阅读说明：本技术 检测VKORC1基因rs9923231位点多态性的人工模拟核酸分子信标与试剂盒 (Artificial mimic nucleic acid molecular beacon and kit for detecting rs9923231 site polymorphism of VKORC1 gene ) 是由 葛猛 潘世让 余倩 杜柏均 王宏伟 于 2019-02-21 设计创作，主要内容包括：本发明公开了一种VKORC1基因rs9923231位点多态性的分型检测方法与试剂盒。本发明采用VKORC1基因特异性的引物SEQ1和SEQ2扩增VKORC1基因片段,同时在VKORC1基因特异性的引物界定的扩增区域内设计VKORC1基因特异性人工模拟核酸分子信标SEQ3-FAM和SEQ4-VIC。本发明提供的基于基因特异性PCR结合人工模拟核酸分子信标判断VKORC1基因rs9923231位点多态性的方法不仅准确率高,而且还具有检测速度快、操作简单、结果判读客观、闭管反应污染少等优点,非常适合于在临床中大规模开展。(The invention discloses a typing detection method and a kit for rs9923231 site polymorphism of VKORC1 gene. The invention adopts primers SEQ1 and SEQ2 specific to VKORC1 gene to amplify a VKORC1 gene fragment, and designs VKORC1 gene specific artificial simulated nucleic acid molecular beacons SEQ3-FAM and SEQ4-VIC in an amplification region defined by the primers specific to the VKORC1 gene. The method for judging rs9923231 site polymorphism of VKORC1 gene based on gene specificity PCR combined with artificial simulated nucleic acid molecular beacon provided by the invention not only has high accuracy, but also has the advantages of high detection speed, simple operation, objective result interpretation, less closed tube reaction pollution and the like, and is very suitable for large-scale clinical development.)

1. The molecular beacon for detecting rs9923231 site polymorphism of a human VKORC1 gene consists of a molecular beacon A and a molecular beacon B;

the sequence of the molecular beacon A is a sequence 2 in a sequence table, wherein the 2 nd position of the sequence 2 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues;

the sequence of the molecular beacon B is a sequence 3 in a sequence table, wherein the 2 nd position of the sequence 3 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues.

2. The molecular beacon of claim 1, wherein: and fluorescent groups and quenching groups are marked at two ends of the molecular beacon A and the molecular beacon B, and the fluorescent groups marked by the molecular beacon A and the molecular beacon B are different.

3. The molecular beacon of claim 2, wherein: the molecular beacon A is marked with FAM fluorophore; the molecular beacon B is marked with a VIC fluorescent group.

4. A kit for detecting rs9923231 site polymorphism of human VKORC1 gene, which comprises the molecular beacon of any one of claims 1-3 and a primer pair capable of amplifying the recognition sequence of the circular region of the molecular beacon of any one of claims 1-3 from human genome.

5. The kit of claim 4, wherein: the primer pair consists of a single-stranded DNA shown in a sequence 4 in a sequence table and a single-stranded DNA shown in a sequence 5 in the sequence table.

6. A kit for detecting rs9923231 site polymorphism of human VKORC1 gene, comprising the molecular beacon of any one of claims 1-3 or the kit of parts of claims 4 or 5.

7. Use of the molecular beacon of any one of claims 1 to 3 or the kit of parts of claims 4 or 5 or the kit of parts of claim 6 for detecting a polymorphism at site rs9923231 of the human VKORC1 gene;

or, the use of a molecular beacon according to any one of claims 1 to 3 or a kit of parts according to claim 4 or 5 or a kit according to claim 6 for predicting or aiding in the prediction of warfarin treatment dosage in a patient to be tested.

8. A method for detecting rs9923231 site polymorphism of human VKORC1 gene, comprising the following steps: detecting a sample to be detected by using the molecular beacon as claimed in any one of claims 1 to 3 or the kit as claimed in claim 4 or 5, and determining rs9923231 site polymorphism of VKORC1 gene in the sample to be detected according to the change of fluorescence signals in the sample to be detected.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a typing detection method and a kit for rs9923231 site polymorphism of VKORC1 gene.

Background

Deep Vein Thrombosis (DVT) is a blood clot that forms in the deep vein, often in the large veins, such as the femoral or popliteal veins. It is commonly manifested as pain, swelling, and fever in the relevant areas. Blood clots may migrate to the lungs, forming a pulmonary embolism. Factors of DVT formation include surgery, trauma, cancer, obesity, smoking, hormonal birth control, antiphospholipid syndrome and genetic disorders, among others. With age, its incidence is increasing.

Currently, anticoagulant (blood thinners) treatment of DVT is a commonly used method. Typical drugs include low molecular weight heparin, warfarin, coumarin, or direct oral anticoagulants. Among them, warfarin is the first generation anticoagulant widely used, often for the treatment of thrombosis, prevention of stroke. However, the therapeutic response of patients to warfarin is affected by different factors, such as age, sex, vitamin K intake and drug dosage. In addition, certain genetically deficient individuals experience poor therapeutic response, which in clinical practice can lead to internal bleeding and stroke. The whole genome association research proves that certain genes are related to warfarin drug metabolism, and interact with other factors such as environment and the like to jointly influence the administration dosage of warfarin treatment.

Vitamin K epoxyreductase complex 1 (VKORC 1) is a small transmembrane protein of the endoplasmic reticulum, plays a major role in the vitamin K pathway, and is the target protein for warfarin. The presence of non-coding mutations in the VKORC1 gene leads to differential expression of the protein, which determines the drug dose in the patient. Rs9923231(G/A) of VKORC1 has been found to be an important label associated with warfarin dosage. A is a low dose, highly sensitive type, and G is a high dose, non-sensitive type. The detection of this site is an important criterion for the treatment of different patients.

At present, methods for detecting gene polymorphism mainly include a PCR-Sanger sequencing method, a chip hybridization method, a high-resolution melting curve method and the like. Although these methods can detect gene polymorphisms to some extent, they have considerable limitations. The Sanger sequencing method has more steps, needs PCR post-treatment, is complex to operate, is easy to cause pollution, and cannot meet clinical requirements. The chip hybridization method is complicated in operation, and detection thereof depends on expensive equipment and instruments, resulting in high cost. The high-resolution dissolution curve method has high requirements on instruments, can be used only by a machine which is provided with high-resolution software and is sensitive to temperature, and has difficulty in clinical popularization. The fluorescent quantitative PCR based on the Taqman hydrolysis probe cuts off the probe to generate a fluorescent signal by utilizing the exonuclease activity of Taq enzyme, and the fluorescent quenching is not thorough due to the fact that a fluorescent group and a quenching group of the Taqman probe are not close to each other closely, and a background fluorescent signal exists. In addition, the Taqman probe has poor single base mismatch recognition capability, easily generates a non-specific fluorescent signal, interferes result interpretation, and further influences the detection accuracy. Therefore, a simple, convenient, high-sensitivity, accurate and reliable method for detecting gene polymorphism is urgently needed clinically.

The Molecular Beacon (Molecular Beacon) is in a hairpin type in spatial structure and consists of a circular region and a stem region, wherein the circular region is complementary with a target DNA sequence and is about 15-35 nucleotides long, the stem region is about 5-7 nucleotides long, the stem region is formed by a complementary sequence which has higher GC content and is irrelevant with the target sequence, and the 5 'end of the Molecular Beacon is marked with a fluorescent group (F) and the 3' end of the Molecular Beacon is marked with a quenching group (Q). In the case of molecular beacons, the fluorescent group is close to the quencher group (about 7-10 nm) in the free state. At the moment, fluorescence resonance energy transfer occurs, so that fluorescence emitted by the fluorescent group is absorbed by the quenching group and emitted in a thermal form, the fluorescence is almost completely quenched, and the fluorescence background is extremely low. When the circular region of the molecular beacon is hybridized with target DNA with completely complementary sequence to form a double-stranded hybrid, the stem region of the molecular beacon is pulled apart, and the distance between the fluorescent group and the quenching group is increased. According to Foerster's theory, the efficiency of central fluorescence energy transfer is inversely proportional to the 6 th power of the distance between the two, and therefore, the fluorescence of the molecular beacon is almost 100% recovered after hybridization, and the detected fluorescence intensity is proportional to the amount of target DNA in solution (FIG. 1). Thus, the ideal molecular beacon is more efficient than the Taqman hydrolysis probe. However, the introduction of a stem region in the molecular beacon, which is not related to the target sequence, often results in some non-specific interaction between the molecular beacon and the template sequence, which leads to an increase in background signal, and thus, affects the detection efficiency. To eliminate this background signal, high requirements are imposed on the design of the molecular beacon, especially on the sequence design of the stem region. In addition, studies have shown that molecular beacons have a good effect for detecting gene mutations (including single-base mismatches, deletions, or insertion mutations) when the sequence of the loop region is short, but in practice, in many cases, the sequence of the loop region is too long due to the low GC content of a specific target sequence region, thereby affecting the detection efficiency. Therefore, it is often difficult to obtain an ideal molecular beacon.

The development of base-directed modification, i.e., artificial, mimetic, non-natural nucleotide pairs, studies has been in the recent 40 years, in which isocytosine deoxynucleotide-isoguanine deoxynucleotide (isoC-isoG) and its derivatives 5-methylisocytosine deoxynucleotide-isoguanine deoxynucleotide (iso)^MeC-isoG) is classical. The work on the nucleotide pairs in isoC-isoG was first carried out by the American famous synthetic biologist Benner SA, whose team realized the entire central principle of replication, transcription and even translation of isocytosine deoxynucleotide-isoguanine deoxynucleotide (isoC-isoG) artificial expanded nucleic acids in vitro. As shown in FIG. 2, isoC and isoG are isomers of natural nucleotides C and G, respectively, which can perfectly pair themselves but cannot form a pair with natural nucleotides.

In addition to the above manual modification of base structure, there is a large class of non-natural nucleic acids based on modification of base sugar rings, such as Locked Nucleic Acids (LNA). LNA, which broadly refers to an oligonucleotide sequence containing one or more LNA monomers (locked nucleotides), is an artificial mimic nucleic acid that has been rapidly developed in recent years and has been widely used in the fields of molecular diagnostics, gene therapy, and the like. As shown in fig. 3, a methylene bridge is formed between the 2 '-O and 4' -C of the pentose ring of the LNA monomer. LNA does not alter the base pairing of natural nucleic acids, but has greater affinity and greater mismatch recognition relative to natural nucleic acids.

Disclosure of Invention

The invention aims to provide a novel typing detection method and a kit for rs9923231 polymorphic site of VKORC1 gene based on a molecular beacon of artificial simulated nucleic acid.

In order to achieve the above objects, the present invention provides a molecular beacon for detecting rs9923231 site polymorphism of human VKORC1 gene.

The molecular beacon for detecting rs9923231 site polymorphism of human VKORC1 gene consists of a molecular beacon A and a molecular beacon B;

the sequence of the molecular beacon A is a sequence 2 in a sequence table, wherein the 2 nd position of the sequence 2 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues;

the sequence of the molecular beacon B is a sequence 3 in a sequence table, wherein the 2 nd position of the sequence 3 is a 5-methyl isocytosine deoxynucleotide residue, the 3 rd position is an isoguanine deoxynucleotide residue, the 15 th position is a locked nucleotide residue, the 29 th position is a 5-methyl isocytosine deoxynucleotide residue, the 30 th position is an isoguanine deoxynucleotide residue, and the rest nucleotide residues are natural nucleotide residues.

The 7 th to 25 th sites of the molecular beacon A and the molecular beacon B are both circular region sequences, and the 1 st to 6 th sites and the 26 th to 31 th sites are both stem region sequences.

The circular regions of the molecular beacon A and the molecular beacon B are both targeted to the rs9923231 site of the VKORC1 gene. Wherein the molecular beacon A targets the 'G' at the rs9923231 site of VKORC1 gene; the molecular beacon B targets the 'A' of rs9923231 site of VKORC1 gene.

Furthermore, two ends of the molecular beacon A and the molecular beacon B are also marked with a fluorescent group and a quenching group, and the fluorescent groups marked by the molecular beacon A and the molecular beacon B are different. The molecular beacon A and the molecular beacon B can be the same or different in labeled quenching group.

In each molecular beacon, the fluorescence emitted by the fluorophore can be absorbed by the quencher. The fluorescent group and the quenching group can be respectively positioned at the 5 'terminal and the 3' terminal of the basic molecular beacon, and the positions of the fluorescent group and the quenching group can be exchanged as long as the requirement that the fluorescence emitted by the fluorescent group in the basic molecular beacon in a free state can be quenched by the quenching group is met.

Further, the fluorophore may be FAM, Hex, TET, Cy3, JOE; the quencher group can be Dabcyl, TAMRA. In the invention, the 5 'end of the molecular beacon A is marked with FAM fluorescent group, and the 3' end is marked with Dabcyl quenching group; the 5 'end of the molecular beacon B is marked with a VIC fluorescent group, and the 3' end is marked with a Dabcyl quenching group.

In order to achieve the above objects, the present invention further provides a kit for detecting rs9923231 site polymorphism of human VKORC1 gene.

The kit for detecting rs9923231 site polymorphism of human VKORC1 gene comprises the molecular beacon and a primer pair which can be amplified from human genome and contains a recognition sequence of the circular region of the molecular beacon.

In the above-mentioned kit, the primer pair is composed of a single-stranded DNA represented by sequence 4 in the sequence table and a single-stranded DNA represented by sequence 5 in the sequence table.

In the above kit, the molecular beacon and the primer pair are packaged independently. The molar ratio of the molecular beacon A to the molecular beacon B in the molecular beacon can be 1: 1; the molar ratio of the two single-stranded DNAs in the primer pair may be 1: 1. The molar ratio of the molecular beacon A and the molecular beacon B in the kit to the two single-stranded DNAs of the primer pair can be 2:2:5: 5.

In order to achieve the purpose, the invention also provides a kit for detecting rs9923231 site polymorphism of human VKORC1 gene.

The kit for detecting rs9923231 site polymorphism of human VKORC1 gene comprises the molecular beacon or the kit.

The kit can also comprise positive quality control, negative quality control and other reagents. The other reagents can be reaction buffer, dNTPs and MgCl₂Solution, DNA polymerase and/or nuclease-free water. The positive quality control comprises a recombinant plasmid 1, a recombinant plasmid 2 and a recombinant plasmid 3. The recombinant plasmid 1 is obtained by replacing a DNA fragment between EcoRV and SmaI recognition sequences in an escherichia coli cloning vector pUC57 with a DNA fragment shown in a sequence 1 (the site rs9923231 of a VKORC1 gene in the sequence 1 is G)A plasmid; the recombinant plasmid 2 is obtained by replacing a DNA fragment between EcoRV and SmaI recognition sequences in an escherichia coli cloning vector pUC57 with a DNA fragment shown in a sequence 1 (the site rs9923231 of a VKORC1 gene in the sequence 1 is A); the recombinant plasmid 3 is obtained by mixing the recombinant plasmid 1 and the recombinant plasmid 2 according to a molar ratio of 1: 1. The negative quality control can be specifically nuclease-free water. The DNA polymerase can be EX Taq DNA polymerase.

In order to achieve the above objects, the present invention also provides a novel use of the above molecular beacon or the above kit.

The invention provides application of the molecular beacon or the reagent set in detecting rs9923231 site polymorphism of human VKORC1 gene.

The invention also provides application of the molecular beacon or the reagent set in predicting or assisting in predicting warfarin treatment dosage of a patient to be detected.

In order to achieve the above object, the present invention finally provides a method for detecting rs9923231 site polymorphism of human VKORC1 gene.

The method for detecting rs9923231 site polymorphism of human VKORC1 gene comprises the following steps: and detecting a sample to be detected by using the molecular beacon or the reagent set, and determining rs9923231 site polymorphism of VKORC1 gene in the sample to be detected according to the change of a fluorescence signal in the sample to be detected.

In the method, the step of detecting the sample to be detected by using the molecular beacon or the kit of reagents is to detect the DNA of the sample to be detected by using the molecular beacon or the kit of reagents.

The method for determining rs9923231 site polymorphism of VKORC1 gene in a sample to be detected according to change of a fluorescence signal in the sample to be detected comprises the following steps:

if the sample to be detected releases the FAM fluorescence signal, does not release the VIC fluorescence signal, and the value of the FAM fluorescence signal is continuously increased, the genotype of the rs9923231 locus of the VKORC1 gene of the sample to be detected is or is a candidate for the GG genotype;

if the sample to be detected releases the VIC fluorescence signal, does not release the FAM fluorescence signal, and the VIC fluorescence signal value is continuously increased, the genotype of the rs9923231 locus of the VKORC1 gene of the sample to be detected is or is selected as the AA genotype;

and if the sample to be detected releases the VIC fluorescence signal and the FAM fluorescence signal, and both the FAM fluorescence signal value and the VIC fluorescence signal value are continuously increased, determining that the genotype of the gene rs9923231 site of the VKORC1 gene of the sample to be detected is the AG genotype or the candidate is the AG genotype.

The GG genotype refers to a homozygote of G at the base of rs9923231 site of VKORC1 gene on two homologous chromosomes of the DNA of a sample to be detected;

the AA genotype refers to a homozygote of A at the base of rs9923231 site of VKORC1 gene on two homologous chromosomes of the DNA of a sample to be detected;

the AG genotype refers to a heterozygote of A and G of bases of rs9923231 sites of VKORC1 genes on two homologous chromosomes of DNA of a sample to be detected.

In the above method, the sample to be tested may be a blood sample of a patient to be tested (e.g. a deep venous thrombosis patient).

In the above molecular beacon or kit or use or method, the rs9923231 site of the VKORC1 gene is located at the 51 st site of the sequence 1.

Compared with the prior art, the invention has the following beneficial effects: the method for judging rs9923231 site polymorphism of VKORC1 gene based on gene specificity PCR combined with artificial simulated nucleic acid molecular beacon provided by the invention not only has high accuracy, but also has the advantages of high detection speed, simple operation, objective result interpretation, less closed tube reaction pollution and the like, and is very suitable for large-scale clinical development.