A multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material and its application

Abstract

The present invention provides a multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material and its application, belonging to the field of biological detection technology. The nucleotide sequence of the multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material provided by the present invention is shown as SEQ ID NO.1-SEQ ID NO.18. Using the primer set provided by the present invention to detect whether a Turner syndrome patient carries Y chromosome material has the advantages of low detection cost, high efficiency, simple operation and easy promotion, strong specificity, high detection sensitivity, and short detection cycle. It can screen Y chromosome material before growth hormone treatment of Turner syndrome patients, and provide a diagnostic basis for whether Turner syndrome patients need preventive gonadectomy.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material and an application thereof. Background Art

[0002] Turner syndrome, also known as congenital ovarian hypoplasia syndrome, is caused by the complete or partial loss of one X chromosome in all or part of the somatic cells, or other structural abnormalities of the X chromosome. The incidence rate in live female babies is 1 / 2000-1 / 4000, which is one of the common human chromosomal abnormalities. The typical clinical features of Turner syndrome mainly include short stature, gonadal dysgenesis, and special signs such as cubital valgus, webbed neck, shield chest, and low back hairline. About 50% of Turner syndrome patients have a karyotype of 45,X, 20%-30% are mosaics, and the rest have X chromosome structural abnormalities (such as chromosome long arm or short arm deletion 46,X,del (Xq) or 46,X,del (Xp), X chromosome long arm or short arm equal arm 46,X,i (Xq) or 46,X,i (Xp), ring X chromosome 46,X,r (X)). In addition, 5% of Turner syndrome patients contain a Y chromosome, and another 3% of Turner syndrome patients contain an unrecognizable marker chromosome (mar), whose characteristics and origin cannot be identified using traditional cytogenetic banding technology. It may be derived from a pure Y chromosome or a mosaic Y chromosome.

[0003] The chance of developing gonadoblastoma in patients with Turner syndrome is low (about 1%), but if the patient contains a Y chromosome or a fragment derived from the Y chromosome, the risk of developing gonadoblastoma increases by 5%-30%, so timely screening and identification of these patients is particularly important. However, clinically, it is only recommended to use molecular detection technology to screen the Y chromosome sequence for individuals with masculinization characteristics but negative Y chromosome material in traditional cytogenetics and FISH analysis, or Turner syndrome patients with marker chromosomes or ring chromosomes. Therefore, timely detection of whether Turner syndrome patients contain Y chromosomes or fragments derived from the Y chromosome, and then determining whether to preventively remove bilateral gonads, can prevent the occurrence of gonadoblastoma.

[0004] The complete Y chromosome can be directly detected by karyotype analysis, while the mar and Y chromosome fragments derived from the Y chromosome cannot be identified and require the assistance of other molecular detection methods. The commonly used detection methods currently include: fluorescence in situ hybridization (FISH), chromosome microarray (CMA), whole genome sequencing, etc. These technologies generally have the disadvantages of complex operation, high detection cost, and long detection cycle. Multiplex PCR is a technology that can amplify multiple DNA target fragments simultaneously in the same reaction system. Compared with ordinary PCR and other molecular detection technologies, multiplex PCR has the advantages of simple operation, short detection cycle, low cost, and high detection efficiency, and is particularly suitable for multi-site detection of trace samples. At present, there is no report in this field on the use of multiplex PCR to detect Y chromosome material in patients with Turner syndrome. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a multiplex PCR primer set for detecting whether Turner syndrome patients carry Y chromosome material, design multiple pairs of Y chromosome primers, and use the multiplex PCR detection method to quickly screen Y chromosome material from the peripheral blood genome, thereby providing a diagnostic basis for whether Turner syndrome patients should undergo preventive gonadectomy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material, and the nucleotide sequences of the primer set are shown in SEQ ID NO.1-SEQ ID NO.18.

[0008] The present invention also provides an application of the primer set in preparing a product for detecting whether a Turner syndrome patient carries Y chromosome material.

[0009] The present invention also provides an application of the primer set in preparing a product for assessing the risk of gonadoblastoma in patients with Turner syndrome.

[0010] Preferably, the product includes reagents and kits.

[0011] Preferably, the detection includes the following steps: obtaining sample DNA, performing multiplex PCR amplification using the above-mentioned primer set, and observing the results by electrophoresis separation; if the electrophoresis results show only two bands of 499 bp and 294 bp, it indicates that the patient is a Turner syndrome patient with negative Y chromosome material; if the electrophoresis results show other bands besides the two bands, it indicates that the patient is a Turner syndrome patient carrying Y chromosome material.

[0012] Preferably, the reaction system of the multiplex PCR is as follows: 5 μl of primer set mixture, 25 μl of multiplex PCR buffer, 1 μl of multiplex PCR DNA polymerase, 100 ng of genomic DNA, and water to 50 μl; the final concentration of each upstream and downstream primer in the primer set mixture is 0.1 μM.

[0013] Preferably, the reaction procedure of the multiplex PCR is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 90 s, extension at 72°C for 60 s, for a total of 30 cycles; and complete extension at 72°C for 10 min.

[0014] The present invention also provides a kit for detecting whether a Turner syndrome patient carries Y chromosome material, comprising the above primer set.

[0015] Preferably, a normal control and a blank control are also included.

[0016] Preferably, the normal control includes male samples with normal karyotype and female samples with normal karyotype.

[0017] Beneficial effects of the present invention:

[0018] The 9 pairs of primers designed by the present invention cover different regions of the Y chromosome, which is conducive to the detection of Y chromosome materials at different locations and improves the reliability of the detection. The use of the primer set provided by the present invention to detect whether a Turner syndrome patient carries Y chromosome material has the following advantages:

[0019] Low detection cost: Compared with FISH and whole genome sequencing, the multiplex PCR method used in the present invention has a lower detection cost.

[0020] High efficiency: FISH only detects the SRY gene, while the present invention detects 9 genes (including the SRY gene) in different regions of the Y chromosome, with a wider range and higher efficiency. Moreover, a negative FISH detection of SRY does not mean that there is no Y chromosome material (studies have reported that when Turner syndrome patients lack SRY but contain other Y chromosome materials, it can still lead to the occurrence of gonadoblastoma).

[0021] Easy to operate and popularize: Only PCR instrument and gel electrophoresis equipment are needed to complete the test, and no other special equipment is required. The experimental operation is simple and easy to learn, which is conducive to popularization and use. The FISH experiment is complicated and requires professionals to operate the test, and a fluorescence microscope is required to observe the experimental results.

[0022] Strong specificity: The 9 pairs of primers designed have strong specificity, no mixed bands, and no false positive or false negative amplification.

[0023] High detection sensitivity: The amount of genomic DNA required for the detection of the present invention can be as low as 1 ng.

[0024] Short detection cycle: Extract the patient's genome from peripheral blood, then perform multiple PCR tests, and finally observe the results by electrophoresis. The whole process takes only about 5 hours to complete. Whole genome sequencing and chromosome microarray technology require large equipment such as sequencers, and test samples are often sent to sequencing companies for testing, resulting in a long detection cycle, usually taking about 1 month at the fastest, and cannot be diagnosed quickly and in time.

[0025] In summary, the primer set provided by the present invention is used to detect whether a Turner syndrome patient carries Y chromosome material, which has the advantages of low detection cost, high efficiency, simple operation and easy promotion, strong specificity, high detection sensitivity and short detection cycle. It can screen Y chromosome material in Turner syndrome patients before growth hormone treatment, and provide a diagnostic basis for whether Turner syndrome patients need preventive gonadectomy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The locations of the nine genes detected by multiplex PCR on chromosome Y;

[0027] Figure 2 It is the PAGE electrophoresis diagram of multiplex PCR, where M is 100 bp DNA Ladder, 1 is a male with normal karyotype (46, XY), 2 is a female with normal karyotype (46, XX), and 3 is a blank control;

[0028] Figure 3 is the sensitivity test result of the multiplex PCR reaction system, where M is a 100 bp DNA ladder, 1-7 correspond to 100 ng DNA, 50 ng DNA, 10 ng DNA, 1 ng DNA, 0.1 ng DNA, 0.01 ng DNA, 0.001 ng DNA, and 8 is a blank control;

[0029] Figure 4Single gene PCR electrophoresis diagram of 9 genes, including M, 100bp DNALadder, 1, TSPY (118bp), 2, SRY (180bp), 3, SHOX (294bp), 4, RPS4Y2 (311bp), 5, blank control, 6, HSFY1 (431bp), 7, SPRY3 (499bp), 8, DDX3Y (522bp), 9, CDY1 (705bp), 10, TBL1Y (932bp), 11, blank control;

[0030] Figure 5-13 These are the sequencing comparison results of PCR products of TSPY, SRY, SHOX, RPS4Y2, HSFY1, SPRY3, DDX3Y, CDY1, and TBL1Y genes;

[0031] Fig.14 This is the electrophoresis diagram of peripheral blood genome of patients with Turner syndrome;

[0032] Fig.15 These are the results of multiplex PCR detection of Y chromosome material in clinical samples. In Figure A, M is a 100 bp DNA Ladder, 1 is a male with normal karyotype, 2 is a female with normal karyotype, 3 is a blank control, 4 is a male dwarf with karyotype 46,X,+mar, 5 is a Turner syndrome patient with karyotype 45,X

[40] / 47XYY

[21] , 6 and 7 are both Turner syndrome patients with karyotype 45,X,+mar; In Figure B, M is a 100 bp DNA Ladder, 1 is a male with normal karyotype, 2 is a female with normal karyotype, 3 is a blank control, 4 is a female patient with congenital adrenal hyperplasia carrying a Y chromosome, 5 and 6 are both Turner syndrome patients with 45,X monosomy, and 7 is a Turner syndrome patient with 45,X / 46,XX mosaicism. DETAILED DESCRIPTION

[0033] The present invention provides a multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material, and the nucleotide sequence of the primer set is shown as SEQ ID NO.1-SEQ ID NO.18.

[0034] The primer sequences, PCR product sizes and positions on chromosome Y of the multiplex PCR primer set designed by the present invention are shown in Tables 1 and Figure 1 shown.

[0035] Table 1 Related information of the multiplex PCR primer set of the present invention

[0036]

[0037] The present invention uses SHOX (Yp11.2 and Xp22.33) and SPRY3 (Yq12 and Xq28) shared by X and Y chromosomes as positive reference genes, wherein SHOX is located in the pseudo-autosomal 1 region (PAR1 region) of the short arms of X and Y chromosomes, and SPRY3 is located in the pseudo-autosomal 2 region (PAR2 region) of the long arms of X and Y chromosomes. In addition, the testis-specific protein (TSPY) gene is a specific gene encoded on the Y chromosome, located in the gonadoblastoma region (GBY) of the short arm of the Y chromosome, and is directly related to the occurrence and development of gonadoblastoma. This region has multiple tandem repeat sequences (TSPY1, TSPY2, TSPY3, TSPY4, TSPY8, TSPY9, TSPY10) with highly similar sequences. The TSPY sequence primer designed by the present invention can simultaneously identify multiple tandem repeat sequences of TSPY.

[0038] The present invention also provides an application of the above primer set in preparing a product for detecting whether a Turner syndrome patient carries Y chromosome material or in preparing a product for assessing the risk of gonadoblastoma in a Turner syndrome patient.

[0039] In the present invention, the product preferably includes reagents and kits. The detection preferably includes the following steps: obtaining sample DNA, performing multiple PCR amplification using the above primer set, and observing the results by electrophoresis separation; if the electrophoresis result shows only two bands of 499 bp and 294 bp, it indicates that the patient is a Turner syndrome patient with negative Y chromosome material, and if the electrophoresis result shows other bands other than the two bands, it indicates that the patient is a Turner syndrome patient carrying Y chromosome material.

[0040] The present invention does not specifically limit the specific method of obtaining sample DNA, and the sample is preferably peripheral blood. In the present invention, the reaction system of the multiplex PCR is preferably: 5 μl of primer set mixture (10×Primer Mix), 25 μl of multiplex PCR buffer, 1 μl of multiplex PCR DNA polymerase, 100 ng of genomic DNA, and water supplemented to 50 μl in 50 μl; the final concentration of each primer upstream and downstream in the primer set mixture is 0.1 μM. The present invention does not specifically limit the specific sources of the above-mentioned substances. The water is preferably sterile deionized water. In the present invention, the reaction procedure of the multiplex PCR is preferably: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 90 s, extension at 72°C for 60 s, for a total of 30 cycles; thorough extension at 72°C for 10 min.

[0041] The present invention also provides a kit for detecting whether a Turner syndrome patient carries Y chromosome material, comprising the above-mentioned primer set, and preferably, also comprising a normal control and a blank control, wherein the normal control preferably comprises a male peripheral blood sample with a normal karyotype and a female peripheral blood sample with a normal karyotype, and the blank control is preferably sterile deionized water.

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] Peripheral blood was collected from males with normal karyotype (46, XY) and females with normal karyotype (46, XX) using EDTA anticoagulation blood collection tubes. 200 μl of peripheral blood samples were used to extract genomic DNA. The specific extraction steps were performed according to the instructions of the Tiangen Blood Genomic DNA Extraction Kit (DP348). Nanodrop 2000 was used to test the concentration and purity of the samples, that is, the A260 / A280 of the samples was between 1.8-2.0, and A260 / A230>2.0.

[0045] The primer sequences shown in Table 1 were used to perform multiple PCR amplification on the extracted DNA, and sterile deionized water was used as a blank control. The multiple PCR reaction system (50 μl) was: 5 μl of 9 pairs of primer mixtures (10×PrimerMix) (where the final concentration of each primer upstream and downstream was 0.1 μM), 25 μl of multiple PCR buffer, 1 μl of multiple PCR DNA polymerase, 100 ng of genomic DNA, and sterile deionized water to 50 μl. The multiple PCR reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 90 s, extension at 72°C for 60 s, for a total of 30 cycles; complete extension at 72°C for 10 min, and finally storage at 4°C.

[0046] Prepare a 20ml system of 5% non-denaturing polyacrylamide gel, including 3.3ml of 30% Acrylamide, 4ml of 5×TBE, 140μl of 10% ammonium persulfate, 13μl of TEMED, and make up to 20ml with deionized water. Take 5μl of multiple PCR products and load them for non-denaturing polyacrylamide gel electrophoresis. First, electrophorese at a low voltage of 10V. After the sample enters the gel, electrophorese at a voltage of 100V for 60min. After the electrophoresis, use Gel-Green (1:10000) to slowly shake on a shaker for 10min to stain, and finally use a gel imager to observe and take pictures. The results of multiple PCR electrophoresis are as follows: Figure 2 shown.

[0047] Depend on Figure 2It can be seen that the multiplex PCR products of males with normal karyotype (46, XY) have 9 bands, which are TBL1Y (932bp), CDY1 (705bp), DDX3Y (522bp), SPRY3 (499bp), HSFY1 (431bp), RPS4Y2 (311bp), SHOX (294bp), SRY (180bp) and TSPY (118bp) from top to bottom. Therefore, if the Y chromosome is intact, all 9 bands can be amplified. The multiplex PCR products of females with normal karyotype (46, XX) have 2 bands, which are SPRY3 (499bp) and SHOX (294bp) from top to bottom. There are no amplified bands in the blank control group, indicating that there is no DNA contamination in the reagents during the operation. Among them, SHOX (Yp11.2 and Xp22.33) and SPRY3 (Yq12 and Xq28) shared by X and Y chromosomes are used as positive reference genes. According to the electrophoresis results, the bands of the 9 items amplified by the multiplex PCR reaction can be separated independently in the non-denaturing polyacrylamide gel electrophoresis, and the bands that match the size of the corresponding target fragments are amplified, indicating that there is no cross-reaction between the primers in the multiplex PCR detection method constructed by the present invention and the specificity is strong. Therefore, the multiplex PCR reaction system established using the 9 pairs of primers designed by the present invention can be used for the detection of Y chromosome material in female patients with Turner syndrome.

[0048] Example 2

[0049] Multiplex PCR sensitivity test

[0050] In a 50 μl multiplex PCR reaction system, a sensitivity test was performed using a genomic DNA sample of a male with normal karyotype (46, XY). The amount of genomic DNA used was 100 ng, 50 ng, 10 ng, 1 ng, 0.1 ng, 0.01 ng, 0.001 ng, 0 ng (sterile deionized water, blank control). The rest was the same as in Example 1. The results are shown in FIG. Figure 3 As shown. Figure 3 It can be seen that the amount of genomic DNA required for the detection of the present invention can be as low as 1 ng.

[0051] Example 3

[0052] 9 pairs of primers specificity verification:

[0053] Before using the multiplex PCR reaction system, a single primer PCR test was performed to detect specificity, using genomic DNA of a male with normal karyotype (46, XY) as a template, and the concentrations of both upstream and downstream primers were 0.1 μM. The specific steps of the multiplex PCR were the same as those in Example 1. The electrophoresis results are shown in Figure 4As shown, the single primer PCR product has only one specific amplification electrophoresis band. The band size shown in the electrophoresis band position is consistent with the size of the designed 9 pairs of primer PCR products, and there is no mixed band. The Sanger sequencing results of the 9 pairs of primers PCR products are compared (NCBIBLAST) as shown Figure 5-13 As shown, the sequences of the PCR amplified fragments of the 9 pairs of primers are all fragments of the 9 target genes, and there are no other non-specific amplified sequences.

[0054] Example 4

[0055] Peripheral blood was collected from 36 patients with Turner syndrome who visited the Department of Endocrinology and Genetics and Metabolism of the Affiliated Hospital of Jining Medical College from 2013 to 2021 to extract genomic DNA, and multiplex PCR was performed to detect Y chromosome material. Among them, the karyotype of 2 patients showed mar, and the karyotype of 1 patient carried Y chromosome mosaicism. Two healthy males (46, XY) and two females (46, XX) with normal chromosome karyotypes were collected as normal karyotype controls. In addition, a male dwarf with a karyotype of 46, X, + mar and a female patient with congenital adrenal hyperplasia (CAH) were also tested as examples of the application of the present invention to other diseases involving the Y chromosome. The study was approved by the Human Ethics Committee of the Affiliated Hospital of Jining Medical College. All patients and their families have been informed of the purpose of this study and have signed informed consent.

[0056] The specific steps of the genomic DNA extraction method, multiplex PCR amplification method, and electrophoresis method are the same as those in Example 1. After obtaining DNA from peripheral blood of patients with Turner syndrome and testing the concentration and purity of the samples using Nanodrop 2000, the integrity of the extracted genomic DNA was analyzed using 0.8% agarose gel electrophoresis. The results of peripheral blood genomic electrophoresis of 6 patients were as follows: Fig.14 As shown, it is used to illustrate that the peripheral blood genomic DNA extracted by the present invention has good integrity.

[0057] Test results: After multiple PCR amplification and electrophoresis, some results are as follows Fig.15 Among them, only two target bands of SHOX and SPRY3 were amplified in 35 patients with Turner syndrome, and no other Y chromosome-specific bands were found, indicating that the 35 patients with Turner syndrome did not contain Y chromosome material, and the mar of the two patients with Turner syndrome that contained mar may come from other chromosomes ( Fig.15 A, lanes 6 and 7). One patient with Turner syndrome whose karyotype was 45,X

[40] / 47XYY

[21] had 9 amplified bands, indicating that the patient had a complete Y chromosome ( Fig.15A, Lane 5). In a male dwarf patient with 46,X,+mar, in addition to the SHOX and SPRY3 bands, four fragments of TSPY, SRY, DDX3Y and TBL1Y, which are unique to the Y chromosome, were amplified, while CDY1, HSFY1 and RPS4Y2 were not amplified, indicating that the Y chromosome of this patient has a fragment deletion near the CDY1 (Yq11.23), HSFY1 (Yq11.222) and RPS4Y2 (Yq11.223) genes, and that the patient's mar is very likely to come from the Y chromosome ( Fig.15 A, Lane 4). Nine bands were amplified from a female patient with congenital adrenal hyperplasia, indicating that the female patient had a Y chromosome ( Fig.15 B, lane 4).

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> The Affiliated Hospital of Jining Medical College <120> A multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material and its application <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <400> 1 ttctgttctg gtcttgctgc tgtccttg 28 <210> 2 <211> twenty four <212> DNA <213> Artificial Sequence <400> 2 tgccgtgtgc catcctcgct atgt 24 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <400> 3 gacctcgtcg gaaggcgaag atgct 25 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 tggctgcgtt gatgggcggt aagt 24 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <400> 5 cctcattcct cttgctggct cacatactg 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 tgtcctccac tgttgtgcta tctcacttcc 30 <210> 7 <211> twenty two <212> DNA <213> Artificial Sequence <400> 7 tgtttcttcg gcctttctag tg 22 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 cacattggtc cgtggcaac 19 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <400> 9 gtggaggagc aagtacagcg agcagtaa 28 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <400> 10 taggttcttc gtcggcaaca gcagtagg 28 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <400> 11 agttcttcgc tcacgctcta tcttccatcc 30 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <400> 12 gaatgagagt gccacaggtt gaggagaca 29 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <400> 13 gtgaagaagc agacagaagc gaggtgat 28 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <400> 14 ggcagtagca gagataagac ttggaagcag 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <400> 15 gcagcagatc agcaggacac ggtggtcttc 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <400> 16 aggcaggatg gatgcaccta gtccaatcgc 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <400> 17 gctccactga tgatgaagac aactgtgctg 30 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <400> 18 gctggcaggg tgtaaagact gaagagga 28

Claims

1. A multiplex PCR primer set for detecting whether a Turner syndrome patient carries Y chromosome material, characterized in that: The nucleotide sequences of the primer set are shown in SEQ ID NO.1-SEQ ID NO.

18.

2. Use of the primer set according to claim 1 in preparing a product for detecting whether a Turner syndrome patient carries Y chromosome material.

3. Use of the primer set according to claim 1 in the preparation of a product for assessing the risk of gonadoblastoma in patients with Turner syndrome.

4. The use according to claim 2 or 3, characterized in that: The product includes a reagent or a kit.

5. The use according to claim 2, characterized in that: The detection comprises the following steps: obtaining sample DNA, performing multiplex PCR amplification using the primer set described in claim 1, and observing the results by electrophoresis separation; if only two bands of 499 bp and 294 bp appear in the electrophoresis results, it indicates that the patient is a Turner syndrome patient with negative Y chromosome material; if other bands other than the two bands appear in the electrophoresis results, it indicates that the patient is a Turner syndrome patient carrying Y chromosome material.

6. The use according to claim 5, characterized in that: The reaction system of the multiplex PCR is calculated as follows: 5 μl of primer set mixture, 25 μl of multiplex PCR buffer, 1 μl of multiplex PCR DNA polymerase, 100 ng of genomic DNA, and water to 50 μl; the final concentration of each upstream and downstream primer in the primer set mixture is 0.1 μM.

7. The use according to claim 5, characterized in that: The reaction procedure of the multiplex PCR is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 90 s, extension at 72°C for 60 s, for a total of 30 cycles; and complete extension at 72°C for 10 min.

8. A kit for detecting whether a patient with Turner syndrome carries Y chromosome material, characterized in that: Comprising the primer set according to claim 1.

9. The kit according to claim 8, characterized in that Normal control samples and blank control samples were also included.

10. The kit according to claim 9, characterized in that The normal control samples include male samples with normal karyotype and female samples with normal karyotype.

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