A kit for guiding the combined use of early screening and nutritional intervention for gestational diabetes mellitus

Abstract

This invention provides a set of primers and their applications for detecting SNP loci related to early prediction and nutritional intervention of gestational diabetes mellitus. Using time-of-flight nucleic acid mass spectrometry, it can rapidly complete the genotyping of 20 SNPs in a single reaction well without involving probes, fluorescent labels, or gel electrophoresis. The detection is accurate, rapid, and cost-effective. The product of this invention can predict the risk of gestational diabetes mellitus at an early stage and guide individualized folic acid intervention to reduce the risk of gestational diabetes mellitus. At the same time, it can improve existing gestational diabetes mellitus, combining the combined use of early prediction and nutritional intervention for gestational diabetes mellitus.

Description

Technical Field

[0001] This application belongs to the field of disease screening and nutritional intervention technology. Specifically, this application relates to a kit for guiding the combined use of early screening and nutritional intervention for gestational diabetes. Background Technology

[0002] Gestational diabetes mellitus (GDM), one of the most common complications of pregnancy, is clinically defined as an abnormality in glucose metabolism that first appears or is diagnosed during pregnancy. It has adverse effects on the short-term and long-term health of both the pregnant woman and her offspring. Short-term effects include an increased risk of adverse pregnancy outcomes such as gestational hypertension, preeclampsia, macrosomia, and cesarean section. In the long term, although blood glucose levels return to normal in most women with GDM after delivery, epidemiological data show that approximately 2.5%–16.7% of individuals develop type 2 diabetes within one year postpartum, and the incidence of type 2 diabetes rises to over 40% within 1–10 years postpartum.

[0003] Currently, screening for gestational diabetes mellitus (GDM) (OGTT) mainly focuses on the period between 24 and 28 weeks. If high-risk individuals could be identified and intervened earlier, as early as 20 weeks, the risk of adverse pregnancy outcomes due to GDM could be effectively reduced. However, at present, there is still a lack of economical, feasible, and effective methods for early prediction of GDM. As a complex metabolic disease driven by both genetic and environmental factors, the genetic basis of GDM is receiving increasing attention. In 2025, Liu et al. reported a genome-wide association study (GWAS) covering 121,556 pregnant women, identifying 19 independent single nucleotide polymorphisms (SNPs) significantly associated with GDM. Polymorphism (SNP) sites: rs10466351 (MTNR1B gene), rs10758593 (GLIS3 gene), rs10882102 (HHEX-IDE gene), rs11127048 (GCKR gene), rs12694904 (GALNT5 gene), rs2882298 (PCSK1 gene), rs4716672 (MNX1 gene), rs4746822 (HKDC1 gene), rs4776024 (MYO5C gene), rs582 The following loci were identified: rs6113722 (FOXA2 gene), rs61160304 (PAX4 gene), rs73069940 (CTBP1 gene), rs741037 (GCK gene), rs76435632 (UBE2E2 gene), rs7851502 (FCN1 gene), rs9366994 (GLP1R gene), rs9368222 (CDKAL1 gene), and rs9650069 (SLC30A8 gene), of which 13 loci were reported for the first time. Regarding risk prediction, the combined model integrating clinical characteristics and genetic information before 20 weeks of gestation demonstrated the best predictive performance, with an AUC of 0.729 and an accuracy of 0.835 (NatCommun. 2025, 16(1):4178).

[0004] Folic acid, also known as vitamin B9, is essential for human cell growth and division, and is a necessary factor in the synthesis of proteins and nucleic acids. However, the human body cannot synthesize folic acid and must obtain it from external sources. MTHFR (methylenetetrahydrofolate reductase) is a key enzyme in folic acid metabolism, catalyzing the reduction of tetrahydrofolate to 5-methyltetrahydrofolate, and providing methyl groups in homocysteine ​​metabolism to maintain normal metabolism. The MTHFR gene exhibits polymorphism. Mutations at the C677T site (rs1801133) of the MTHFR gene result in three genotypes in the population: wild-type CC, heterozygous mutant CT, and homozygous mutant TT. Compared to the CC type, the enzyme activity of the heterozygous mutant CT genotype can be reduced by up to 65%, and the enzyme activity of the homozygous mutant TT genotype can be reduced by up to 30%. Reduced MTHFR enzyme activity leads to a limited ability of the body to convert folic acid to 5-methyltetrahydrofolate, ultimately causing hyperhomocysteinemia, abnormal nucleic acid synthesis and methylation, and resulting in various diseases. Studies have found that homocysteine ​​(Hcy) levels in CDM patients are significantly higher than in the control group, and Hcy is negatively correlated with folic acid levels and positively correlated with insulin resistance, which may be related to the occurrence of GDM (Chinese Journal of Maternal and Child Health, 2021, 32(3):427-429.). A prospective cohort study showed that among 14,553 pregnant women followed up, 824 cases of GDM were reported. Compared with women with insufficient total folic acid intake (<400μg / day), the RR value (95% CI) of GDM was 0.83 (0.72~0.95) (P=0.007) for women with adequate total folic acid intake (≥400μg / day). Adequate folic acid supplementation before pregnancy can reduce the risk of GDM (Diabetes Care, 2019, 42(6):1034-1041.). A case-control study included 49,611 women with GDM and 137,821 healthy women, showing that preconception folic acid intake could reduce the risk of GDM by 27%, with an adjusted OR (95% CI) of 0.73 (0.69–0.79) (P<0.001) (Sci Rep, 2021, 11(1):7335.). Another study selected 8,649 women from the Tongji Maternal and Child Health Cohort. After excluding those with pre-gestational diabetes, glucose intolerance, multiple pregnancies, miscarriages, and those without accurate folic acid intake information (dosage and duration unclear), 4,353 women were included. The incidence of GDM was 8.6%. Preconception and pregnancy administration of 400 μg / day of folic acid was shown to prevent neural tube defects, but daily intake of ≥800 μg of folic acid from preconception to mid-pregnancy may have a higher risk of GDM (Diabetes Care, 2019, 42(7):e113-e115.).Therefore, the current "Chinese Guidelines for Medical Nutrition Therapy for Diabetes (2022 Edition)" recommends that supplementing with an additional 400 μg of folic acid daily on top of a balanced diet before and during early pregnancy is beneficial in reducing the risk of GDM. However, supplementing with more than 800 μg of folic acid may increase the risk of GDM. A single-center prospective cohort study found that pregnant women with MTHFR C677T genotype GDM who took 800 μg of folic acid daily during mid-to-late pregnancy (vs. the usual 400 μg) achieved normal OGTT on average about 27 days earlier, suggesting that individualized "genotype-dosage" intervention can improve existing GDM (Front Endocrinol (Lausanne). 2023, 14:1007192.). This demonstrates that folic acid plays an important role in the development and progression of GDM. Individualized folic acid supplementation based on MTHFR C677T genotype can effectively reduce the risk of GDM and the time to recovery for patients with existing GDM, thereby reducing the harm caused by GDM.

[0005] Currently, there are several kits for detecting GDM susceptibility genes. For example, the patent with application number 201710827583.9 uses nucleic acid mass spectrometry to detect 12 loci, including rs7903146 ​​(TCF7L2 gene), rs12255372 (TCF7L2 gene), rs5219 (KCNJ11 gene), rs7754840 (CDKAL1 gene), rs4402960 (IGF2BP2 gene), rs1387153 (MTNR1B gene), rs10830963 (MTNR1B gene), rs2237892 (KCNQ1 gene), rs1801278 (IRS1 gene), rs1799884 (GCK gene), rs1801282 (PPARG gene), and rs1063192 (CDKN2B gene). Patent application number 201910383683.6 detected two loci, rs290487 (TCF7L2 gene) and rs108309632 (MTNR1B gene), using quantitative real-time PCR. Patent application number 202310209406.X detected six loci on the mitochondrial genome, m.73A>G, m.185G>A, m.16051A>G, m.16092T>A, m.16291C>T, and m.6228C>T, using first-generation sequencing. The patent application with application number 202310222385.5 detected eight loci using first-generation sequencing: rs1393210449 (MUC5AC gene), rs4566357 (COL4A4 gene), rs13059843 (ACAP2 gene), rs147522534 (ACAP2 gene), rs10830962 (MTNR1B gene), rs11188519 (CC2D2B gene), rs3769644 (COL4A4 gene), and rs11642441 (SMG1 gene). The relevant gene loci involved in these patents are mostly summaries of previous domestic and foreign research results. Many of them have not been studied or verified in a very large sample size (N≥10,000 cases) of Chinese population, so the evidence is not convincing enough. At the same time, they all detect loci related to early prediction of GDM, but do not detect loci related to nutritional (such as folic acid) intervention. Moreover, most of the kits have poor detection timeliness and high cost, making them unsuitable for large-scale population screening.

[0006] Therefore, it is necessary to develop a gene testing kit that simultaneously covers high-evidence-level gene loci for early prediction of GDM in the Chinese population and gene loci related to nutritional intervention, while being simple to operate and low-cost, in order to effectively reduce the harm of GDM to pregnant women and their offspring. Summary of the Invention

[0007] To address the aforementioned technical problems in existing technologies, this invention proposes a set of primers and their applications for detecting SNP loci related to early prediction and nutritional intervention in GDM. Using time-of-flight nucleic acid mass spectrometry, it can rapidly complete genotyping of 20 SNPs in a single reaction well, without involving probes, fluorescent labeling, or gel electrophoresis. This method is accurate, rapid, and cost-effective. It allows for early prediction of GDM risk and guides individualized folic acid intervention to reduce the risk of GDM, while also improving existing GDM. This integrated approach of early prediction and nutritional intervention provides a scientific basis for effectively reducing the harm of GDM to pregnant women and their offspring.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention selected 19 gene polymorphism sites related to early prediction of GDM and 1 gene polymorphism site related to nutrient (folate) intervention, specifically rs10466351 (MTNR1B gene), rs10758593 (GLIS3 gene), rs10882102 (HHEX-IDE gene), rs11127048 (GCKR gene), rs12694904 (GALNT5 gene), rs2882298 (PCSK1 gene), rs4716672 (MNX1 gene), rs4746822 (HKDC1 gene), and rs4776024 (…). MYO5C gene), rs58299944 (SLC39A11 gene), rs6113722 (FOXA2 gene), rs61160304 (PAX4 gene), rs73069940 (CTBP1 gene), rs741037 (GCK gene), rs76435632 (UBE2E2 gene), rs7851502 (FCN1 gene), rs9366994 (GLP1R gene), rs9368222 (CDKAL1 gene), rs9650069 (SLC30A8 gene), and rs1801133 (MTHFR gene).

[0010] Based on the 20 polymorphic sites mentioned above, a set of amplification primers and a single-base extension primer sequence for amplifying one mutation site were designed and synthesized. The specific amplification primer sequences are shown in Table 1, and the single-base extension primer sequences are shown in Table 2.

[0011] Table 1. Sequences of upstream and downstream primer pairs for PCR amplification at each site

[0012] Table 2 Single-base extension primer sequences for each site

[0013] The specific operation process is as follows: 1. Blood sample DNA extraction: 1) Blood sample collection and preservation: The collector should wear sterile gloves, wipe the person being sampled with an alcohol swab, pack the collected blood collection tubes into a sealed bag, and then write the person's information on the collected sample. The sample should be stored at 2~8℃ for no more than one month.

[0014] 2) Blood sample DNA extraction: Genomic DNA was extracted from blood samples using a 96-bead genomic DNA extraction reagent (DE1796D) and a magPure-96plus fully automated nucleic acid extractor.

[0015] 3) Determine DNA concentration and purity: Refer to the standard operating instructions of the Nanodrop2000 UV spectrophotometer. The A260 / A280 of the DNA should be between 1.6 and 2.0, and the concentration should be above 10 ng / μL.

[0016] 2. PCR amplification reaction: 1) Prepare PCR Mix: Take the reagents contained in the kit out of the refrigerator, equilibrate to room temperature, fully dissolve each component, vortex to mix, centrifuge quickly for 10 seconds, and prepare PCR Mix according to Table 3.

[0017] Table 3 PCR Mix Reaction Solution System

[0018] 2) After vortexing the prepared PCR Mix, prepare the reaction system (5μL) for multiplex PCR amplification: 3μL PCR Mix, 1μL upstream and downstream primer mix, and 1μL template DNA are added sequentially to a 384-well plate. Seal the plate tightly with the sealing film to prevent sample evaporation, vortex to mix, and then centrifuge.

[0019] 3) Place the 384-well plate in an PCR instrument for amplification reaction. Reaction conditions: 95℃ pre-denaturation for 2 min, (95℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min) for 45 cycles, 72℃ for 5 min, and hold at 4℃; obtain PCR amplification products, centrifuge, and set aside.

[0020] 3. SAP digestion: Remove the PCR amplification product from step 2, centrifuge, remove the sealing film, and place on an ice plate at 4°C for later use. Prepare SAPMix according to Table 4, vortex to mix, and add 2 μL to each well of a 384-well plate. Seal tightly with the sealing film to prevent sample evaporation, vortex to mix, and centrifuge. Place the sealed 384-well plate in a PCR instrument for SAP digestion reaction under the following conditions: 37°C for 40 min, 85°C for 5 min, and hold at 4°C. Obtain the SAP digestion product, centrifuge, and set aside.

[0021] Table 4 SAP Reaction Solution System

[0022] 4. Single base extension reaction Prepare the single-base reaction system according to Table 5, vortex and mix well, and add 1.06 μL to each well of a 384-well plate. Then add 0.94 μL of the corresponding single-base extension primer Mix to each well. Seal the plate tightly with sealing film to prevent sample evaporation, vortex and mix well, and centrifuge. Place the sealed 384-well plate on a PCR instrument for extension reaction. The reaction conditions are shown in Table 6.

[0023] Table 5 iPLEX reaction solution system

[0024] Table 6. Amplification-related parameters of the iPLEX reaction instrument

[0025] 5. Resin purification Evenly press the resin into each well of a 384-well pre-prepared plate, approximately 9 mg of resin per well, and set aside to air dry. Gently peel off the sealing film from the 384-well plate containing the single-base extension product obtained in step 4, add 11 μL of ddH2O to each well, seal the plate, and briefly centrifuge to remove the ddH2O to the bottom of the plate. Invert the PCR plate onto the resin-dried template, rotate it 180°, and gently tap the resin into the PCR wells. If there is excess resin, gently tap it out of the PCR plate for recovery. Shake the PCR plate with the added resin at 60 rpm / min for 20 minutes to ensure complete purification. Centrifuge at 4000 rpm / min for 5 minutes before analysis.

[0026] 6. MassARRY's computer lab session First, import the edited site assay into the Typer analysis software. Then, select the reaction well position for the sample to be detected. Next, import the corresponding sample name for the 384 plate. Finally, connect the detection chip. Place the 384 plate and the detection chip in the corresponding positions on the mass spectrometer, and then start the time-of-flight mass spectrometry detection.

[0027] 7. Result Interpretation Single-base extension products were detected by mass spectrometry to obtain their molecular weight. The analysis software calculated the difference between the molecular weight of the extension primer itself and the possible molecular weight of the extension product, comparing this difference with the molecular weights of pre-set extension primers for each mutation site and the ddNTPs of possible mutated bases. Through automatic analysis, the software compared the signal intensities of A, T, G, and C, as well as the differences in quality between adjacent signals (ddATP = 271.2 Da, ddCTP = 247.2 Da, ddGTP = 287.2 Da, ddTTP = 327.1 Da), to obtain template sequence information. The quality of PCR amplification and single-base extension during the process of obtaining the corresponding extended bases was evaluated. Based on the peak quality, the software automatically classified it into five quality levels: A, B, C, D, and N, with quality decreasing sequentially. N represents no extension reaction by the extension primers; ideally, the result is A.

[0028] The beneficial effects of this invention are: This invention offers a convenient and rapid method using time-of-flight nucleic acid mass spectrometry (TOFMS). It enables the rapid genotyping of 20 SNPs related to early prediction and nutritional intervention of GDM within a single reaction well, without involving probes, fluorescent labeling, or gel electrophoresis. This method boasts high throughput, high efficiency, high accuracy, and low cost, making it suitable for large-scale population screening. Furthermore, the kit can predict the early risk of GDM and guide individualized folic acid intervention to reduce this risk. It can also improve existing GDM. This kit combines early prediction and nutritional intervention for GDM, providing a scientific basis for effectively reducing the harm of GDM to pregnant women and their offspring. Attached Figure Description

[0029] Figure 1 Mass spectrometry peaks of nucleic acid from genotype 1 at the rs1801133 site of the MTHFR gene in replicate well 1; Figure 2 Mass spectrometry peaks of nucleic acid from genotypes at the rs1801133 site of the MTHFR gene in repeat well 2; Figure 3 Mass spectrometry peaks of nucleic acid from a repeat well 3 for the MTHFR gene rs1801133 site. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] Example 1 The nucleic acid composition provided in this embodiment for detecting genes related to early prediction of GDM and nutritional intervention based on nucleic acid mass spectrometry technology includes an amplification primer pair for performing multiplex PCR amplification of genes related to early prediction of GDM and nutritional intervention, and an extension primer pair for performing single-base extension reaction.

[0032] The amplification primer set includes the following primer pairs: rs61160304 upstream primer, whose nucleotide sequence is shown in SEQ ID NO:1; rs61160304 downstream primer, whose nucleotide sequence is shown in SEQ ID NO:2; rs9650069 upstream primer, whose nucleotide sequence is shown in SEQ ID NO:3; rs9650069 downstream primer, whose nucleotide sequence is shown in SEQ ID NO:4; rs9366994 upstream primer, whose nucleotide sequence is shown in SEQ ID NO:5; rs9366994 downstream primer, whose nucleotide sequence is shown in SEQ ID NO:6; rs9368222 upstream primer, whose nucleotide sequence is shown in SEQ ID NO:7; rs9368222 downstream primer, whose nucleotide sequence is shown in SEQ ID NO:8; and rs11127048 upstream primer, whose nucleotide sequence is shown in SEQ ID NO:8. The nucleotide sequence of the downstream primer rs11127048 is shown in SEQ ID NO:10; the nucleotide sequence of the upstream primer rs2882298 is shown in SEQ ID NO:11; the nucleotide sequence of the downstream primer rs2882298 is shown in SEQ ID NO:12; the nucleotide sequence of the upstream primer rs7851502 is shown in SEQ ID NO:13; the nucleotide sequence of the downstream primer rs7851502 is shown in SEQ ID NO:14; the nucleotide sequence of the upstream primer rs6113722 is shown in SEQ ID NO:15; the nucleotide sequence of the downstream primer rs6113722 is shown in SEQ ID NO:16; the nucleotide sequence of the upstream primer rs58299944 is shown in SEQ ID NO:17; the nucleotide sequence of the downstream primer rs58299944 is shown in SEQ ID NO:16. The nucleotide sequence of the upstream primer rs10466351 is shown in SEQ ID NO:19; the nucleotide sequence of the downstream primer rs10466351 is shown in SEQ ID NO:20; the nucleotide sequence of the upstream primer rs10758593 is shown in SEQ ID NO:21; the nucleotide sequence of the downstream primer rs10758593 is shown in SEQ ID NO:22; the nucleotide sequence of the upstream primer rs1801133 is shown in SEQ ID NO:23; the nucleotide sequence of the downstream primer rs1801133 is shown in SEQ ID NO:24; and the nucleotide sequence of the upstream primer rs4716672 is shown in SEQ ID NO:25.The nucleotide sequences of the following primers are listed: rs4716672 (downstream primer, SEQ ID NO:26); rs10882102 (upstream primer, SEQ ID NO:27); rs10882102 (downstream primer, SEQ ID NO:28); rs12694904 (upstream primer, SEQ ID NO:29); rs12694904 (downstream primer, SEQ ID NO:30); rs73069940 (upstream primer, SEQ ID NO:31); rs73069940 (downstream primer, SEQ ID NO:32); rs76435632 (upstream primer, SEQ ID NO:33); and rs76435632 (downstream primer, SEQ ID NO:33). The nucleotide sequence of the upstream primer rs4746822 is shown in SEQ ID NO:35; the nucleotide sequence of the downstream primer rs4746822 is shown in SEQ ID NO:36; the nucleotide sequence of the upstream primer rs741037 is shown in SEQ ID NO:37; the nucleotide sequence of the downstream primer rs741037 is shown in SEQ ID NO:38; the nucleotide sequence of the upstream primer rs4776024 is shown in SEQ ID NO:39; and the nucleotide sequence of the downstream primer rs4776024 is shown in SEQ ID NO:40.

[0033] In addition, the single-base extension primer set includes the following sequences: rs61160304 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:41 of the sequence listing; rs9650069 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:42 of the sequence listing; rs9366994 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:43 of the sequence listing; rs9368222 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:44 of the sequence listing; rs11127048 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:45 of the sequence listing; rs2882298 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:46 of the sequence listing; rs7851502 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:47 of the sequence listing; and rs6113722 single-base extension primer, the nucleotide sequence of which is shown in SEQ ID NO:47 of the sequence listing. The nucleotide sequences of the single-base extension primers are shown in SEQ ID NO:48; rs58299944 (single-base extension primer), rs10466351 (single-base extension primer), rs10758593 (single-base extension primer), rs1801133 (single-base extension primer), rs4716672 (single-base extension primer), rs10882102 (single-base extension primer), rs12694904 (single-base extension primer), and rs73069940 (single-base extension primer). The nucleotide sequence of the single-base extension primer rs76435632 is shown in SEQ ID NO:57; the nucleotide sequence of the single-base extension primer rs4746822 is shown in SEQ ID NO:58; the nucleotide sequence of the single-base extension primer rs741037 is shown in SEQ ID NO:59; and the nucleotide sequence of the single-base extension primer rs4776024 is shown in SEQ ID NO:60.

[0034] Example 2 I. To verify the effectiveness of the amplification primers and extension primers in the GDM early prediction and nutritional intervention gene detection kit of the present invention, two clinical blood samples were selected and numbered YF-01 and YF-02, respectively. Each sample was set up with 3 replicates, and SNP genotyping was performed on a nucleic acid mass spectrometry platform. The process is as follows: Step 1, Blood DNA Extraction: Blood DNA was extracted from the blood using the Blood Genomic DNA Extraction Kit from Jiaxing Medpulse Technology Co., Ltd. PK lysis buffer was added to proteinase K dry powder to prepare a 40 mg / mL proteinase K lysis buffer. The mixture was inverted several times to mix. 10 μL of blood was transferred to a centrifuge tube, and 400 μL of lysis buffer and 20 μL of proteinase K were added. The mixture was incubated at 70°C for 1 hour. Then, 200 μL of the incubated liquid was added to the corresponding well in the extraction kit containing the lysis adsorption buffer. The pre-prepared plate was placed in the corresponding position on the nucleic acid extractor, and a magnetic rod sleeve was placed in the corresponding position. The appropriate automated extraction program was selected, and extraction was started. The extraction parameters are shown in Table 7. After the extraction program was completed, the nucleic acid eluent was aspirated into a centrifuge tube. The liquid in the centrifuge tube contained genomic DNA.

[0035] Table 7 Extraction Program Parameters

[0036] Step 2: Prepare PCR Mix (ddH2O 1.8μL, 10×PCR Buffer 0.5μL, MgCl2 0.4μL, dNTP 0.1μL, PCR enzyme 0.2μL). After vortexing and mixing, add the following to a 384-well plate according to the multiplex PCR amplification reaction system (5μL): PCR Mix 3μL, upstream and downstream primer mix 1μL, template DNA (10ng / μL) 1μL. Seal the plate tightly with sealing film to prevent sample evaporation, vortex and centrifuge. Place the 384-well plate in a Bio-Rad T100 384-well amplification reaction. Reaction conditions: 95℃ pre-denaturation for 2 min, (95℃ 30s, 56℃ 30s, 72℃ 1min) 45 cycles, 72℃ 5min, and hold at 4℃. Obtain the PCR amplification product, centrifuge, and set aside.

[0037] Step 3, SAP digestion: Take the PCR amplification product from Step 2, centrifuge, remove the sealing film, and place on an ice plate at 4℃ for later use; prepare SAP Mix (ddH2O 1.53μL, SAP Buffer 0.17μL, SAP enzyme 0.3μL), vortex to mix, and add 2μL to each well of a 384-well plate. Seal tightly with the sealing film to prevent sample evaporation, vortex to mix, and centrifuge; place the sealed 384-well plate on a Bio-RadT100 384-well plate for SAP digestion reaction under the following conditions: 37℃ for 40 min, 85℃ for 5 min, and hold at 4℃; obtain the SAP digestion product, centrifuge for later use.

[0038] Step 4, Single-base extension reaction: Prepare iPLEX Mix (ddH2O 0.7395μL, iPLEX Buffer 0.2μL, Termination Mix 0.1μL, iPLEX enzyme 0.0205μL), vortex to mix, and add 1.06μL to each well of a 384-well plate. Then add 0.94μL of the corresponding single-base extension primer Mix to each well. Seal the plate tightly with sealing film to prevent sample evaporation, vortex to mix, and centrifuge. Place the sealed 384-well plate on the Bio-RadT100 384-well plate for the extension reaction. Reaction conditions: 95℃ pre-denaturation for 30s, (52℃ 5s, 80℃ 5s) 5 replicates, [95℃ denaturation for 5s, (52℃ 5s, 80℃ 5s) 5 replicates] 40 replicates; 72℃ for 3min, 4℃ hold; obtain the extension product, centrifuge for later use.

[0039] Step 5, Resin Purification: Gently peel off the sealing film from the 384-well plate containing the single-base extension product obtained in Step 4. Add 11 μL of ddH2O to each well, shake to mix, centrifuge, and place on an ice plate at 4°C for later use. Place the 6MG 384 plate on a clean A4 sheet of paper. Use a small spoon to take an appropriate amount of purification resin, and use a plastic plate to repeatedly push and flatten the resin from side to side, compacting it to ensure uniform resin content in each well. Invert the 384 plate onto the 6MG 384 plate, swap the two plates so that the 6MG plate is on top, and tap the back of the 6MG plate to allow the resin to fall into the 384-well plate containing the single-base extension product. After sealing with the sealing film, rotate the plate on a shaker at 60 rpm / min for 20 minutes to ensure thorough purification.

[0040] Step 6, Chip Spotting: Place the 384-well plate from Step 5 in a centrifuge and centrifuge at 4000 rpm / min for 5 min. Remove the sealing membrane and set aside. Start the MassARRAY Nanodispenser RS1000 spotting instrument and transfer the resin-purified extension product onto the 384-well SpectroCHIP chip. Analyze the spotted chip using MALDI-TOF and use TYPER4.0 software for typing and outputting the detection results.

[0041] II. The above test results are analyzed as follows: SNP genotyping was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (Matrix-Assisted Laser Desorption / ionization Time-of-Flight Mass Spectrometry). The genotyping results were output using TYPER 4.0 software, as shown in Tables 8 and 9. Tables 8 and 9 show the SNP locus genotyping results for Examples YF-01 and YF-02, respectively. As can be seen from Tables 8 and 9, the genotypes of all 20 loci provided in Example 1 were accurately detected, with a detection rate of 100%. The genotypes of all loci in three replicates were compared, and the concordance rate reached 100%. The mass spectra of three replicates of the rs1801133 locus in sample YF-01 are randomly selected and illustrated in the attached figures. See the attached figures for details. Figures 1-3 .

[0042] Table 8. SNP locus gene detection results of sample YF-01

[0043] Table 9. SNP locus gene detection results of sample YF-02

[0044] III. Interpretation of the above test results: A multigene risk assessment model was constructed using the risk allele types and effect values ​​(Beta values) of 19 loci provided in Supplementary Data 4 of Liu et al.'s study (Nat Commun. 2025, 16(1):4178) (shown in Table 10) to predict the risk of GDM. The predictive performance of the combined model was improved when clinical characteristics before 20 weeks of gestation were integrated with the multigene risk assessment model. Based on the genotyping results of the MTHFR gene C677T locus (rs1801133), individualized folic acid nutritional intervention recommendations before and after the onset of GDM were provided. Table 11 shows the interpretation of the gene detection results of the example samples YF-01 and YF-02.

[0045] Table 10. Risk allele types and effect sizes for 19 GDM-related gene loci.

[0046] Table 11. Predicted risk of GDM and nutritional intervention recommendations for two samples.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A test reagent, characterized by comprising: The testing reagent includes reagents for detecting polymorphic sites of 19 risk genes for gestational diabetes and 1 polymorphic site of a nutritional intervention gene. The genes include MTNR1B, GLIS3, HHEX-IDE, GCKR, GALNT5, PCSK1, MNX1, HKDC1, MYO5C, SLC39A11, FOXA2, PAX4, CTBP1, GCK, UBE2E2, FCN1, GLP1R, CDKAL1, SLC30A8, and MTHFR.

2. The agent of claim 1, wherein The polymorphic sites include rs10466351, rs10758593, rs10882102, rs11127048, rs12694904, rs2882298, rs4716672, rs4746822, rs4776024, rs58299944, rs6113722, rs61160304, rs73069940, rs741037, rs76435632, rs7851502, rs9366994, rs9368222, rs9650069, and rs1801133.

3. Use of a test agent for the manufacture of a kit for the joint prediction of the risk of developing gestational diabetes mellitus and of a nutritional intervention during pregnancy, characterized in that, The detection reagent is the reagent described in claim 1.

4. The application according to claim 3, characterized in that, The detection reagent includes PCR amplification primer pairs and single-base extension primers. The PCR amplification sequences are shown in SEQ ID NO:1-SEQ ID NO:40, and the single-base extension primers are shown in SEQ ID NO:41-SEQ ID NO:

60.

5. The application according to claim 3, characterized in that, The detection reagent is one or more of the following: DNA microarray or chip, specific PCR primers or probes, and targeted high-throughput sequencing reagents.

6. A reagent kit, characterized in that, The kit contains the reagent as described in claim 1.

7. The reagent kit according to claim 6, characterized in that, The kit also includes DNA extraction reagents, PCR amplification reagents, SAP reagents, and resin purification reagents.

8. A set of molecular markers, characterized in that, The molecular markers include rs10466351, rs10758593, rs10882102, rs11127048, rs12694904, rs2882298, rs4716672, rs4746822, rs4776024, rs58299944, rs6113722, rs61160304, rs73069940, rs741037, rs76435632, rs7851502, rs9366994, rs9368222, rs9650069, and rs1801133.

9. A set of primers, characterized in that, The primer set includes PCR amplification primer pairs and single-base extension primers. The PCR amplification sequences are shown in SEQ ID NO:1-SEQ ID NO:40, and the single-base extension primers are shown in SEQ ID NO:41-SEQ ID NO:

60.

10. The use of the primer set according to claim 9 in the preparation of a kit for jointly predicting the risk of gestational diabetes mellitus and pregnancy nutritional intervention.

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