Kit for detecting autosomal and sex chromosome number abnormalities

Abstract

The present application relates to the field of molecular biology diagnosis, and particularly relates to a kit for detecting autosomal and sex chromosome number abnormalities and application thereof. The kit comprises primer pairs shown in SEQ ID. NO. 1-236. The present application simultaneously amplifies short tandem repeat sequences on multiple chromosomes and chromosome-specific detection sites in one PCR reaction, analyzes the PCR products through high-resolution technology such as capillary electrophoresis, and determines whether the chromosome number is normal or abnormal through the calculation of the detection edge area. Starting from a DNA sample, the result can be obtained within 3 hours, and all autosomal and sex chromosome numbers can be simultaneously detected through one PCR experiment, and the detection technical process is simple and easy to standardize.

Description

Technical Field

[0001] This invention relates to the field of molecular biological diagnostics, and specifically to a kit for detecting abnormalities in the number of autosomes and sex chromosomes, and its application. Background Technology

[0002] According to statistics, 80% of miscarriages occur in the first twelve weeks of pregnancy, or the first trimester. The miscarriage rate then drops rapidly. Among all pregnant women, about 10% to 20% have had a miscarriage. The miscarriage rate is positively correlated with the age of the pregnant woman. Among them, 5% of women have had consecutive miscarriages. More than 60% of early miscarriages are caused by chromosomal abnormalities in the embryo.

[0003] Embryonic chromosomal abnormalities can be classified into numerical abnormalities, mosaicism, and structural abnormalities. Among them, trisomy is the most common numerical abnormality, followed by polyploidy and monosomy of the X chromosome. Structural abnormalities, such as balanced translocations, Robertsonian translocations, inversions, and overlaps, are less common. Numerical abnormalities account for about 85% of all chromosomal abnormalities.

[0004] Among trisomy chromosomal abnormalities, chromosome 16 has the highest incidence, followed by chromosomes 22, 15, and 21. Chromosomal abnormalities 13 and 18, and other chromosomes, have lower incidences. Trisomy chromosomal abnormalities occur when a specific chromosome has three sets of copies, and are a type of aneuploidy. The cause is incomplete meiosis during sperm and egg formation, resulting in gametes having a second copy of a specific chromosome. This leads to an extra complete copy of that chromosome in the embryo at the time of fertilization, resulting in trisomy. The detailed mechanism by which chromosomal abnormalities cause miscarriage is not yet fully understood. Two current hypotheses suggest that the mother's immune system may be able to recognize fetal chromosomal abnormalities, thus terminating the pregnancy; another hypothesis suggests that chromosomal abnormalities in the fetus itself cause the loss of genes necessary for development, leading to developmental arrest.

[0005] Chromosomal abnormality diagnosis includes conventional karyotype analysis, gene chip analysis, fluorescence in situ hybridization (FISH), and multiplex ligation probe amplification (MLPA). Karyotype analysis is the most commonly used and considered the gold standard. Karyotype analysis involves treating chromosomes with proteases, staining them, and then sorting and numbering them according to characteristics such as chromosome length and centromere position. The abnormality is diagnosed based on the structure or number of chromosomes. Karyotype analysis requires a large sample volume, necessitating the in vitro culture of amniotic fluid cells. Cell growth rates vary, and chromosome staining and sorting are time-consuming and labor-intensive, typically taking one to two weeks to obtain results. It demands high levels of expertise in amniotic fluid extraction and chromosome identification, resulting in significant time and labor costs.

[0006] Gene chip analysis is a commonly used tool for chromosome analysis of aborted tissue. Thousands or tens of thousands of nucleic acid probes are placed on a chip of a few square centimeters. After the DNA in the sample binds to the probes, it is detected by fluorescence or current. A single test can provide a large amount of gene sequence information. It has the advantages of high throughput and automation, but it also has the disadvantages of insufficient sensitivity and poor reproducibility.

[0007] Fluorescence in situ hybridization (FISH) involves hybridizing a fluorescently labeled nucleic acid probe with the nucleic acid sequence in the sample according to complementary base pairing and observing the results under a fluorescence microscope. The detection process is cumbersome, time-consuming, and has low sensitivity and specificity. It is also limited by the resolution of the microscope and requires high interpretation skills from the operator.

[0008] Multiplex ligation probe amplification (MLPA) is a novel technique developed in recent years for the qualitative and semi-quantitative analysis of DNA sequences. The basic principle of MLPA involves hybridization between probes and target DNA sequences, followed by ligation, PCR amplification, and separation of the products by capillary electrophoresis and data collection. The ligation reaction is highly specific; the ligase can only connect the two probe segments into a single, complete nucleic acid strand when both probes are completely hybridized to the target sequence (i.e., the target sequence and the probe's specific sequence are perfectly complementary). Conversely, if the target sequence and probe sequences are not perfectly complementary, even a difference of only one base will lead to incomplete hybridization, preventing the ligation reaction from proceeding. If the target sequence contains a point mutation, deletion, or amplification mutation, the corresponding probe's amplification peak will be absent, decreased, or increased, indicating the presence of copy number abnormalities or point mutations in the target sequence. MLPA requires a high concentration of the DNA to be tested, is susceptible to sample contamination, involves expensive reagents, and demands highly skilled operators. The entire experimental procedure takes approximately two days, making it more time-consuming than other detection methods.

[0009] Therefore, an improved method is needed to accurately, specifically, and more quickly detect the number of chromosomes in an individual and whether there are any abnormalities. The test results can help identify chromosomal abnormalities such as Turner syndrome, Down syndrome, Edwards syndrome, and male Klinefelter syndrome, preventing the transmission of defective genes to the next generation, reducing the suffering of patients and their families, promoting eugenics, and improving population quality. Simultaneously, it can serve as an auxiliary diagnostic tool for early miscarriage. For women experiencing early miscarriage, the test results can help determine the number of chromosomes in the miscarriage tissue, clarify the cause of the miscarriage, provide genetic counseling and guidance for future pregnancies, and offer important reference indicators for families experiencing miscarriage. Summary of the Invention

[0010] The purpose of this invention is to address the above-mentioned problems by providing a kit for detecting abnormalities in the number of autosomes and sex chromosomes, and its application.

[0011] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A kit for detecting numerical abnormalities of human autosomes and sex chromosomes, the kit comprising: (1) Primer pairs for detecting short tandem repeat sequence sites on the following autosomes: Chromosome 1: D1S551, D1S3736, D1S1596, D1S1646, D1S1668; Chromosome 2: D2S441, D2S1394, D2S1328, D2S1399, D2S1766; Chromosome 3: D3S4529, D3S1766, D3S2460, D3S3053, D3S2398; Chromosome 4: D4S1627, D4S2366, D4S1647, D4S 3243, D4S2431, Chromosome 5: D5S2845, D5S2849, D5S1725, D5S2501, D5S816, Chromosome 6: D6S1017, D6S1053, D6S1056, D6S2410, D6S1040, Chromosome 7: D7S1818, D7S817, D7S3047, D7S821, D7S3058, Chromosome 8: D8S1048, D8S1130, D8S1113, D8S592, D8S1179, Chromosome 9: D9S922, D9S2150, D9S938, D9S930, D9S934, chromosome 10; D10S1426, D10S1430, D10S2470, D10S1239, D10S1425, chromosome 11: Chromosomes D11S2001, D11S1981, D11S1999, D11S2002, D11S1998; Chromosome 12: D12S374, D12S1301, D12S1052, D12S1064, D12S378; Chromosome 13: D13S305, D13S634, D13S801, D13S628, D13S796; Chromosome 14: D14S750, D14S588, D14S1433, D14S749, D14S611; Chromosome 15: Chromosomes D15S659, D15S643, D15S818, D15S660, D15S1515; Chromosome 16: D16S753, D16S3253, D16S767, D16S2624, D16S2621; Chromosome 17: D17S2196, D17S974, D17S1294, D17S1299, D17S1534; Chromosome 18: D18S976, D18S1002, D18S535, D18S390, D18S386; Chromosome 19: D19S559, D19S586, D19S591, D19S714, D19S245; Chromosome 20: D20S477, D20S1143, D20S482, D20S480, D20S164.Chromosome 21: D21S1432, D21S1414, D21S1437, D21S1411, D21S1809, D21S1412, D21S1446; Chromosome 22: D22S686, D22S689, D22S685, D22S691, D22S532; (2) Primer pairs for detecting short tandem repeat sequence sites and specific detection sites on the following sex chromosomes: DXYS267, HPRT, AMEL, TAF9L, ZFX / Y, SRY; (3) Reagents used for multiple polymerase chain reaction (PCR).

[0012] Furthermore, the primer pairs described above contain a non-specific sequence GTTTGTGTCTTCTT at the 5' end of either the upstream or downstream primer.

[0013] Furthermore, the primer pairs described above contain at least one fluorescent group; the fluorescent group is selected from any one of FAM, VIC, HEX, JOE, TAMRA, NED, PET, and ROX.

[0014] Furthermore, the specific primer pair sequences are shown below: A method for detecting normal or abnormal numbers of human autosomes and sex chromosomes using the above-mentioned kit includes the following steps: (a) Mix the DNA sample to be tested and the primer pair shown in SEQ ID NO.1-236 in a PCR reaction tube; (b) The mixture obtained in step (a) is subjected to a polymerase chain reaction; (c) Use high-resolution technology to separate the amplification products with different fluorescence and different fragment lengths produced in step (b) and generate detection peak area information; (d) Confirm the number of chromosomes in the DNA sample from the product of step (c), and calculate the detection peak ratio R by dividing the peak area of ​​the short tandem repeat sequence site detection. (e) Based on the detection peak ratio R determined in step (d), determine whether the number of autosomes and sex chromosomes is normal or abnormal.

[0015] The advantages of this invention are: (1) A single multiplex PCR amplification reaction simultaneously detects short tandem repeat sequence sites on autosomes and sex chromosomes, combined with capillary electrophoresis analysis, yielding results within 3 hours. Compared to karyotype analysis methods based on sex chromosome number, this invention significantly shortens the detection time, simplifies the detection process, and facilitates standardization.

[0016] (2) When performing multiplex PCR amplification, the detection peak is prone to bifurcation after capillary fragment analysis. Therefore, the present invention adds a non-specific sequence GTTTGTGTCTTCTT to the 5' end of the upstream or downstream primer of the short tandem repeat sequence primer pair. This can avoid the bifurcation of the detection peak generated during capillary electrophoresis analysis, which affects the calculation of the detection peak area R and the interpretation of chromosome number. Therefore, the non-specific sequence can increase the accuracy of chromosome number quantification.

[0017] (3) In addition to quantifying chromosome number, the detection results of this invention can identify normal chromosomes, autosomal trisomy, 45,X, 47,XXX, 47,XXY, 47,XYY, 48XXYY, 48,XXXY, 69,XXX, 69,XXY, and 69XYY. It can also indicate whether the sample is mosaic, uniparental diploid, or whether the sample is contaminated by the mother. Uniparental diploidity and maternal contamination are determined by comparing amplified fragments of short tandem repeat sequences of the individual with those of both parents.

[0018] (4) Applicable to DNA extracted from all samples, including amniotic fluid, chorionic villus tissue, whole blood, saliva and dried blood spot samples. The DNA loading volume is 1-100 ng, with high sensitivity. Even at a sample concentration as low as 0.5 ng / ul, it can still accurately detect normal and abnormal chromosome numbers. Attached Figure Description

[0019] Figure 1The images show the electrophoresis results of chromosomes 1, 2, 3, 4, 5, and 6 in individuals with normal chromosome numbers, using different fluorescence spectra (FAM, VIC, NED, and PET). These images reveal the analytical results of NC01 in the examples, distinguishing short tandem repeat sequences through different fluorescence and fragment sizes. The analytical results, based on the detection peak ratio R of the short tandem repeat sequence sites, are interpreted as 1:1 or a single detection peak, indicating a normal number of autosomes and sex chromosomes.

[0020] Figure 2 The images show the electrophoresis results of chromosomes 7, 8, 9, 10, 11, and 12 in individuals with normal chromosome numbers, under different fluorescence spectra (FAM, VIC, NED, and PET). This document discloses the analysis results of NC01 in the embodiments, distinguishing short tandem repeat sequences through different fluorescence and fragment sizes. The analysis results are interpreted based on the detection peak ratio R of the short tandem repeat sequence site as 1:1 or a single detection peak; TAF9L is 1:1, indicating a normal number of autosomes and sex chromosomes.

[0021] Figure 3 The images show the electrophoresis results of chromosomes 13, 16, 18, 21, and 22, and sex chromosomes in different fluorescence spectra (FAM, VIC, NED, PET) for individuals with normal chromosome numbers. They reveal the analytical results of NC01 in the examples, distinguishing short tandem repeat sequences through different fluorescence and fragment sizes. The analytical results are interpreted as a 1:1 ratio (R) or a single detection peak based on the detection peak ratio R at the short tandem repeat sequence site. For TAF9L, the ratio is 1:1, indicating a normal number of autosomes and sex chromosomes.

[0022] Figure 4 The images show the electrophoresis results of chromosomes 14, 15, 17, 19, 20, and 21 in individuals with normal chromosome numbers, under different fluorescence spectra (FAM, VIC, NED, and PET). They reveal the analytical results of NC01 in the examples, distinguishing short tandem repeat sequences through different fluorescence and fragment sizes. The analytical results are interpreted as a 1:1 ratio or a single detection peak based on the detection peak ratio R at the short tandem repeat sequence site; TAF9L is 1:1, indicating a normal number of autosomes and sex chromosomes.

[0023] Figure 5 The image shows the electrophoresis results of individuals with 69 chromosomes (XXY) on chromosomes 1, 2, 3, 4, 5, and 6 in different fluorescence spectra (FAM, VIC, NED, PET). It reveals the analysis results of PC12 in this example, distinguishing short tandem repeat sequences by different fluorescence and fragment sizes. The analysis results are interpreted based on the peak ratio R of the short tandem repeat sequence sites as a single detection peak, or 1:2, 2:1, or 1:1:1. HPRT shows a single detection peak or 1:1, and TAF9L shows 3:2, indicating a triploid genotype.

[0024] Figure 6 The image shows the electrophoresis results of individuals with 69 chromosomes (XXY) on chromosomes 7, 8, 9, 10, 11, and 12 in different fluorescence spectra (FAM, VIC, NED, PET). It reveals the analysis results of PC12 in this example, distinguishing short tandem repeat sequences by different fluorescence and fragment sizes. The analysis results are interpreted based on the peak ratio R of the short tandem repeat sequence sites as a single detection peak, or 1:2, 2:1, or 1:1:1. HPRT shows a single detection peak or 1:1, and TAF9L shows 3:2, indicating a triploid genotype.

[0025] Figure 7 The electrophoresis results of XXY individuals with 69 chromosomes and sex chromosomes at FAM, VIC, NED, and PET fluorescence spectra in different fluorescent media (69, 13, 16, 18, 21, 22, 18, 21, 22, 18, 29, 20 ...

[0026] Figure 8 The image shows the electrophoresis results of individuals with a chromosome number of 69 and XXY in different fluorescence spectra (FAM, VIC, NED, and PET) at chromosomes 14, 15, 17, 19, 20, and 21. It reveals the analytical results of PC12 in this example, distinguishing short tandem repeat sequences by different fluorescence and fragment sizes. The analytical results are interpreted based on the peak ratio R of the short tandem repeat site as a single detection peak, or 1:2, 2:1, or 1:1:1. HPRT is a single detection peak or 1:1, and TAF9L is 3:2, indicating a triploid genotype. Detailed Implementation

[0027] Example 1 1. Preparation of reaction solution and primer mixture Table 1. Preparation of Reaction Solution Components Table 2 Final concentrations of each primer in primer mixture 1 Table 3 Final concentrations of each primer in primer mixture 2 Table 4 Final concentrations of each primer in primer mixture 3 Table 5 Final concentrations of each primer in primer mixture 4 Example 2: PCR Amplification and Result Analysis 1. Sample processing: Human genomic DNA was extracted for subsequent PCR reactions. The DNA concentration was 0.5 ng / uL to 50 ng / uL, and the ratio of OD260nm / OD280nm was between 1.6 and 2.0.

[0028] 2. Preparation of amplification reagents: (1) Take out the reaction solution and primer mixture 1-4 from the kit, thaw at room temperature, mix by inverting, and then briefly centrifuge with a microcentrifuge to allow all liquids to settle to the bottom of the tube.

[0029] (2) Preparation of amplification reagents: Prepare amplification reagents according to Table 6 below. Table 6 Amplification Reagent Preparation Table (3) Shake the prepared reagent evenly with a shaker, and then briefly centrifuge with a micro centrifuge to allow the liquid to settle to the bottom of the tube.

[0030] (4) Add 23 μL of the prepared amplification reagent to each PCR reaction tube and transfer it to the sample processing area for sample loading.

[0031] 3. Sample addition: Add 2 μL of the genomic DNA of the test sample, normal male control genomic DNA, normal female control genomic DNA, and blank control to the corresponding PCR reaction tube containing 23 μL of amplification reagent. After capping the PCR reaction tube, centrifuge briefly.

[0032] 4. PCR amplification and capillary electrophoresis analysis: (1) Place the PCR reaction tubes into the PCR instrument and set the reaction program according to the method in Table 7. Table 7 Amplification Reaction Procedure (2) Capillary electrophoresis analysis of PCR amplification products: ABI3130, ABI3730, ABI3500Dx or ABISeqStudio Genetic Analyzer were used for detection. Take 1uL of PCR amplification product, 1uL of 500LIZ Size Standard and 9uL of Hi-Di Formamide, mix well and denature at 95℃ for 3min, then immediately place on ice for 2min and then detect.

[0033] 5. Software Analysis: After the reaction procedure was completed, the fragment size and detection peak area of ​​the PCR amplification products were analyzed using GeneMapper software.

[0034] 6. Calculation of peak ratio: For autosomal detection sites, including short tandem repeat sequences on chromosomes 1 to 22 and sex chromosome detection sites AMEL, TAF9L, DXYS267, and HPRT, the detection peak ratio (R) needs to be calculated. For the same detection site, the detection peak ratio (R) is the detection peak area of ​​the shortest fragment divided by the detection peak area of ​​the longer fragment. The table below shows the reference range for the detection peak ratio (R).

[0035] 7. Result Interpretation: 7.1 Interpretation of Autosomal Results (1) Normal number of autosomes: Among the detection sites of the corresponding autosomes, at least two sites show a 1:1 ratio of detection peak area, and the remaining sites show a single detection peak.

[0036] (2) Autosomal trisomy: At least two of the detection sites on the same chromosome show a peak area ratio of 1:2, 2:1 or 1:1:1, while the remaining sites show a single detection peak.

[0037] 7.2 Interpretation of Sex Chromosome Results (1) Based on the peak area ratio of sex chromosomes, the type is determined according to the following table: (2) If all autosomes are trisomic, and the sex chromosomes are TAF9L 1:1 XXX or TAF9L 3:2 XXY or TAF9L 3:1 XYY, the sample is judged as triploid (69,XXX or 69,XXY or 69,XYY).

[0038] Example 3 Reagent Performance Verification 1. Conformity rate of positive reference materials Twenty positive reference samples were tested, as shown in the table below. High, medium, and low concentrations were tested, with concentrations of 50 ng / uL, 25 ng / uL, and 5 ng / uL, respectively. Each concentration was tested three times repeatedly, and three batches of reagents were tested. The positive concordance rate of the test results was 100%.

[0039] Table 8 Number of autosomes and sex chromosomes in positive reference samples 2. Normal reference sample compliance rate Four negative reference samples were tested, as shown in the table below. High, medium, and low concentrations were tested, with concentrations of 50 ng / uL, 25 ng / uL, and 5 ng / uL, respectively. Each concentration was tested three times repeatedly, and three batches of reagents were tested. The test results showed that the compliance rate of the normal reference samples was 100%.

[0040] Table 9 Negative Reference Material Autosome and Sex Chromosome Types 3. Repeatability Four repeatability reference samples were tested at concentrations of 25 ng / uL and 5 ng / uL, respectively. Each concentration was tested ten times. Three batches of reagents were tested. The interpretation of the number of autosomes and sex chromosomes was correct. The test results met the requirements and the repeatability was consistent.

[0041] Table 10 Reproducible Reference Material Autosomal and Sex Chromosome Types 4. Minimum detection limit Twenty-two reference samples were tested to the detection limit, as shown in the table below. The concentrations were diluted to 5 ng / µL, 2.5 ng / µL, and 0.5 ng / µL, and each was tested 20 times. Three batches of reagents were tested, and the number of replicates of the test results all met the requirements.

[0042] Table 11 Detection Limit Reference Samples: Autosomal and Sex Chromosome Types 5. National Reference Material Testing A set of "National Reference Material for Chromosomal Copy Number Variation Detection" developed by the China National Institutes for Food and Drug Control was tested. The results showed that the chromosome number abnormalities within the reagent's detection range met the requirements of the national reference material type. The results are shown in the table below.

[0043] Example 4 1. We collected 150 samples of amniotic fluid, aborted chorionic villus tissue, and EDTA-anticoagulated whole blood. Karyotype analysis was used as a reference method to verify the consistency between the results.

[0044] 2. Amniotic fluid cells, chorionic villus tissue, and EDTA-anticoagulated whole blood samples were collected. Human genomic DNA was extracted using a MagCore nucleic acid extractor and a MagCore Genomic DNA WholeBlood Kit. The concentration and purity of the DNA were detected using a micro spectrophotometer. The concentration of the DNA in 150 samples ranged from 0.5 ng / uL to 50 ng / uL, and the ratio of OD260nm to OD280nm was between 1.6 and 2.0.

[0045] 3. Following the steps in Example 2, DNA samples were added and reacted using a PCR instrument. 4. Following the steps in Example 2, the results were analyzed. The results are shown in the table below. All samples met the copy number requirements. Among them, there were 51 cases of trisomy 21, 27 cases of trisomy 18, 8 cases of trisomy 13, 12 cases of Klinefelter syndrome XXY, 18 cases of monosomy X, 15 normal female samples, and 19 normal male samples (Table 12).

[0046] Table 12 Detection results of 150 samples The above description is merely a preferred embodiment of the present invention. It should be understood that the foregoing description of the embodiments is given only by way of example, and various modifications can be made by those skilled in the art. The above specification, embodiments, and experimental results provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those skilled in the art can make various modifications and alterations without departing from the principles and spirit of the present invention. Therefore, all equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A kit for detecting numerical abnormalities of human autosomes and sex chromosomes, characterized in that: The kit contains: (1) Primer pairs for detecting short tandem repeat (STTR) sites on autosomes, wherein the STTR sites include the following: chromosome 1: D1S551, D1S3736, D1S1596, D1S1646, D1S1668; chromosome 2: D2S441, D2S1394, D2S1328, D2S1399, D2S1766; chromosome 3: D3S4529, D3S1766, D3S2460, D3S3053, D3S2398; chromosome 4: D4S1627, D4S2366, D4S1647, D4S3243, D4S2431; chromosome 5: D5S2845, D5S2849. Chromosomes D5S1725, D5S2501, D5S816; Chromosome 6: D6S1017, D6S1053, D6S1056, D6S2410, D6S1040; Chromosome 7: D7S1818, D7S817, D7S3047, D7S821, D7S3058; Chromosome 8: D8S1048, D8S1130, D8S1113, D8S592, D8S1179; Chromosome 9: D9S922, D9S2150, D9S938, D9S930, D9S934; Chromosome 10: D10S1426, D10S1430, D10S2470, D10S1239, D10S 1425, Chromosome 11: D11S2001, D11S1981, D11S1999, D11S2002, D11S1998; Chromosome 12: D12S374, D12S1301, D12S1052, D12S1064, D12S378; Chromosome 13: D13S305, D13S634, D13S801, D13S628, D13S796; Chromosome 14: D14S750, D14S588, D14S1433, D14S749, D14S611; Chromosome 15: D15S659, D15S643, D15S818, D15S660, D15S15 15. Chromosomes 16: D16S753, D16S3253, D16S767, D16S2624, D16S2621; Chromosome 17: D17S2196, D17S974, D17S1294, D17S1299, D17S1534; Chromosome 18: D18S976, D18S1002, D18S535, D18S390, D18S386; Chromosome 19: D19S559, D19S586, D19S591, D19S714, D19S245; Chromosome 20: D20S477, D20S1143, D20S482, D20S480, D20S164.Chromosome 21: D21S1432, D21S1414, D21S1437, D21S1411, D21S1809, D21S1412, D21S1446; Chromosome 22: D22S686, D22S689, D22S685, D22S691, D22S532; (2) Primer pairs for detecting short tandem repeat sequence sites and specific detection sites on sex chromosomes, wherein the short tandem repeat sequence sites and specific detection sites include: DXYS267, HPRT, AMEL, TAF9L, ZFX / Y, SRY; (3) Reagents used in multiple polymerase chain reactions; The specific primer pair sequences are shown in SEQ ID NO. 1~236.

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