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CN-121975935-A - Primer composition for detecting hereditary thrombolysis related gene mutation and application thereof

CN121975935ACN 121975935 ACN121975935 ACN 121975935ACN-121975935-A

Abstract

The invention provides a primer composition for detecting genetic thrombolysis related gene mutation and application thereof, and relates to the technical field of gene detection. The primer composition comprises PCR amplification primers for specifically amplifying 24 single nucleotide polymorphism sites of the following 22 genes and extension primers for single base extension of the 24 single nucleotide polymorphism sites. The primer composition constructs a detection system which is adaptive to the genetic characteristics of specific groups and gives consideration to pathogenic factors and risk factors by selecting twenty-two genes covering key systems such as coagulation, anticoagulation and the like and twenty-four specific sites, effectively overcomes the race heterogeneity limitation of conventional indexes, and can simultaneously capture the major mutation and the polygenic minor accumulation effect, thereby comprehensively analyzing the genetic susceptibility of individuals and providing a systematic and accurate basis for accurate screening and diagnosis of hereditary thrombosis.

Inventors

  • LI XIA
  • QIAN GANG
  • LI ZHIKAI
  • GUO HUIMIN

Assignees

  • 浙江迪谱诊断技术有限公司

Dates

Publication Date
20260505
Application Date
20260123

Claims (10)

  1. 1. A primer composition for detecting genetic thrombolytic related gene mutations, wherein the primer composition comprises a PCR amplification primer for specifically amplifying 24 single nucleotide polymorphic sites of the following 22 genes and an extension primer for single base extension of the 24 single nucleotide polymorphic sites, wherein the 24 single nucleotide polymorphic sites comprise: Rs146922325 and rs199469469 of PROC gene, rs199469503 of PROS1 gene, rs1799963 of F2 gene, rs6025 and rs4524 of F5 gene, rs16984852 of THBD gene, rs2227589 of SERPINC1 gene, rs1801133 of MTHFR gene, rs1799762 of PAI-1 gene, rs8176719 of ABO gene, rs8178847 of APOH gene, rs1799983 of NOS3 gene, rs2036914 of F11 gene, rs2066865 of FGG gene, rs6088735 of EDEM2 gene, rs12445050 of PLCG gene, rs867186 of PROCR gene, rs2288904 of SLC44A2 gene, rs8176592 of TFPI gene, rs7188250 of FTO gene, rs1805081 of NPC1 gene, rs1054533 of F2RL3 gene and rs1047891 of CPS1 gene.
  2. 2. The primer composition for detecting a mutation of a gene associated with hereditary thrombosis according to claim 1, wherein said PCR amplification primer comprises a nucleotide sequence shown as SEQ ID NO. 1 to SEQ ID NO. 48 and/or, The extension primer comprises a nucleotide sequence shown as SEQ ID NO. 49-73.
  3. 3. The primer composition for detecting a mutation in a gene associated with hereditary thrombosis as claimed in claim 1, wherein the rs146922325 locus and the rs199469469 locus of the PROC gene are located in the same amplicon sequence amplified by the same pair of PCR amplification primers, and an extension primer for the rs146922325 locus is bound to one strand of the amplicon, and an extension primer for the rs199469469 locus is bound to the complementary strand of the amplicon, The extension primer comprises a first extension primer and a second extension primer which are combined with the same DNA template chain aiming at the rs8176719 site of the ABO gene, wherein the nucleotide sequences of the first extension primer and the second extension primer are different so as to be respectively complementarily paired with target sequences containing different genotypes of the rs77641731 adjacent SNP sites.
  4. 4. A genetic thrombolytic gene detection kit comprising the primer composition for detecting genetic thrombolytic gene mutations according to any one of claims 1-3; preferably, the kit further comprises a PCR reaction reagent, an SAP enzyme, a single base extension reaction reagent, and a chip cleaning resin; Preferably, the kit is configured to detect 24 single nucleotide polymorphism sites of the 22 genes simultaneously in a single reaction well.
  5. 5. A method for detecting a genetic thrombolysis-related gene polymorphism, characterized in that the detection is performed by using the primer composition according to any one of claims 1 to 3 or the kit according to claim 4; preferably, the detection method comprises: Extracting genome DNA of a sample to be detected; using the genome DNA as a template, and performing multiplex PCR amplification by using PCR amplification primers in the primer composition to obtain PCR amplification products containing the 24 single nucleotide polymorphism sites; Performing digestion treatment on the PCR amplification product by utilizing SAP enzyme; taking the digested product as a template, and performing single-base extension reaction by using an extension primer in the primer composition to obtain an extension product; After desalting and purifying the extension product, detecting by using a matrix-assisted laser dissociation adsorption time-of-flight mass spectrometer, and determining genotypes of the 24 single nucleotide polymorphism sites according to molecular weight differences of mass spectrum signals; Preferably, the multiplex PCR amplification procedure comprises a 95℃pre-denaturation followed by a first stage cycle of 95℃denaturation, 60℃annealing, 72℃extension followed by a second stage cycle of 95℃denaturation, 65℃annealing, 72℃extension, and finally 72℃post-extension; Preferably, the single base extension reaction procedure includes 95℃pre-denaturation followed by 40 cycles of amplification, where each cycle includes 95℃denaturation followed by 5 internal cycles of 52℃annealing and 80℃extension, and finally 72℃post-extension.
  6. 6. A method of assessing the risk of developing venous thromboembolism comprising: Obtaining genotype data of 24 single nucleotide polymorphism sites of an individual to be tested obtained by using the primer composition according to any one of claims 1-3 or the detection method according to claim 5, and obtaining risk alleles of the 24 sites and corresponding ratio; calculating the polygenic risk score of the individual to be tested by using a weighting algorithm according to the genotype data and the ratio; The clinical characteristic information of the individual to be tested is obtained, wherein the clinical characteristic information at least comprises at least one of age, sex, active tumor history, history of previous venous thromboembolism, activity limited condition, recent wound or operation history and acute infection condition; Inputting the polygenic risk score and the clinical characteristic information into a pre-constructed logistic regression model, and calculating to obtain the probability of the venous thromboembolism morbidity risk of the individual to be detected; Preferably, the calculation formula of the polygenic risk score is: ; Wherein n represents the total number of loci, is 24, beta i represents the weight coefficient of the risk allele of the ith locus, G i represents the number of the risk alleles of the ith locus, and takes on the values of 0, 1 or 2, wherein 0 represents that the risk alleles are not carried, 1 represents that the risk alleles are carried, and 2 represents that the risk alleles are carried; Preferably, the logistic regression model is trained based on a sample set comprising a case group and a control group; preferably, the logistic regression model divides the population risk into a low risk, a medium risk and a high risk level, wherein if the polygenic risk score of the individual to be tested is positioned at the rear 20% of the population distribution, the individual to be tested is judged to be high in genetic risk; Preferably, the construction process of the logistic regression model comprises the steps of dividing a sample into a training set and a testing set, optimizing parameters of logistic regression on the training set by adopting grid search of hierarchical sampling and 5-fold cross verification, and constructing the model by the determined optimal parameters; Preferably, the clinical profile information includes age, gender, history of active tumors, history of past venous thromboembolism, limited activity, history of recent trauma or surgery, and acute infection.
  7. 7. An apparatus for evaluating the risk of developing venous thromboembolism, comprising: An acquisition module for detecting genotype data of 24 single nucleotide polymorphism sites of the obtained individual to be detected by using the primer composition according to any one of claims 1-3 or the detection method according to claim 5; The calculating module is used for calculating the polygenic risk score of the individual to be detected by using a weighting algorithm according to the genotype data and the ratio; The acquisition module is also used for acquiring clinical characteristic information of the individual to be tested, wherein the clinical characteristic information at least comprises at least one of age, sex, active tumor history, past venous thromboembolism history, activity limited condition, recent wound or operation history and acute infection condition; the evaluation module is used for inputting the polygenic risk score and the clinical characteristic information into a pre-constructed logistic regression model, and calculating to obtain the probability of the venous thromboembolism incidence of the individual to be tested.
  8. 8. A computer device, characterized in that it comprises a processor and a memory, the memory storing a computer program, the processor being adapted to execute the computer program to carry out the method of assessing the risk of developing venous thromboembolism as claimed in claim 6.
  9. 9. A computer storage medium, characterized in that it stores a computer program which, when executed on a processor, implements the method of assessing the risk of developing venous thromboembolism according to claim 6.
  10. 10. Use of a primer composition according to any one of claims 1-3 for the preparation of a kit or a gene chip for assessing the risk of developing venous thromboembolism.

Description

Primer composition for detecting hereditary thrombolysis related gene mutation and application thereof Technical Field The invention relates to the technical field of gene detection, in particular to a primer composition for detecting genetic thrombolysis related gene mutation and application thereof. Background The thrombus-prone disease refers to a pathological state that the thrombus is easy to form due to the weakening of an anticoagulation mechanism or the strengthening of a coagulation mechanism of an organism caused by hereditary or acquired factors, and the clinical manifestation of the thrombus-prone disease is mainly Venous Thromboembolism (VTE). Repeated attacks of thrombotic events caused by thrombolysis significantly increase disability and mortality of patients, severely compromising human health. Research shows that a considerable part of the difference of individuals on VTE susceptibility is attributed to genetic factors, the incidence rate of hereditary thrombosis patients is obviously increased compared with that of natural people, and clinical diagnosis and treatment are more complicated. Hereditary thrombosis is usually closely related to variation of various genes, and involves abnormality of systems such as anticoagulant protein, coagulation factor and fibrinolytic protein. Therefore, detection of related genes can provide reference basis for early screening, prevention and auxiliary diagnosis of diseases. Currently, diagnosis of hereditary thrombosis is largely dependent on laboratory tests, common means including measurement of anticoagulant protein levels in plasma and nucleic acid-based gene detection. In clinical risk assessment, multi-factor assessment scales, such as Caprini scale for surgical patients and Padura scale for medical inpatients, are often used, and the thrombus risk of the patient is managed in a layered manner by combining laboratory detection results. In addition, with the development of molecular biology techniques, techniques including Polymerase Chain Reaction (PCR), first generation sequencing, and high throughput sequencing (NGS) have been applied to the detection of related genetic loci. However, existing detection and assessment systems still have a number of limitations. Firstly, the measurement of the plasma protein level is easy to be interfered by various factors such as anticoagulation drug treatment, physiological state change of a patient and the like, so that false negative or false positive appears in a detection result, and the stability is insufficient. Secondly, the traditional PCR or the first generation sequencing technology has low flux, and is difficult to meet the requirement of simultaneous detection of multiple genes and multiple sites related to hereditary thrombosis, while the high-flux sequencing technology has high flux, but has the advantages of high cost, complex operation and long detection period, and is difficult to popularize in large-scale clinical screening. More importantly, in the aspect of genetic factor screening, the existing clinical assessment scale is often based on the genetic background of European and American population, mainly focuses on high-frequency mutation of FII G20210A, FV Leiden and the like in western population, but ignores gene loci which have higher mutation frequency and obvious pathogenic significance in other populations (such as Chinese population), so that risk assessment of specific population is not accurate enough. In summary, although various detection means exist at present, a specialized detection product which can simultaneously achieve high throughput, low cost, simple operation and specific genetic characteristics of a specific crowd is still lacking in the market. The existing polygenic genetic risk score (PRS) is applied to other disease researches, but the combined application in the field of hereditary thrombosis is not seen. The existing single detection means or general risk assessment model is difficult to comprehensively and accurately quantify the comprehensive morbidity risk of an individual, and cannot completely meet the actual requirements of clinic on accurate medical treatment and early intervention. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a primer composition for detecting genetic thrombolysis related gene mutation and application thereof, wherein twenty-two genes adapting to genetic characteristics of specific groups are selected to have twenty-four specific sites, so that a plurality of key physiological systems are covered, pathogenic factors and risk factors are considered, the race heterogeneity limitation of conventional indexes is effectively overcome, and the comprehensive analysis and accurate screening of the genetic thrombolysis are realized. In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: In a first aspect, the inventio