Iron nutrition deficiency or overload risk assessment method for gestational diabetes mellitus patient

Abstract

The invention discloses an iron nutrition deficiency or overload risk assessment method for gestational diabetes patients, and relates to molecular biology. The primer group for evaluating the iron nutrition deficiency or overload risk of the gestational diabetes mellitus patient is provided, the primer group is used for amplifying the mononucleotide polymorphic site of the hepcidin coding gene, and the primer length is 18-30 bp. And the single nucleotide polymorphic sites comprise rs10416533, rs7251432, rs8101606, rs55863037 and rs2293689, and the single nucleotide polymorphic sites comprise rs10416533, rs7251432, rs8101606, rs55863037, the primer group is as shown in SEQ ID NO: 1-15. The invention provides a detection kit comprising the primer group and a method for evaluating iron nutrition deficiency or overload risk of gestational diabetes mellitus patients by adopting the detection kit. The method has the advantages of ultrahigh flexibility, full-automatic data analysis, high PCR multiple number and high conversion success rate. The insertion and deletion INDEL and translocation fusion genes can be effectively detected, and the accuracy rate gt is high; the yield is 99.7%. The sample flux and the cost performance are high.

Description

Technical Field

[0001] The present invention relates to molecular biology, and in particular to a method for assessing the risk of iron deficiency or overload in patients with gestational diabetes based on the detection of hepcidin encoding gene polymorphism during pregnancy using the MassARRAY® MALDI-TOF System (time-of-flight mass spectrometry biochip system). Background Art

[0002] Iron is potentially toxic. It causes oxidative stress by catalyzing the production of reactive oxygen species, leading to β-cell damage and dysfunction. Iron also affects hypoxia-inducible factor, which can downregulate glucose transporters and reduce glucose tolerance. In living systems, both iron deficiency and iron overload can be harmful. Pregnant women are particularly susceptible to iron deficiency and related adverse pregnancy outcomes (Rawal S, et al. A longitudinal study of iron status during pregnancy and the risk of gestational diabetes: findings from a prospective, multiracialcohort. Diabetologia 2017;60(2):249-57]), such as premature birth and vaginal bleeding. However, iron overload may be a source of free radical development, which may cause oxidative damage to DNA, lipids and proteins. During pregnancy, oxidative stress caused by iron supplementation may harm the fetus and increase the risk of congenital defects, premature birth and low birth weight in the short term.

[0003] Studies have shown that iron metabolism is related to insulin resistance in women with GDM, suggesting that changes in iron metabolism may be involved in the pathogenesis of GDM by aggravating stress hyperglycemia. Studies have shown that hepcidin levels in women with GDM are significantly increased (AmstadBencaiova G, et al. Serum hepcidin and iron status parameters in pregnant women and the association with adverse maternal and fetal outcomes: a studyprotocol for a prospective cohort study. BMJ open 2019;9(11):e032280. doi:10.1136 / bmjopen-2019-032280). Hepcidin is a 25-amino acid peptide secreted by the liver and excreted by the kidneys. Hepcidin is a key regulator of iron homeostasis, controlling the expression of ferroportin on the surface of enterocytes and macrophages (Nemeth E, et al. Hepcidin regulates cellular iron efflux by binding toferroportin and inducing its internalization. Science (New York, NY) 2004;306(5704):2090-3. doi: 10.1126 / science.1104742 [published Online First: 2004 / 10 / 30]). Hepcidin blocks the entry of iron into the bloodstream by binding to ferroportin on the plasma membrane of cells in various tissues, inducing its internalization and degradation (Hamed HM, et al. The association of TMPRSS6 gene polymorphismwith iron status in Egyptian children (a pilot study). BMC pediatrics 2024;24(1):105. doi: 10.1186 / s12887-024-04573-w).Certain polymorphisms in the promoter region of the hepcidin encoding gene (HAMP) are associated with low expression of the hormone, thereby increasing serum iron levels (Hamed HM, et al. The association of TMPRSS6 gene polymorphism with iron status in Egyptian children (a pilot study). BMC pediatrics 2024;24(1):105. doi: 10.1186 / s12887-024-04573-w). Therefore, there is a need for a method that can achieve personalized and precise iron supplementation during pregnancy by screening for hepcidin encoding gene polymorphisms. Summary of the invention

[0004] The first object of the present invention is to provide a primer set for assessing the risk of iron deficiency or overload in patients with gestational diabetes.

[0005] The second object of the present invention is to provide a detection kit for evaluating the risk of iron deficiency or overload in patients with gestational diabetes.

[0006] The third purpose of the present invention is to solve the problem of expensive gene polymorphism sequencing in existing risk assessment products for iron deficiency or overload in patients with gestational diabetes, and to provide a method for assessing the risk of iron deficiency or overload in patients with gestational diabetes based on the MassARRAY® MALDI-TOF System (time-of-flight mass spectrometry biochip system) using the detection kit for detecting the iron nutritional status of patients with gestational diabetes.

[0007] The primer set for assessing the risk of iron deficiency or overload in patients with gestational diabetes is used to amplify the single nucleotide polymorphism (SNP) site of the hepcidin encoding gene, and the primer length is 18 to 30 bp (base pairs) to ensure the specificity and sensitivity of the primers.

[0008] The single nucleotide polymorphism sites include rs10416533, rs7251432, rs8101606, rs55863037, and rs2293689.

[0009] The primer set is shown in SEQ ID NOs: 1-15.

[0010] The detection kit for assessing the risk of iron deficiency or overload in patients with gestational diabetes mellitus comprises:

[0011] 1) Primer set as described above;

[0012] 2) The reaction solution for multiplex PCR has the following components:

[0013]

[0014] A method for detecting genotyping of iron deficiency or overload in patients with gestational diabetes mellitus, using the detection kit, combined with PCR amplification and MassARRAY nucleic acid mass spectrometry technology, the method comprising the following steps:

[0015] 1) Collect samples and extract genomic DNA;

[0016] 2) using the primer set to perform PCR amplification on the target fragment of the genomic DNA, including at least one gene fragment amplification, at least one product alkaline phosphatase treatment and at least one single-base primer extension reaction amplification, to obtain an amplification product mixture;

[0017] 3) Purifying the amplified product mixture through resin to obtain the amplified product;

[0018] 4) Use MassARRAY Analyzer Compac mass spectrometry to detect and analyze the amplified products and output the results.

[0019] In step 4), the MassARRAY Analyzer Compac is a genotyping detection system exclusively developed and produced by Sequenom, Inc. of the United States.

[0020] The above-mentioned samples include human peripheral blood, but are not limited to peripheral blood.

[0021] A non-diagnostic time-of-flight mass spectrometry-based method for assessing the risk of iron deficiency or overload in women with gestational diabetes mellitus includes:

[0022] 1) Obtaining the test kit for assessing the risk of iron deficiency or overload in patients with gestational diabetes;

[0023] 2) using the genotyping detection method to obtain the genotypes of the five loci;

[0024] 3) weighting the risk of iron deficiency or overload in patients with gestational diabetes based on the genotype of the locus, calculating the score associated with the risk of iron deficiency or overload according to the weight formula, and analyzing the iron nutrition status based on the score.

[0025] The weight formula is as follows:

[0026] Score = tts1*45.101+tts2*27.726-tts3*32.697-tts4*46.657+tts5*50.577+age*1.922, where TTS-001, TTS-002, TTS-003, TTS-004, and TTS-005 represent the genotype assignments of rs10416533, rs7251432, rs8101606, rs55863037, and rs2293689, respectively.

[0027] The iron nutrition status of the gestational diabetes patient is obtained by comparing the score with the critical value. The score higher than the minimum value indicates a high risk of iron deficiency, the score higher than the maximum value indicates a high risk of iron overload, and the score between the minimum and maximum values ​​indicates a low risk. Specifically: the minimum value of the standardized score is set to 16, and the maximum value is set to 83. The risk of iron deficiency or overload of the sample is judged according to the score: below 16 is a high risk of iron deficiency, above 83 is a high risk of iron overload, and the score between 16 and 83 is a low risk.

[0028] The product after multiple PCR amplification in the present invention is added with a SNP sequence specific extension primer to extend one base at the SNP site. Different genotypes extend different bases, and then use an instantaneous nanosecond (10 -9 s) Strong laser excitation separates the bases according to their mass-to-charge ratio in a non-electric field drift region, and distinguishes different genotypes based on the different masses of the extended bases and the different times they take to reach the detector in the vacuum tube.

[0029] Compared with the prior art, the present invention has the following outstanding advantages:

[0030] 1) Ultra-high sensitivity: the detection limit is 0.4ng.

[0031] 2) Ultra-high flexibility: self-designed multiplex PCR, common primer synthesis, universal kit, the chip can be used multiple times, there is no need to optimize PCR conditions, new detection can be quickly established under standard conditions, and it only takes two days from design to results.

[0032] 3) High-quality data: Fully automatically analyze data, complete genotyping reports, and provide mass spectrometry to obtain sample status and result credibility analysis.

[0033] 4) High PCR multiplex number and transformation success rate: 10-36 multiplex PCR can produce good results, and the highest can reach 40 multiplex; >95% of SNP sites can be analyzed and studied using this platform.

[0034] 5) High accuracy: By designing primers in both forward and reverse directions, it can effectively detect insertion and deletion INDELs and translocation fusion genes with an accuracy rate of >99.7%.

[0035] 6) High sample throughput: more than 3,000 samples can be processed and 100,000 genotypes can be typed every day.

[0036] 7) High cost-effectiveness: 10 to 40 SNP sites can be measured simultaneously without fluorescent labeling, and the cost of consumables and sample usage are very low.

[0037] 8) Low DNA sample quantity and quality requirements: Only 10 ng DNA is required for each PCR reaction.

[0038] 9) The present invention provides a primer set, a kit and a method for evaluating gene mutations at risk of iron deficiency or overload in patients with gestational diabetes, which can detect polymorphisms of hepcidin encoding genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the mass spectrometry cluster diagram of different bases at site rs10416533.

[0040] Figure 2 This is the mass spectrometry cluster diagram of different bases at site rs7251432.

[0041] Figure 3 This is the mass spectrometry cluster diagram of different bases at site rs8101606.

[0042] Figure 4 This is the mass spectrometry cluster diagram of different bases at site rs55863037.

[0043] Figure 5 This is the mass spectrometry cluster diagram of different bases at site rs2293689.

[0044] Figure 6 This is a basic flow chart of time-of-flight mass spectrometry. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. On the contrary, the present invention encompasses any substitutions, modifications, equivalent methods and schemes made on the essence and scope of the present invention as defined by the claims. Further, in order to enable the public to have a better understanding of the present invention, some specific details are described in detail in the following detailed description of the present invention. For those skilled in the art, the present invention can be fully understood without the description of these details.

[0046] Example 1

[0047] Design of primer combinations

[0048] The present invention is based on the optimization selection of 5 SNP site systems screened out in the hepcidin coding gene, combined with the characteristics of its SNP site system, and designs a PCR primer composition for amplifying a DNA fragment containing 5 SNP sites on the hepcidin gene, the primer length range is mostly 20-35bp, the PCR amplification product length is between 100 and 200bp, the GC content is preferably 40% to 60%, and the primer itself is avoided as much as possible to produce a hairpin structure and a continuous arrangement of more than 5 purine or pyrimidine nucleotides. In addition, since mass spectrometry detection uses a multiplex PCR method, there are many primers in the same reaction system, so it is necessary to pay special attention to the complementarity between primers, and it is necessary to absolutely avoid the complementarity of the 3' end of the SNP extension primer with other primers exceeding 3 bases, otherwise non-specific extension will occur, greatly interfering with the test results. In addition, the designed primers should have no obvious homology with other sequences in the genome to prevent erroneous test results. The 5' end of the front primer and the back primer has an additional 10bp linker sequence: ACGTTGGATG, commonly known as the "protective base". The protective base sequence increases the molecular weight of the PCR primer and can prevent the remaining PCR primer from entering the mass spectrometry detection window to avoid interfering with the detection effect. The design principle for single-base extension primers is: the first base amplified by the primer is the base to be tested, and hairpin structures and repeated nucleotide sequences are avoided.

[0049] The invention is used to detect hepcidin metabolism-related gene polymorphisms, namely rs10416533, rs7251432, rs8101606, rs55863037 and rs2293689. The primer set is shown in SED ID NO.1-15.

[0050] Example 2

[0051] The usage of the kit of the present invention is described in detail below in conjunction with Example 1. Example 2 is implemented under the technical premise of the present invention and provides a detailed implementation method and specific operation process.

[0052] S1: DNA extraction:

[0053] 1. Sample processing: Add 2 times the volume of Buffer TBP to 200ul of pregnant woman's whole blood, mix thoroughly, and leave at room temperature for 1 min until the red blood cells are completely lysed. Centrifuge at 8,000rpm for 1 min and discard the supernatant. Resuspend the precipitate with 500μL TE Buffer, centrifuge at 8,000rpm for 1 min, discard the supernatant, wash once with TE Buffer until the precipitate is white, and then add 200 μL PBS solution.

[0054] 2. Add 20μL Proteinase K and mix well. Then add 200μL Buffer DL, shake and mix well, and incubate in a 56℃ water bath for 10 min. When the mixture becomes clear and transparent, the lysis is complete.

[0055] 3. Add 200 μL of anhydrous ethanol to the above centrifuge tube and mix thoroughly by inversion.

[0056] 4. Place the adsorption column in the collection tube, use a pipette to add all the solution and translucent fibrous suspension into the adsorption column, let it stand for 2 minutes, then centrifuge at 10,000 rpm at room temperature for 1 minute, and discard the waste liquid in the collection tube.

[0057] 5. Place the adsorption column back into the collection tube, add 500 μL GW Solution to the adsorption column, centrifuge at 10,000 rpm for 30 seconds and discard the waste liquid.

[0058] 6. Place the adsorption column back into the collection tube, add 700 μL Wash Solution to the adsorption column, centrifuge at 10,000 rpm for 30 seconds, and discard the waste liquid.

[0059] 7. Repeat step 6 once.

[0060] 8. Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes at room temperature to remove the remaining WashSolution. Open the cover of the adsorption column and leave it at room temperature for several minutes to completely dry the remaining WashSolution in the adsorption material.

[0061] 9. Take out the adsorption column, put it into a new 1.5 ml centrifuge tube, add 50 μL CE Buffer and let it stand for 3 minutes. Centrifuge at 12,000 rpm at room temperature for 2 minutes to collect the DNA solution.

[0062] S2: DNA quality test:

[0063] 1. Take 5 μL DNA solution, 1% agarose, 1X TAE buffer solution for electrophoresis (voltage 120-180 V) detection. A single band indicates that the DNA is intact and not degraded. A clear band indicates that the concentration can meet the PCR requirements.

[0064] 2. Detect the concentration and purity with a spectrophotometer. Take 1 μL to check the OD value. If the OD260 / 280 is between 1.7 and 2.0, it means the DNA quality is good. If it is less than 1.7, there is protein contamination, and if it is greater than 2.0, there is RNA contamination.

[0065] S3: Primer Design

[0066] Primer design software Sequenom's Genotyping Tools and Massarray Assay Design software were used to design PCR amplification primers and single-base extension primers for the sites to be tested. The specific amplification primer sequences and single-base extension primer sequences are shown in Table 1.

[0067] Table 1 Primer set sequences

[0068]

[0069] 1. Primer length 18-30 bp, PCR product 80-200 bp;

[0070] 2. Tm value 55-65℃, annealing temperature around 60℃;

[0071] 3.GC content 40% to 70%;

[0072] 4. Pay special attention to avoid the presence of primer dimers and non-specific amplification.

[0073] S4: PCR amplification

[0074] 1. Multiplex PCR technology was used. In a 384-well plate, the total volume of each reaction system was 5 μL. The reaction system is shown in Table 2.

[0075] 2. PCR reaction conditions:

[0076]

[0077] S5: Alkaline phosphatase treatment of PCR products

[0078] After the PCR reaction is completed, the PCR product is treated with SAP (shrimp alikaiine phosphtase) to remove the dNTPs in the reaction. The SAP reaction solution is prepared according to the order in Table 1 (taking 384 samples as an example)

[0079] Table 2 Composition of SAP reaction solution

[0080]

[0081] Note: The above 384-well reaction solution has a 4% excess.

[0082] 1. Take a row of 12 tubes as a dispensing container, dispense 66 μL of SAP reaction solution into each well of the 12 tubes, centrifuge the 12 tubes briefly, use a 10 μL pipette (multichannel pipette) to draw the SAP reaction solution from the 12 tubes and dispense it into 384-well PCR reaction plates, add 2 μL SAP reaction solution to each well, seal the film, and centrifuge.

[0083] 2. Place the 384-well PCR reaction plate with SAP reaction solution added into the PCR instrument and run the SAP reaction program.

[0084] 3. SAP program: 37℃, 40 min; 85℃, 5 min; 4℃, forever.

[0085] 4. After the SAP reaction program is completed, remove the 384-well PCR reaction plate from the PCR instrument and centrifuge it briefly for later use.

[0086] S6: Extension reaction

[0087] Prepare iPlex reaction reagents according to 3.

[0088] Table 3 iPlex reaction reagents

[0089]

[0090] Note: The above 384-well reaction solution has a 4% excess.

[0091] 1. Take a row of 12 tubes and dispense 66 μL of the iPlex reaction solution into each well. After a brief centrifugation, use a 10 μL dispenser to dispense into a 384-well PCR reaction plate, add 2 μL into each well, seal the plate, and centrifuge.

[0092] 2. Place the 384-well plate with iPlex reaction solution into the PCR instrument and run the reaction program named "extension".

[0093]

[0094] 3. After the reaction is completed, take out the 384-well plate and centrifuge it briefly for later use.

[0095] S7: Product purification

[0096] 1. Take 6 mg of resin and evenly cover it on a 384-well resin scraper. Scrape off the excess resin and let it sit for 20 min.

[0097] 2. Centrifuge the 384-well plate at 1000 rpm for 1 min after the reaction is completed, add 25 μL of deionized water to each well, invert it on the resin plate (make sure it is fixed and cannot be shifted), then invert the resin plate and buckle it on the 384-well plate, tap it to make the resin fall into the 384-well plate, and seal the film.

[0098] 3. With the long axis of the 384-well plate as the axis, turn the 384-well plate over for 20 minutes, and centrifuge at 3500 rpm for 5 minutes for later use.

[0099] S8: Mass spectrometry detection

[0100] 1. Nanodispenser SpectroCHIP chip spotting, transfer the test sample from the 384-well reaction plate to the MassARRAY SpectroCHIP chip with a surface covered with matrix.

[0101] 2. MassARRAY Analyzer Compac Mass spectrometry detection, MassARRAY Analyzer Compac is a high-performance mass spectrometer with high throughput, high sensitivity and high resolution. Place the sampled SpectroCHIP chip into the mass spectrometer.

[0102] 3. After the sample is transferred to the SpectroCHIP chip, it can be placed in the mass spectrometer for detection. Each detection point only takes 3 to 5 seconds and the analysis is fully automatic.

[0103] 4. TYPER software analyzes the experimental results and obtains typing data. TYPER is a software specifically used for mass spectrometry data analysis, which can automatically process the data output by the mass spectrometer and perform typing analysis.

[0104] Embodiment 3:

[0105] In order to verify that the kit can accurately detect the polymorphism of the five SNP sites of the hepcidin gene, three samples to be tested that have been tested using the kit of the present invention were simultaneously subjected to first generation sequencing verification.

[0106] As shown in Table 4, for the detection of 5 SNP sites (rs10416533, rs7251432, rs8101606, rs55863037, rs2293689), the detection results using the kit of the present invention are completely consistent with the first-generation sequencing results. This shows that the kit of the present invention can accurately detect the polymorphism of the corresponding SNP sites. It has high accuracy and reliability and can be comparable to the traditional first-generation sequencing method. This consistency is very important for fields such as gene detection and genetic analysis to ensure the accuracy and repeatability of the results. Figure 1 This is the mass spectrometry cluster diagram of different bases at site rs10416533. Figure 2 This is the mass spectrometry cluster diagram of different bases at site rs7251432. Figure 3 This is the mass spectrometry cluster diagram of different bases at site rs8101606. Figure 4 This is the mass spectrometry cluster diagram of different bases at site rs55863037. Figure 5 This is the mass spectrometry cluster diagram of different bases at site rs2293689.

[0107] Table 4 Comparison of the results of the two detection methods

[0108]

[0109] * Invention: Detection results using the kit involved in the present invention;

[0110] # Generation 1: The first generation of sequencing results.

[0111] The MassARRAY® MALDI-TOF System (time-of-flight mass spectrometry biochip system) used in the present invention is a genotyping detection system exclusively developed and produced by Sequenom, Inc., USA. It is also the only device that directly performs SNP typing detection using the principle of time-of-flight mass spectrometry. Its main features are: after multiple PCR amplification, SNP sequence-specific extension primers are added to extend one base at the SNP site, and different genotypes extend different bases, and then an instantaneous nanosecond (10 -9 s) Strong laser excitation separates the bases according to their mass-to-charge ratio in a non-electric field drift region. Different genotypes are distinguished based on the different masses of the extended bases and the different times they take to reach the detector in the vacuum tube. The basic process of time-of-flight mass spectrometry is as follows: Figure 6 As shown. Multiple PCR specific amplification primers and extension primers are designed according to the selected polymorphic sites. The PCR reaction amplifies the gene sequence containing the polymorphic site. The specific extension primer extends 1 base at the SNP site. After specific treatment, the sample is co-crystallized with the chip matrix, and the sample is instantly amplified in nanoseconds (10 -9 s) Strong laser excitation, the matrix transfers the absorbed laser pulse energy to the sample to be analyzed and separates it according to the mass-to-charge ratio. The ion capture instrument collects and stores the pulse signal, and performs mass spectrometry analysis on it. By comparing the signal intensity of A, T, G and C and the difference in mass between adjacent signals (ddATP=271.2Da, ddCTP=247.2Da, ddGTP=287.2Da, ddTTP=327.1Da), the information of the template sequence can be obtained. The accuracy and reliability of the kit of the present invention are verified by first-generation sequencing. It provides a powerful tool for the study of hepcidin gene polymorphism and helps to further reveal the association between hepcidin gene and disease.

[0112] Embodiment 4:

[0113] The collected DNA samples were tested using the sample preparation, primer design (Table 1), mass spectrometry detection and data analysis methods of the present invention to obtain mutation information of 5 sites in the samples;

[0114] In the first stage, 39 samples of gestational diabetes were collected and their genotypes were determined.

[0115] In the second stage, the database was sorted based on file retrieval, the score information related to the sample genotypes was collected, and the score corresponding to the genotype of each sample was calculated.

[0116] In the third stage, a linear regression model was used to fit the scores obtained in the second stage to obtain the following formula, which was used to calculate the scores of the samples based on the genotype assignments of the five loci and age:

[0117] Score = tts1*45.101+tts2*27.726-tts3*32.697-tts4*46.657+tts5*50.577+age*1.922, where TTS-001, TTS-002, TTS-003, TTS-004, and TTS-005 represent the genotype assignments of rs10416533, rs7251432, rs8101606, rs55863037, and rs2293689, respectively.

[0118] In the fourth stage, the scores were standardized to make the results more comparable and interpretable: standardized score (Score) = exp(Score). The minimum standardized score was set to 16 and the maximum was set to 83. The risk of iron deficiency or overload of the sample was determined based on the score: below the critical value of 16 was a high risk of iron deficiency, above 83 was a high risk of iron overload, and between 16 and 83 was a low risk.

[0119] The present invention provides a primer set, a kit and a method for evaluating gene mutations at risk of iron deficiency or overload in patients with gestational diabetes, which can detect hepcidin encoding gene polymorphisms, and provides a new idea and method for evaluating the risk of iron deficiency.

[0120] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A primer set for assessing the risk of iron deficiency or overload in patients with gestational diabetes, characterized in that It is used to amplify the single nucleotide polymorphism site of the hepcidin encoding gene, and the primer length is 18 to 30 bp.

2. The primer set for assessing the risk of iron deficiency or overload in patients with gestational diabetes mellitus according to claim 1, characterized in that The single nucleotide polymorphism sites include rs10416533, rs7251432, rs8101606, rs55863037, and rs2293689.

3. The primer set for screening risk prediction of iron deficiency during pregnancy as claimed in claim 1, characterized in that The primer set is shown in SEQ ID NOs: 1-15.

4. A test kit for assessing the risk of iron deficiency or overload in patients with gestational diabetes, characterized in that include: 1) A primer set as claimed in any one of claims 1 to 3; 2) The reaction solution for multiplex PCR has the following components: 10~30 ng / μL template DNA 1μL; 10 μM Primer F 0.25 μL; 10 μM Primer F 0.25 μL; 25 mM dNTPs (mix) 0.1 μL; 10× PCR Buffer 0.5μL; 25 mM MgCl2 0.4 μL; 5 U / μL HotStar Taq 0.1μL; Add ddH2O to 5μL.

5. A method for genotyping and detecting iron deficiency or overload in patients with gestational diabetes, characterized in that Using the detection kit as claimed in claim 4, the method comprises the following steps: 1) Collect samples and extract genomic DNA; 2) using the primer set to perform PCR amplification on the target fragment of the genomic DNA, including at least one gene fragment amplification, at least one product alkaline phosphatase treatment and at least one single-base primer extension reaction amplification, to obtain an amplification product mixture; 3) Purifying the amplified product mixture through resin to obtain the amplified product; 4) MassARRAY Analyzer Compac mass spectrometry was used to detect the amplified products, and TYPER was used to analyze and output the results to obtain the typing data.

6. A method for genotyping and detecting iron deficiency or overload in patients with gestational diabetes mellitus as claimed in claim 5, characterized in that The sample includes but is not limited to human peripheral blood.

7. A method for assessing the risk of iron deficiency or overload in patients with gestational diabetes, characterized in that The method comprises: 1) Obtaining the test kit for assessing the risk of iron deficiency or overload in patients with gestational diabetes as described in claim 4; 2) obtaining the genotypes of the five loci using the genotyping detection method as described in claim 5; 3) weighting the risk of iron deficiency or overload in patients with gestational diabetes based on the genotype of the locus, calculating the score associated with the risk of iron deficiency or overload according to the weight formula, and analyzing the iron nutrition status based on the score.

8. A method for assessing the risk of iron deficiency or overload in patients with gestational diabetes mellitus as claimed in claim 7, characterized in that: The weight formula is as follows: Score = tts1*45.101+tts2*27.726-tts3*32.697-tts4*46.657+tts5*50.577+age*1.922, where TTS-001, TTS-002, TTS-003, TTS-004, and TTS-005 represent the genotype assignments of rs10416533, rs7251432, rs8101606, rs55863037, and rs2293689, respectively.

9. A method for assessing the risk of iron deficiency or overload in patients with gestational diabetes mellitus as claimed in claim 7 or 8, characterized in that: The iron nutrition status of the gestational diabetes patient is obtained by comparing the score with the critical value. The score higher than the minimum value indicates a high risk of iron deficiency, the score higher than the maximum value indicates a high risk of iron overload, and the score between the minimum and maximum values ​​indicates a low risk. Specifically: the minimum value of the standardized score is set to 16, and the maximum value is set to 83. The risk of iron deficiency or overload of the sample is judged according to the score: below 16 is a high risk of iron deficiency, above 83 is a high risk of iron overload, and the score between 16 and 83 is a low risk.

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