CN108179199A - Kit and its primer special combination based on two generation sequencing technologies detection X-STR locus - Google Patents

Abstract

The invention discloses the kit based on two generation sequencing technologies detection X str locus seats and its primer special combinations.Primer combination provided by the invention, is made of 32 kinds of DNA moleculars shown in sequence in sequence table 1 to sequence 32.It is demonstrated experimentally that using the STR partings of 16 X str locus seats in the genomic DNA of the kit detection women mouth epithelial cells provided by the invention based on two generation sequencing technologies detection X str locus seats, genotyping result is accurate.Kit provided by the invention has important application value.

Description

Kit for detecting X-STR loci based on next-generation sequencing technology and its special primers combination

technical field

The invention relates to the technical field of forensic medicine, in particular to a kit for detecting an X-STR locus based on a next-generation sequencing technology and a special primer combination thereof.

Background technique

In modern forensic science, DNA physical evidence plays an important role, and will become more and more important with the development of technology. At present, DNA physical evidence mainly uses capillary electrophoresis (capillary electrophoresis, CE) to detect length polymorphisms of STR loci. However, CE can only type the length of the locus, and all alleles with the same number of nucleotides are considered to be the same allele, which can easily lead to some alleles with different sequences but the same length being identified as the same allele Genes, and the sequence differences in the flanking parts cannot be detected and utilized, which seriously affect the identification of different individuals in the case. In recent years, next generation sequencing (NGS) technology has been gradually applied to forensic STR typing research. Compared with CE typing, NGS technology has many advantages in STR analysis: 1. It can obtain the complete base sequence of STR, and the difference between STR loci of different individuals is clear at a glance; 2. It can cover all STR loci Each base of each base and ensure the accuracy of the data through the high sequencing depth of the output; 3. The input amount of the sample is low, which is more advantageous for the typing of trace samples and highly degraded samples; 4. Openness, discovery ability and The ability to find new information is better, which means new types and new mutations for STR loci; 5. Low cost, by adding labels to each sample material once, multiple samples can be sequenced in parallel at the same time, both This saves time and reduces sequencing costs.

Human X-chromosome short tandem repeats (X-chromosome short tandem repeats, X-STR) refer to the short tandem repeat fragments existing in the non-recombination region on the X chromosome. The locus has the characteristics of sex-linked inheritance, and its genetic characteristics are different from autosomes and Y chromosomes, showing sex-linkage characteristics, and it has important reference value in the identification of certain kinship relationships. For some mixed male and female specimens, since male X-STR has only one allele, it cannot completely cover the female X-STR allele peak, and X-STR typing can achieve better results than autosomal STR typing . For the identification of father-daughter relationship and same-father-sister relationship, X-STR typing has special value. If the X-STR phenotypes of the two identified persons are different, the relationship can be ruled out. At the same time, X-STR also plays a certain role in the identification of certain relatives, such as granddaughters, nieces, etc.

Currently available commercial CE kits for the X chromosome include Investigator Argus X-12Kit (Fa.Qiagen) and Mentype1Argus X-8PCR amplification kit (Biotype AG, Dresden, Germany), etc., but only the length polymorphism can be detected , has certain limitations. Illumina has also launched a next-generation sequencing kit containing X-STR, the ForenSeq ^TM DNA Signature Prep Kit, but it only contains 7 X-STR loci (DXS10135, DXS8378, DXS7132, DXS10074, DXS10103, HPRTB and DXS7423 ), and the data analysis program supporting the kit can only analyze the sequencing data of the inherent loci in the kit, which has certain limitations. In view of the particularity of X-STR and the limitations of CE, it is of great value to develop a kit for detecting X-STR loci based on next-generation sequencing technology.

Contents of the invention

The purpose of the present invention is to perform X-STR typing.

The present invention first protects a combination of primers, which can be composed of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13. Primer 14, Primer 15, Primer 16, Primer 17, Primer 18, Primer 19, Primer 20, Primer 21, Primer 22, Primer 23, Primer 24, Primer 25, Primer 26, Primer 27, Primer 28, Primer 29, Primer 30, Primer 31 and Primer 32.

The primer 1 can be the following A1) or A2):

A1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing;

A2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1 and has the same function as Sequence 1.

The primer 2 can be as follows A3) or A4):

A3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing;

A4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2.

The primer 3 can be as follows A5) or A6):

A5) a single-stranded DNA molecule shown in sequence 3 in the sequence listing;

A6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 3 and has the same function as Sequence 3.

The primer 4 can be as follows A7) or A8):

A7) a single-stranded DNA molecule shown in sequence 4 in the sequence listing;

A8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 4 and has the same function as Sequence 4.

The primer 5 can be as follows A9) or A10):

A9) the single-stranded DNA molecule shown in sequence 5 in the sequence listing;

A10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 5 and has the same function as Sequence 5.

The primer 6 can be as follows A11) or A12):

A11) the single-stranded DNA molecule shown in sequence 6 in the sequence listing;

A12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 6 and has the same function as sequence 6.

The primer 7 can be as follows A13) or A14):

A13) the single-stranded DNA molecule shown in the sequence 7 in the sequence listing;

A14) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 7 and has the same function as Sequence 7.

The primer 8 can be as follows A15) or A16):

A15) a single-stranded DNA molecule shown in sequence 8 in the sequence listing;

A16) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 8 and has the same function as Sequence 8.

The primer 9 can be as follows A17) or A18):

A17) the single-stranded DNA molecule shown in sequence 9 in the sequence listing;

A18) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 9 and has the same function as Sequence 9.

The primer 10 can be as follows A19) or A20):

A19) the single-stranded DNA molecule shown in the sequence 10 in the sequence listing;

A20) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 10 and has the same function as sequence 10.

The primer 11 can be as follows B1) or B2):

B1) a single-stranded DNA molecule shown in sequence 11 in the sequence listing;

B2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 11 and has the same function as sequence 11.

The primer 12 can be as follows B3) or B4):

B3) a single-stranded DNA molecule shown in sequence 12 in the sequence listing;

B4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 12 and has the same function as sequence 12.

The primer 13 can be as follows B5) or B6):

B5) a single-stranded DNA molecule shown in sequence 13 in the sequence listing;

B6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 13 and has the same function as sequence 13.

The primer 14 can be as follows B7) or B8):

B7) a single-stranded DNA molecule shown in sequence 14 in the sequence listing;

B8) A DNA molecule having the same function as sequence 14 after one or several nucleotide substitutions and/or deletions and/or additions to sequence 14.

The primer 15 can be as follows B9) or B10):

B9) a single-stranded DNA molecule shown in sequence 15 in the sequence listing;

B10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 15 and has the same function as sequence 15.

The primer 16 can be as follows B11) or B12):

B11) a single-stranded DNA molecule shown in sequence 16 in the sequence listing;

B12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 16 and has the same function as sequence 16.

The primer 17 can be as follows B13) or B14):

B13) a single-stranded DNA molecule shown in sequence 17 in the sequence listing;

B14) A DNA molecule having the same function as the sequence 17 after one or several nucleotide substitutions and/or deletions and/or additions to the sequence 17.

The primer 18 can be as follows B15) or B16):

B15) a single-stranded DNA molecule shown in sequence 18 in the sequence listing;

B16) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 18 and has the same function as sequence 18.

The primer 19 can be as follows B17) or B18):

B17) a single-stranded DNA molecule shown in sequence 19 in the sequence listing;

B18) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 19 and has the same function as sequence 19.

The primer 20 can be as follows B19) or B20):

B19) a single-stranded DNA molecule shown in sequence 20 in the sequence listing;

B20) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 20 and has the same function as sequence 20.

The primer 21 can be the following C1) or C2):

C1) a single-stranded DNA molecule shown in sequence 21 in the sequence listing;

C2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 21 and has the same function as sequence 21.

The primer 22 can be as follows C3) or C4):

C3) a single-stranded DNA molecule shown in sequence 22 in the sequence listing;

C4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 22 and has the same function as sequence 22.

The primer 23 can be as follows (C5) or C6):

C5) a single-stranded DNA molecule shown in sequence 23 in the sequence listing;

C6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 23 and has the same function as sequence 23.

The primer 24 can be as follows (C7) or C8):

C7) a single-stranded DNA molecule shown in sequence 24 in the sequence listing;

C8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 24 and has the same function as sequence 24.

The primer 25 can be as follows (C9) or C10):

C9) a single-stranded DNA molecule shown in sequence 25 in the sequence listing;

C10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 25 and has the same function as sequence 25.

The primer 26 can be as follows (C11) or C12):

C11) a single-stranded DNA molecule shown in sequence 26 in the sequence listing;

C12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 26 and has the same function as sequence 26.

The primer 27 can be as follows (C13) or C14):

C13) a single-stranded DNA molecule shown in sequence 27 in the sequence listing;

C14) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 27 and has the same function as sequence 27.

The primer 28 can be as follows (C15) or C16):

C15) a single-stranded DNA molecule shown in sequence 28 in the sequence listing;

C16) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 28 and has the same function as sequence 28.

The primer 29 can be as follows (C17) or C18):

C17) a single-stranded DNA molecule shown in sequence 29 in the sequence listing;

C18) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 29 and has the same function as sequence 29.

The primer 30 can be as follows (C19) or C20):

C19) a single-stranded DNA molecule shown in sequence 30 in the sequence listing;

C20) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 30 and has the same function as sequence 30.

The primer 31 may be D1) or D2) as follows:

D1) a single-stranded DNA molecule shown in sequence 31 in the sequence listing;

D2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 31 and has the same function as sequence 31.

The primer 32 can be as follows D3) or D4):

D3) a single-stranded DNA molecule shown in sequence 32 in the sequence listing;

D4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 32 and has the same function as sequence 32.

In the primer combination, the primer 1, the primer 2, the primer 3, the primer 4, the primer 5, the primer 6, the primer 7, the primer 8, the primer 9 , the primer 10, the primer 11, the primer 12, the primer 13, the primer 14, the primer 15, the primer 16, the primer 17, the primer 18, the primer 19 , the primer 20, the primer 21, the primer 22, the primer 23, the primer 24, the primer 25, the primer 26, the primer 27, the primer 28, the primer 29 , The molar ratio of the primer 30, the primer 31 and the primer 32 may specifically be 1:1:1:1:1:1:1:1:2:2:2:2:1:1:2 :2:1:1:2:2:1:1:1:1:1:1:1:1:1:1:1:1.

The present invention also protects a compound amplification system based on the X-STR gene locus, which may include any of the above-mentioned primer combinations.

The primer 1, the primer 2, the primer 3, the primer 4, the primer 5, the primer 6, the primer 7, the primer 8, the primer 13, the primer 14, The primer 17, the primer 18, the primer 21, the primer 22, the primer 23, the primer 24, the primer 25, the primer 26, the primer 27, the primer 28, Specifically, the concentrations of the primer 29, the primer 30, the primer 31 and the primer 32 in the multiplex amplification system may be 0.3 μM.

The concentration of the primer 9, the primer 10, the primer 11, the primer 12, the primer 15, the primer 16, the primer 19 and the primer 20 in the multiple amplification system is specific It can be 0.6 μM.

The composite amplification system may also include reagents required for PCR amplification reactions; the "reagents required for PCR amplification reactions" does not include primers required for PCR amplification reactions.

Any of the multiplex amplification systems described above may include Master Mix, an aqueous solution of primer combinations and human genome DNA (as a template).

Any of the multiplex amplification systems mentioned above can be 20 μL, including 10 μL Master Mix, 4 μL aqueous solution of primer combination and 1 ng human genomic DNA (as template).

Any of the multiple amplification systems described above can be 20 μL, specifically, it can be composed of 10 μL Master Mix, 4 μL primer combination aqueous solution, 2.5 μL genomic DNA aqueous solution of female oral epithelial cells with a concentration of 0.425 ng/mL, and 3.5 μL ddH ₂ O.

Any of the above-mentioned Master Mix can specifically be a product of the Physical Evidence Appraisal Center of the Ministry of Public Security.

The present invention also protects a kit, which can contain any of the above-mentioned primer combinations or any of the above-mentioned multiplex amplification systems; the use of the kit can be as follows (h1) or (h2) or (h3) Or (h4): (h1) X-STR typing; (h2) SNP typing; (h3) Indel typing; (h4) detection of genetic markers.

The present invention also protects the preparation method of any of the above-mentioned multiplex amplification systems or the kit; the preparation method may include the step of individually packaging each primer in any of the above-mentioned primer combinations.

The present invention also protects the application of any of the above-mentioned primer combinations or the above-mentioned composite amplification system in the preparation of a kit; the use of the kit can be as follows (h1) or (h2) or (h3) or (h4): (h1) X-STR typing; (h2) SNP typing; (h3) Indel typing; (h4) detection of genetic markers.

The present invention also protects the application of any of the above-mentioned primer combinations or any of the above-mentioned composite amplification systems in detecting X-STR typing and/or SNP typing and/or Indel typing and/or detecting genetic markers.

The present invention also protects a method for detecting the genotype of the X-STR locus of the sample to be tested, which may specifically include the following steps:

(1) using the genomic DNA of the sample to be tested as a template, using any one of the primer combinations described above to perform PCR amplification to obtain a PCR amplification product;

(2) The PCR amplification product is subjected to next-generation sequencing, and the genotype of the X-STR locus is determined according to the sequencing result.

上述方法中，所述X-STR基因座可为DXS6804、DXS10079、GATA31E08、GATA172D05、DXS7423、DXS7424、DXS8378、DXS9895、DXS7132、DXS7133、DXS6789、DXS101、DXS6800、GATA165B12、DXS9902和DXS6799中的16个、任意15, Any 14, Any 13, Any 12, Any 11, Any 10, Any 9, Any 8, Any 7, Any 6, Any 5, Any 4, Any 3 , any 2 or any 1.

In the above method, the sample to be tested may be human oral epithelial cells.

Compared with Illumina's ForenSeq ^TM DNA Signature Prep Kit, the kit for detecting X-STR loci based on next-generation sequencing technology provided by the present invention contains 16 X-STR loci, providing more new genetic information. Moreover, the maximum amplicon lengths of all X-STR loci in the composite amplification system provided by the present invention are less than 300 bp, which is beneficial to the analysis of degraded samples. Experiments have proved that the STR typing of 16 X-STR loci in the genomic DNA of female oral epithelial cells is detected using the kit for detecting X-STR loci based on next-generation sequencing technology provided by the present invention, and the typing results are accurate. The kit provided by the invention has important application value.

Description of drawings

Figure 1 is the length of the PCR amplification product of the primers corresponding to each X-STR locus in the hg19 human reference genome.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Agencourt AMPure XP 5mL Kit is the product of Beckman Coulter Company, the product catalog number is A63880. NanoDrop 2000 quantitative instrument is a product of Thermo Fisher Scientific Company. The Miseq FGx next-generation sequencer is a product of Illumina. TruSeq DNA PCR-Free HT Library Prep Kit is a product of Illumina, and its catalog number is FC-121-3003. KAPA Library Quantification Kit is a product of KAPA Company, the product catalog number is KK4824.

Example 1. Preparation of a kit for detecting the X-STR locus based on next-generation sequencing technology

1. Preparation of primer combinations

The primer set consisted of 32 primers for the detection of 16 X-STR loci. The name of each X-STR locus, the name of the primer corresponding to the amplified X-STR locus, the nucleotide sequence of the primer, and the range of allelic genotypes are shown in Table 1. The primers corresponding to each X-STR locus are the PCR amplification products in the hg19 human reference genome (for information on the hg19 human genome, see the website http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/hg19.2bit) See Table 2 for the length and nucleotide sequence.

Table 1

Table 2

2. Preparation of kits for detecting X-STR loci based on next-generation sequencing technology

The kit for detecting the X-STR locus based on next-generation sequencing technology includes a primer mix. The primer mixture is formed by mixing the 32 primers prepared in step 1.

Example 2. Application of the kit for detecting the X-STR locus based on next-generation sequencing technology

1. DNA sample preparation

Genomic DNA of a female oral epithelial cell sample was extracted, and then diluted with ultrapure water to obtain an aqueous solution of genomic DNA of female oral epithelial cells with a concentration of 0.425 ng/μL.

2. Library preparation

1. PCR amplification

Using the aqueous genomic DNA solution of female oral epithelial cells obtained in Step 1 as a template and the primer mixture prepared in Step 2 of Example 1 as primers, PCR amplification was performed to obtain PCR amplification products.

The reaction system was 20 μL, consisting of 10 μL Master Mix (product of the Physical Evidence Identification Center of the Ministry of Public Security), 4 μL primer mix, 2.5 μL genomic DNA aqueous solution of female oral epithelial cells, and 3.5 μL ddH ₂ O. In this reaction system, primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 13, primer 14, primer 17, primer 18, primer 21, primer 22, primer 23, Primer 24, Primer 25, Primer 26, Primer 27, Primer 28, Primer 29, Primer 30, Primer 31 and Primer 32 were all at a concentration of 0.3 μM, Primer 9, Primer 10, Primer 11, Primer 12, Primer 15, Primer 16 , Primer 19 and Primer 20 were all at a concentration of 0.6 μM.

Reaction program: 11min at 95°C; 30s at 94°C, 2min at 60°C, 1min at 72°C, 28 cycles; 60min at 60°C; store at 4°C.

2. Purification and quantification

(1) Take the PCR amplification product and purify it according to the instructions of Agencourt AMPure XP 5mL Kit.

(2) After step (1), the PCR amplification product was quantified using a NanoDrop 2000 quantifier to obtain a PCR purified product.

3. Library preparation

Take the PCR purified product, and follow the instructions of the TruSeq DNA PCR-Free HT Library Prep Kit to perform end repair, end repair product purification, A-tail ligation, adapter ligation and ligation product purification in sequence, and then follow the instructions of the KAPA Library Quantification Kit Steps for library quantification and library normalization to complete library preparation.

3. Loading test

Take the library prepared in step 2, and use the sequencing reagent Miseq Reagent Nano Kit v2 (product of illumina company) to perform sequencing on the Miseq FGx next-generation sequencer.

The experimental results are shown in Table 3. The results show that the genomic DNA of the female oral epithelial cell sample has been completely STR typed, which can fully meet the requirements of forensic STR testing.

table 3

Example 3. Verification of the accuracy of the kit for detecting the X-STR locus based on next-generation sequencing technology

1. DNA sample preparation

Genomic DNA of a female oral epithelial cell sample was extracted, and then diluted with ultrapure water to obtain an aqueous solution of genomic DNA of female oral epithelial cells with a concentration of 0.425 ng/μL.

Second, capillary electrophoresis detection

Take 1ng of the genomic DNA of female oral epithelial cells obtained in Step 1, and perform capillary electrophoresis detection according to the instructions of DNATyper X19 (product of the Physical Evidence Identification Center of the Ministry of Public Security), to obtain the allelic genotype of the locus. The typing results are detailed in column 2 of Table 4.

Third, next generation sequencing technology detection

The genomic DNA of the female oral epithelial cells obtained in step 1 was detected according to the method in Example 2, and the allelic genotype of the locus was obtained. The typing results are detailed in column 3 of Table 4.

The results showed that the genetic locus overlapped with DNATyper X19 of the kit for detecting the X-STR locus based on the next-generation sequencing technology prepared in Example 1 was completely consistent with the typing results in the genomic DNA of female oral epithelial cells obtained in step 1.

Table 4

Note: "--" means it does not exist.

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Claims (10)

1. primer combination, by primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15 , Primer 16, Primer 17, Primer 18, Primer 19, Primer 20, Primer 21, Primer 22, Primer 23, Primer 24, Primer 25, Primer 26, Primer 27, Primer 28, Primer 29, Primer 30, Primer 31 and Primer 32 composition; The primer 1 is the following A1) or A2): A1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing; A2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1 and has the same function as Sequence 1; The primer 2 is as follows A3) or A4): A3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing; A4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2; The primer 3 is as follows A5) or A6): A5) a single-stranded DNA molecule shown in sequence 3 in the sequence listing; A6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 3 and has the same function as sequence 3; The primer 4 is as follows A7) or A8): A7) a single-stranded DNA molecule shown in sequence 4 in the sequence listing; A8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 4 and has the same function as sequence 4; The primer 5 is as follows A9) or A10): A9) the single-stranded DNA molecule shown in sequence 5 in the sequence listing; A10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 5 and has the same function as sequence 5; The primer 6 is as follows A11) or A12): A11) the single-stranded DNA molecule shown in sequence 6 in the sequence listing; A12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 6 and has the same function as sequence 6; The primer 7 is as follows A13) or A14): A13) the single-stranded DNA molecule shown in the sequence 7 in the sequence listing; A14) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 7 and has the same function as sequence 7; The primer 8 is as follows A15) or A16): A15) a single-stranded DNA molecule shown in sequence 8 in the sequence listing; A16) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 8 and has the same function as sequence 8; The primer 9 is as follows A17) or A18): A17) the single-stranded DNA molecule shown in sequence 9 in the sequence listing; A18) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 9 and has the same function as sequence 9; The primer 10 is as follows A19) or A20): A19) the single-stranded DNA molecule shown in the sequence 10 in the sequence listing; A20) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 10 and has the same function as sequence 10; The primer 11 is as follows B1) or B2): B1) a single-stranded DNA molecule shown in sequence 11 in the sequence listing; B2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 11 and has the same function as sequence 11; The primer 12 is as follows B3) or B4): B3) a single-stranded DNA molecule shown in sequence 12 in the sequence listing; B4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 12 and has the same function as sequence 12; The primer 13 is as follows B5) or B6): B5) a single-stranded DNA molecule shown in sequence 13 in the sequence listing; B6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 13 and has the same function as sequence 13; The primer 14 is as follows B7) or B8): B7) a single-stranded DNA molecule shown in sequence 14 in the sequence listing; B8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 14 and has the same function as sequence 14; The primer 15 is as follows B9) or B10): B9) a single-stranded DNA molecule shown in sequence 15 in the sequence listing; B10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 15 and has the same function as sequence 15; The primer 16 is as follows B11) or B12): B11) a single-stranded DNA molecule shown in sequence 16 in the sequence listing; B12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 16 and has the same function as sequence 16; The primer 17 is as follows B13) or B14): B13) a single-stranded DNA molecule shown in sequence 17 in the sequence listing; B14) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 17 and has the same function as sequence 17; The primer 18 is as follows B15) or B16): B15) a single-stranded DNA molecule shown in sequence 18 in the sequence listing; B16) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 18 and has the same function as sequence 18; The primer 19 is as follows B17) or B18): B17) a single-stranded DNA molecule shown in sequence 19 in the sequence listing; B18) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 19 and has the same function as sequence 19; The primer 20 is as follows (B19) or B20): B19) a single-stranded DNA molecule shown in sequence 20 in the sequence listing; B20) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 20 and has the same function as sequence 20; The primer 21 is the following C1) or C2): C1) a single-stranded DNA molecule shown in sequence 21 in the sequence listing; C2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 21 and has the same function as sequence 21; The primer 22 is the following C3) or C4): C3) a single-stranded DNA molecule shown in sequence 22 in the sequence listing; C4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 22 and has the same function as sequence 22; The primer 23 is as follows (C5) or C6): C5) a single-stranded DNA molecule shown in sequence 23 in the sequence listing; C6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 23 and has the same function as sequence 23; The primer 24 is as follows (C7) or C8): C7) a single-stranded DNA molecule shown in sequence 24 in the sequence listing; C8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 24 and has the same function as sequence 24; The primer 25 is as follows (C9) or C10): C9) a single-stranded DNA molecule shown in sequence 25 in the sequence listing; C10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 25 and has the same function as sequence 25; The primer 26 is the following C11) or C12): C11) a single-stranded DNA molecule shown in sequence 26 in the sequence listing; C12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 26 and has the same function as sequence 26; The primer 27 is as follows (C13) or C14): C13) a single-stranded DNA molecule shown in sequence 27 in the sequence listing; C14) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 27 and has the same function as sequence 27; The primer 28 is as follows (C15) or C16): C15) a single-stranded DNA molecule shown in sequence 28 in the sequence listing; C16) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 28 and has the same function as sequence 28; The primer 29 is as follows (C17) or C18): C17) a single-stranded DNA molecule shown in sequence 29 in the sequence listing; C18) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 29 and has the same function as sequence 29; The primer 30 is as follows (C19) or C20): C19) a single-stranded DNA molecule shown in sequence 30 in the sequence listing; C20) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 30 and has the same function as sequence 30; The primer 31 is the following D1) or D2): D1) a single-stranded DNA molecule shown in sequence 31 in the sequence listing; D2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 31 and has the same function as sequence 31; The primer 32 is as follows D3) or D4): D3) a single-stranded DNA molecule shown in sequence 32 in the sequence listing; D4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 32 and has the same function as sequence 32. 2. The primer combination according to claim 1, characterized in that: said primer 1, said primer 2, said primer 3, said primer 4, said primer 5, said primer 6, said primer 7 , the primer 8, the primer 9, the primer 10, the primer 11, the primer 12, the primer 13, the primer 14, the primer 15, the primer 16, the primer 17 , the primer 18, the primer 19, the primer 20, the primer 21, the primer 22, the primer 23, the primer 24, the primer 25, the primer 26, the primer 27 , the molar ratio of the primer 28, the primer 29, the primer 30, the primer 31 and the primer 32 is 1:1:1:1:1:1:1:1:1:2:2:2 :2:1:1:2:2:1:1:2:2:1:1:1:1:1:1:1:1:1:1:1:1:1. 3. A multiple amplification system based on the X-STR loci, comprising the primer combination according to claim 1 or 2. 4. composite amplification system as claimed in claim 3, is characterized in that: The primer 1, the primer 2, the primer 3, the primer 4, the primer 5, the primer 6, the primer 7, the primer 8, the primer 13, the primer 14, The primer 17, the primer 18, the primer 21, the primer 22, the primer 23, the primer 24, the primer 25, the primer 26, the primer 27, the primer 28, The concentration of the primer 29, the primer 30, the primer 31 and the primer 32 in the multiplex amplification system is 0.3 μM; The concentration of the primer 9, the primer 10, the primer 11, the primer 12, the primer 15, the primer 16, the primer 19 and the primer 20 in the multiple amplification system is 0.6 μM. 5. composite amplification system as claimed in claim 3 or 4, is characterized in that: described composite amplification system also comprises the reagent needed for carrying out PCR amplification reaction; Described " carries out the reagent required for PCR amplification reaction " Primers required for PCR amplification reactions are not included. 6. A test kit, which contains the primer combination described in claim 1 or 2 or the composite amplification system described in any one of claims 3 to 5; the purposes of the test kit are as follows (h1) or (h2) or ( h3) or (h4): (h1) X-STR typing; (h2) SNP typing; (h3) Indel typing; (h4) detection of genetic markers. 7. The preparation method of the composite amplification system according to any one of claims 3 to 5 or the kit according to claim 6, comprising the step of packaging each primer in the primer combination according to claim 1 or 2 separately. 8. the described primer combination of claim 1 or 2, or, the arbitrary described compound amplification system of claim 3 to 5, the application in preparation kit; The purposes of described kit are as follows (h1) or (h2 ) or (h3) or (h4): (h1) X-STR typing; (h2) SNP typing; (h3) Indel typing; (h4) detection of genetic markers. 9. The primer combination according to claim 1 or 2, or, the composite amplification system described in any one of claims 3 to 5, when detecting X-STR typing and/or SNP typing and/or Indel typing and/or Application in detection of genetic markers. 10. A method for detecting the genotype of the X-STR locus of a sample to be tested, comprising the steps of: (1) using the genomic DNA of the sample to be tested as a template, using the primer combination described in claim 1 or 2 to carry out PCR amplification to obtain a PCR amplification product; (2) The PCR amplification product is subjected to next-generation sequencing, and the genotype of the X-STR locus is determined according to the sequencing result.