the fluorescence labeling method to label a fluorescent dye at the 5' end of the primer, the PCR product can emit optical signals with specific wavelength under the laser excitation state, the optical signals can be collected by electrophoresis detection through a genetic analyzer (ABI 3130/ABI 3500/ABI 3730 series one by one), and the detection is carried out through the collected optical signals.
The invention also provides a kit comprising the primer group in the scheme.
In the present invention, the kit preferably further comprises a PCR reaction premix and deionized water. In the present invention, the kit preferably further comprises a positive control DNA 9948. In the invention, the primer group, the PCR reaction premixed solution and the deionized water form a reaction system of the kit.
In the present invention, in the primer set,
SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.2-0.25. mu.M, and more preferably 0.238. mu.M; SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.17-0.23 μ M, and more preferably 0.198 μ M; SEQ ID NO: 5 and SEQ ID NO: 6 is independently 0.1-0.16. mu.M, and more preferably 0.140. mu.M; SEQ ID NO: 7 and SEQ ID NO: 8 is 0.13-0.18. mu.M, and preferably 0.150. mu.M; SEQ ID NO: 9 and SEQ ID NO: 10 is independently 0.11-0.15. mu.M, more preferably 0.128. mu.M; SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.08-0.12. mu.M, more preferably 0.106. mu.M; SEQ ID NO: 13 and SEQ ID NO: 14 is independently 0.11 to 0.15. mu.M, and more preferably 0.130. mu.M; SEQ ID NO: 15 and SEQ ID NO: 16 is 0.13-0.18. mu.M, and preferably 0.150. mu.M; SEQ ID NO: 17 and SEQ ID NO: 18 is independently 0.11 to 0.18. mu.M, and more preferably 0.135. mu.M; SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.2-0.25. mu.M, and more preferably 0.225. mu.M; SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.45 to 0.52. mu.M, more preferably 0.478. mu.M; SEQ ID NO: 23 and SEQ ID NO: 24 is independently 0.14-0.19. mu.M, more preferably 0.168. mu.M; SEQ ID NO: 25 and SEQ ID NO: 26 is 0.08 to 0.13. mu.M, and preferably 0.112. mu.M.
In the present invention, the PCR reaction premix preferably includes hot-start DNA polymerase, dNTP, magnesium ions, potassium ions, Tris buffer and enhancer; the PCR reaction premix takes deionized water as a solvent, and preferably comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl 250 to 100mM (NH)4)2SO40.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction. In one embodiment of the present invention, the recipe for the PCR reaction premix is shown in Table 2.
TABLE 2 PCR reaction premix formula of the inventive example
H in Table 22O is deionized water which is highly deionized water, the resistivity reaches 18.2M omega, and the interference of external ions on an amplification system is completely eliminated.
In the invention, the PCR reaction premix has the characteristics of high speed, high sensitivity, strong adaptability and the like, and can not freeze at the temperature of-20 ℃, thereby effectively preventing the influence of freeze thawing on the performance of the reagent. The sensitivity of the PCR reaction premix liquid is as high as 0.03ng DNA, the effective amplification time is reduced to be within 1.5h, the PCR reaction premix liquid has excellent amplification effect on DNA from different sources (blood, blood stains, semen, seminal stains, saliva stains, hair, tissues, nails, body fluid and the like), and meanwhile, the PCR reaction premix liquid also has very good amplification effect on special blood cards, saliva cards and FTA cards.
In the invention, the positive control DNA9948 is used as an amplification standard substance for testing the quality of an amplification system. In the preferred embodiment of the invention, the positive control can be correctly typed, and the sensitivity is as high as 0.03ng and is far lower than the standard 0.125 ng. The 9948 fractal pattern amplified by the present invention is shown in FIG. 1 and Table 3.
Table 3: 9948 genotyping
| Genetic loci | 9948 type |
| Amelogenin | X，Y |
| D18S51 | 15，18 |
| |
29，30 |
| D3S1358 | 15，17 |
| FGA | 24，26 |
| D8S1179 | 12，13 |
| vWA | 17 |
| CSF1PO | 10，11 |
| D16S539 | 11 |
| D7S820 | 11 |
| D13S317 | 11 |
| D5S818 | 11，13 |
| D2S1338 | 23 |
In the present invention, the kit preferably further comprises an internal molecular weight standard and an allelic ladder. In the present invention, the molecular weight internal standard and the allele ladder are detection reagents of the kit.
In the present invention, the correspondence between the allelic ladder and the STR locus is specifically found in table 4:
TABLE 4 allelic ladder and correspondence of the STR loci
The allele ladder 13A of the invention covers all the most common alleles and most rare alleles of each locus, and is more convenient for comparing genotyping results.
In the present invention, the molecular weight internal standard is BTY-550; the BTY-550 comprises the following DNA fragments: 65. 75, 100, 139, 150, 160, 200, 250, 300, 340, 400, 450, 490, 500, 540, and 550. In the present invention, the internal molecular weight standard is labeled with the fluorescent dye ORG, representing orange. The molecular weight internal standard can effectively distinguish the sizes of 65-550 bp DNA fragments.
The invention also provides application of the primer group and the kit in the scheme in forensic individual identification, forensic DNA database construction, suspect family investigation or judicial genetic relationship identification.
In the present invention, the application preferably comprises the steps of:
putting a sample to be detected into an amplification system containing the primer group in the scheme, performing PCR amplification to obtain an amplification product, and detecting the amplification product;
when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;
when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;
the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 60sec, 71 ℃,20 sec, 28 cycles; 60 deg.C for 10 min.
In the present invention, the sample to be detected preferably includes human blood, blood stain, semen, seminal stain, saliva, salivary stain, hair, tissue, nail or body fluid.
In the present invention, the standard amplification system for PCR amplification is 25. mu.l, and comprises 5. mu.l of the mixture of the primer sets, 10. mu.l of the PCR reaction premix, 7.5. mu.l of deionized water, and 2.5. mu.l of the positive control DNA 9948. The amplification procedure for the standard amplification system is shown in Table 5.
TABLE 5 amplification procedure for the Standard amplification System
The method for detecting the amplification product of the present invention preferably includes: carrying out electrophoretic detection on the amplification product; the electrophoresis detection equipment is preferably ABI3500 XL; in the specific implementation process of the invention, HiDi formamide, molecular weight internal standard BTY-500 and PCR amplification product or allele ladder 13A are mixed to obtain a mixture; taking the volume of HiDi formamide as 8.5 μ l, and measuring the molecular weight internal standard BTY-5000.5 μ l and PCR amplification product or allele step 13A 1 μ l; after the mixture was dispensed into a 96-well plate, centrifugation was carried out to remove air bubbles, and the 96-well plate was placed in a sample application tray and then in a 3500Xl genetic analyzer at a sample application voltage of 1.8kVolts for 12sec, after which electrophoresis was started.
The present invention preferably further comprises analyzing the results of the electrophoresis on GeneMapper _ IDX software after said electrophoresis. The positive control 9948 typing map is shown in FIG. 1, and the allelic ladder map is shown in FIG. 2; molecular weight internal standard BTY-550 is shown in FIG. 3.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Two hair portions from suspected father and son are subjected to direct multiplex amplification, and 13 STR loci are detected. The amplification method adopts a direct amplification method, namely a section of hair with hair follicles is taken and directly put into an amplification system, so that the hair follicles are completely immersed in liquid, the standard amplification program is adopted in the amplification program, an ABI Proflex PCR instrument is adopted in an amplification instrument, a 3500xl genetic analyzer is adopted in a genetic analyzer, and GeneMapper-ID-X analysis software is adopted in analysis software. The operation steps of this embodiment are as follows:
first, 0.5cm above the hair follicles was cut short with scissors, and the cut hair with follicles was placed in a 200. mu.l PCR tube.
Preparing 10 mul amplification system according to the standard reaction system of the invention: mu.l of the primer mixture, 4. mu.l of the reaction premix, and 4. mu.l of deionized water were added to the PCR tube with cut hair. Performing PCR amplification on an ABI Proflex PCR instrument by the following amplification program: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 60sec, 71 ℃,20 sec, 28 cycles; 60 deg.C for 10 min; storing at constant temperature of 10 ℃.
Detecting and data analyzing by genetic analyzer
After PCR amplification is finished, the amplification product is detected on ABI3500 XL. 8.5. mu.l of HiDi formamide + 0.5. mu.l of molecular weight internal standard BTY-500+ 1. mu.l of PCR product/1. mu.l of allele step 13A; after being divided into 96-well plates, the plates were centrifuged to remove air bubbles, and the 96-well plates were placed in a sample tray and then in a 3500Xl genetic analyzer at a sample injection voltage of 1.8kVolts for 12sec, after which electrophoresis was started.
Analysis was performed on GeneMapper IDX software after the end of electrophoresis, see fig. 4: a suspected gene fractal graph; FIG. 5: a suspected father genotyping map; the results of the suspected parent and child gene typing are shown in Table 6.
TABLE 6 suspected paternal genotyping
| Genetic loci | Suspected father genotype | Suspected female genotype |
| D3S1358 | 12,13 | 13,14 |
| TH01 | 11,12 | 12,19 |
| D21S11 | 12 | 12,13 |
| D18S51 | 19,24 | 19,23 |
| PentaE | 17,18 | 18,20 |
| Amelogenin | X，Y | X，Y |
| D5S818 | 15,16 | 15 |
| D13S317 | 20,21 | 20,21 |
| D7S820 | 13,14 | 13,14 |
| D16S539 | 21,22 | 22,23 |
| CSF1PO | 14,17 | 13,17 |
| D2S1338 | 19,23 | 19,21 |
| D6S1043 | 10,12 | 10,14 |
By analyzing and comparing the obtained genotypes of the 13 STR loci, the invention can find that the genetic information of the 13 STR loci of the suspected father and son accords with the genetic rule, and can judge the relationship between the father and the son.
Example 2: application of kit of the invention in forensic medicine
The invention is mainly used for the construction of DNA database of criminals and the identification of the identity of criminal suspects in the field of forensic science.
The national forensic DNA database is a national DNA database of illegal criminals established by the ministry of public security in China, breaks through 5000 thousands of data in the database at present, and is a main tool for case detection of a public security system. The database is usually built by adopting a method of directly amplifying and detecting blood samples, the detection material in the identification work of the identity of the criminal suspect is usually a complex detection material, and the amplification detection method is adopted for detection after DNA is extracted. The invention can take two different inspection materials into consideration, and is convenient for the construction of DNA databases in China and the identification of the identity of the criminal suspect. The detailed part of the operation is as follows:
firstly, it is known that the material to be detected by the criminal is usually blood card, which can be directly amplified. Using a 1mm diameter punch, a 1mm diameter blood piece was punched directly from the dried blood card and placed in a 200. mu.l PCR tube.
Secondly, unknown criminals are generally case field inspection materials which are complex, and the detection analysis of data is generally carried out by a method of firstly extracting DNA and then amplifying. DNA was extracted by the Chelex-100 and magnetic bead method. (the extraction method refers to DNA extraction of Zhengxiufen 'forensic DNA analysis' Chapter four).
Thirdly, the preparation and amplification of the reaction system, referring to the standard system of the invention, 10 mul of reaction system is prepared, see table 8 below. Performing PCR amplification on an ABI Proflex PCR instrument by the following amplification program: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 60sec, 71 ℃,20 sec, 28 cycles; 60 deg.C for 10 min; storing at constant temperature of 10 ℃.
TABLE 7 reaction system of this example
Fourthly, detecting by a genetic analyzer and analyzing data
After PCR amplification is finished, the amplification product is detected on ABI3500 xl. 8.5. mu.l of HiDi formamide + 0.5. mu.l of molecular weight internal standard BTY-500+ 1. mu.l of PCR product/1. mu.l of allele step 44Y; after being divided into 96-well plates, the plates were centrifuged to remove air bubbles, and the 96-well plates were placed in a sample tray and then in a 3500Xl genetic analyzer at a sample injection voltage of 1.8kVolts for 12sec, after which electrophoresis was started.
And after the electrophoresis is finished, analyzing on GeneMapper _ IDX software, exporting the analyzed data to a CODIS format file after the analysis is finished, and uploading the exported data to a national DNA database. FIG. 6 is a blood card sample typing chart, and FIG. 7 is a DNA extraction sample typing chart. The invention detects the direct amplification sample and the extracted DNA sample, has correct result and good graphic balance, and can completely meet the daily application of the forensic.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A primer group for amplifying 13 STR loci of human simultaneously, wherein the 13 STR loci comprise 12 autosomal STR loci and 1 individual identification STR locus; the 12 autosomal STR loci are respectively: D10S1248, Penta E, CSF1PO, D2S1338, D18S51, D1S1656, D12S391, D6S477, FGA, D15S659, D8S1132 and Penta D; the 1 individual identification STR loci are as follows: amelogenin;
the nucleotide sequence of the upstream primer for amplifying the D10S1248 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying Penta E is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying CSF1PO is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying the D2S1338 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying the D18S51 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying the Amelogenin is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying the D1S1656 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying the D12S391 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying the D6S477 is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying FGA is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying the D15S659 is shown as SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22;
the nucleotide sequence of the upstream primer for amplifying the D8S1132 is shown in SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24;
the nucleotide sequence of the upstream primer for amplifying Penta D is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 26.
2. The primer set of claim 1, wherein the 13 STR loci are grouped into five groups, wherein,
the first group includes: D10S1248 and Penta E;
the second group includes: CSF1PO, D2S1338 and D18S 51;
the third group includes: amelogenin, D1S1656 and D12S 391;
the fourth group includes: D6S477 and FGA;
the fifth group includes: D15S659, D8S1132 and Penta D;
the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.
3. A kit comprising the primer set of claim 1 or 2.
4. The kit of claim 3, further comprising a PCR reaction premix and deionized water.
5. The kit of claim 3 or 4, further comprising an internal molecular weight standard and an allelic ladder.
6. The kit according to claim 3, wherein in the primer set,
SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.2-0.25 mu M;
SEQ ID NO: 3 and SEQ ID NO: 4 is independently 0.17-0.23 mu M;
SEQ ID NO: 5 and SEQ ID NO: 6 is 0.1-0.16 mu M independently;
SEQ ID NO: 7 and SEQ ID NO: the concentration of the primers shown in 8 is 0.13-0.18 mu M independently;
SEQ ID NO: 9 and SEQ ID NO: 10 is independently 0.11-0.15 mu M;
SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.08-0.12 mu M;
SEQ ID NO: 13 and SEQ ID NO: 14 is independently 0.11-0.15 mu M;
SEQ ID NO: 15 and SEQ ID NO: 16 is independently 0.13-0.18 mu M;
SEQ ID NO: 17 and SEQ ID NO: 18 is independently 0.11-0.18 mu M;
SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.2-0.25 mu M;
SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.45-0.52 mu M;
SEQ ID NO: 23 and SEQ ID NO: 24 is independently 0.14-0.19 μ M;
SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.08-0.13. mu.M.
7. The kit according to claim 4, wherein the PCR reaction premix solution uses deionized water as a solvent and comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl250 to 100mM (NH)4)2SO40.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction.
8. The kit of claim 5, wherein the allelic ladder and the STR locus correspond in relationship to one another as:
in the first group: D10S1248 corresponds to 10,11,12,13,14,15,16,17, 18; penta E corresponds to 5,6,7,8,9,10,11,12,13,14,15,16,17,19,20,21,22,23,24, 26;
in the second group: CSF1PO corresponds to 6,7,8,9,10,11,12,13,14,15, 16; D2S 1338: 16,17,18,20,21,23,24,25, 26; D18S51 corresponds to 8,9,10,11,12,13,14,15,16,17,18,19,20,21,23,24,26, 27;
in the third group: amelogenin corresponds to X, Y; D1S1656 corresponds to 10,11,12,13,14,15,16,17, 18; D12S391 corresponds to 15,16,17,20,21,23,24,25,26, 27;
in the fourth group: D6S477 corresponds to 11,14,15,16,18,19, 20; FGA corresponds to 16,17,18,19,20,21,23,24,25,26,27,29,30,31.2,43.2,44.2,45.2, 46.2;
in the fifth group: D15S659 corresponds to 10,12,13,14,15,16,17,18,19, 20; D8S1132 corresponds to 16,17,18,19,20,21,22,23,24, 25; penta D corresponds to 5,6,7,8,9,10,11,12,13,14,15, 16.
9. Use of the primer set of claim 1 or 2 and the kit of any one of claims 3 to 8 for forensic individual identification, forensic DNA database construction, suspect pedigree screening or forensic genetic relationship identification.
10. The application according to claim 9, characterized in that it comprises the following steps:
placing a sample to be detected into an amplification system containing the primer group of claim 1 or 2, performing PCR amplification to obtain an amplification product, and detecting the amplification product;
when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;
when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;
the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃, 60sec, 71 ℃,20 sec, 28 cycles; 60 deg.C for 10 min.
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Application publication date: 20210528 |