A double fluorescent labeling method for the detection of 62 Y chromosome short tandem repeat sequences by multiplex amplification

Abstract

The present invention discloses a 62 Y chromosome short tandem repeat sequence composite amplification detection kit using a dual fluorescent labeling method, and uses eight groups of dual fluorescent dyes to label and prepare 62 Y-STRs locus composite amplification primer sets, and simultaneously amplifies 62 Y-STRs sites. The present invention uses eight groups of fluorescent dyes to label, has 62 Y-STRs site information, covers all directory sites of my country's Y chromosome DNA database, is suitable for a variety of test materials, efficiently provides site information, and has strong applicability; uses FRET dual fluorescent labeling technology, so that the fluorescent dyes ATTO 550, RHO12, RHO13, and RHO14 with longer wavelengths still have efficient fluorescent signals, improves the sensitivity of detection, and amplification efficiency; has good species specificity, has good amplification reaction for PCR amplifiers of different brands, and has strong applicability.

Description

Technical Field

[0001] The invention relates to a 62 Y chromosome short tandem repeat sequence composite amplification detection kit adopting a double fluorescence labeling method, and belongs to the technical field of molecular genetics. Background Art

[0002] Short tandem repeats (STRs), also known as microsatellites or simple sequence repeats (SSRs), are a type of DNA tandem repeats that are widely present in the genomes of eukaryotic organisms, with a core sequence of 2-6 base repeat units. STR loci are large in number and widely distributed, accounting for about 3% of the entire genome (International Human Genome Sequencing Consortium, 2001), and are highly polymorphic. Their polymorphism mainly stems from the difference in the number of core sequence repeats between individuals, and this difference follows Mendel's law of inheritance in the genetic process. Therefore, STR amplification detection technology is widely used in individual identification, kinship identification, and population genetics research. Y-STR refers to short tandem repeats that exist on the male Y chromosome, and only exist on the Y chromosome, so it is paternal inheritance. Due to the characteristics of Y-STR, there are many important applications in human genetics, such as forensic evidence investigation, kinship identification, historical investigation, tracing human migration patterns through male pedigrees, and family research.

[0003] At present, Y-STR composite amplification technology is the main auxiliary means for forensic individual identification and paternity testing, and plays an important role in sexual assault cases, mixed sample differentiation, and family screening through Y-STR. With the development of science and technology, the construction of Y-STR databases has gradually received more attention. Many countries use this technology to establish Y-STR DNA databases, that is, to analyze the Y-STR data of prisoners and criminal suspects and enter them into the database to facilitate comparison and screening. With the wide application, the increase in data comparison, and the characteristics of Y-STR paternal inheritance, users have higher requirements for the number of loci, information content, and compatibility of the test kit. The Y-STRs detection kits currently on sale in the domestic market have a relatively small number of loci, and the number of rapidly mutating Y-STRs (hereinafter referred to as (RM) Y-STRs) contained is small, which is not conducive to the rapid determination of the geographical scope in the case, resulting in a large waste of resources.

[0004] In the Y-STRs individual identification DNA fluorescence detection kit, the most common detection method is the detection method that combines multiplex PCR amplification and capillary electrophoresis. That is, after using four or five sets of primer complexes labeled with single fluorescent dyes to specifically amplify the Y-STRs loci using the polymerase chain reaction method (hereinafter referred to as the PCR method), the specific amplification products and the standard products of known standard length labeled with single fluorescent dyes are mixed and then electrophoresed synchronously. After selecting the spectrum with the same fluorescent dye as the labeled primer on the genetic analysis sequencer, capillary electrophoresis is performed, and the system matching collection software is used to collect information, and then the analysis software is used to perform genotyping analysis on the electrophoresis results.

[0005] Currently, the primers in the Y-STRs fluorescence detection kits on the domestic market are usually labeled with four groups such as 6-FAM, VIC, NED and PET, and five groups such as 6-FAM, VIC, NED, TAZ and SID single fluorescent dyes. The excitation wavelengths of fluorescent dyes such as VIC, NED, PET are all greater than 540nm. Because the longer the wavelength, the lower the energy, the products amplified using single fluorescent labeled primers such as VIC, NED, PET emit relatively weak fluorescence during electrophoresis, which is not conducive to the signal collection of the laser detector in the genetic analyzer, and it is easy to cause the loss of Y-STRs locus information. Summary of the invention

[0006] The present invention provides a 62 Y chromosome short tandem repeat sequence composite amplification detection kit adopting a double fluorescent labeling method. The present invention adopts eight groups of double fluorescent dye labeling methods to establish a 62 Y-STRs composite amplification detection system, which can simultaneously amplify 62 Y-STRs sites and provide 62 Y-STRs site information at the same time. It has the characteristics of high sensitivity, fast amplification speed and large amount of information, and can efficiently perform individual information identification.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A 62 Y chromosome short tandem repeat sequence composite amplification detection kit using a dual fluorescent labeling method, using eight sets of dual fluorescent dyes to label and prepare 62 Y-STRs loci composite amplification primer sets, and simultaneously amplifying 62 Y-STRs loci;

[0009] The above-mentioned multiplex amplification kit simultaneously detects 62 Y-STRs gene loci, including: DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS5 33. DYS576, DYS643, DYS460, DYS549, DYF387S1a, DYF387S1b, DYS449, DYS518, DYS627, DYS570, DYS5 27a, DYS527b, DYS447, DYS444, DYS557, DYS596, DYS446, DYS510, DYS622, DYS443, DYS587, DYS522, Y GATA A10, DYS520, DYS552, DYS593, DYS531, DYS459a, DYS459b, DYS508, DYS388, DYS617, DYS645, DYS713, D YS630, DYS404S1a, DYS404S1b, DYS626, DYS526a, DYS526b, rs771783753, rs759551978, rs199815934.

[0010] The Y-STR allele fragment length selected in this application is within the range of 75-600bp; the individual recognition ability is above 0.95; it has regular three, four or five base repeats; the gene mutation rate is low, less than 0.2%; the allele discrimination rate is high; and the site has a high degree of species specificity.

[0011] The present application uses dual fluorescent dyes to label the upstream primers to form a composite amplification detection kit. The amplification system of the present invention includes a primer mixture of 62 Y-STRs loci, a reaction buffer, and a hot-start Taq polymerase.

[0012] The kit includes a composite primer set of 110 specific primers for simultaneously amplifying 62 Y-STRs loci, a reaction mixture, and a hot-start Taq polymerase;

[0013] The 110 specific primers include 55 upstream primers labeled with different fluorescent dyes and 55 downstream primers labeled without fluorescent dyes, and the sequence numbers are from PM NO.001 to PM NO.110; among them, DYS385a and DYS385b amplification primers are the same, DYS389I and DYS389II amplification primers are the same, DYF387S1a and DYF387S1b amplification primers are the same, DYS527a and DYS527b amplification primers are the same, DYS459a and DYS459b amplification primers are the same, DYS404S1a and DYS404S1b amplification primers are the same, and DYS526a and DYS526b amplification primers are the same.

[0014] In order to achieve repetitive, sensitive and efficient amplification in the multiplex PCR process, the primers for the locus must be designed and selected with high specificity, and the analytical errors due to homology must be eliminated. The entire set of primers in this application is designed according to uniform parameters to make the annealing temperature of the primers as consistent as possible; the primer binding region avoids the SNP site, the amplicon avoids the deletion site, and the upstream primer of each gene locus is labeled with a dual fluorescent dye.

[0015] The 110 specific primers are divided into seven groups. The first, second and third groups have the first base at the 5' end of the primer labeled with FAM, TET and HEX in sequence; the fourth, fifth, sixth and seventh groups have the first base at the 5' end of the primer labeled with ATTO 550, RHO12, RHO13 and RHO14 in sequence, and then use FAM to label a base in the middle sequence between the 5' and 3' of the primer;

[0016] Group 1: rs771783753, DYS481, DYS389, DYS635, DYS533, DYS627, DYS527, DYS576, primer numbers: PM NO.001-PM NO.016;

[0017] Group II: DYS460, DYS458, DYS19, DYS387, DYS444, DYS385, DYS593, primer numbers: PMNO.017-PM NO.030;

[0018] Group 3: rs199815934, DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS522, primer numbers: PM NO.031-PM NO.046;

[0019] Group 4: DYS456, DYS570, DYS390, DYS438, GATAH4, DYS449, DYF404S1, primer numbers: PMNO.047-PM NO.060;

[0020] Group 5: rs759551978, DYS459, DYS388, DYS437, DYS447, DYS391, DYS643, DYS557, DYS596, primer numbers: PM NO.061-PM NO.078;

[0021] Group VI: DYS531, DYS630, DYS622, DYS552, DYS587, DYS713, DYS446, DYS626, primer numbers: PM NO.079-PM NO.094;

[0022] Group 7: DYS645, DYS526, YGATA A10, DYS510, DYS617, DYS443, DYS520, DYS508, primer numbers: PM NO.095-PM NO.110;

[0023] Primer sequences are shown in Table 2.

[0024] The reaction mixture consists of: MgCl2 10.25 mM, KCl 150 mM, Tris-HCl 125 mM, BSA 13 mg / ml, dATP 4.0 mM, dTTP 4.0 mM, dCTP 4.0 mM, dGTP 4.0 mM.

[0025] This kit is suitable for amplification of different samples and has a large annealing temperature range. The amplification system can be amplified using the following program on various reaction thermal cyclers: 95℃3min, 95℃5s, 61℃90s, 28-30 cycles; final extension at 60℃ for 20min; maintain at 4℃ for PCR amplification; perform capillary electrophoresis on the amplified product on a genetic analyzer and collect data, and then use GeneMapper analysis software for data analysis.

[0026] The samples for extracting DNA from the above samples mainly include human semen stains, blood, body fluids, hair, tissues, bloodstains, etc. The main methods for extracting DNA genomes are magnetic bead extraction or Chelex100 method; filter paper, cotton swabs, saliva cards, gauze, FTA cards and other samples can also be used for direct amplification.

[0027] The above-mentioned test kit can be used in paternity screening, individual identification, paternity testing, Y chromosome DNA database construction and other aspects of the Chinese population.

[0028] The present invention uses a fluorescence resonance energy transfer system (hereinafter referred to as FRET, Fluorescence Resonance Energy Transfer) to improve the problem of weakened fluorescence signals in the long-wave range, in order to improve the fluorescence intensity and the sensitivity of the kit. FRET consists of two fluorescent groups, a fluorescent donor and the other a fluorescent acceptor, and the two can transfer energy within a certain distance range. When the distance between the fluorescent donor and the fluorescent acceptor is less than 10nm, the energy is non-radiatively transferred to the fluorescent acceptor group through the interaction within the molecule after the electron excites the fluorescent donor group. The energy transfer quenches the fluorescence intensity of the fluorescent donor and reduces its excited state life, while increasing the fluorescence emission intensity of the fluorescent acceptor group. The present invention simultaneously adopts the FRET system and eight groups of dual-fluorescence-labeled Y-STRs primer mixed systems, which not only improves the detection sensitivity in the long-wavelength range, but also greatly increases the number of Y-STRs sites by the use of eight groups of dual fluorescence, and can simultaneously amplify 62 Y-STRs sites, which not only include all the sites commonly used for Y-STRs genotyping, but also include a large number of (RM) Y-STRs sites. The loci information of this application and related Y-STRs fluorescence detection kits at home and abroad are shown in the table below.

[0029]

[0030]

[0031]

[0032] Note: + indicates included loci, - indicates excluded loci

[0033] The technologies not mentioned in the present invention are all referred to the prior art.

[0034] The beneficial effects of the present invention are:

[0035] 1. Because it uses eight groups of fluorescent dyes to mark, it has 62 Y-STRs site information, covering all the catalog sites in my country's Y chromosome DNA database, and is suitable for a variety of samples, providing site information efficiently, and has strong applicability.

[0036] 2. The FRET dual fluorescence labeling technology is used to ensure that the fluorescent dyes ATTO 550, RHO12, RHO13, and RHO14 with longer wavelengths still have efficient fluorescence signals, thereby improving the sensitivity of detection and amplification efficiency; by optimizing the amplification system, the overall amplification time is controlled at around 70 minutes, with a fast amplification speed and good balance.

[0037] 3. The primers in this kit have good species specificity and have no amplification reaction on the genomes of animals that humans often come into contact with in life, such as chickens, ducks, dogs, cats, pigs, etc.; when designing the primers, the Tm values ​​of the primers are kept as consistent as possible, and they have good amplification reactions for PCR amplifiers of different brands and have strong applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The allele typing standard diagram of the kit in Example 1;

[0039] Figure 2 The typing results of 9948 pairs of amplified positive standards in Example 1;

[0040] Figure 3 It is the amplification map of the kit Y20210616-5 in Example 2;

[0041] Figure 4 It is the amplification map of the kit Y20210616-6 in Example 2;

[0042] Figure 5 , Figure 6 and Figure 7 These are the results of the three concentration gradient amplifications of 0.5 ng, 0.25 ng and 0.125 ng of the DNA positive standard 9948 amplified by the kit in Example 3. DETAILED DESCRIPTION

[0043] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0044] A 62 Y chromosome short tandem repeat sequence composite amplification detection kit using a double fluorescent labeling method, comprising: a composite primer set of 110 specific primers for simultaneously amplifying 62 Y-STRs loci, a reaction mixture and a hot start Taq polymerase; eight sets of double fluorescent dyes are used to label and prepare a composite amplification primer set for 62 Y-STRs loci, and 62 Y-STRs loci are amplified simultaneously;

[0045] 62 Y-STRs gene loci, including: DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS 576, DYS643, DYS460, DYS549, DYF387S1a, DYF387S1b, DYS449, DYS518, DYS627, DYS570, DYS527a , DYS527b, DYS447, DYS444, DYS557, DYS596, DYS446, DYS510, DYS622, DYS443, DYS587, DYS522, Y GATA A10, DYS520, DYS552, DYS593, DYS531, DYS459a, DYS459b, DYS508, DYS388, DYS617, DYS645, DYS713, D YS630, DYS404S1a, DYS404S1b, DYS626, DYS526a, DYS526b, rs771783753, rs759551978, rs199815934.

[0046] The composition of the reaction mixture is shown in Table 1.

[0047] Table 1 Composition of reaction mixture

[0048]

[0049]

[0050] The composite primer set sequence of 110 specific primers is numbered: PM NO.001 to PM NO.110; among them, DYS385a and DYS385b amplification primers are the same, DYS389I and DYS389II amplification primers are the same, DYF387S1a and DYF387S1b amplification primers are the same, DYS527a and DYS527b amplification primers are the same, DYS459a and DYS459b amplification primers are the same, DYS404S1a and DYS404S1b amplification primers are the same, and DYS526a and DYS526b amplification primers are the same.

[0051] The 110 primers (55 pairs) were divided into seven groups. The first base at the 5'-end of the primers in the first, second and third groups was labeled with FAM, TET and HEX respectively; the first base at the 5'-end of the primers in the fourth, fifth, sixth and seventh groups was labeled with ATTO 550, RHO12, RHO13 and RHO14 respectively, and then a base in the middle sequence between the 5'-end and 3'-end of the primers was labeled with FAM.

[0052] Group 1: rs771783753, DYS481, DYS389, DYS635, DYS533, DYS627, DYS527, DYS576, primer numbers: PM NO.001-PM NO.016.

[0053] The second group: DYS460, DYS458, DYS19, DYS387, DYS444, DYS385, DYS593, primer numbers: PMNO.017-PM NO.030.

[0054] The third group: rs199815934, DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS522, primer numbers: PM NO.031-PM NO.046.

[0055] The fourth group: DYS456, DYS570, DYS390, DYS438, GATAH4, DYS449, DYF404S1, primer numbers: PMNO.047-PM NO.060.

[0056] The fifth group: rs759551978, DYS459, DYS388, DYS437, DYS447, DYS391, DYS643, DYS557, DYS596, primer numbers: PM NO.061-PM NO.078.

[0057] Group 6: DYS531, DYS630, DYS622, DYS552, DYS587, DYS713, DYS446, DYS626, primer numbers: PM NO.079-PM NO.094.

[0058] Group 7: DYS645, DYS526, YGATA A10, DYS510, DYS617, DYS443, DYS520, DYS508, primer numbers: PM NO.095-PM NO.110.

[0059] Using the professional primer design software Oligo7, primers were designed on both wings of the core repeat region of 62 Y-STRs gene loci, so that the Tm value of each primer was close to 60°C, and the amplification product was between 75-600bp. Each primer was tested and optimized until the amplification efficiency was high, no mismatches occurred, and no non-specific amplification peaks were generated. Then a composite amplification test was performed, and no non-specific peaks were generated between 57°C and 63°C. The primer sequences are shown in Table 2.

[0060] Table 2 Primer sequence list

[0061]

[0062]

[0063]

[0064]

[0065] In order to ensure that the peak height of the amplified loci meets the requirements, a series of tests were conducted on the primer concentration and amplification components, and finally the primer addition amount and the components of the amplification buffer were determined.

[0066] The kit consists of two parts, including a pre-reaction amplification kit and a post-reaction electrophoresis kit. The components of each kit are shown in Table 3.

[0067] Table 3 Components of each kit

[0068]

[0069] The composition of the reaction mixture is 2.5X buffer components. The allele typing standard for analysis provided by the present invention is a product set of allele plasmids corresponding to each Y-STRs locus primer amplification. DNA positive standard 9948 was purchased from Suzhou Xinhai Biotechnology Co., Ltd., and hot start Taq DNA polymerase was purchased from Bao Biotechnology Co., Ltd. Figure 1 It is the Allelic Ladder electrophoresis pattern.

[0070] It has been verified that the kit of the present invention has good amplification efficiency within the annealing temperature range of 57° C.-63° C., and therefore has good amplification efficiency on PCR instruments of different brands (ABI9700, ABI veriti, E1000, etc.).

[0071] When amplifying the 9948 positive standard, prepare the reaction mixture according to Table 4.

[0072] Table 4 Amount of each component added in the preparation of PCR system

[0073] Components 25μL system 2.5×PCR Mix 10.0μL Y62 Plex primers 5.0μL Taq DNA polymerase 1.0μL 9948 positive DNA 2.5μL PCR Grade Water Add to a total volume of 25 μL Total reaction volume 25μL

[0074] The amplification system can obtain good results on various PCR amplification instruments (ABI9700, ABI veriti, E1000, etc.) using the following program: 95℃3min, 95℃5s, 61℃90s, 28 cycles; 60℃ final extension for 20min; 4℃ hold for PCR amplification; 1ul+0.15ul molecular weight internal standard+9ul deionized formamide are mixed with the obtained PCR amplification product, and capillary electrophoresis is performed on the ABI3130XL genetic analyzer. Electrophoresis parameters: injection time 10s, injection voltage: 3.0Kv; other parameters are machine default. Use collection software to collect electrophoresis data, and use GeneMapper software for data analysis. Figure 2 This is the 9948 amplification detection map, which is completely consistent with the theoretical typing.

[0075] In the case of amplifying FTA blood cards, prepare the reaction mixture according to Table 5.

[0076] Table 5 Amount of each component added in the preparation of PCR system

[0077] Components 25μL system 2.5×PCR Mix 10.0μL Y62Plex primers 5.0μL Taq DNA polymerase 1.0μL FTA card or filter paper 1.0 or 1.2mm aperture 1-2 pieces PCR Grade Water Add to a total volume of 25 μL Total reaction volume 25μL

[0078] Amplification was performed using the E1000 PCR amplifier produced by GE according to the amplification program: 95°C for 3 min, 95°C for 5 s, 61°C for 90 s, 28 cycles; final extension at 60°C for 20 min; PCR amplification was performed at 4°C; 1ul of the obtained PCR amplification product was mixed with 0.15ul of molecular weight internal standard and 9ul of deionized formamide, and then capillary electrophoresis was performed on the ABI3130XL genetic analyzer. Electrophoresis parameters: injection time 10s, injection voltage: 3.0Kv; other parameters were machine default. The electrophoresis data was collected using the collection software, and the data was analyzed using the GeneMapper software. Figure 3 , Figure 4 This is the FTA card amplification detection map. It can be seen from the figure that the present application can use FTA cards, filter paper and other test materials for direct amplification.

[0079] The present invention also tests the detection sensitivity of the kit, and finds the minimum DNA concentration that can detect all Y-STRs loci typing by amplifying different concentrations of 9948 positive DNA. Refer to Table 4 for the configuration of the amplification system. The input amounts of 9948 are 0.5ng, 0.25ng, and 0.125ng, respectively. The maximum input amount of the positive standard should not exceed 5ng, otherwise it will cause electrophoresis failure. The amplification program is 95℃ for 3min, 95℃ for 5s, 61℃ for 90s, 28 cycles; final extension at 60℃ for 20min; PCR amplification at 4℃; the amplified product is subjected to electrophoresis and data analysis using a 3130XL genetic analyzer, as shown in Figure 4. Figure 5-7There is no amplification reaction to the genomes of animals that humans often come into contact with in life, such as chickens, ducks, dogs, cats, pigs, etc. After experiments, the individual recognition ability is above 0.95, and when the DNA concentration is 0.03ng, it can still be accurately typed and the test is completed.

[0080] In summary, the present invention uses eight groups of fluorescent dyes for dual-dye labeling, which not only greatly increases the number of Y chromosome loci, but also avoids the problem of low signal intensity and easy quenching of fluorescent dyes in the long wavelength range, while enhancing the ability of Y-STRs paternal screening and improving the sensitivity and balance of the kit.

Claims

1. A 62 Y chromosome short tandem repeat sequence composite amplification detection kit using a dual fluorescent labeling method, which is characterized in that eight sets of dual fluorescent dyes are used to label and prepare 62 Y-STRs loci composite amplification primer sets, and 62 Y-STRs loci are amplified simultaneously; The above-mentioned multiplex amplification kit simultaneously detects 62 Y-STRs gene loci, including: DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, D YS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS576, DYS643 , DYS460, DYS549, DYF387S1a, DYF387S1b, DYS449, DYS518, DYS627, DYS570, DYS527a, DYS5 27b, DYS447, DYS444, DYS557, DYS596, DYS446, DYS510, DYS622, DYS443, DYS587, DYS522, Y GATA A10, DYS520, DYS552, DYS593, DYS531, DYS459a, DYS459b, DYS508, DYS388, DYS617, DYS645, DYS713, D YS630, DYF404S1a, DYF404S1b, DYS626, DYS526a, DYS526b, rs771783753, rs759551978, rs199815934; 110 specific primers were used to amplify 62 Y-STRs loci simultaneously. The 110 specific primers were divided into seven groups. The first base at the 5' end of the primers in the first, second and third groups was labeled with FAM, TET and HEX in sequence; the first base at the 5' end of the primers in the fourth, fifth, sixth and seventh groups was labeled with ATTO 550, RHO12, RHO13 and RHO14 in sequence, and then a base in the middle sequence between the 5' and 3' of the primers was labeled with FAM. Group 1: rs771783753, DYS481, DYS389, DYS635, DYS533, DYS627, DYS527, DYS576, primer numbers: PM NO.001-PM NO.016; Group II: DYS460, DYS458, DYS19, DYF387, DYS444, DYS385, DYS593, primer numbers: PMNO.017-PM NO.030; Group 3: rs199815934, DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS522, primer numbers: PM NO.031-PM NO.046; Group 4: DYS456, DYS570, DYS390, DYS438, GATAH4, DYS449, DYF404S1, primer numbers: PMNO.047-PM NO.060; Group 5: rs759551978, DYS459, DYS388, DYS437, DYS447, DYS391, DYS643, DYS557, DYS596, primer numbers: PM NO.061-PM NO.078; Group VI: DYS531, DYS630, DYS622, DYS552, DYS587, DYS713, DYS446, DYS626, primer numbers: PM NO.079-PM NO.094; Group 7: DYS645, DYS526, YGATAA10, DYS510, DYS617, DYS443, DYS520, DYS508, primer numbers: PM NO.095-PM NO.110; The primer sequences are as follows:

2. The 62 Y-chromosome short tandem repeat sequence composite amplification detection kit using a dual fluorescent labeling method as claimed in claim 1, characterized in that it comprises a composite primer set of 110 specific primers for simultaneously amplifying 62 Y-STRs loci, a reaction mixture and a hot start Taq polymerase; The 110 specific primers include 55 upstream primers labeled with different fluorescent dyes and 55 downstream primers labeled with non-fluorescent dyes, with sequence numbers ranging from PM NO.001 to PM NO.110; among them, The amplification primers for DYS385a and DYS385b are the same, the amplification primers for DYS389I and DYS389II are the same, the amplification primers for DYF387S1a and DYF387S1b are the same, the amplification primers for DYS527a and DYS527b are the same, the amplification primers for DYS459a and DYS459b are the same, the amplification primers for DYF404S1a and DYF404S1b are the same, and the amplification primers for DYS526a and DYS526b are the same.

3. The 62 Y chromosome short tandem repeat sequence composite amplification detection kit using a dual fluorescent labeling method as described in claim 2 is characterized in that the reaction mixture consists of: MgCl2 10.25mM, KCl 150mM, Tris-HCl 125Mm, BSA 13mg / ml, dATP 4.0mM, dTTP 4.0mM, dCTP 4.0mM and dGTP 4.0mM.

4. The 62 Y chromosome short tandem repeat sequence composite amplification detection kit using a dual fluorescent labeling method as described in any one of claims 1 to 3, characterized in that the amplification program is: 95°C for 3 min, 95°C for 5 s, 61°C for 90 s, 28-30 cycles; final extension at 60°C for 20 min; maintaining at 4°C for PCR amplification; performing capillary electrophoresis on the amplified product on a genetic analyzer and collecting data, and then using GeneMapper analysis software for data analysis.

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