CN106755340B

CN106755340B - Method and system for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci

Info

Publication number: CN106755340B
Application number: CN201611082622.9A
Authority: CN
Inventors: 莫晓婷; 白雪; 张建; 孙辉; 姚伊人; 李万水; 赵兴春
Original assignee: Institute of Forensic Science Ministry of Public Security PRC
Current assignee: Institute of Forensic Science Ministry of Public Security PRC
Priority date: 2016-11-30
Filing date: 2016-11-30
Publication date: 2020-07-07
Anticipated expiration: 2036-11-30
Also published as: CN106755340A

Abstract

The invention provides a method and a system for performing Y-STR typing on a male individual by using 26Y-STR loci, wherein the method comprises the steps of obtaining DNA of the male individual and obtaining the genotypes of the 26Y-STR loci, wherein the 26Y-STR loci are DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA H4, DYS438, DYS439, DYS460, DYS447, DYS527a/b, DYS617, DYS522, DYS444, DYS508 and DYS 533; and obtaining the Y-STR typing result of the male individual according to the genotypes of the 26 loci of the male individual. The typing result can be used for performing family examination, paternity test, detection of male components in male and female mixed samples, detection of male components in a plurality of male mixed samples and the like on the male individual.

Description

Method and system for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci

Technical Field

The invention relates to a typing method and a typing system, in particular to a method and a system for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci.

Background

Tandem Repeat (STR) is a polymorphic genetic marker system widely existing in human chromosomal DNA, and has a wide range (one STR locus exists in 16kb on average), a small core sequence (2-7bp), amplification product lengths of less than 500bp, and allelic locus numbers representing the number of sequence repeats. The allelic fragments of STR loci are concentrated in length, so that multiple STR loci can be amplified in a multiplex manner. Multiple STR loci are amplified in a composite mode, and the accumulated identification capacity can approach or reach the level of DNA fingerprints.

The Y chromosome is a chromosome which is unique to males, and all male individuals in the same father line have the same Y-STR haplotype. The Y-STR test is used as a supplementary means of autosomal test, has important application value in family investigation, paternity test, detection of male components in male and female mixed components and judgment of a plurality of male mixed components, and plays an increasingly important role in DNA test because more than 90% of criminals involved in case crimes are males.

At present, the forensic DNA technology has more functions in the aspect of passive comparison, introduces a Y-STR test method into a case for investigation in the field of investigation science, creates a Y-STR family investigation method, can reduce the investigation range of a case for investigation and provides a directive clue for investigation. The family investigation means that the Y-STR data extracted from the crime scene is compared with families in a certain range, and once the comparison is carried out, the Y-STR data can point to the family where the criminal agent is located.

At present, Henan, Shandong and the like are building libraries in county (city) as a unit, drawing family maps in administrative village as a unit, collecting biological samples on the basis of the family maps, and carrying out Y-STR database construction work. Through automatic comparison of the Y database, the system can quickly point to the family of the criminal suspect, and the detection range is narrowed; rapidly pointing to the family where the unknown corpse is located, and indicating the detection direction; can eliminate the interference of female DNA, obtain clear male information and automatically compare in storage. The Henan Y library adopts a guideline of 'construction while construction to promote construction', and the library is used to successfully solve hundreds of case plans, thereby having great effect.

The patent CN103866019 discloses a Y-STR fluorescence multiplex amplification detection reagent for forensic Y-STR detection, and particularly discloses 21Y-STR loci and amplification primers. However, the design of the primers causes the amplification products of some Y-STR loci to be arranged too closely and even the amplification product of the previous locus spans the range of the next locus, so that the typing cannot be judged, and in addition, the close arrangement of the loci can cause crosstalk and dislocation of the allele typing standard substance during analysis, so that the whole result cannot be analyzed and judged correctly.

How to provide a method and a system for performing Y-STR typing on male individuals, which can solve the above problems, is a problem to be solved.

Disclosure of Invention

The invention provides a method for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci, which can avoid the problems of incapability of judging and reading typing, typing crosstalk and dislocation in the typing process by adopting a specific Y-STR locus combination and specific primers designed aiming at the loci, and can accurately obtain the genotypes of the 26Y-STR loci of the male individuals, thereby providing data support for family investigation, paternal right identification, detection of male components in male and female mixed components, judgment of a plurality of male mixed components and the like of the male individuals.

The invention also provides a system for carrying out Y-STR typing on male individuals by utilizing the 26Y-STR loci, and the system can realize accurate typing on the male individuals aiming at the 26 loci.

The invention also provides a composite detection system, and the detection system can accurately obtain the genotypes of 26Y-STR loci of male individuals.

The invention also provides a detection kit comprising the composite detection system.

The invention provides a method for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci, which comprises the following steps:

1) obtaining DNA of a male individual;

2) obtaining a genotype for 26Y-STR loci of the DNA, the 26Y-STR loci being DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA H4, DYS438, DYS439, DYS460, DYS447, DYS527a/b, DYS617, DYS522, DYS444, DYS508 and DYS533, comprising the step of amplifying the same with amplification primers corresponding to the loci to obtain amplification products;

wherein the amplification primers of locus DYS460 correspond to the nucleotide sequences of SEQ ID No.1 to SEQ ID No. 2; the amplification primers of locus DYS389I/II are the same and have the nucleotide sequences from SEQ ID No.3 to SEQ ID No. 4; the amplification primers of loci DYS390, DYS392, DYS458, DYS437 correspond to the nucleotide sequences of SEQ ID No.5 to SEQ ID No.12, respectively; the amplification primers of locus DYS385a/b are the same and have the nucleotide sequences from SEQ ID No.13 to SEQ ID No. 14; the amplification primers of the loci GATA-H4, DYS522, DYS456, DYS391, DYS447, DYS438, DYS448, DYS393, DYS635, DYS439, DYS19, DYS444 correspond to the nucleotide sequences of SEQ ID nos. 15 to 38, respectively; the amplification primers of the locus DYS527a/b are the same and have nucleotide sequences from SEQ ID No.39 to SEQ ID No. 40; the amplification primers of loci DYS617, DYS508, DYS533 correspond to the nucleotide sequences of SEQ ID No.41 to SEQ ID No.46, respectively;

3) and obtaining the Y-STR typing result of the male individual according to the genotypes of the 26 loci of the male individual.

In the present embodiment, the 26 loci are obtained by the applicant through comprehensive analysis of the living environment, ethnic origin, etc. of a large number of male individuals. The primers aiming at the gene loci are obtained by a large number of experiments of the applicant, so that the problems of incapability of interpretation, typing crosstalk and dislocation in the process of typing the Y-STR of male individuals are successfully solved, and meanwhile, the combination of the specific Y chromosome loci can also realize family investigation, paternity test, male component source inference in male and female mixed samples and/or male component source inference in male and female mixed samples of the male individuals.

Further, in the present embodiment, "obtaining DNA of a male individual" refers to extracting DNA from a sample such as blood, tissue, etc. of the individual, or directly obtaining a blood card containing the DNA of the individual. The 'Y-STR typing result of the male individual' refers to a Y-STR typing result which can be used for performing family investigation, paternity test, source inference of male components in a male and female mixed sample and/or source inference of male components in a male and female mixed sample on the male individual. Wherein the inference of the source of the male component in the mixed sample of males refers to the differentiation of male individuals from non-identical paternal families.

In one embodiment of the invention, the primer is a fluorescently labeled primer.

In another embodiment of the present invention, wherein 2) further comprises the step of analyzing the amplification product using a genetic analyzer after obtaining the amplification product to obtain genotypes of the 27 loci. In the context of the present invention, the genetic analyzer may be one conventionally used by the person skilled in the art, for example an ABI3130 or ABI3500 type genetic analyzer, by

The 26 loci of the PCR amplification product were genotyped using ID-X software or other GeneMapper software.

The invention provides a system for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci, which comprises a DNA acquisition system, a composite detection system and an inference system;

the DNA obtaining system is used for obtaining DNA of the male individual;

the composite detection system is used for obtaining the genotypes of the 26Y-STR loci of the DNA, obtaining the Y-STR typing result of the individual according to the genotypes,

the 26Y-STR loci are DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA H4, DYS438, DYS439, DYS460, DYS447, DYS527a/b, DYS617, DYS522, DYS444, DYS508 and DYS533, and the process for obtaining the genotypes of the 26 loci comprises the step of amplifying the loci by using amplification primers corresponding to the loci to obtain amplification products;

the inference system is used for performing family investigation, paternity test, male component source inference in male and female mixed samples and/or male component source inference in male and female mixed samples on the male individual according to the Y-STR typing result of the individual.

Further, the multiplex detection system is also used for analyzing the amplification product using a genetic analyzer after obtaining the amplification product to obtain the genotypes of the 26 loci.

Further, the genetic analyzer uses typing standards for alleles of each locus to determine the genotype of each locus in the male individual's DNA.

The invention provides a composite detection system, which comprises male individual DNA, 26 loci and amplification primers,

the composite detection system is used for obtaining the amplification products of 26Y-STR loci of the DNA by using the amplification primers, obtaining the genotypes of 26 loci of the DNA of a male individual from the amplification products, further obtaining the Y-STR typing result of the individual from the genotypes,

the 26Y-STR loci are DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA H4, DYS438, DYS439, DYS460, DYS447, DYS527a/b, DYS617, DYS522, DYS444, DYS508 and DYS533,

the amplification primers consist of amplification primers corresponding to the 26 loci, wherein the amplification primers of locus DYS460 correspond to the nucleotide sequences of SEQ ID No.1 to SEQ ID No. 2; the amplification primers of locus DYS389I/II are the same and have the nucleotide sequences from SEQ ID No.3 to SEQ ID No. 4; the amplification primers of loci DYS390, DYS392, DYS458, DYS437 correspond to the nucleotide sequences of SEQ ID No.5 to SEQ ID No.12, respectively; the amplification primers of locus DYS385a/b are the same and have the nucleotide sequences from SEQ ID No.13 to SEQ ID No. 14; the amplification primers of the loci GATA-H4, DYS522, DYS456, DYS391, DYS447, DYS438, DYS448, DYS393, DYS635, DYS439, DYS19, DYS444 correspond to the nucleotide sequences of SEQ ID nos. 15 to 38, respectively; the amplification primers of the locus DYS527a/b are the same and have nucleotide sequences from SEQ ID No.39 to SEQ ID No. 40; the amplification primers of the loci DYS617, DYS508, DYS533 correspond to the nucleotide sequences of SEQ ID No.41 to SEQ ID No.46, respectively.

In the scheme of the invention, the DNA polymerase can be one or more of Fast Start DNA polymerase, Taq DNA polymerase and Hotstart DNA polymerase.

In an embodiment of the present invention, the present invention provides a method for typing 26 loci using the composite detection system, comprising: 1) using the obtained male individual DNA as a template; 2) performing a multiplex PCR amplification reaction using the male individual DNA with the amplification primer as a template to obtain an amplification product; 3) the amplification products were analyzed using a genetic analyzer to obtain genotypes at 26 loci.

In the protocol of the present invention, the 26 loci information is shown in table 1:

TABLE 1

Genetic loci	Repetitive sequence	5' labelling
			DYS19	TAGA	ROX
DYS385a/b	GAAA	HEX
			DYS389I/II	(TCTG)(TCTA)	FAM
DYS390	(TCTA)(TCTG)	FAM
			DYS391	TCTA	TAMRA
DYS392	TAT	FAM
			DYS393	AGAT	ROX
DYS437	TCTA	HEX
			DYS448	AGAGAT	TAMRA
DYS456	AGAT	TAMRA
			DYS458	GAAA	HEX
DYS635	TSTA	ROX
			GATA-H4	TAGA	HEX
DYS438	TTTTC	TAMRA
			DYS439	AGAT	ROX
DYS460	ATAG	FAM
			DYS447	TAAWA	TAMRA
DYS527ab	GAAA	ROX
			DYS617	TAT	TAMRA
DYS522	GATA	HEX
			DYS444	TAGA	ROX
DYS508	TATC	HEX
			DYS533	ATCT	FAM

The amplification primer sequences provided by the invention are as follows. The amplification primers and the corresponding loci are shown in the following table 2, wherein PCRU represents an upstream primer, and PCRL represents a downstream primer;

TABLE 2

The scheme of the invention has the following advantages:

1. the detection method and the detection system of the invention adopt specific STR locus combination and primer sequences, so that the distribution among all the sites on the finally obtained typing map is uniform, the specificity of the primers is high, complete STR typing can be obtained, the peak type is sharp and clear, the balance is good, no Pull-up peak and stutter band exist, no non-specific artificial product appears, and the detection method and the detection system can completely meet the requirements of forensic Y-STR inspection.

2. The detection method and the detection system of the invention are used for simultaneously detecting that the specific 26Y-STR loci have high paternity test capability, and male individuals are subjected to family investigation and male component source inference in male and female mixed samples and/or male component source inference in male and female mixed samples.

3. The scheme of the invention can obtain an intuitive detection result by using the fluorescent marker and utilizing a conventional genetic analyzer according to the difference of the molecular weight and the fluorescent color of the gene to be amplified, and the result has 100 percent of accuracy compared with the existing detection system.

4. The scheme provided by the invention can effectively provide accurate scientific basis for family investigation, paternity identification and the like from the gene level.

Drawings

FIG. 1 shows the allelic arrangement pattern for each locus of the present application (where the first line is blue fluorescence, the second line is green fluorescence, the third line is yellow fluorescence, and the fourth line is red fluorescence).

FIG. 2 shows a graph of the typing results obtained for one sample using the system of the present invention.

FIG. 3 shows a typing map of an allelic typing standard (ladder) obtained using the system of patent CN103866019 (wherein the first line is blue fluorescence, the second line is green fluorescence, the third line is black fluorescence, and the fourth line is red fluorescence).

FIG. 4 shows a typing map of an allelic typing standard (ladder) obtained using the system of the present application (wherein the first line is blue fluorescence, the second line is green fluorescence, the third line is black fluorescence, and the fourth line is red fluorescence).

Figure 5 shows a diagram of the allele typing of locus DYS460 obtained using the system of patent CN103866019 (where the first line is blue fluorescence, the second line is green fluorescence, the third line is black fluorescence and the fourth line is red fluorescence).

Detailed Description

193 human anticoagulant (male 193) was used in the following examples and was provided by the national center for physical evidence identification.

The methods used in the following examples are conventional unless otherwise specified, and the reagent consumables and apparatus used are shown in Table 3 below:

TABLE 3

Example 1 verification of the accuracy of the method and system of the invention for Y-STR typing of male individuals

In this example, the male individuals are 193 males who are anticoagulated, and the individual sources of the male individuals are known, but the individual sources of the male individuals are not known in the implementation process of the present application example 1, and the method and the system for Y-STR typing are performed by the method and the system, including:

1) obtaining DNA of a male individual by using a DNA obtaining system in the system, 2) obtaining genotypes of 26Y-STR loci of the DNA by using a composite detection system in the system, and obtaining a Y-STR typing result of the male individual according to the genotypes, 3) performing family investigation, paternal right identification, male component source in a male and female mixed sample and/or male component source inference in the male and female mixed sample on the male individual according to the Y-STR typing result of the individual by using an inference system in the system.

In this embodiment, the composite detection system is configured to obtain amplification products of the 26Y-STR loci of the DNA by using the amplification primers, obtain genotypes of the 26 loci of the DNA of the male individual from the amplification products, and further obtain a Y-STR typing result of the individual from the genotypes;

the 26Y-STR loci are DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA H4, DYS438, DYS439, DYS460, DYS447, DYS527a/b, DYS617, DYS522, DYS444, DYS508 and DYS 533;

1. 193 blood cards containing the above 193 male individual DNAs were obtained as experimental samples, and standard DNA9948 was used as a positive control sample.

2. And (3) typing the 26 loci of the sample by using the composite detection system, wherein the typing comprises the following steps: performing multiplex PCR amplification on a DNA template by using the amplification primers to obtain an amplification product; the amplification products were genotyped at 26 loci using a genetic analyzer. The specific process is as follows:

2.1 primer pool configuration

A pool of amplification primers configured wherein the amplification primers for the 26 loci are as described above; the primer sequences provided by the invention are synthesized by Shanghai biological engineering technology service company Limited.

Diluting the synthesized primers to 100. mu.M with 1 × TE buffer solution, mixing the upstream and downstream primers of 26 loci at equal ratio to obtain 50. mu.M primers, adding different volumes of the 23 PCR primers into a new centrifuge tube to serve as a 23-fold PCR primer pool, wherein the final concentration of the primers at each STR locus is shown in Table 4 below:

TABLE 4

STR loci	Final concentration (μmol/L)
		DYS460	0.15
DYS389I/II	0.3
		DYS390	0.23
DYS533	0.2
		DYS392	0.5
DYS508	0.3
		DYS437	0.3
DYS385a/b	0.3
		GATA-H4	0.3
DYS522	0.3
		DYS456	0.15
DYS458	0.2
		DYS391	0.24
DYS447	0.26
		DYS438	0.4
DYS448	0.2
		DYS617	0.5
DYS393	0.15
		DYS635	0.3
DYS439	0.3
		DYS19	0.4
DYS444	0.26
		DYS527a/b	0.34

2.2 multiplex PCR reactions

This example used a model 9700 PCR amplification apparatus for multiplex PCR reactions.

(1) A PCR mix (25. mu.L system) was prepared as shown in Table 5 below.

TABLE 5

Name of reagent	Concentration of
		PCR primer pool	12.5μL
Tris-HCl	20mM
		KCl	50mM
MgCl₂	1.6mM
		Bovine serum albumin	0.8mg/ml
Tween-20	0.2％
		Glycerol	3.2％
NaN₃	0.02％
		dNTP	200μM
Taq DNA polymerase	2U
		Male individual DNA template card	0.25mm²
Total of	25μL

(2) Amplification procedure

The thermal cycle parameters of the PCR amplification process are ① 95 deg.C, 11min, ② 28 cycles, each cycle is 94 deg.C for 1min, 60 deg.C for 1min, 72 deg.C for 1min, ③ 60 deg.C for 60min, and ④ 25 deg.C for heat preservation.

2.3 typing of PCR products

Preparation of samples to be typed:

1. preparation of electrophoretic loading mixtures, internal standards and deionized formamide were prepared in the following ratios to make up the loading mixtures: 10 μ l of Typer500 internal standard +1000 μ l of deionized formamide, mixed well.

2. Add 10. mu.l of the loading mixture and 1. mu.l of the amplification product to each tube and mix well.

Denaturation at 3.95 ℃ for 3 min, cooling immediately on ice for 3 min, and electrophoresis.

Detection was performed on a ABI3130XL model genetic analyzer. Data were collected using ABI3130XL Date Collection software 3.1 and the results of electrophoresis were analyzed using GeneMapper 3.3 software to obtain the genotypes of the 26 loci.

2.4, analysis of results

A representative typing result obtained by typing 193 DNA samples using the system of the present invention is shown in fig. 2, in order to verify the accuracy of the typing result, 100 DNA samples were randomly extracted from 193 DNA samples, 26 loci were sequenced (sequencing by beijing maioden biotechnology limited), all typing results obtained by using the composite detection system of the present embodiment were consistent with the sequencing result, and the consistency reached 100%, which proves that the typing result of the composite detection system of the present invention is accurate.

3. And obtaining the Y-STR typing result of the male individual according to the genotypes of the 26 loci of the male individual, and carrying out paternal relatives identification. The paternal relative identification results obtained from the 193 specimens by the method of this example were consistent with the known paternal relative identification results of individuals, indicating that the method of the present invention can be used to identify paternal relative of male individuals.

Furthermore, the genotypes of the 26 loci can also be used for family examination of male individuals, source estimation of male components in a male and female mixed sample, and/or source estimation of male components in a male and female mixed sample.

Example 2

The present Y-STR typing system and the existing system

Comparison of plus kit in pedigree judgment

Compared with a patent CN103866019 detection system, the Y-STR typing system provided by the invention increases 5Y-STR loci, increases the resolving power for different family males, and in addition, the increased Y-STR loci all belong to low mutation rate loci, so that 26Y-STR loci in the system all belong to medium and low mutation rate loci, and are more suitable for family investigation, and the high mutation loci easily cause error elimination in the family investigation.

Examples are as follows:

in 2016, 30 days, vaginal swab and anal swab are extracted from Dongchun Maji (female, 12 years old) in paper shop, Henan, Hezhou province, the vagina and anus are killed by rape, and after positive sperm spot test, two test specimens are DNA tested to obtain autosomal DNA and Y chromosome DNA maps of the same male in 2016, 7 and 31 days. Since there is no definite suspect in the case, and the case is remote and the foreign population is small, the analysis of the infringement object of the case is definite, and the possibility of working on the case for the local person is high. Therefore, the Y-STR family investigation method is determined to be applied to investigate the periphery of the case. The special case group immediately deploys blood samples of male members in villages around the case collection places according to families, and 1400 blood samples are tested by adopting the Y-STR typing system of the application by 8 months and 3 days in 2016. The query that the Min has only one-step mutation at the DYS527ab site from 26 sites of the Y-STR typing and the physical evidence Y-STR typing of the blood sample of the Min Zhao-Chicun Min in the Hei Fang town of Ru Zhou cannot exclude the suspicion of the Min family.

Obtained from commercial sources

The plus kit further verifies that the Minself-protected blood sample and the material evidence have mutations at three sites, wherein DYS627 and DYS570 belong to one-step mutation, and DYF387S1 is a two-step mutation. Statistical results are shown in the table below, Min-self-protected blood samples and material evidence of mutations at four sites, typically with a tolerance greater than 3, rule out the family suspicion, i.e., using

The plus kit will most likely exclude the Minsurname family from error.

However, we carefully analyze that three of the four mutated sites belong to rapid mutation sites, and besides the two-step mutation of DYF387S1, DYS570, DYS627 and DYS527ab belong to one-step mutation, so we judge that the suspicion of this family cannot be excluded.

The patent group adopts the Y-STR typing system to find the Min family, and on one hand, the Min family members in the village take blood and check the Min family members, and finally the suspect is locked as the Min family member Min Stan.

The following table shows the typing results for the DNATYPRY 26 kit, with FW as evidence and T1 as Minchive.

Typing of the plus kit, FW is material evidence and T1 is Minico.

Example 3 comparison of the detection System of the invention with the detection System of patent CN103866019

1. The detection system and the detection system in the patent CN103866019 have the following size ranges of amplification products of each allele by primers designed aiming at each locus:

patent CN 103866019:

the present application (wherein FIG. 1 shows the amplification product layout at each locus of the present application):

as can be seen from the black line, the amplification products of DYS460 and DYS389II in CN103866019 are arranged too tightly, which causes the following two problems:

problem 1), allelic typing standard (ladder) typing errors.

Figure 3 shows a genotyping graph of the allelic typing standard (ladder) obtained using the system of patent CN103866019 and it can be seen that, in patent CN103866019 shown, the allelic typing standards of loci DYS438 and DYS389II were typed out of alignment, the first peak of DYS438 should be 7, indicated in red box, the first peak identified in error, which resulted in all subsequent alignments due to this error. It is well known to those skilled in the art that mistyping of an allelic typing standard will necessarily result in failure to correctly type a sample.

FIG. 4 shows the genotyping chart of the allelic typing standard (ladder) obtained by using the system of the present application, and it can be seen that the allelic loci are uniformly arranged, the primer specificity is high, complete STR typing can be obtained, the peak shape is sharp and clear, and the balance is good.

Problem 2), which is the typing of the previous locus, is not interpretable within the scope of the next locus.

For example, the allelic distribution of DYS460 is 7,8,9,10,11,12,13(https:// YHRD. org/tools/marker/DYS460, YHRD database). We tested the locus DYS460 of 812 male samples and found that the typing was 13 for 4 of the samples and 14 for two of the samples, as shown in the table below.

LOCUS:DYS460

Allelic typing	Number of samples	Frequency of
			8	1	0.00123152709359606
9	195	0.240147783251232
			10	328	0.403940886699507
11	268	0.330049261083744
			12	14	0.0172413793103448
13	4	0.00492610837438424
			14	2	0.00246305418719212

GD:0.67155324454175

For patent CN103866019, when DYS460 was typed 13, the position of the corresponding peak was 120bp, and 14 corresponded to 124 bp. And the range size of the DYS458 is 121bp-164bp, so that the system of the patent CN103866019 is used, the DYS460 allele 14 is in the range of the DYS458, and the DYS458 has two peaks, so that the judgment cannot be accurately carried out. The sample classified by DYS460 as 14 would be in the position of the red box shown in fig. 5. Thus, for some allelic types of the DYS460 locus, the system of patent CN103866019 was not able to be accurately typed. In the present embodiment, however, since the applicant has redesigned the primers for each locus allele and the combination and arrangement of the particular loci so that the above problems do not occur, it is clear from the above range of amplification product sizes for each allele of the present application that the DYS460 allele 14 does not erroneously occur at the position of the next locus DYS 389I.

2. The stability of the detection system of the invention is compared with that of the detection system of the patent CN103866019

The applicant verifies through experiments that the detection system of the present invention can stably detect the alleles of each locus within one week after the primer pool (primer mix) and the PCR buffer solution are mixed, while the CN103866 detection system can gradually decrease the amplification products corresponding to at least the DYS389II locus due to the formation of dimers between the primers after the primer pool and the PCR buffer solution are mixed, and the DYS389II locus suffers from dysallelic typing deletion due to the amplification products being lower than the detection limit in two hours, as shown in the following table, the proportion of success of allelic typing continuously decreases with time, and the genotyping of all alleles can be substantially performed after one day, while the system of the present invention can still perform successful genotyping of all alleles after one week, and the PCR systems of the above systems all adopt conventional enzymes and reagents:

for locus DYS389 II:

compared with the detection system disclosed by the patent CN103866019, the detection system disclosed by the invention has high detection stability, can more accurately and completely obtain the typing of each allele of the male individual gene locus, and can provide accurate and complete data for subsequent family investigation, paternity test, male component detection in male and female mixed samples and male component detection in a plurality of male mixed samples of the male individual.

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<210>8

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>8

agacgctcca aaggaccca 19

<210>9

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>9

aactcagcaa caggaatga 19

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>10

ctatggttct ggcattacaa g 21

<210>11

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>11

gactatgggc gtgagtgcat 20

<210>12

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>12

aaacagagga agaccctgtc attc 24

<210>13

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>13

ctgaagagct agacaccat 19

<210>14

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>14

atcgcattcc aattacatag t 21

<210>15

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>15

ttagcacttt cagcacatca c 21

<210>16

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>16

gactctttcc tctgatggtg 20

<210>17

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>17

gcccatccaa atcattcata at 22

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>18

agccggtgct ggaagacaga 20

<210>19

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>19

atcgttggga ccttgtgata atg 23

<210>20

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>20

attagggttc tctagaggga cag 23

<210>21

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>21

gctgttccct attcattcaa tcat 24

<210>22

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>22

ctatagggag acggaataaa a 21

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>23

attcgtgtta tctctgcctt 20

<210>24

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>24

tgactggtgt gtcacagcat g 21

<210>25

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>25

agtccatgac agctgatgc 19

<210>26

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>26

gtggcagacg cctataatcc 20

<210>27

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>27

tgacgttgtc aaagagcttc aa 22

<210>28

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>28

tttattccgc tgtgttggag a 21

<210>29

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>29

gctggagtca ttcctaatgt gg 22

<210>30

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>30

aaactttgag gtatgtctca ta 22

<210>31

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>31

ctgtcatgga atgctctctt g 21

<210>32

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>32

aatctgccca aatatcca 18

<210>33

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>33

atgctcgagt tgttatgg 18

<210>34

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>34

ctgaagtggc ttggaattc 19

<210>35

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>35

ctagcgtcaa tctctgcacc tg 22

<210>36

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>36

ctgaatgact actgagtttc t 21

<210>37

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>37

atgtgtatga aaggtgtgaa cca 23

<210>38

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>38

tgtacggctg tctaagggat c 21

<210>39

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>39

gtcgttatcg caaacatag 19

<210>40

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>40

gatgcattct aggaagatta 20

<210>41

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>41

tgtaggcagg ttttatggag g 21

<210>42

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>42

agtccgggag tgatagcatt ag 22

<210>43

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>43

ccaaattcca gcttattatt cc 22

<210>44

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>44

gcagagagta gatatagatg ataga 25

<210>45

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>45

ctgtctattc atctaacatc tttgtcatc 29

<210>46

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>46

gagatcacca atgaaatgta tttattc 27

Claims

1. A method for Y-STR typing of male individuals using 26Y-STR loci, the method comprising:

1) obtaining DNA of the male individual;

2) obtaining a genotype for the 26Y-STR loci of the DNA, the 26Y-STR loci being DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA H4, DYS438, DYS439, DYS460, DYS447, DYS527a/b, DYS617, DYS522, DYS444, DYS508, and DYS533, comprising the step of amplifying the same with amplification primers corresponding to the loci to obtain amplification products;

2. The method of claim 1, wherein the primer is a fluorescently labeled primer.

3. The method of claim 1, wherein 2) further comprises the step of analyzing the amplification product using a genetic analyzer after obtaining the amplification product to obtain the genotypes of the 26 loci.

4. A system for carrying out Y-STR typing on male individuals by utilizing 26Y-STR loci is characterized by comprising a DNA acquisition system, a composite detection system and an inference system;

the DNA obtaining system is used for obtaining DNA of the male individual;

5. The system of claim 4, wherein the primer is a fluorescently labeled primer.

6. The system of claim 5, wherein the multiplex detection system is further configured to analyze the amplification products after obtaining the amplification products using a genetic analyzer to obtain the genotypes of the 26 loci.

7. A composite detection system, comprising: male individual DNA, amplification primers for 26 loci,

8. A detection kit comprising the composite detection system according to claim 7.