A highly resistant 44-y-chromosome short tandem repeat sequence composite amplification detection kit

Abstract

The application discloses a high-tolerant 44 Y-chromosome short tandem repeat sequence composite amplification detection kit. The kit adopts a six-group double fluorescent dye method to label 44 STR gene locus composite amplification primer groups, and performs one-pot amplification on 41 Y-STR and 3 Y-Indel. The kit contains recommended 20 core loci, 15 preferred loci and some alternative loci, meets the requirements of the Ministry of Public Security Y-STR DNA database construction, and has higher cumulative individual identification and cumulative non-paternity exclusion rate. According to the Chinese population data expansion allele typing, the application selects high polymorphism sites suitable for Chinese population genetics, has strong individual identification ability and strong tolerance. The amplification system and program are optimized, the adaptability of the kit to detection materials, the detection sensitivity, the amplification efficiency and the inhibition tolerance are greatly improved, site information can be efficiently provided, and the kit has good species specificity and detection ability for male samples in mixed samples.

Description

Technical Field

[0001] This invention relates to a highly tolerant multiplex amplification test kit for 44 Y chromosome short tandem repeat sequences, which relates to forensic genetic testing technology and belongs to the field of molecular genetics technology. Background Technology

[0002] The Y chromosome is a sex chromosome that determines the sex of an individual. It is the smallest acrocentric chromosome in the G group, containing 60 million base pairs, only one-third the size of the X chromosome. The Y chromosome is unique to males and can only be stably passed from male parents to male offspring, leaving a strong genetic lineage; therefore, the Y chromosome exhibits a paternal inheritance pattern. Human Y-STR genetic markers refer to short tandem repeat sequences located in the non-recombining region of the Y chromosome.

[0003] STR (short tandem repeat) genetic markers are a class of repeating sequences of up to several tens of nucleotides, consisting of repeating units of 2 to 6 nucleotides. They have high haplotype diversity and can be used for forensic individual identification, paternity testing, and DNA genealogy construction.

[0004] Autosomal STRs have shown significant effectiveness in individual identification and kinship determination, but their limitation lies in the poor expandability of information on other kinship relationships of an individual's autosomes besides parents and children.

[0005] The haplotype paternal inheritance characteristics of Y-chromosome STRs can provide auxiliary means for forensic individual identification and paternity testing. They have unique application value in areas such as paternal family paternity testing, identification of male components in mixed lesions, analysis of mixtures of different male individuals, and investigation of male pedigrees of criminal suspects. In sex crime cases, Y-STR testing is often used for large-scale screening to identify the suspect's family and then, based on autosomal STR testing, quickly pinpoint the suspect.

[0006] In recent years, numerous scholars both domestically and internationally have conducted genetic surveys and studies on populations from different families and regions. The results show that Y-STR genetic markers exhibit significant polymorphism. Detection of Y-STR and Y-Indel loci allows for the acquisition of more haplotype information using Y-STR. Even when Y-STR typing is inconsistent among individuals of the same paternal lineage or when Y-STR typing is similar among individuals of different paternal lineages, the low mutation rate of Y-Indels can be used for more accurate family tracing and identification. Therefore, public security organs nationwide are not only establishing a Y-chromosome DNA database for criminals but also a database for male family tracing systems. However, currently available fluorescent detection kits for Y-STRs in the domestic market have poor tolerance and are easily affected by various inhibitory factors, often leading to failure to elute peaks and typing failure. Summary of the Invention

[0007] This invention provides a highly tolerant 44 Y-chromosome short tandem repeat (STR) multiplex amplification detection kit. Employing six fluorescent dye labeling methods, a 44-STR multiplex amplification detection system is established, capable of simultaneously amplifying 44 STR loci and providing information on all 44 STR loci. It features high sensitivity, fast amplification speed, and a large amount of information, enabling efficient individual information identification. It exhibits extremely high tolerance to different inhibitors and can be used for paternal kinship identification, criminal suspect family line searches, and Y-STR database establishment, among other applications.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A highly tolerable multiplex amplification kit for 44 Y-chromosome short tandem repeat sequences (STRs) is developed. A primer set for multiplex amplification of 44 STR loci is prepared using a six-dye method, simultaneously amplifying all 44 STR loci. The 44 Y-STR loci include 41 Y-chromosome STR loci and 3 Y-chromosome indels. The 41 Y-chromosome STR loci are: DYS481, DYS389, DYS635, DYS533, DYS627, DYS527, DYS522, DYS460, DYS458, DYS19, DY387S1, DYS593, DYS385, DYS444, DYS393, DYS549, DYS439, DYS392, D... YS448, DYS518, DYS645, DYS596, DYS456, DYS570, DYS390, DYS438, Y_GATA_H4, DYS449, DYF404S1, DYS576, DYS437, DYS447, DYS388, DYS643, DYS391, DYS557. Among them, DYS385, DYS387, DYS527, DYS389, and DYS404S1 have palindromic structures, showing two peaks, with a / b distinction, and are double-copy loci, so they are all counted as two loci; 3 Y chromosome indels: rs759551978, rs771783753, rs199815934.

[0010] Tolerance to different inhibitors was tested. In the amplification system, the final concentration of hemoglobin reached 800 μM, and all loci showed normal peak elution and accurate genotyping. Similarly, the final concentrations of heme, EDTA, humic acid, and indigo were all tested. All loci showed normal peak elution and accurate genotyping.

[0011] This invention employs both the FRET system and a hybrid system of six sets of fluorescently labeled Y-chromosome STR primers. This not only improves detection sensitivity over a long wavelength range, but also significantly increases the number of Y-chromosome STR loci by using six sets of dual fluorescence, enabling the simultaneous amplification of 44 STR loci. The composite primer set of 79 specific primers for simultaneously amplifying the 44 STR loci includes 39 upstream primers labeled with different fluorescent dyes and 40 downstream primers without fluorescent labels, with sequence numbers ranging from AMNO.01 to AM NO.79.

[0012] The sites detected in the above fluorescent multiplex amplification system were labeled with five different fluorescent molecules. Each group was considered to have the same fluorescent label. In the first and second groups, the first base at the 5' end of the primer was labeled with FAM and HEX, respectively. In the third, fourth, and fifth groups, the first base at the 5' end of the primer was labeled with ATTO 550, RHO12, and ATTO 590, respectively. Then, FAM was used to label one base in the middle sequence between the 5' and 3' ends of the primer. The five combinations are as follows:

[0013] Group 1: DYS481, DYS389, DYS635, DYS533, DYS627, DYS522, DYS527; Primer numbers: AMNO.01-AM NO.15;

[0014] Group 2: DYS460, DYS458, DYS19, DY387S1, DYS593, DYS385, DYS444, rs759551978, rs771783753, rs199815934; Primer numbers: AM NO.16-AM NO.29, AM NO.74-AM NO.79;

[0015] Group 3: DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS645, DYS596; Primer numbers: AM NO.30-AM NO.45;

[0016] Group 4: DYS456, DYS570, DYS390, DYS438, Y_GATA_H4, DYS449, DYF404S1; Primer numbers: AM NO.46-AM NO.59;

[0017] Group 5: DYS576, DYS437, DYS447, DYS388, DYS643, DYS391, DYS557; Primer numbers: AMNO.60-AM NO.73.

[0018] The specific primer sequences and their final concentrations in the above-mentioned multiplex amplification kit are shown in the table below:

[0019] Table 1 Primer sequences and final concentrations for each gene locus

[0020]

[0021]

[0022]

[0023] The upstream primer marker types are shown in Table 1. The loci are arranged as follows: Figure 1 As shown.

[0024] The molecular weight internal standard Orange-600s used in this amplification system was labeled with orange 647N, which is the sixth group of fluorescent labels. Figure 2 As shown.

[0025] The aforementioned molecular weight internal standard Orange-600s consists of the following 31 fragments: 60bp, 80bp, 100bp, 114bp, 119bp, 140bp, 160bp, 180bp, 203bp, 214bp, 220bp, 240bp, 260bp, 280bp, 300bp, 314bp, 320bp, 340bp, 360bp, 380bp, 400bp, 414bp, 420bp, 460bp, 480bp, 500bp, 520bp, 540bp, 560bp, 580bp, and 600bp.

[0026] The amplification reaction system of the above-mentioned multiplex amplification kit in 25 μl is as follows: 8-10 μl of 2.5×PCR Mix, 5 μl of Y44 primer mixture, 1 μl of Taq DNA polymerase, 2 μl of template DNA, and deionized water to make up to 25 μl. The primer mixture is a mixture of amplification primers for 44 STR loci. The 2.5×PCR Mix components are: 35 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 75 mM KCl, 2.5 mg / ml BSA, 6.2 mM dNTPs, 6.00% DMSO, 80 mM betaine, and 3 mM (NH4)2SO4.

[0027] The amplification and detection method of the above-mentioned multiplex amplification reagent includes the following steps: ① Sample preparation: extract genomic DNA from the sample as an amplification template or directly use an extract-free sample as an amplification template; ② Perform PCR amplification on the genomic DNA of the sample obtained in step ① using amplification primers with sequences as shown in AMNO.01-AMNO.79; ③ Detect the fluorescence signal of the amplification product using a genetic analyzer; ④ Collect fluorescence signal data and analyze the DNA typing results of the 44 loci obtained using gene analysis software.

[0028] The template DNA mentioned above includes extracted samples and direct amplification samples. Extracted samples mainly include male semen stains, blood, bodily fluids, hair, tissue, bloodstains, etc., with the primary DNA extraction methods being magnetic bead extraction or the Chelex 100 method. Direct amplification samples include human blood or oral cells collected using one or more carriers selected from filter paper, blood cards, cotton swabs, and FTA cards. The preferred amount of DNA template in the sample is 0.5 ng to 4 ng.

[0029] The above-mentioned multiplex amplification reagent is suitable for amplification of different samples and has a wide annealing temperature range. The amplification system can be amplified using the following program on various reaction thermal cyclers: 95℃ for 3 min; 95℃ for 5 s, 60℃ for 90 s, 28-30 cycles; final extension at 60℃ for 12 min; hold at 4℃.

[0030] The above-mentioned multiplex amplification reagents were used to detect the fluorescence signal of the amplification products using a genetic analyzer; the fluorescence signal data were collected, and the DNA typing results of the 44 loci were analyzed using gene analysis software.

[0031] The above-mentioned multiplex amplification reagents, with the addition of a positive internal control (IPC) at a fixed position, do not change their amplification efficiency with changes in template concentration, and can quickly assess sample quality. If the sample is degraded or contains inhibitors, the sample quality can be quickly assessed.

[0032] The aforementioned multiplex amplification kit also includes allele ladders for 44 STR loci, DNA standards, fluorescent molecular weight internal standards Oranger-600s, and 6Dye (FRET) Matrix.

[0033] The aforementioned multiplex amplification kit can be applied to father-son relationship identification, identification of male components in mixed spots, analysis of mixtures of different male individuals, identification of male individuals in large-scale disasters, and in family lineage investigations in some homicide and rape cases, it can quickly narrow down the investigation scope and accurately pinpoint the perpetrator's family lineage.

[0034] Any techniques not mentioned in this invention are based on existing technologies.

[0035] The beneficial effects of this invention are:

[0036] 1. This study employs six sets of fluorescent dyes to label 44 STR loci, simultaneously amplifying 32 low-mutation-rate Y-STR loci and 9 rapidly mutating Y-STR loci, including three Y-indel loci with near-zero mutation rates in the Chinese population. It incorporates 20 core Y-STR loci and 15 preferred Y-STR loci as required by the Ministry of Public Security's database requirements, exhibiting good data comparison compatibility and facilitating rapid comparison in male pedigree screening.

[0037] 2. Adding a positive internal control (IPC) at a fixed position ensures that its amplification efficiency does not change with the template concentration, allowing for rapid evaluation of sample quality and PCR reaction efficiency.

[0038] 3. Employing FRET dual fluorescent labeling technology, the detection sensitivity is high, and accurate typing can still be obtained even with DNA as low as 40 pg.

[0039] 4. The sample adaptability is good. Common biological samples such as blood, saliva, hair, semen stains, muscle, nails, and cartilage in men can all be identified after extraction. FTA cards and saliva cards can also be directly amplified.

[0040] 5. Through optimization of the amplification system, the system has high specificity and good stability. After repeated verification, no non-specific amplification products were generated, and the signal intensity was stable, making the overall amplification time less than 60 minutes.

[0041] 6. The primers in this kit have good species specificity and do not amplify the genomes of animals that humans frequently come into contact with, such as chickens, ducks, dogs, cats, and pigs.

[0042] 7. When testing mixed samples, it exhibits strong tolerance to high concentrations of female samples. No amplification was observed in 3ug female samples. In a 3ug female sample containing 0.1ng male sample (female to male ratio of 30,000:1), 41 Y chromosome STR loci and 3 Y-indel loci were effectively detected without locus loss.

[0043] 8. When testing tolerance to different inhibitors, the final concentrations of hemoglobin, heme, EDTA, humic acid, and indigo in the amplification system reached 800 μM, 600 μM, 1 mM, 160 mg / L, and 160 mM, respectively. All loci showed normal peak elution and accurate genotyping. Overall, the kit demonstrated strong tolerance to inhibitors such as hemoglobin, heme, EDTA, humic acid, and indigo. Attached Figure Description

[0044] Figure 1 A schematic diagram showing the arrangement of the 44 Y-STR loci;

[0045] Figure 2 This is a schematic diagram of the internal standard for the molecular weight of Oranger-600s;

[0046] Figure 3 This is a standard diagram for allele typing.

[0047] Figure 4 Genotyping map of positive standard 9948;

[0048] Figure 5 Genotyping map of loci directly amplified in male sample 1 (FAT card);

[0049] Figure 6 Genotyping map of loci directly amplified from male sample 2 (saliva card);

[0050] Figure 7 The amplification results were obtained for the sensitivity detection of the DNA positive standard 9948 at 1 ng, 0.2 ng, 0.08 ng, and 0.04 ng.

[0051] Figure 8 The kit in this embodiment of the invention amplifies the genotyping patterns of other species of samples, including pigs, chickens, dogs, cattle, sheep, and fish.

[0052] Figure 9 This is a schematic diagram of the amplification of a mixed sample consisting of 3ug female samples and 0.1ng male samples;

[0053] Figure 10 To amplify the genotyping maps of loci containing different concentrations of hemoglobin inhibitors;

[0054] Figure 11 To amplify the genotyping maps of loci containing different concentrations of heme inhibitors;

[0055] Figure 12 To amplify the genotyping maps of loci containing different concentrations of EDTA inhibitors;

[0056] Figure 13 To amplify the locus genotyping maps containing different concentrations of humic acid inhibitors;

[0057] Figure 14 To amplify the locus typing maps containing different concentrations of indigo inhibitors;

[0058] Figure 15 The allele frequencies of each locus detected by this kit in the Han Chinese population. Detailed Implementation

[0059] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0060] The application principle of the present invention will be described in detail below with reference to the accompanying drawings.

[0061] Extensive data research and analysis were conducted on Y-STR loci. This kit selects the following 44 STR loci, including 41 Y-chromosome STR loci: DYS481, DYS389, DYS635, DYS533, DYS627, DYS527, DYS522, DYS460, DYS458, DYS19, DY387S1, DYS593, DYS385, DYS444, DYS393, DYS549, DYS439, DYS 392, DYS448, DYS518, DYS645, DYS596, DYS456, DYS570, DYS390, DYS438, Y_GATA_H4, DYS449, DYF404S1, DYS576, DYS437, DYS447, DYS388, DYS643, DYS391, DYS557; 3 Y chromosome indels: rs759551978, rs771783753, rs199815934. The GenBank accession number, core repeat sequence, allele core repeat region range, and 9948 genotyping for each locus are shown in Table 2.

[0062] Table 2. Information on relevant loci of the Y44 kit.

[0063]

[0064]

[0065] Note that in the “repetitive structure”, the lowercase letter to the right of the brackets [] represents the number of times the sequence in the brackets [] to the left of it is repeated.

[0066] Based on the above locus information, experimental parameters were confirmed and optimized.

[0067] 1. Confirm the combination of 6 fluorescent markers with dual fluorescent labels.

[0068] This kit uses a six-color fluorescent labeling combination with dual fluorescent labels, employing FAM, HEX, ATTO550, RHO12, ATTO 590, and 647N to establish a fluorescent multiplex amplification system. ATTO 647N is the fluorophore selected as the molecular weight internal standard.

[0069] To further improve detection sensitivity in the long wavelength range, the number of Y chromosome STR loci was increased. This kit uses a FRET system and six sets of fluorescent labels. The sites to be detected in the fluorescent multiplex amplification system are labeled with five different fluorescent molecules, with identical fluorescent labels considered as a group. Specifically, the first and second groups label the first base of the primer's 5' end with FAM and HEX, respectively; the third, fourth, and fifth groups label the first base of the primer's 5' end with ATTO 550, RHO12, and ATTO 590, respectively, and then use FAM to label one base in the 5'-terminus and 3'-terminus of the primer. After repeated experimental verification, the optimal fluorescent dye combination scheme was determined, and the five combinations are shown below.

[0070] DYS481, DYS389, DYS635, DYS533, DYS627, DYS522, and DYS527 form the first group, labeled using FAM; DYS460, DYS458, DYS19, DY387S1, DYS593, DYS385, DYS444, rs759551978, rs771783753, and rs199815934 form the second group, labeled using HEX; DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS645, and DYS596 form the third group, labeled using FAM-ATTO. The first group of upstream primers was labeled with FAM-ATTO550 dual fluorescent markers; the second group, consisting of DYS456, DYS570, DYS390, DYS438, Y_GATA_H4, DYS449, and DYF404S1, was labeled with FAM-RHO12 dual fluorescent markers; the third group, consisting of DYS576, DYS437, DYS447, DYS388, DYS643, DYS391, and DYS557, was labeled with FAM-ATTO550 dual fluorescent markers. The labeling types for each upstream primer are shown in Table 1.

[0071] The molecular weight internal standard Orange-600s used in this amplification system is labeled with orange 647N, which is the sixth group of fluorescent labels. The molecular weight internal standard Orange-600s consists of the following 31 fragments: 60bp, 80bp, 100bp, 114bp, 119bp, 140bp, 160bp, 180bp, 203bp, 214bp, 220bp, 240bp, 260bp, 280bp, 300bp, 314bp, 320bp, 340bp, 360bp, 380bp, 400bp, 414bp, 420bp, 460bp, 480bp, 500bp, 520bp, 540bp, 560bp, 580bp, and 600bp.

[0072] 2. Design specific primers

[0073] Primers were designed using the professional primer design software Oligo7 to target the core repeat regions of 44 STR gene loci, with multiple primer pairs designed for each locus. Each primer had a Tm value close to 60℃, and the amplified products ranged from 75 to 600 bp. Amplification and optimization experiments were conducted on individual primer pairs to select primers with high amplification efficiency, no mismatches, and no nonspecific amplification. Multiplex amplification tests were then performed, and primers that might cause nonspecific amplification were redesigned based on the results until no nonspecific amplification occurred. The primer concentration in the multiplex amplification system was adjusted according to the amplification efficiency of the primer pairs.

[0074] The specific primer nucleotide sequences and concentrations are shown in Table 1.

[0075] 3. Optimization of the amplification system

[0076] Through repeated experiments, the final composition of the 2.5×PCR Mix used in this amplification system is as follows: 35mM Tris-HCl (pH 8.0), 6mM MgCl2, 75mM KCl, 2.5mg / ml BSA, 6.2mM dNTPs, 6.00% DMSO, 80mM betaine, and 3mM (NH4)2SO4.

[0077] After optimization, the amplification system of this kit is shown in Table 3:

[0078] Table 3 PCR amplification system of the reagent kit

[0079]

[0080] 4. Optimization of amplification conditions

[0081] After determining the enzyme content and concentrations of components such as magnesium ions in this kit system, the thermal cycling parameters were optimized. Gradient experiments were conducted with five different amplification cycle numbers: 27, 28, 29, 30, and 32 cycles. The optimal number of amplification cycles for amplifying positive templates was determined to be 28 cycles. The annealing temperature was also optimized after determining the cycle number. With 28 amplification cycles, annealing temperatures were set at 58℃, 59℃, 60℃, 62℃, and 64℃, with 60℃ being the optimal temperature. This also accommodates temperature differences between different PCR instruments. The specific PCR amplification program is shown in Table 4.

[0082] Table 4. PCR amplification program for the Y44 kit.

[0083]

[0084] 5. Construction of allele typing standards

[0085] Allelic standards are essential for genotyping and are crucial for the reproducibility and accuracy of STR genotyping. The Chinese population differs from other populations due to racial and geographical variations, resulting in different allelic distributions. Therefore, allelic gradient standards specific to the Chinese population are required for testing. Collecting currently prevalent genotypes allows for individual genotyping. The allelic genotypes and 9948 positive standard genotypes collected in this invention are shown in Table 5.

[0086] Table 5. Control DNA9948 typing and Y44 Plus Allelic Ladder information

[0087]

[0088]

[0089] Specific applications of this reagent kit:

[0090] Example 1: DNA, blood card, and saliva card amplification

[0091] Using 1 ng DNA 9948, male blood card and saliva card as templates, the reaction reagents were mixed, vortexed and centrifuged according to the amplification system and then subjected to PCR amplification.

[0092] The obtained PCR amplification product was mixed with 1 μL of the PCR product, 0.15 μL of the molecular weight internal standard Oranger-600s, and 9 μL of deionized formamide. The mixture was then subjected to capillary electrophoresis on an ABI 3130XL genetic analyzer. Electrophoresis parameters: injection time 10 s, injection voltage 3.0 kV; other parameters were left at machine default. Electrophoresis data were collected using data collection software, and GeneMapper software was used for data analysis and to generate maps.

[0093] Depend on Figure 2-6 The results show that the system of this invention can amplify different types of samples to obtain clear and accurate typing maps, and both the molecular weight internal standard and the allele typing standard can accurately calibrate the typing.

[0094] Example 2: Sensitivity Detection

[0095] The sensitivity of this kit was tested using different concentrations of 9948 as templates for amplification. The detection chromatograms are shown below. Figure 7 As shown, the minimum detection sensitivity of this system is 40 pg, at which all subtypes can be completely detected.

[0096] Example 3: Species-Specific Detection

[0097] This kit was used for species-specific detection of different species. DNA templates were taken from chickens, pigs, dogs, cattle, sheep, and fish. The detection patterns are shown below. Figure 8 As shown, this system did not exhibit amplification of bands in other species, indicating good species specificity.

[0098] Example 4: Verification of Amplification Capability of Mixed Samples

[0099] A two-sample pooling simulation experiment was performed using this kit. Male sample 9948 and female sample 9947 were mixed at a ratio of 1:30000 as amplification templates. The amplification result was obtained as follows (i.e., the amplification result of 3 μg of female DNA template containing 0.1 ng of male DNA template), as shown in the image. Figure 9 As shown.

[0100] Example 5: Inhibitor Tolerance Test

[0101] The kit was tested for tolerance to different inhibitors by adding different concentrations of inhibitors to the amplification system. The resulting detection chromatograms after amplification are shown below. Figure 10-14 As shown, the final concentrations of hemoglobin, heme, EDTA, humic acid, and indigo in the amplification system reached 800 μM, 600 μM, 1 mM, 160 mg / L, and 160 mM, respectively. All loci showed normal peak elution and accurate genotyping. This indicates that the overall kit exhibits strong tolerance to inhibitors such as hemoglobin, heme, EDTA, humic acid, and indigo.

[0102] In summary, this invention, by employing six groups of fluorescent dyes for dual-dye labeling, not only greatly increases the number of Y chromosome loci but also avoids the problem of low fluorescent dye signal intensity and easy quenching in the long wavelength range. At the same time, it enhances the ability to identify individuals and improves the sensitivity, uniformity, and inhibitor resistance of the kit.

Claims

1. A highly tolerable multiplex amplification kit for 44 Y-chromosome short tandem repeat sequences, comprising primer sets for multiplex amplification of 44 Y-STR loci using a six-set dual-fluorescent dye labeling method, capable of simultaneously amplifying 44 Y-STR loci. The kit is characterized by: 44 Y-STR loci including 41 Y-chromosome STR loci and 3 Y-chromosome indels; the 41 Y-chromosome STR loci are: DYS481, DYS389, DYS635, DYS533, DYS627, DYS527, DYS522, and DYS460. DYS458, DYS19, DYF387, DYS593, DYS385, DYS444, DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS645, DYS596, DYS4 56, DYS570, DYS390, DYS438, Y_GATA_H4, DYS449, DYF404S1, DYS576, DYS437, DYS447, DYS388, DYS643, DYS391 and DYS557, among which, DYS385, DYS387, DYS527, DYS389, and DYS404S1 have palindromic structures, exhibiting two peaks, with a / b distinctions, and are double-copy loci; therefore, they are all counted as two loci. The three Y chromosome indels are rs759551978, rs771783753, and rs199815934. The amplification system achieved an inhibitory concentration of over 800 μM for hemoglobin; over 600 μM for heme; over 1 mM for EDTA; over 160 mg / L for humic acid; and over 160 mM for indigo. The primer sequences for the 44 Y-STR sites are as follows: Amplification primers were labeled with fluorescent dyes, and the amplification primers for the 44 STR loci were divided into the following five groups: Group 1: DYS481, DYS389, DYS635, DYS533, DYS627, DYS522, DYS527; Group 2: DYS460, DYS458, DYS19, DY387S1, DYS593, DYS385, DYS444, rs759551978, rs771783753, rs199815934; Group 3: DYS393, DYS549, DYS439, DYS392, DYS448, DYS518, DYS645, DYS596; Group 4: DYS456, DYS570, DYS390, DYS438, Y_GATA_H4, DYS449, DYF404S1; Group 5: DYS576, DYS437, DYS447, DYS388, DYS643, DYS391, DYS557; Five sets of amplification primers were labeled with five different fluorescent dyes. The first and second sets of primers were labeled with FAM and HEX respectively at the first base of the 5' end. The third, fourth and fifth sets of primers were labeled with ATTO 550, RHO12 and ATTO 590 respectively at the first base of the 5' end. Then, FAM was used to label one base of the middle sequence of the 5'-end and 3'-end of the primers.

2. The highly tolerable 44 Y chromosome short tandem repeat sequence multiplex amplification detection kit according to claim 1, characterized in that: It also contains allele ladders for 44 Y-STR loci, DNA standards 9948 and 6 Dye (FRET) Matrix, and molecular weight internal standard Orange-600s.

3. The highly tolerable 44 Y chromosome short tandem repeat sequence multiplex amplification detection kit according to claim 2, characterized in that: The molecular weight internal standard Orange-600s consists of the following 31 fragments: 60 bp, 80 bp, 100 bp, 114 bp, 119 bp, 140 bp, 160 bp, 180 bp, 203 bp, 214 bp, 220 bp, 240 bp, 260 bp, 280 bp, 300 bp, 314 bp, 320 bp, 340 bp, 360 bp, 380 bp, 400 bp, 414 bp, 420 bp, 460 bp, 480 bp, 500 bp, 520 bp, 540 bp, 560 bp, 580 bp, and 600 bp. The fluorochrome labeling of the molecular weight internal standard Orange-600s is 647N.

4. The highly tolerable 44 Y chromosome short tandem repeat sequence multiplex amplification detection kit according to any one of claims 1-3, characterized in that: The amplification reaction system includes: 8-10 μL of 2.5×PCR Mix, 5 μL of Y44 primer mixture, 1 μL of Taq DNA polymerase, 2 μL of template DNA, and deionized water to make up to 25 μL; wherein, the Y44 primer mixture is a mixture of amplification primers for 44 STR loci; the 2.5×PCR Mix components include: 35 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 75 mM KCl, 2.5 mg / ml BSA, 6.2 mM dNTPs, 6.00% DMSO, 80 mM betaine, and 3 mM (NH4)2SO4.

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