STR primer group, kit and method for detecting polyploidy, UPD and maternal pollution in sample

Abstract

The invention belongs to the field of gene detection, and provides an STR primer group, a kit and a method for detecting polyploid, UPD and maternal pollution in a sample, the primer group comprises a first primer group, the first primer group comprises 178 pairs of primers, and the nucleotide sequences of the 178 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO.356. The kit comprises an amplification reaction solution, dNTP, DMSO, nuclease-free water and the STR primer group. By adopting the primer group and the kit disclosed by the invention, the polyploidy of a sample, the monoparental diploid of chromosome level and whole genome level and the condition of maternal pollution can be detected by the method at the same time. And Cnv-seq or PGT-A can be configured for use, so that the problem that polyploidy and single-parent diploid maternal pollution cannot be detected by the two technologies at present can be effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, relates to STR genetic marker technology with good application value in human genome, and specifically relates to an STR primer set, a kit and a method for detecting polyploidy, UPD and maternal contamination in a sample. Background Art

[0002] The genome copy number variation sequencing technology (CNV-seq) based on high-throughput sequencing can detect abnormal chromosome numbers, large fragment deletions / duplications, and pathogenic copy number variations, and is widely used in prenatal diagnosis, assisted reproduction, and pediatric genetic disease auxiliary diagnosis. However, polyploidy, uniparental diploidy, and maternal cell contamination in samples cannot be detected by CNV-seq, and short tandem repeat sequences (STR) are needed to make up for the shortcomings of CNV-seq.

[0003] Preimplantation chromosomal aneuploidy testing (PGT-A) is a genetic test for embryonic chromosomal aneuploidy before implantation during assisted reproduction. This technology can guide the selection of high-quality embryos with euploid chromosomes for transplantation. However, during the testing process, it is also faced with the situation that polyploidy and uniparental diploidy at the chromosome level cannot be directly detected. Short tandem repeats (STR) analysis is also required to overcome the above defects.

[0004] At present, STR loci detection mostly adopts PCR amplification followed by electrophoresis separation, or multi-color fluorescent dye labeling is usually used when building a multi-locus fluorescent labeling composite amplification detection system. The principle is that the length ranges of allele fragments of loci labeled with the same fluorescent dye must not overlap, and the length ranges of allele fragments of loci labeled with different fluorescent dyes can overlap. In this way, different loci can be distinguished according to the color of fluorescence and the size region of allele fragments. However, due to the limitation of electrophoresis resolution and the type of fluorescent dyes, the number of STR loci that can be detected at one time is limited by this scheme, and it is impossible to detect hundreds of STR loci at one time, and it cannot effectively detect uniparental disomy at the chromosome level.

[0005] Therefore, there is a need to provide a method that can simultaneously make up for the defects of CNV-seq and PGT-A that cannot detect polyploidy, UPD (uniparental disomy) and maternal contamination in samples. Summary of the invention

[0006] In order to solve the defect that CNV-seq and PGT-A cannot detect polyploidy, UPD (uniparental disomy) and maternal contamination in samples, the present invention discloses an STR primer set, a kit and a method for detecting polyploidy, UPD and maternal contamination in samples.

[0007] In a first aspect, the present invention provides an STR primer set for detecting polyploidy, UPD and maternal contamination in a sample, the primer set comprising a first primer set, the first primer set comprising 178 pairs of primers, the nucleotide sequences of the forward primers in the 178 pairs of primers are shown in SEQ ID NO.1-SEQ ID NO.178, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.179-SEQID NO.356.

[0008] In an improved embodiment, the primer set also includes a second primer set, which includes 10 pairs of primers, the nucleotide sequences of the forward primers in the 10 pairs of primers are shown in SEQ ID NO.357-SEQ ID NO.366, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.367-SEQ ID NO.376.

[0009] The second aspect of the present invention provides a method for designing the STR primer set described in the first aspect, the design method comprising: In the STR database, for all STR loci of 23 pairs of human chromosomes, STR loci containing polynucleotides are removed; STR loci with polymorphic information content PIC>0.2 are retained; STR loci with repeating unit base numbers of 3, 4, 5 and 6 are retained; STR loci with a total length greater than 150 bp are removed; STR loci with a GC content greater than 60% within 200 bp upstream and downstream are removed; STR loci with 200 bp upstream and downstream sequences having homology in the human genome are removed, and 178 STR loci are obtained, and primers are designed for each of the STR loci to obtain a first primer set.

[0010] In an improved embodiment, the design method further includes extracting STR loci with a sequencing depth ≤ a sequencing depth threshold from the 178 STR loci as low-depth STR loci, and designing primers for each of the low-depth STR loci to obtain a second primer set.

[0011] The third aspect of the present invention provides a kit for detecting polyploidy, UPD and maternal contamination in a sample, the kit comprising an amplification reaction solution, an enhancer dNTP, an enhancer DMSO, nuclease-free water and the STR primer set described in the first aspect, wherein the amplification reaction solution comprises a hot start polymerase, Tris buffer, KCL, MgCl 2 NH 4 ) 2 SO 4 , DNTP, betaine, Triton, BSA, Tween and glycerol.

[0012] A fourth aspect of the present invention provides a method for detecting polyploidy, UPD and maternal contamination in a sample, comprising: S1. Obtain gDNA or whole genome amplification products of the sample to be tested, wherein the sample to be tested includes offspring samples and offspring mother samples when performing maternal contamination judgment, and the sample to be tested is an offspring sample when performing polyploidy judgment and UPD judgment; S2. Using the primer set described in the first aspect or the kit described in the second aspect, perform multiple targeted amplification and purification on the gDNA or the whole genome amplification product, construct a DNA library through the purified products after multiple targeted amplification, and perform sequencing to obtain sequencing data; S3. According to the sequencing data, the polyploidy, UPD and maternal contamination of the sample to be tested are judged Compared with the prior art, the beneficial effects that can be achieved by at least one of the above technical solutions adopted in the embodiments of this specification include at least: the STR primer set, kit and detection method for detecting polyploidy, UPD and maternal contamination in the sample of the present invention can realize the ploidy identification of gDNA samples and the identification of maternal contamination. At the same time, the triploidy and uniparental diploidy of the cell whole genome amplification product can be analyzed. The present invention is simple to operate, compatible with CNV-Seq and PGT-A, and can make up for the defect that CNV-seq cannot detect polyploidy and maternal contamination. The high-throughput STR detection scheme can also make up for the defect that hundreds of STR loci can be detected simultaneously due to the troubles of dyes and professional instruments based on PCR-capillary electrophoresis or fluorescent PCR-capillary electrophoresis. DETAILED DESCRIPTION

[0013] The following describes the implementation methods of the present application through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and the features of the embodiments can be combined with each other in the absence of conflict. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0014] One embodiment of the present invention provides an STR primer set for detecting polyploidy, UPD and maternal contamination in a sample. The primer set is designed based on all STR loci for 23 pairs of human chromosomes in the STR database through the following process: 1. Remove the STR loci containing the polynucleotide; 2. Retain the STR loci with polymorphic information content PIC>0.2; retain the STR loci with repeating unit base numbers of 3, 4, 5 and 6; 3. Remove STR loci with a total length greater than 150 bp; 4. Remove STR loci with GC content greater than 60% within 200 bp upstream and downstream; 5. Remove the STR loci whose upstream and downstream 200 bp sequences have homology in the human genome.

[0015] Through the above operation, 178 STR loci can be obtained, and primers are designed for each STR locus to obtain a first primer set, wherein the first primer set includes 178 pairs of primers, the nucleotide sequences of the forward primers in the 178 pairs of primers are shown in SEQ ID NO.1-SEQ ID NO.178, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.179-SEQ ID NO.356.

[0016] Among them, 178 STR basis loci are included: D1S1656, GATA23G09, TTTA063, GATA26G09, AAT259, AAT252, Human_STR_99081 / F13B, AATA011, TPOX, D2S441, GAAT1A 5, GATA8F07, GATA176C01, GATA8H05, GATA194A05, AAT203, D3S1358, ATCT053, GATA146D07, ATC4D07, AAT071, ATA22E01, GATA22G05, ATC T018, GATA24H01, GATA2F11, ATA26B08, GATA8A05, ATT077P, FGA, ATA20G07, GATA63C02, GATA51A07, D5S818, CSF1PO, AAT013, SE30, ATC0 33, GATA11B08, D6S1043, GATA23F08, ATA6C09P, D6S1009, G15833, D6S494, TATT019, TAT028, D7S820, ATA55A05, TATG002, D7S1818, D7S18 20, D7S3052, ATT023, Human_STR_1395783, GATA156H01, AAT076, D8S1179, LPL, MFD455-AAT052, GATA165A11, GATA89A11, ATA42G04, ATC C001, ATA24F10, TTA007, D10S1248, TH01, ATA25D12, GATA46A12, AAT228, D11S2362, D11S4465, D11S1981, D11S1392, D11S2363, D11S4952 , D11S1998, AAT262, ATA73C05, GATA63D12, GATA5H03, vWA, ATA5A09, GATA11C08, D13S317, AGAT113Z, D13S1493, D13S801, D13S1824, D13 S796, D14S1434, ATA70B03, GGAA30H04, D14S1280, D14S121, D14S748, D14S125, D14S588, D14S617, D14S1426, TAT032z, GATA85D02, PentaE. D15S1513, D15S822, D15S1232, D15S659, D15S643, D15S1507, D15S816, D15S1515, ATA 67B07, GATA151C03, D16S539, GAAT2C03, GATA25A04, ATA58A02, AGAT060, D18S51, D18S86 2. D18S878, D18S391, D18S865, D18S1371, D19S433, TGA012, AAT257, GATA81E09, TATT031 , D20S482, D20S604, D20S1145, D20S607, D20S1085, D20S469, D20S1082, GGAA3C07, Penta D. D21S1432, D21S1409, D21S1442, D21S226, D21S1413, D21S11, AGAT120, UT7136, D22S1045, Human_STR_1547748, DXS7132, DXS10074, DXS8378, HPRTB, DXS981, DXS10135, DYS19, DYS391 , DYS439, DYS438, DYS437, DYS392, DYS393, DYS456, DYS458, DYS388, DYS426, Y-GATA-H4, DYS 435, DYS436, DYS462, DYS549, DYS533, DYS570, DYS643, DYS434, DYS460, DYS461 and Y-GATA-A10.

[0017] Further, when designing primers for the screened STR loci, STR loci with lower sequencing depths are extracted according to the sequencing depth threshold, and STR loci including GATA8F07, GATA89A11, DXS10074, YGATAA10 and DYS19 are obtained. These five STR loci are defined as low-depth STR loci, and a method of designing several more pairs of primers for each low-depth STR locus is used to improve its sequencing depth. That is, it can be understood that primers are designed for each low-depth STR locus to form a second primer set, and the second primer set includes 10 pairs of primers. The nucleotide sequence of the forward primer in the 10 pairs of primers is shown in SEQ ID NO.357-SEQIDNO.366, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.367-SEQ ID NO.376. The 10 pairs of primers of the second primer set are combined with the 178 pairs of primers of the first primer set to obtain 188 pairs of primers, and the 188 pairs of primers constitute a primer composition with relatively uniform amplification.

[0018] The second embodiment of the present invention provides a kit, which includes the primer set shown in the above embodiment, and further includes: an amplification reaction solution, an enhancer dNTP, an enhancer DMSO and nuclease-free water, wherein the amplification reaction solution includes a high-fidelity hot-start polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl 2 , 50~100mM (NH 4 ) 2 SO 4 , 50-200um DNTP, 1000~1500mM betaine, 0.19%~0.38% Triton, 2~3mg / ml BSA, 5%~15% Tween and 2.5%~7.5% glycerol. It should be noted that the components of the enhancer dNTP and DNTP are the same. The difference is that since the DNTP used in conventional PCR amplification has a concentration range, a high dntp concentration will affect the amplification efficiency. However, because multiple PCR is used, only an additional part needs to be added in the first round to promote the multiple PCR reaction. Therefore, a part is named DNTP separately for distinction.

[0019] The third embodiment of the present invention provides a method for detecting polyploidy, UPD and maternal contamination in a sample, comprising: S1. Obtain gDNA or whole genome amplification products of the sample to be tested, wherein the sample to be tested includes offspring samples and offspring mother samples when judging maternal contamination, and the sample to be tested is an offspring sample when judging polyploidy and UPD; the sample to be tested is selected from any one of peripheral blood, saliva, hair follicles and blastocyst cells, wherein blastocyst cells can be derived from abortion tissue and non-embryonic tissue in in vitro cultured embryos. gDNA is extracted from peripheral blood, saliva, and hair follicles, and the whole genome amplification product is obtained by WGA amplification of blastocyst cells.

[0020] S2. Use the primer set described in the first aspect or the kit described in the second aspect to perform multiple targeted amplification and purification on the gDNA or the whole genome amplification product, construct a DNA library through the purified products after multiple targeted amplification and perform sequencing to obtain sequencing data.

[0021] S3. According to the sequencing data, the polyploidy, UPD and maternal contamination of the sample to be tested are judged, including: S31, aligning the data to be tested with the reference genome sequence fragments of the STR locus corresponding to each primer in the primer set, calculating the copy number and the number of reads of each STR locus, calculating the proportion of the number of reads with a ratio of 1:1:1 in all the STR loci, and performing polyploidy judgment on the sample to be tested according to the threshold and the proportion.

[0022] Specifically, the target region of the sequencing data can be located, that is, it is compared with the reference genome sequence fragment (i.e., the sequence of the STR locus) used to design primers, and the copy number and corresponding read number of each genome repeat unit after the comparison are calculated. Among them, the STR locus is divided into two cases, the first is a homozygous site, which has only one repeat unit; the second is a heterozygous site, which has two repeat units, and the corresponding read number ratio is divided into (1:1, 1:1:1, 2:1), among which the site with a ratio of 1:1:1 accounts for a small proportion in normal diploid samples, but is common in polyploid samples. Therefore, the read number ratio can be used to judge the ratio of the 1:1:1 site to determine whether the sample to be tested is polyploid.

[0023] Among them, the threshold setting for polyploidy judgment can be shown in Table 1 below: Table 1: Thresholds and ploidy categories for polyploidy determination

[0024] It should be noted that in the last item of Table 1 above, when the reads ratio cannot be determined, it is not considered when determining the ploidy.

[0025] S32. Perform UPD judgment on the sample to be tested according to the heterozygosity of STR loci. Specifically, for uniparental disploid (UPD) samples, since all homologous chromosomes are derived from the same parent, the uniparental disploid at the chromosome level and the uniparental disploid at the whole genome level lack heterozygosity, making all sites homozygous sites. Then, the heterozygosity of the STR loci of the chromosome is used to analyze whether the sample is uniparental disploid.

[0026] S33, calculating the proportion of maternal contamination in the sample to be tested according to the calculated proportion of maternal specific loci in the sequencing data. Specifically, when a sample has maternal contamination, there will be maternal specific loci in the STR loci in the sequencing data, and the proportion of maternal contamination in the sample can be determined by calculating the proportion of maternal specific loci.

[0027] The present invention provides a detailed description of the above primer set, kit and detection method through the following examples: Example 1: Screening STR loci and designing primers for each STR locus The screening of STR loci was based on the genome version hg19. Specific primers were designed according to the STR loci, and genomic DNA was used as the detection object. A large number of experimental screening, optimization and verification were carried out, and finally 188 pairs of primers with high amplification efficiency and good specificity were selected. The nucleotide sequences of the 188 pairs of primers are shown in SEQ ID NO.1-SEQ ID NO.376.

[0028] Example 2: A DNA library is established and sequenced to obtain sequencing data, and the sequencing data is analyzed to obtain the polyploidy results of the sample.

[0029] (1) In this example, a triploid sample is used as a sample to be tested, a gDNA sample of the triploid sample is obtained, and the concentration of the gDNA sample is detected: a first-round PCR reaction system and program, and a second-round PCR reaction system and program are set. The first-round PCR reaction system is shown in Table 2 below, and the reaction program is shown in Table 3 below; the second-round PCR reaction system is shown in Table 4 below, and the reaction program is shown in Table 5 below; Table 2: First round PCR reaction system

[0030] The concentration of gDNA samples was quantified using Qubit (Thermo Fisher).

[0031] Table 3: First round PCR reaction program

[0032] When performing the first round of PCR reaction, place the PCR tube containing the reaction reagent into the PCR instrument, set the PCR program according to the table above, and perform the first round of amplification. After amplification, perform magnetic bead purification, that is, use vortex mixing to perform instant centrifugation, add 27 μl of AMPure XP magnetic beads equilibrated at room temperature to 30μl of PCR product, pipette or vortex mixing, and purify. After purification, elute the product with 24ul of nuclease-free water.

[0033] Table 4: Second round PCR reaction system

[0034] Table 5: Second round PCR reaction program

[0035] When performing the second round of PCR reaction, place the PCR tube containing the reaction reagent into the PCR instrument, set the PCR program according to the table above, and perform the second round of amplification. After the second round of amplification, perform magnetic bead purification, that is, centrifuge the PCR tube instantaneously, add 36 μl of AMPure XP magnetic beads equilibrated at room temperature to 30 μl of PCR product, purify the product, elute with 24ul of nuclease-free water after purification, and transfer 20ul of supernatant to a new PCR tube.

[0036] (2) DNA library quantification and quality control First, use Qubit Fluorometer to detect the concentration of the product after two amplifications and purification in (1). The normal range is 5ng / ul-50ng / ul. The concentration is mainly related to the quality of the template.

[0037] Secondly, take 10 μl of the library (concentration of 1-2 ng / uL) sample and use the Qsep100 fully automatic nucleic acid protein analysis system to measure the length and purity of the library fragments. The fragment distribution range of a normal library is about 350 bp.

[0038] (3) Sequencing of DNA library The machine was operated according to the instructions of the miseq DX sequencing reagent. The raw data after sequencing was in fastq format. Short fragment sequences less than 50 bp were removed from the files after the machine, and then the genotypes of each locus were analyzed using gangSTR software.

[0039] (4) Count the proportion of 1:1:1 genotypes to determine whether the sample is polyploid. The results of partial genome testing are shown in Table 6 below. Table 6: Results of some loci detected in triploid samples

[0040] Statistical calculations were performed using the statistical method in Table 6 above: a total of 141 loci were detected in this sample, of which 13 loci could not determine the genotype, 19 1:1:1 loci and 2 2:1 loci, for a total of 66, accounting for 51% of the loci for which the genotype could be determined. The proportion of 1:1:1 loci and 2:1 loci in normal diploid samples did not exceed 15%, so this sample was determined to be a triploid sample.

[0041] Example 3: Using the uniparental disomy of chromosome 6 as the sample to be tested, according to the library construction and sequencing steps in Example 2 above, a DNA library is established and sequenced to obtain sequencing data, and the sequencing data is analyzed to obtain the uniparental disomy results of the sample. The results are shown in Table 7 below; Table 7: Results of uniparental disomy test for chromosome 6

[0042] According to Table 7 above, the STR loci of chromosome 6 of this sample are all homozygous and lack heterozygous sites, so this sample is determined to be uniparental diploid of chromosome 6.

[0043] Example 4: Peripheral blood, saliva, and hair follicles of a mother and her child were used as samples, and gDNA was extracted respectively. The mother's gDNA and the child's gDNA were diluted to the same concentration, and the mother's and child's gDNA were mixed at a volume ratio of 25% and 30%. According to the library construction and sequencing steps in Example 2 above, a DNA library was established and sequenced to obtain sequencing data, and the sequencing data was analyzed to obtain the maternal contamination results of the samples.

[0044] Among them, the proportion of maternal source pollution is based on the following formula: , the final contamination ratio is the average of all the sites' contamination ratios. The calculation results are shown in Tables 8 and 9 below: Tables 8 and 9 only show the available sites among the 178 sites, listing the cases of 30% contamination and 25% contamination. The method of the present invention can detect 20% contamination.

[0045] Table 8: Test results of maternal contamination (30%)

[0046] Table 9: Test results of maternal contamination (25%)

[0047] Example 5: A triploid gDNA sample was used as a test sample, diluted to 18 pg, and then amplified using an MDA whole genome amplification kit. The kit of the present invention was used to build a library and sequence the product after whole genome amplification to obtain sequencing data. The sequencing data was analyzed to obtain the test results of the sample. The results of some sites are shown in Table 10 below: Table 10: Detection results of ploidy gDNA samples

[0048] According to the statistical calculation of the loci in Table 10 above, there are 144 non-homozygous loci in this sample, of which 20 are 1:1:1 loci, accounting for 13.8%. The proportion of 1:1:1 loci in normal diploid samples is less than 10%, so this sample is judged to be triploid.

[0049] The embodiments of the present invention achieve the following technical effects: the primer set, the kit and the analysis method of the present invention can detect 178 STR loci at the same time, and compared with the conventional PCR method, the throughput is high, the signal value is more, and therefore it is more reliable and accurate. At the same time, the method can detect polyploidy of the sample, uniparental diploidy at the chromosome level and the whole genome level, and maternal contamination. Due to the limitation of the throughput, the PCR method cannot detect UPD at the chromosome level, but can only detect UPD at the whole genome level.

[0050] At the same time, the primer set and kit of the present invention, when used with Cnv-seq or PGT-A, can effectively solve the problem that these two current technologies cannot detect polyploidy and uniparental diploid maternal contamination.

[0051] Obviously, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the embodiments of the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A STR primer set for detecting polyploidy, UPD and maternal contamination in a sample, characterized in that: The primer set includes a first primer set, which includes 178 pairs of primers. The nucleotide sequences of the forward primers in the 178 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO.178, and the nucleotide sequences of the reverse primers are shown as SEQ ID NO.179-SEQ ID NO.

356.

2. The STR primer set according to claim 1, characterized in that: The primer set also includes a second primer set, which includes 10 pairs of primers. The nucleotide sequences of the forward primers in the 10 pairs of primers are shown in SEQ ID NO.357-SEQ ID NO.366, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.367-SEQ ID NO.

376.

3. A method for designing a STR primer set according to claim 1 or 2, characterized in that: The design method comprises: In the STR database, for all STR loci of 23 pairs of human chromosomes, STR loci containing polynucleotides are removed; STR loci with polymorphic information content PIC>0.2 are retained; STR loci with repeating unit base numbers of 3, 4, 5 and 6 are retained; STR loci with a total length greater than 150 bp are removed; STR loci with a GC content greater than 60% within 200 bp upstream and downstream are removed; STR loci with 200 bp upstream and downstream sequences having homology in the human genome are removed, and 178 STR loci are obtained, and primers are designed for each of the STR loci to obtain a first primer set.

4. The method for designing a STR primer set according to claim 3, characterized in that: 178 STR base loci included: D1S1656, GATA23G09, TTTA063, GATA26G09, AAT259, AAT252, Human_STR_99081 / F13B, AATA011, TPOX, D2S441, GAAT1A5, G ATA8F07, GATA176C01, GATA8H05, GATA194A05, AAT203, D3S1358, ATCT053, GATA146D07, ATC4D07, AAT071, ATA22E01, GATA22G05, ATCT01 8, GATA24H01, GATA2F11, ATA26B08, GATA8A05, ATT077P, FGA, ATA20G07, GATA63C02, GATA51A07, D5S818, CSF1PO, AAT013, SE30, ATC033, GATA11B08, D6S1043, GATA23F08, ATA6C09P, D6S1009, G15833, D6S494, TATT019, TAT028, D7S820, ATA55A05, TATG002, D7S1818, D7S1820 , D7S3052, ATT023, Human_STR_1395783, GATA156H01, AAT076, D8S1179, LPL, MFD455-AAT052, GATA165A11, GATA89A11, ATA42G04, ATCC0 01, ATA24F10, TTA007, D10S1248, TH01, ATA25D12, GATA46A12, AAT228, D11S2362, D11S4465, D11S1981, D11S1392, D11S2363, D11S4952, D11S1998, AAT262, ATA73C05, GATA63D12, GATA5H03, vWA, ATA5A09, GATA11C08, D13S317, AGAT113Z, D13S1493, D13S801, D13S1824, D13S 796, D14S1434, ATA70B03, GGAA30H04, D14S1280, D14S121, D14S748, D14S125, D14S588, D14S617, D14S1426, TAT032z, GATA85D02, PentaE, D15S1513, D15S822, D15S1232, D15S659, D15S643, D15S1507, D15S816, D15S1515, ATA67B07, GATA151C03, D16S539, GAAT2C03, GATA25A04, ATA58A02, AGAT060, D18S51, D18S862, D18S878, D18S391, D18S865, D18S1371, D19S433, TGA012, AAT257, GATA81E09, TATT031, D20S482, D20S604, D20S1145, D20S607, D20S1085, D20S469, D20S1082, GGAA3C07, Penta D, D21S1432, D21S1409, D21S1442, D21S226, D21S1413, D21S11, AGAT120, UT7136, D22S1045, Human_STR_1547748, DXS7132, DXS10074, DXS8378, HPRTB, DXS981, DXS10135, DYS19, DYS391, DYS439, DYS438, DYS437, DYS392, DYS393, DYS456, DYS458, DYS388, DYS426, Y-GATA-H4, DYS435, DYS436, DYS462, DYS549, DYS533, DYS570, DYS643, DYS434, DYS460, DYS461 and Y-GATA-A10.

5. The method for designing a STR primer set according to claim 4, characterized in that: The design method further comprises: STR loci with sequencing depth ≤ sequencing depth threshold among 178 STR loci were extracted as low-depth STR loci, and primers were designed for each of the low-depth STR loci to obtain a second primer set, wherein the low-depth STR loci included GATA8F07, GATA89A11, DXS10074, YGATAA10 and DYS19.

6. A kit, characterized in that: The invention comprises an amplification reaction solution, an enhancer dNTP, an enhancer DMSO, nuclease-free water and the STR primer set as claimed in claim 1 or 2, wherein the amplification reaction solution comprises a hot-start polymerase, Tris buffer, KCL, MgCl2, (NH4)2SO4, DNTP, betaine, Triton, BSA, Tween and glycerol.

7. A method for detecting polyploidy, UPD and maternal contamination in a sample, characterized in that: include: Obtaining gDNA or whole genome amplification products of the sample to be tested, wherein the sample to be tested includes offspring samples and offspring mother samples when performing maternal contamination judgment, and the sample to be tested is an offspring sample when performing polyploidy judgment and UPD judgment; Using the primer set according to claim 1 or 2 or the kit according to claim 6 to perform multiple targeted amplification and purification on the gDNA or the whole genome amplification product, constructing a DNA library through the purified products after multiple targeted amplification and sequencing to obtain sequencing data; According to the sequencing data, the polyploidy, UPD and maternal contamination of the sample to be tested are judged, including: Compare the data to be tested with the reference genome sequence fragments of the STR loci corresponding to each primer in the primer set, calculate the number of reads of each STR locus, calculate the proportion of the number of reads with a ratio of 1:1:1 in all the STR loci, and perform polyploidy judgment on the sample to be tested according to the threshold and the proportion; Performing UPD judgment on the sample to be tested according to the heterozygosity of the STR locus; The proportion of maternal contamination in the sample to be tested is calculated based on the calculated proportion of maternal-specific loci in the sequencing data.

8. The method for detecting polyploidy, UPD and maternal contamination in a sample according to claim 7, characterized in that: The sample to be tested is selected from any one of peripheral blood, saliva, hair follicles and blastocyst cells.

9. The method for detecting polyploidy, UPD and maternal contamination in a sample according to claim 7, characterized in that: Multiplex targeted amplification includes two rounds of PCR reactions. The first round of PCR reaction system includes: 40-50ng of gDNA or whole genome amplification product, 5ul of primer set, 10ul of amplification reaction solution, 3.5ul of enhancer dNTP and 2.5ul of enhancer DMSO, and the system volume is supplemented to 30u with nuclease water; The second round PCR reaction system includes: 13.5ul of the first round PCR amplification product, 2.5ul of enhancer DMSO, 2ul of nuclease water, 2ul of universal adapter and 10ul of amplification reaction solution.

10. The method for detecting polyploidy, UPD and maternal contamination in a sample according to claim 9, characterized in that: The first round of PCR amplification program was: 95°C for 3-4 min; 15-20 cycles (98°C for 20 s, 60°C for 2-5 min) 72°C for 5-10 min, and 16°C hold; The second round of PCR amplification program was 95°C for 3-4 min; 6-15 cycles (98°C for 20 s, 58°C for 1-3 min) and 72°C for 30 s-2 min, and maintained at 16°C.

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