Primer probe group, kit and detection method for high homocysteine urine disease susceptibility gene detection

Abstract

The invention relates to a primer probe group, a kit and a detection method for high homocysteine urine disease susceptibility gene detection, and belongs to the technical field of gene polymorphism detection. In order to solve the problems of narrow detection range, insufficient genetic interpretation ability, deficiency of individualized screening strategy and the like in the existing HCU screening technology, the invention provides a primer probe group for high homocysteine urine disease susceptibility gene detection, which comprises a primer probe group for detecting Tgt of CBS gene c.154 site in a detected sample, a primer probe group for detecting CBS gene c.154 site, a primer probe group for detecting CBS gene c.154 site, a primer probe group for detecting CBS gene c.154 site, and a primer probe group for detecting CBS gene c.154 site, c mutation: Ggt at c.457 site; a mutation and Ggt at IVS 13-111 sites; the primer probe group is used for detecting C mutation. Comprehensive and accurate detection of the high homocysteine urinary disease susceptibility gene is realized, a reliable molecular marker is provided for genetic counseling and family management through early risk prediction, accurate diagnosis and individualized intervention, and the molecular marker has important clinical application value in the aspects of improving diagnosis efficiency and sensitivity and improving patient prognosis.

Description

Technical Field

[0001] This invention belongs to the field of gene polymorphism detection technology, and particularly relates to primer and probe sets, kits and detection methods for detecting susceptibility genes for hyperhomocysteinuria. Background Technology

[0002] Homocystinuria (HCU) is an autosomal recessive inherited metabolic disorder caused by mutations in the cystathionine β-synthase (CBS) gene. As the key rate-limiting enzyme in the conversion of homocysteine ​​to cystathionine, reduced or absent CBS activity directly leads to the abnormal accumulation of homocysteine ​​(Hcy) in the body, resulting in significantly elevated Hcy levels in plasma and urine. Long-term hyperhomocysteinemia can cause serious clinical consequences such as neurological abnormalities, intellectual disability, skeletal deformities, lens dislocation, and atherosclerosis. Therefore, early identification and intervention are of significant clinical importance in preventing irreversible organ damage.

[0003] Current HCU screening technologies mainly include biochemical testing and conventional genetic testing. Biochemical testing provides quantitative assessment by analyzing the concentrations of Hcy, methionine, and their metabolic ratios in blood or urine. However, this method only reflects metabolic endpoints and cannot reveal the root cause of disease. Furthermore, the results are easily affected by diet and physiological state, resulting in insufficient specificity. Conventional genetic testing focuses on some high-frequency mutation sites in the CBS gene (such as p.I278T and p.G307S), using PCR amplification and Sanger sequencing for screening. While suitable for identifying known mutations in specific populations, its ability to detect rare or novel mutations is limited, and its sensitivity is low. In recent years, high-throughput sequencing (NGS) technology has been gradually applied to single-gene disease screening, but most studies are still based on gene panels or target region capture protocols, limiting the detection range and making it difficult to comprehensively cover potential pathogenic variants across the entire genome.

[0004] The existing technology system has three core defects: First, the detection range is narrow, and rare mutations or new variants are easily missed, leading to missed diagnoses for some patients; second, the genetic interpretation ability is insufficient, and some positive results lack functional verification or pathogenicity evidence, affecting clinical decision-making; third, the individualized screening strategy is lacking, and the mutation spectrum of different patients varies significantly, making it difficult for existing methods to meet the needs of systematic evaluation. Summary of the Invention

[0005] To address the problems of narrow detection range, insufficient genetic interpretation ability, and lack of personalized screening strategies in existing HCU screening technologies, this invention provides a primer and probe set, kit, and detection method for detecting susceptibility genes for hyperhomocysteinuria.

[0006] The technical solution of the present invention:

[0007] The primer and probe set for susceptibility gene detection of hyperhomocysteinuria includes a primer and probe set for detecting T>C mutation at c.154 site, G>A mutation at c.457 site, and G>C mutation at IVS13-111 site of the CBS gene in the tested samples.

[0008] Furthermore, the primer-probe set used to detect the T>C mutation at c.154 site consists of upstream and downstream primers with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, and a probe as shown in SEQ ID No. 3; the primer-probe set used to detect the G>A mutation at c.457 site consists of upstream and downstream primers with nucleotide sequences as shown in SEQ ID No. 4 and SEQ ID No. 5, and a probe as shown in SEQ ID No. 6; and the primer-probe set used to detect the G>C mutation at IVS13-111 site consists of upstream and downstream primers with nucleotide sequences as shown in SEQ ID No. 7 and SEQ ID No. 8, and a probe as shown in SEQ ID No. 9.

[0009] Furthermore, the probe shown in SEQ ID No. 3 has a fluorescent group ROX at its 5' end and a quencher group BHQ2 at its 3' end; the probe shown in SEQ ID No. 6 has a fluorescent group CY5 at its 5' end and a quencher group BHQ2 at its 3' end; and the probe shown in SEQ ID No. 9 has a fluorescent group FAM at its 5' end and a quencher group BHQ1 at its 3' end.

[0010] A kit for detecting susceptibility genes for hyperhomocysteinuria, comprising the primer and probe set as described in any one of claims 1-3 for detecting the T>C mutation at c.154 site, the G>A mutation at c.457 site, and the G>C mutation at IVS13-111 sites of the CBS gene in the test sample.

[0011] Furthermore, it also contains a PCR premix, which contains DNA polymerase, dNTPs, and magnesium. 2+ Ions, fluorescent dyes, and buffer solutions.

[0012] Furthermore, it also contains a negative control and a positive control, wherein the negative control is physiological saline and the positive control is a recombinant DNA plasmid containing all target gene target sequence polymorphic sites.

[0013] A detection method for detecting susceptibility genes in hyperhomocysteinuria, comprising the following steps:

[0014] Step 1: Extract total RNA from the tested samples using TRIZOL;

[0015] Step 2: Reverse transcribe total RNA to generate cDNA;

[0016] Step 3: Use primer and probe sets to perform qRT-PCR amplification of the target gene and quantify it by fluorescence signal;

[0017] Step 4: Sanger Validation. Sequencing is performed on each successfully amplified PCR product to verify pathogenic point mutations.

[0018] The primer-probe set comprises the upstream and downstream primers with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2 for detecting the T>C mutation at the c.154 site of the CBS gene, and the probe shown in SEQ ID No. 3; the upstream and downstream primers with nucleotide sequences as shown in SEQ ID No. 4 and SEQ ID No. 5 for detecting the G>A mutation at the c.457 site, and the probe shown in SEQ ID No. 6; or the upstream and downstream primers with nucleotide sequences as shown in SEQ ID No. 7 and SEQ ID No. 8 for detecting the G>C mutation at the IVS13-111 site, and the probe shown in SEQ ID No. 9.

[0019] The probe shown in SEQ ID No. 3 has a fluorescent group ROX at its 5' end and a quencher group BHQ2 at its 3' end; the probe shown in SEQ ID No. 6 has a fluorescent group CY5 at its 5' end and a quencher group BHQ2 at its 3' end; the probe shown in SEQ ID No. 9 has a fluorescent group FAM at its 5' end and a quencher group BHQ1 at its 3' end.

[0020] Furthermore, the reverse transcription reaction system described in step two is as follows: 1 μg total RNA, 10 μL 2×TS Reaction Mix, 1 μL TransScript RT / RI Enzyme Mix, 1 μL gDNA remover, and RNase-Free Water to make up the final volume of 20 μL; the reverse transcription reaction program is as follows: react at 42℃ for 15 minutes; react at 85℃ for 5 seconds.

[0021] Furthermore, the reaction system for qRT-PCR described in step three is as follows: 10 μL of 2×Probe qPCR MasterMix, 0.4 μL of upstream primer, 0.4 μL of downstream primer, 1 μL of Taq Man probe, 2 μL of cDNA, and RNase-Free Water to make up the difference, so that the final volume is 20 μL.

[0022] The qRT-PCR reaction procedure is as follows: pre-denaturation at 95℃ for 5 minutes, denaturation at 95℃ for 10 seconds → annealing / extension at 60℃ for 30 seconds, and repeat the denaturation → annealing / extension steps 40 times; only one set of primers and probes is added for each qRT-PCR reaction.

[0023] Furthermore, in step four, the Sanger validation uses the same primers as the PCR amplification for bidirectional Sanger sequencing; the total volume of the sequencing reaction system is 10 μL, including: 1 μL of BigDye Terminator v3.1 Ready Reaction Mix, 1.5 μL of 5× sequencing buffer, 1 μL of primers, 2 μL of purified PCR product, and nuclease-free water to a final volume of 10 μL; the sequencing cycle conditions are as follows: 96℃ for 1 min; 25 cycles: 96℃ for 10 s, 50℃ for 5 s, 60℃ for 4 min; incubation at 4°C.

[0024] The beneficial effects of this invention are:

[0025] This invention focuses on the precise detection and clinical intervention of hyperhomocysteinuria. Through systematic analysis of blood samples from patients and their parents using whole-exome sequencing (WES) technology, it has for the first time discovered three previously unreported heterozygous CBS gene variants (c.154T>C, c.457G>A, and IVS13-111G>C). These variants are not recorded in existing literature or population databases, significantly enriching the genetic variation spectrum of this disease and providing crucial clues for elucidating its molecular pathogenesis. By comparing and integrating with gene databases from healthy populations, combined with bioinformatics analysis and functional prediction, this invention further screened mutation sites with high pathogenicity associations. Their pathogenicity was validated in the patient population, laying an important foundation for subsequent gene diagnosis and precision medicine.

[0026] Targeting the aforementioned key mutation sites, this invention has developed a specific primer and probe set capable of accurately detecting the T>C mutation at c.154, the G>A mutation at c.457, and the G>C mutation at IVS13-111 of the CBS gene, effectively covering rare mutations and novel variants, and significantly reducing the risk of missed diagnoses. The accompanying kit integrates PCR premix, negative control, and positive control. The DNA polymerase, dNTPs, magnesium ions, fluorescent dye, and buffer in the premix work synergistically to ensure efficient and stable amplification. The control settings enhance the reliability and reproducibility of the test results through a standardized process. The detection method employs a standardized process of "total RNA extraction → reverse transcription to generate cDNA → qRT-PCR amplification → Sanger verification," combined with a well-defined reaction system and procedure, supporting qualitative electrophoresis or quantitative fluorescence analysis, providing multi-dimensional evidence for clinical diagnosis.

[0027] The implementation of this invention enables comprehensive and accurate detection of susceptibility genes for hyperhomocysteinuria, providing reliable molecular markers for genetic counseling and family management through early risk prediction, precise diagnosis, and individualized intervention. Its technological advantages are not only reflected in improved diagnostic efficiency and sensitivity, but also in demonstrating significant clinical application value in reducing irreversible organ damage and improving patient prognosis. Attached Figure Description

[0028] Figure 1 A comparison of Sanger sequencing data of the c.154 site of the CBS gene in the parents of patients with hyperhomocysteinuria.

[0029] Figure 2 Sanger sequencing comparison of the CBS gene c.457 site in the parents of patients with hyperhomocysteinuria;

[0030] Figure 3 A comparison of Sanger sequencing data of the IVS13-111 sites of the CBS gene in the parents of patients with hyperhomocysteinuria. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0032] Example 1

[0033] This embodiment provides a method for screening key pathogenic mutation sites in hyperhomocysteinuria.

[0034] Characteristic mutation sites were screened from samples of patients with hyperhomocysteinuria using whole-exome sequencing (WES) and bioinformatics analysis. The specific process is as follows:

[0035] Blood samples were collected from patients with hyperhomocysteinuria and their parents, and genomic DNA was extracted. Whole-exome sequencing was used to sequence the samples, obtaining gene sequence information from exon regions. The sequenced sequences were compared with the human reference genome to identify variant sites in the patient samples. Bioinformatics tools such as SIFT, PolyPhen-2, REVEL, and CADD online tools were used to predict protein function, and these variant sites were annotated and analyzed, including assessing the frequency of variants and predicting their impact on protein function.

[0036] The database search results are as follows:

[0037] c.154T>C (p.Cys52Arg, exon 3) was not included in gnomAD, 1000 Genomes, ExAC, or dbSNP in the final annotation, indicating it is an extremely rare / unreported variant. The amino acid substitution Cys→Arg is a strongly non-conserved substitution. Cys52, located within the catalytic domain of the CBS protein, is a conserved cysteine ​​residue involved in disulfide bonds and fold stability. SIFT: Damaging; PolyPhen-2: Probably damaging; REVEL: >0.8 (high pathogenicity score); CADD: >25 (significant functional impact), suggesting it may lead to localized folding disruption, impaired iron-sulfur cluster assembly, and significantly reduced CBS enzyme activity. Final determination: Consists of ACMG: PM2+PP3+ (strongly non-conserved substitution of conserved residues) with a tendency towards pathogenicity. In human clinical practice, Cys→Arg substitution in CBS proteins is known to frequently lead to a severe HCU phenotype.

[0038] c.457G>A (p.Gly153Arg, exon 5) Final annotation database search: also not found in gnomAD / ExAC / 1000g / ClinVar, belonging to unreported new or very low-frequency variants. Amino acid substitution: Gly→Arg, one of the most strongly non-conserved substitutions. The Gly153 site is located in the key flexible loop region of the classic CBS catalytic domain, which is the core of folding and catalytic activity. SIFT: Damaging; PolyPhen-2: Probably damaging; REVEL: 0.85–0.9 (highly pathogenic); CADD: >28 (significantly affects protein function) Summary of structural-functional impact: This region is highly sensitive to CBS activity. The rigid loss of Gly will disrupt the spatial conformation of the catalytic center, thereby significantly interfering with PLP coenzyme binding, and is expected to lead to a severe decrease in enzyme activity. Final judgment: Consistent with ACMG: PM2+PP3+PP2 (drastic substitution of highly conserved residues), tending to be pathogenic, and highly consistent with the classic homocystinuria phenotype.

[0039] IVS13-111G>C (Intron 13, −111 bp to exon 14 donor site) Final annotation database search: Not found in gnomAD / dbSNP / 1000g, belonging to rare non-coding variants. Splicing effect prediction (combined SpliceAI, MaxEntScan): Far from the splice site (−111 bp); does not significantly disrupt the original splice signal; overall prediction: low probability of splicing effect (SpliceAI Δ < 0.1); Functional effect summary: This site is more likely to belong to the intronic passenger variant with low probability of effect, unlikely to cause disease independently, but in the context of compound heterozygosity, it still needs to be carefully interpreted in conjunction with phenotype. Final judgment: ACMG: PM2 (low frequency), but no clear functional evidence, significance undetermined (VUS).

[0040] The patient carries three heterozygous variants in the CBS gene: c.154T>C (p.Cys52Arg) predisposing to pathogenicity; c.457G>A (p.Gly153Arg) predisposing to pathogenicity; and IVS13-111G>C: VUS, with a low probability of splicing influence. Both missense variants are located on highly conserved residues in the CBS catalytic domain and are strongly non-conserved substitutions, predicted to cause significant damage to protein structure and enzyme activity. These three variants are not found in the Genome Aggregation Database (gnomAD) or other public databases, supporting their rarity. The clinical phenotype (extremely high homocysteine, white matter lesions, and oculomotor system manifestations) is completely consistent with classic homocystinuria, suggesting that the two missense variants are pathogenic genotypes, and the VUS may be a paraphyletic variant.

[0041] Taking all these factors into account, these three sites have high accuracy and specificity.

[0042] Figure 1 A comparison of Sanger sequencing data of the c.154 site of the CBS gene in the parents of patients with hyperhomocysteinuria. Figure 1 The results showed that the father had a T>C heterozygous variant at the c.154 site of the CBS gene, and the mother carried the wild-type allele at this site.

[0043] Figure 2 Sanger sequencing comparison of the CBS gene c.457 site in the parents of patients with hyperhomocysteinuria; Figure 2 The results showed that the patient's mother had a G>A heterozygous variant at the c.457 site of the CBS gene, and the father carried the wild-type allele at this site.

[0044] Figure 3 Sanger sequencing comparison of the IVS13-111 sites of the CBS gene in the parents of patients with hyperhomocysteinuria; Figure 3 The results showed that the father had a G>C heterozygous variant at the IVS13-111 locus of the CBS gene, and the mother carried the wild-type allele at this locus.

[0045] By combining patients' clinical symptoms and family genetic information, characteristic mutation sites highly associated with hyperhomocysteinuria were screened. After a series of rigorous screening and validation processes, three previously unreported heterozygous variant sites in the CBS gene—c.154T>C, c.457G>A, and IVS13-111G>C—were finally identified. These sites may play a key role in the development and progression of the disease, providing a reliable basis for the detection and diagnosis of hyperhomocysteinuria.

[0046] Example 2

[0047] This embodiment provides a primer and probe set for detecting susceptibility genes for hyperhomocysteinuria, including a primer and probe set for detecting T>C mutation at c.154 site, G>A mutation at c.457 site, and G>C mutation at IVS13-111 site of the CBS gene in the tested sample.

[0048] The nucleotide sequences of the primer-probe sets used to detect the T>C mutation at c.154 are shown in SEQ ID Nos. 1-3; the nucleotide sequences of the primer-probe sets used to detect the G>A mutation at c.457 are shown in SEQ ID Nos. 4-6; and the nucleotide sequences of the primer-probe sets used to detect the G>C mutation at IVS13-111 are shown in SEQ ID Nos. 7-9. All three probes are labeled with the fluorescent group FAM at their 5' end and the quencher group BHQ2 at their 3' end.

[0049] Example 3

[0050] This embodiment provides a kit for detecting susceptibility genes for hyperhomocysteinuria, containing the primer and probe set provided in Example 2 for detecting the T>C mutation at c.154, the G>A mutation at c.457, and the G>C mutation at IVS13-111 in the CBS gene in the test sample, as well as PCR premix, negative control, and positive control. The PCR premix contains DNA polymerase, dNTPs, and magnesium. 2+ Ions, fluorescent dyes, and buffer solutions were used. The negative control was physiological saline, and the positive control was a recombinant DNA plasmid containing all polymorphic sites of the target gene sequence.

[0051] Example 4

[0052] This embodiment provides a detection method for detecting susceptibility genes for hyperhomocysteinuria, the steps of which are as follows:

[0053] Step 1: Extract total RNA from the tested samples using TRIZOL;

[0054] (1) Add 1 mL of TRIZOL lysis buffer, transfer the mixture to an EP tube, and label the groups accordingly;

[0055] (2) Place the EP tube in a high-speed centrifuge and centrifuge at 12000g for 15 minutes at 4°C. After centrifugation, collect the supernatant and store it in another EP tube. Discard the unlysaturated tissue precipitate.

[0056] (3) Carefully add 200 μL of chloroform to the EP tube using a pipette, vortex and mix well, place at room temperature for 10 minutes, then place in a pre-cooled (4℃) low-temperature centrifuge and centrifuge at 12000g for 15 minutes; at this time, it will separate into three layers: the upper layer is aqueous RNA, the middle layer contains DNA and protein, and the bottom layer is organic phase. Transfer 400 μL of the supernatant to a new EP tube.

[0057] (4) Add 600 μL of isopropanol, shake 5 times to mix evenly, let stand at room temperature for 10 minutes, and then centrifuge at 12000g for 10 minutes in a pre-cooled (4℃) low-temperature centrifuge to obtain RNA precipitate. Discard the supernatant.

[0058] (5) Wash the RNA with 1 mL of 75% ethanol, place it in a pre-cooled (4°C) low-temperature centrifuge, centrifuge at 7500g for 5 minutes, discard the supernatant, remove the liquid and dry it after opening the lid;

[0059] (6) Add 20 μL of DEPC water to each EP tube to dissolve the RNA precipitate;

[0060] (7) Take 1 μL of RNA solution and test the RNA concentration using a Nanodrop 2000 nucleic acid concentration analyzer. Observe that the 260 / 280 OD value is between 1.8 and 2.0 and the curve is a single peak, indicating that the RNA extraction was successful and the purity is acceptable.

[0061] (8) Proceed to the next experiment or store at -80℃.

[0062] Step 2: Reverse transcribe total RNA to generate cDNA;

[0063] The reaction system for reverse transcription is as follows:

[0064] Total RNA 1μg

[0065] 2×TS Reaction Mix 10μL

[0066] TransScript RT / RI Enzyme Mix 1μL,

[0067] gDNA remover 1μL,

[0068] Make up the volume with RNase-Free Water to bring the final volume to 20 μL.

[0069] After carefully adding the sample to the 8-tube PCR instrument and tightening the cap, centrifuge briefly and then place it in the PCR instrument to set the running conditions and begin reverse transcription.

[0070] The reverse transcription reaction procedure was as follows: react at 42°C for 15 minutes; react at 85°C for 5 seconds.

[0071] Step 3: Use the primer and probe set provided in Example 2 to perform qRT-PCR amplification of the target gene and quantify it by fluorescence signal;

[0072] The reaction system for qRT-PCR is as follows:

[0073] 2×Probe qPCR MasterMix 10μL,

[0074] The upstream primer was 0.4 μL, and the final concentration in the system was 0.2 μM.

[0075] 0.4 μL of downstream primer was used, resulting in a final concentration of 0.2 μM in the system.

[0076] 1 μL of TaqMan probe was added, resulting in a final concentration of 0.25 μM in the system.

[0077] 2 μL of cDNA

[0078] Make up the volume with RNase-Free Water to bring the final volume to 20 μL.

[0079] The qRT-PCR reaction procedure is as follows: pre-denaturation at 95℃ for 5 minutes, denaturation at 95℃ for 15 seconds → annealing / extension at 60℃ for 25 seconds, and repeat the denaturation → annealing / extension steps 40 times; only one set of primers and probes is added for each qRT-PCR reaction.

[0080] Each qRT-PCR reaction uses only one primer-probe set. The primer-probe set consists of the upstream and downstream primers (nucleotide sequences shown in SEQ ID No. 1 and SEQ ID No. 2) for detecting the T>C mutation at c.154, and the probe (as shown in SEQ ID No. 3); the upstream and downstream primers (nucleotide sequences shown in SEQ ID No. 4 and SEQ ID No. 5) for detecting the G>A mutation at c.457, and the probe (as shown in SEQ ID No. 6); or the upstream and downstream primers (nucleotide sequences shown in SEQ ID No. 7 and SEQ ID No. 8) for detecting the G>C mutation at IVS13-111, and the probe (as shown in SEQ ID No. 9).

[0081] The probe shown in SEQ ID No. 3 has a fluorescent group ROX at its 5' end and a quencher group BHQ2 at its 3' end; the probe shown in SEQ ID No. 6 has a fluorescent group CY5 at its 5' end and a quencher group BHQ2 at its 3' end; the probe shown in SEQ ID No. 9 has a fluorescent group FAM at its 5' end and a quencher group BHQ1 at its 3' end.

[0082] Step 4: Sanger Validation. Sequencing was performed on each successfully amplified PCR product to verify the pathogenic point mutation; DNA was extracted using a Tianlong commercial kit. The specific steps are as follows:

[0083] (1) Add 20 μL of proteinase K, 300 μL of lysis buffer and 200 μL of sample to a 1.5 mL nuclease-free centrifuge tube, vortex for 15 s and centrifuge for 5 s.

[0084] (2) 60℃ metal bath for 20 minutes, mix several times during the period (mix once every 8 minutes), and invert about 20 times each time;

[0085] (3) Let stand at room temperature for 3 minutes; then centrifuge briefly to remove any residual liquid from the tube cap;

[0086] (4) Gently open the tube cap, add 300 μL of anhydrous ethanol, mix thoroughly, and centrifuge briefly;

[0087] (5) Take all the solution from the centrifuge tube and add it to the adsorption column. Cover the tube and centrifuge at 8000 rpm for 1 min. Discard the waste liquid and put the adsorption column back into the collection tube.

[0088] (6) Gently open the tube cap, add 600 μL of washing solution 1, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0089] (7) Gently open the tube cap, add 600 μL of washing solution 2, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0090] (8) Repeat step 7 once;

[0091] (9) Centrifuge at 12000 rpm for 2 min;

[0092] (10) After centrifugation, place the adsorption column into a new 1.5 mL EP tube, open the cap, and let it stand at room temperature for 5 min;

[0093] (11) Add 100 μL of eluent to the adsorption column (preheated to 60 °C), let stand at room temperature for 5 min, and centrifuge at 12000 rpm for 1 min; collect the solution into an EP tube and store at -20 °C for later use.

[0094] Sanger sequencing was performed using the same primers as for PCR amplification.

[0095] The total volume of the sequencing reaction system was 10 μL, including: 1 μL of BigDye Terminator v3.1 Ready ReactionMix (Thermo Fisher Scientific); 1.5 μL of 5× sequencing buffer; 1 μL of primers; 2 μL of purified PCR product; and nuclease-free water to a final volume of 10 μL. The sequencing conditions were as follows: 96℃ for 1 min; 25 cycles: 96℃ for 10 s, 50℃ for 5 s, 60℃ for 4 min; incubation at 4℃.

[0096] Analysis using Chromas (Technelysium) revealed distinct bimodal peaks at all three loci in bidirectional Sanger sequencing, indicating the presence of true heterozygous variants. A heterozygous variant is defined as a clear superposition of bimodal peaks at the corresponding location, with surrounding peaks exhibiting regular shapes and a good signal-to-noise ratio. Parental carriage of the variant was also examined to assess its co-segregation with the disease phenotype.

Claims

1. A primer probe set for detecting a high homocysteine urine susceptibility gene, characterized by, The primer probe set for detecting the T>C mutation at the c.154 site, the G>A mutation at the c.457 site and the G>C mutation at the IVS13-111 site of the CBS gene in the test sample. 2.The primer probe set for detecting high homocysteine urine susceptibility gene according to claim 1, characterized in that, The primer probe set for detecting the T>C mutation at the c.154 site is an upstream primer and a downstream primer with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, and a probe as shown in SEQ ID No. 3; the primer probe set for detecting the G>A mutation at the c.457 site is an upstream primer and a downstream primer with nucleotide sequences as shown in SEQ ID No. 4 and SEQ ID No. 5, and a probe as shown in SEQ ID No. 6; and the primer probe set for detecting the G>C mutation at the IVS13-111 site is an upstream primer and a downstream primer with nucleotide sequences as shown in SEQ ID No. 7 and SEQ ID No. 8, and a probe as shown in SEQ ID No.

9. 3.The primer probe set for detecting high homocysteine urine susceptibility gene according to claim 2, characterized in that, The 5' end of the probe as shown in SEQ ID No. 3 is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; the 5' end of the probe as shown in SEQ ID No. 6 is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ2; and the 5' end of the probe as shown in SEQ ID No. 9 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

4. A kit for detecting a predisposition to hyperhomocysteinuria, characterized in that, The primer probe set for detecting the T>C mutation at the c.154 site, the G>A mutation at the c.457 site and the G>C mutation at the IVS13-111 site of the CBS gene in the test sample.

5. The kit for the detection of high homocysteine urine susceptibility gene according to claim 4, characterized in that, Also contained is a PCR master mix containing DNA polymerase, dNTPs, magnesium 2+ ions, a fluorescent dye, and a buffer.

6. The kit for the detection of high homocysteine urine susceptibility gene according to claim 5, characterized in that, It also contains a negative control and a positive control, wherein the negative control is physiological saline, and the positive control is a DNA recombinant plasmid containing all target gene target sequence polymorphic sites.

7. A detection method for high homocysteine urine disease susceptibility gene detection, characterized in that, The steps are as follows: Step one, total RNA of the test sample is extracted using TRIZOL; Step two, total RNA is reverse transcribed to generate cDNA; Step three, qRT-PCR is performed using the primer probe set to amplify the target gene, and the fluorescence signal is quantified; Step four, Sanger verification is performed to sequence each successfully amplified PCR product to verify pathogenic point mutations. The primer probe set is an upstream primer and a downstream primer for detecting the T>C mutation at the c.154 site of the CBS gene with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, and a probe as shown in SEQ ID No. 3; an upstream primer and a downstream primer for detecting the G>A mutation at the c.457 site with nucleotide sequences as shown in SEQ ID No. 4 and SEQ ID No. 5, and a probe as shown in SEQ ID No. 6; or an upstream primer and a downstream primer for detecting the G>C mutation at the IVS13-111 site with nucleotide sequences as shown in SEQ ID No. 7 and SEQ ID No. 8, and a probe as shown in SEQ ID No. 9, The 5' end of the probe shown as SEQ ID No. 3 is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; the 5' end of the probe shown as SEQ ID No. 6 is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ2; the 5' end of the probe shown as SEQ ID No. 9 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

8. The detection method for detecting susceptibility genes for hyperhomocysteinuria according to claim 7, characterized in that, The reverse transcription reaction system of step two is: total RNA 1 μg, 2xTS Reaction Mix 10 μL, TransScript RT / RI Enzyme Mix 1 μL, gDNA remover 1 μL, and RNase-Free Water to make up the final volume of 20 μL; the reverse transcription reaction program is: 42°C for 15 minutes; 85°C for 5 seconds.

9. The detection method for detecting susceptibility genes for hyperhomocysteinuria according to claim 8, characterized in that, The reaction system of qRT-PCR of step three is: 2xProbe qPCR MasterMix 10 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, Taq man probe 1 μL, cDNA 2 μL, and RNase-Free Water to make up the final volume of 20 μL; The reaction program of qRT-PCR is: pre-denaturation 95°C for 5 minutes, denaturation 95°C for 10 seconds→ annealing / extension 60°C for 30 seconds, denaturation→ annealing / extension, repeat 40 times; only one set of primer probe group is added for each qRT-PCR reaction.

10. The method of claim 9, wherein the detection method for the high homocysteine urine disease susceptibility gene is characterized by, Sanger verification of step four uses the same primers as PCR amplification for bidirectional Sanger sequencing; The total volume of the sequencing reaction is 10 μL, including: BigDye Terminator v3.1 Ready Reaction Mix 1 μL, 5x sequencing buffer 1.5 μL, primer 1 μL, purified PCR product 2 μL, and nuclease-free water to make up to 10 μL; the cycle sequencing conditions are as follows: 96°C for 1 min; 25 cycles: 96°C for 10 s, 50°C for 5 s, 60°C for 4 min; 4°C for 10 min.

Images