Kit for screening cerebral arterial thrombosis risk and screening method

Abstract

The invention discloses a kit for screening cerebral arterial thrombosis risk and a screening method, and belongs to the technical field of biological detection. The kit for screening the cerebral arterial thrombosis risk is a kit for detecting gene mutation of a C677T site of an MTHFR gene and comprises a forward primer and a reverse primer, the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.2. The kit for screening the cerebral arterial thrombosis risk is a kit for detecting gene mutation of the C677T site of the MTHFR gene. The invention creatively finds that the gene polymorphism of the C677T site of the MTHFR gene has certain relevance with the disease of the cerebral arterial thrombosis, the site mutation is one of high-risk factors of the cerebral arterial thrombosis, and the A1298C site mutation of the MTHFR gene is not obviously associated with the susceptibility of the cerebral arterial thrombosis. The risk of the cerebral arterial thrombosis is evaluated by screening a susceptibility gene locus, the operation is simple, the cost is reduced, the prevention of the cerebral arterial thrombosis is enhanced, and the incidence rate of the cerebral arterial thrombosis is further reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a kit and a screening method for screening the risk of ischemic stroke. Background Art

[0002] Stroke, also known as ischemic stroke, is an acute cerebrovascular disease characterized by high morbidity, mortality, disability, and recurrence rates, making it the leading health threat to the Chinese population. Ischemic stroke accounts for approximately 70% of stroke incidence and predominantly affects the elderly. Risk factors for stroke include hypertension, a history of atrial fibrillation, smoking, diabetes, and dyslipidemia. Despite the widespread use of new treatments such as intravenous thrombolysis and mechanical thrombectomy, the overall prognosis remains unsatisfactory. Therefore, predicting and controlling the development of ischemic stroke is a critical and urgent clinical challenge. Currently, there are two main strategies for preventing the development of ischemic stroke: the "mass approach" and the "high-risk approach." The "mass approach" aims to reduce the prevalence of ischemic stroke by lowering community risk factors through public education and government policies. The "high-risk approach" aims to reduce stroke by identifying individuals at high risk for ischemic stroke and then reducing their risk through appropriate medical treatment.

[0003] Numerous stroke susceptibility genes play a significant role in its onset and prognosis. MTHFR is a key enzyme in folate metabolism. 5,10-methylenetetrahydrofolate (5,10-CH2-THF) is converted to 5-methyltetrahydrofolate (5-MTHF) by MTHFR. 5-MTHF then enters the methyl transfer pathway, where it remethylates homocysteine (Hcy), providing methyl groups for DNA and protein methylation and maintaining low blood homocysteine levels. When the MTHFR gene mutates, 5,10-methylenetetrahydrofolate is unable to successfully donate methyl groups to Hcy through the folate metabolism pathway, resulting in a blockage in the methionine cycle and an accumulation of Hcy in the body, leading to elevated levels. The MTHFR gene is located on chromosome 1 at position 1p36.3 and encodes 656 amino acids, comprising 11 exons and 10 introns. Research has identified nearly 40 polymorphisms in the MTHFR gene. The two most clinically significant and widely studied are C677T and A1298C. The C677T mutation refers to a change in the nucleotide base 677 in exon 4 of the MTHFR gene from cytosine (C) to thymine (T), resulting in a change in the amino acid at that site from alanine to valine. The A1298C mutation refers to an amino acid substitution at the nucleotide base 1298 in exon 7 from adenine (A) to cytosine (C).

[0004] Patent CN113913509A discloses a detection kit and method for detecting gene mutations in molecular markers associated with stroke risk screening. The kit is designed for two stroke-related molecular marker gene mutation sites (C677T and A1298C). Combined with the CRISPR-Cas12b system, it can quickly detect the presence of the corresponding stroke risk-related molecular marker gene mutations in the test sample through changes in fluorescence signals. In this patent, determining the presence of stroke risk requires testing two molecular markers, which is costly and slow. Given the characteristics of ischemic stroke, the development of a rapid screening kit for ischemic stroke risk is of great significance. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides a kit and screening method for screening the risk of ischemic stroke. The kit determines whether there is a risk of ischemic stroke by simply screening for the C677T gene mutation in the MTHFR gene. The detection speed is fast and the detection cost is low.

[0006] The present invention is achieved through the following technical solutions: On the one hand, the present invention discloses a kit for screening ischemic stroke risk. The kit for screening ischemic stroke risk is a kit for detecting gene mutation at the C677T site of the MTHFR gene, comprising a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the reverse primer is shown in SEQ ID NO.2.

[0007] Furthermore, the kit for screening ischemic stroke risk also includes PCR MIX.

[0008] On the other hand, the present invention also discloses a screening method for the kit for screening ischemic stroke risk, comprising the following steps: (1) Extracting sample DNA to obtain a DNA template; (2) performing PCR amplification using the DNA template extracted in step (1) to obtain an amplified product; (3) Perform gene sequencing on the amplified product to determine whether the C677T site of the MTHFR gene has mutated.

[0009] Furthermore, the PCR amplification system in step (2) consists of 1 μL of forward primer, 1 μL of reverse primer, 12.5 μL of PCR MIX, 1 μL of DNA template, and 9.5 μL of ddH2O.

[0010] Furthermore, the PCR amplification procedure in step (2) is as follows: pre-denaturation treatment at 95°C for 5 minutes; denaturation of DNA at 95°C for 10 seconds to unwind the double-stranded DNA molecule into two single strands; 30 seconds at 60°C to allow the primers to bind to both ends of the target DNA sequence; 2 minutes at 72°C, at which temperature the DNA polymerase synthesizes a new DNA chain and synthesizes a complementary chain along the single strand of the template DNA, for 30 cycles; and extension at 72°C for 5 minutes after the last cycle.

[0011] Furthermore, in step (3), the sample is sequenced by using an AB13730xl DNA sequence analyzer to obtain a sequencing peak graph, which is then observed using medcal software to perform genotyping on the gene polymorphism sites.

[0012] Beneficial effects The present invention creatively discovered that the MTHFR gene C677T site gene polymorphism is associated with the incidence of ischemic stroke. This site mutation is one of the high-risk factors for ischemic stroke, while the MTHFR gene A1298C site mutation has not been observed to be significantly associated with ischemic stroke susceptibility. Therefore, the risk of ischemic stroke can be assessed by screening only one susceptibility gene site, which is simple to operate, reduces costs, strengthens the prevention of ischemic stroke, and further reduces the incidence of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the DNA agarose gel electrophoresis diagram of the A1298C site and C677T site of the MTHFR gene; Figure 2 This is the result of gene mutation at A1298C and C677T sites of MTHFR gene. DETAILED DESCRIPTION

[0014] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0015] Referring to the "Stroke Risk Score Card" issued by the country in 2012, it includes 8 core risk factors + 2 warning medical histories. The specific assessment items of the 8 risk factors are: (1) history of hypertension (≥140 / 90mmHg) (1mmHg=0.133kPa) or taking antihypertensive drugs; (2) dyslipidemia or unknown; (3) diabetes; (4) atrial fibrillation or obvious irregular pulse or heart valve disease; (5) smoking; (6) obvious overweight or obesity (body mass index (BMI=weight / height2) ≥26kg / m2); (7) less physical activity (the standard for physical activity is exercise 3 times or more per week, 30 minutes or more each time and for more than 1 year; moderate to heavy manual labor workers are considered to have more physical activity); (8) a history of stroke in the immediate family. The 2 warning medical histories are: (1) a history of stroke; (2) a history of transient ischemic attack (TIA). If a person has ≥3 core risk factors or has had a stroke or transient ischemic attack in the past, he or she is considered to meet the criteria for a high-risk group for ischemic stroke.

[0016] Study subjects: From June 2020 to December 2021, we screened the Beicheng and Dongcheng districts of Liaocheng, Shandong Province, for high-risk ischemic stroke by sifting through community health records. A total of 635 individuals at high risk for ischemic stroke were selected as the experimental group, including 108 patients with prior stroke, 113 patients with prior transient ischemic attack (TIA), and 414 individuals with ≥3 core risk factors (327 males and 308 females). During the same period, 218 healthy individuals undergoing physical examinations at Liaocheng People's Hospital were selected as the healthy control group (110 males and 108 females). Information regarding the included individuals was recorded. This study adhered to medical ethics standards and was approved by the Ethics Committee of Liaocheng People's Hospital (Approval No. 2020016). All participants in the screening voluntarily participated in the study and signed informed consent for clinical research. The 635 individuals identified as being at high risk for ischemic stroke were then divided into three subgroups: a. Those with a current or previous diagnosis of ischemic stroke by imaging (the stroke group); b. Those with a current or previous diagnosis of transient ischemic attack (TIA) (the TIA group); and c. Those with fewer than three core risk factors as defined by the "Stroke Risk Score Card" (the high-risk stroke group).

[0017] The kit for screening ischemic stroke risk of the present invention is a kit for detecting the gene mutation at the C677T site of the MTHFR gene, comprising a forward primer, a reverse primer, and a PCR MIX. The sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the reverse primer is shown in SEQ ID NO.2: SEQ ID NO. 1 (forward primer): 5′-GTGGTCTCTTCATCCCTCGC-3′; SEQ ID NO. 2 (reverse primer): 5′-GGAGCTTATGGGCTCTCCTG-3′.

[0018] In the embodiment of the present invention, the forward primer and reverse primer for detecting the gene mutation at the A1298C site of the MTHFR gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively: SEQ ID NO. 3 (forward primer): 5′-CCTTCCTTGCAAATACATCTTTGTTCTT-3′; SEQ ID NO. 4 (reverse primer): 5′-ACTCCAGCATCACTCACTTTGTG-3′.

[0019] Example 1 Detection of MTHFR gene C677T mutation (1) Extraction of sample DNA A rapid DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract DNA from whole blood samples to obtain DNA templates. The specific operation process is as follows: 1) Take 300 μL of whole blood sample containing anticoagulant, add 750 μL of cell lysis buffer CL, and invert and mix 5 times; 2) Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant and invert the tube onto absorbent paper for 2 minutes to ensure there is sediment at the bottom of the tube. 3) Add 200 μL each of buffer GA and GB and 20 μL of Proteinase K solution and shake well; 4) Set the water bath temperature to 70°C for 15 minutes, shaking several times during the process. 5) Add 200 μL of anhydrous ethanol, shake for 15 seconds to ensure uniform mixing, and quickly centrifuge to remove water droplets on the tube wall.

[0020] 6) Add the resulting solution and precipitate to an adsorption column CB3 and centrifuge at 12,000 rpm for 30 seconds. Discard the supernatant and ensure there is a precipitate at the bottom of the tube. 7) Add 500 μL of anhydrous ethanol and buffer GD to CB3 and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid and place the adsorption column CB3 in a collection tube. 8) Add anhydrous ethanol and 600 μL of rinse solution PW to CB3 and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid and place the adsorption column CB3 in a collection tube. 9) Step (8) is repeated once; 10) Place the adsorption column CB3 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes. Discard the waste liquid and allow it to dry at room temperature. 11) Transfer the adsorption column CB3 to a new centrifuge tube and drop 60 μL of elution buffer TE onto the center of the adsorption membrane. Allow to dry again at room temperature and centrifuge at 12,000 rpm for 2 minutes. Collect the final solution, which is the DNA template. After testing, the DNA concentration of the extracted whole blood samples was 12-100 ng / μL, which was good. The DNA concentration was then diluted to 25 ng / μL by adding ddH2O. The gel electrophoresis results were as follows: Figure 1 As shown, the bands are clear without any other bands, and the position, size and brightness of each band are comparable, indicating that the integrity and purity of the extracted sample DNA are good.

[0021] (2) PCR amplification: PCR amplification was performed using the DNA template extracted in step (1) (the amplification primers were the forward primer shown in SEQ ID NO. 1 and the reverse primer shown in SEQ ID NO. 2) to obtain an amplified product. The PCR amplification system is shown in Table 1 below, and the PCR amplification procedure is shown in Table 2 below; Table 1 PCR amplification system ; Table 2 PCR amplification procedure ; (3) Sample gene sequencing Samples were sequenced using an AB13730xl DNA sequencer to obtain sequencing peaks. Sequencing curves were then inspected to ensure good signal intensity and peak shape, avoiding signal congestion or unclear peaks. The sequencing peaks were then analyzed using medcal software to perform genotyping for polymorphic loci.

[0022] Example 2 Detection of MTHFR gene A1298C mutation The amplification primers in step (2) of Example 2 are the forward primer shown in SEQ ID NO. 3 and the reverse primer shown in SEQ ID NO. 4. The remaining steps and operating conditions are the same as those in Example 1.

[0023] Example 3 Result analysis: The MTHFR gene C677T site and A1298C site in Example 1 and Example 2 were genotyped by Sanger sequencing. Both sites of the MTHFR gene included three types: wild type, heterozygous mutant type and homozygous mutant type. The DNA fragments of the two sites were detected by fluorescent labeling. The mutation status was detected as follows: Figure 2 As shown, the red single peak at the C677T site indicates a homozygous mutation (TT), the red and blue double peaks indicate a heterozygous mutation (CT), and the blue single peak indicates a wild type (CC); the blue single peak at the A1298C site indicates a homozygous mutation (CC), the blue-green double peaks indicate a heterozygous mutation (AC), and the green single peak indicates a wild type (AA). The MTHFR gene sequencing results of the high-risk population for ischemic stroke and the healthy control group are shown in Table 3.

[0024] Statistical analysis showed that the genotype distribution of the C677T site of the MTHFR gene in the high-risk population for ischemic stroke was TT (306), CT (266), and CC (63), with genotype distribution frequencies of 48.2%, 41.8%, and 10%, respectively; the allele distribution was C (392) and T (878), with allele distribution frequencies of 30.9% and 69.1%, respectively; while the genotype distribution in the healthy population was TT (69), CT (114), and CC (35), with genotype distribution frequencies of 31.7%, 52.3%, and 16.1%, respectively; the allele distribution was C (184) and T (252), with allele distribution frequencies of 42.2%, respectively. , 57.8%; the genotype distribution results of the A1298C site of the MTHFR gene in the high-risk population for ischemic stroke were CC (0), AC (168), AA (467), with genotype distribution frequencies of 0, 26.5%, 73.5%, respectively; the allele distribution results were A (1102), C (168), with allele distribution frequencies of 86.8% and 13.2%, respectively; the genotype distribution results in the healthy population were CC (0), AC (71), AA (147), with genotype distribution frequencies of 0, 32.6%, 67.4%, respectively; the allele distribution results were A (365), C (71), with allele distribution frequencies of 83.7% and 16.3%, respectively.

[0025] Table 3 MTHFR gene sequencing results in high-risk ischemic stroke population and healthy control group

[0026] Example 4 Hardy-Weinberg test results Table 4 shows the results of a Hardy-Weinberg equilibrium test for MTHFR gene polymorphisms. The results showed that the genotype distribution at the C677T locus in both the high-risk ischemic stroke population and the healthy controls was consistent with Hardy-Weinberg equilibrium (P>0.05), indicating that the populations included in this study were well representative. However, the calculated genotype distribution at the A1298C locus in both populations was not consistent with Hardy-Weinberg equilibrium (P<0.05).

[0027] Table 4 Hardy-Weinberg genetic equilibrium test table of MTHFR gene locus The present study analyzed the relationship between the MTHFR C677T allele and the A1298C allele polymorphisms in 635 individuals at high risk for ischemic stroke and 218 healthy subjects undergoing physical examinations. Statistical analysis revealed significant differences in the frequencies of the TT genotype and the T allele at the MTHFR C677T locus between the high-risk population and the healthy control group (P<0.05). However, no significant differences were observed in the distribution of the CT genotype or the MTHFR A1298C polymorphism between the two groups (P>0.05).

[0028] Example 5 Relationship between MTHFR gene polymorphism and ischemic stroke in different models Three possible genetic models were constructed to evaluate the relationship between the MTHFR locus and ischemic stroke risk: dominant (CT+TT vs. CC), recessive (CC+CT vs. TT), and codominant (CC+TT vs. CT). The results are shown in Table 5. In the dominant MTHFR C677T model, carriers of the CT and TT genotypes were at a high risk of ischemic stroke, 1.763 times higher than carriers of the CC genotype. In the recessive model, carriers of the TT genotype were at a high risk of ischemic stroke, 2.008 times higher than carriers of the CC and CT genotypes. In the codominant model, carriers of the CC and TT genotypes were at a high risk of ischemic stroke, 1.521 times higher than carriers of the CT genotype. These results suggest that the MTHFR C677T polymorphism is associated with susceptibility to ischemic stroke, and mutations at this locus increase the risk of ischemic stroke.

[0029] Table 5 Relationship between MTHFR polymorphisms and high-risk factors for stroke under different genetic models .

Claims

1. A kit for screening the risk of ischemic stroke, characterized in that: The kit for screening ischemic stroke risk is a kit for detecting the gene mutation at the C677T site of the MTHFR gene, comprising a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the reverse primer is shown in SEQ ID NO.

2.

2. The kit for screening ischemic stroke risk according to claim 1, wherein The kit for screening the risk of ischemic stroke also includes PCR MIX.

3. A screening method using the kit for screening ischemic stroke risk according to claim 1 or 2, characterized in that: The steps include: (1) Extracting sample DNA to obtain a DNA template; (2) performing PCR amplification using the DNA template extracted in step (1) to obtain an amplified product; (3) Perform gene sequencing on the amplified product to determine whether the C677T site of the MTHFR gene has mutated.

4. The screening method according to claim 3, wherein The PCR amplification system in step (2) consists of 1 μL of forward primer, 1 μL of reverse primer, 12.5 μL of PCR MIX, 1 μL of DNA template, and 9.5 μL of ddH2O.

5. The screening method according to claim 3, wherein The PCR amplification procedure in step (2) is as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 10 seconds to unwind the double-stranded DNA into two single strands; 30 seconds at 60°C to allow the primers to bind to both ends of the target DNA sequence; 2 minutes at 72°C, at which temperature the DNA polymerase synthesizes a new DNA chain and synthesizes a complementary chain along the single strand of the template DNA, for 30 cycles; and extension at 72°C for 5 minutes after the last cycle.

6. The screening method according to claim 3, wherein In step (3), the sample was sequenced using an AB13730xl DNA sequence analyzer to obtain a sequencing peak graph, which was then observed using medcal software to perform genotyping on the gene polymorphism sites.

Images