CN110846399B - Cardiovascular disease individualized medication gene detection system kit and application thereof - Google Patents
Cardiovascular disease individualized medication gene detection system kit and application thereof Download PDFInfo
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Abstract
The invention relates to a cardiovascular disease individualized medication gene detection system kit and application thereof, comprising a PCR reaction system and a LDR reaction system. The kit disclosed by the invention adopts a multiple PCR technology and a multiple LDR technology, combines the two technologies, is based on hybridization reaction and ligation reaction, ensures double guarantee, does not need DNA purification in the detection process, reduces the possibility of pollution, and has more accurate result. The accuracy of PCR-LDR detection can be compared favorably with Sanger sequencing, but the diversity of the PCR-LDR detection is higher and the cost is lower.
Description
Technical Field
The invention belongs to the field of gene detection kits and application thereof, and particularly relates to a gene detection system kit for cardiovascular diseases personalized medicine and application thereof.
Background
Cardiovascular diseases seriously jeopardize the health of all humans, with a high priority among many lethal factors. According to the survey, about 2.7 million people in China are suffering from cardiovascular diseases in 2016, the fatality rate of the cardiovascular diseases in rural residents is 309.33/10 ten thousand, the fatality rate in urban residents is 265.11/10 ten thousand, and the overall average fatality rate is more than 43%. The common clinical medicines for cardiovascular diseases comprise aspirin, clopidogrel, warfarin, nitroglycerin and statins. However, the same drug has very significant differences in potency among different people, mainly due to differences in genetic background. 6 genes such as COX-1, GP IIb/IIIa, CYP2C19, CYP2C9, VKORC1, ALDH2 and the like play important roles in the processes of medicine novice, transportation, metabolism, ineffective component excretion and the like, and the single nucleotide polymorphism at a key position can cause the genes to lose functions.
Warfarin is the most widely used antithrombotic drug in clinical applications, but it increases the risk of bleeding and thrombosis in some people. VKORCL and CYP2C9 are the major genetic factors that influence the inter-individual warfarin dosage variation. CYP2C9 has very high polymorphism, and the two major variants are CYP2C9 x 2 and cyp2c9 x 3, which encode proteins with 30% and 80% lower enzymatic activity than wild-type CYP2C9 x 1, respectively. VKORC1 also found multiple SNPs associated with drug dose variation among individuals, with 2 common sites being 1173C >. The onset dose (6.2 mg/d) for the 1173CC type is much higher than the onset dose (4.8 mg/d) for the 1173TT type.
Clopidogrel is an antiplatelet agent used in Acute Coronary Syndrome (ACS) and percutaneous coronary stenting (IF) to reduce the risk of Myocardial Infarction (MI) and stroke in these patients. Clinical studies have shown that some patients are of clopidogrel weak metabolic type, and their CYP2C19 pathway does not inactivate clopidogrel enzyme activity, and thus there is no effect even when taking drugs. Approximately 14% of Chinese people belong to the CYP2C19 poor metabolic form. The 6 types of mutations, CYP2C19 x 2, 3 x 4, 5 x 7, 8, etc., all had lower enzymatic activities than the wild type. CYP2C19 x 17 is the only type of variation with increased enzymatic activity compared to the wild type.
Aspirin resistance is also very common in patients with cardiovascular disease, a condition that increases the risk of ACS and vascular ischemia. 3 and the cause of the GP IIIa PLA2, PEAR1 and PTGS1 variation are related to aspirin resistance. The metabolism of nitroglycerin depends on ALDH2 (Aldehydehydrogenase II), and the protein coded by the ALDH2 gene metabolizes nitroglycerin into NO. One SNP (G1510A) in the coding region of the gene significantly reduces the enzyme activity, and the carriers of the allele are mainly distributed in east Asian populations including China. Neither of these carriers is suitable for treating angina pectoris with nitroglycerin. The SNP locus rs4149056 of the SLCO1B1 gene and the 2 SNP loci rs429358 and rs7412 of the APOE gene affect statin metabolism.
With the increasing attention of clinical medication and genetic differences among individuals, diversified SNP detection technologies are applied to clinical gene detection. DNA sequencing, taqMan probe method and restriction Length polymorphism (RFLP) are the gold standards for traditional SNP typing. Allele-specific PCR, molecular beacons, and mass spectrometry have made SNP typing more accepted and more prevalent. However, these methods differ in sensitivity, specificity and diversity, as well as in the cost of the respective methods. The TaqMan probe method and molecular beacons have sufficient specificity but insufficient diversity. The RFLP process is complex, has low cost, can be operated in a conventional laboratory, but has complex steps and time consumption. Some typing methods rely on special equipment, such as mass spectrometry, which relies on primer color and mass spectrometry. Mass spectrometry has sufficient accuracy and diversity, but the method relies on additional equipment and is relatively costly.
PCR-LDR (ligase detection reaction) is an ideal SNP typing method. Ligase-based techniques rely on the ability of DNA ligase to discriminate single nucleotide differences. When the DNA probe is complementary to the SNP site in the PCR product, the probe can be specifically bound. LDR reaction can achieve the purpose of identifying different SNP sites in a diversity mode based on different lengths of probes. The fluorescently labeled product can be very conveniently isolated and identified on a DNA sequencer. The PCR-LDR method is particularly suitable for detecting the typing of dozens of SNP loci in a single tube, and can greatly reduce the detection cost, so the method becomes a very suitable selectable means for clinical gene detection of cardiovascular diseases.
CN 104313159A discloses that Taq enzyme activity is not eliminated when PCR amplification reaction of a multiple PCR-LDR detection kit of multiple deafness susceptibility genes with high specificity and multiple sites is finished, and excessive Taq enzyme in the system can interfere with downstream LDR reaction, thereby affecting stability and accuracy of LDR result. The detection system and the kit disclosed by the patent application have the advantages that in the multiple PCR stage, a reaction program for inactivating Taq enzyme is set in the PCR program, and the inactivated Taq enzyme cannot interfere with the efficiency of LDR reaction, so that the reliability of a typing result is improved.
Disclosure of Invention
The invention aims to solve the technical problem of providing a cardiovascular disease individualized medication gene detection system kit and application thereof, overcoming the defect that the existing cardiovascular disease gene detection kit only detects a single application direction, and providing more comprehensive and reliable information for doctors and patients by detection results, thereby more reasonably formulating a medication scheme and ensuring medication safety. The invention provides a cardiovascular disease individualized medication gene detection kit based on multiple PCR and multiple LDR technologies, which can be applied to clinically detecting the associated SNP of the metabolic gene of a common medicament of a cardiovascular disease patient and provides reference for reasonable medication.
The invention relates to a gene detection kit for cardiovascular disease individualized medication, which comprises a PCR reaction system and a LDR reaction system;
aiming at 19 related SNP sites of 16 genes related to cardiovascular disease drug metabolism, the PCR reaction system comprises 19 pairs of primers; the LDR reaction system comprises 19 groups of probes, wherein each group of probes comprises 3 LDR probes, one probe is a modification probe (namely a modified probe), and the other two probes are two allele-specific probes of the SNP locus.
19 pairs of primers in the PCR reaction system are PCR oligonucleotide primers for specifically amplifying flanking sequences of target sites; the 19 sets of probes in the LDR reaction system were: LDR oligo nucleotide probe combined with allele specificity and fluorescent Modify probe.
The modification probe comprises 3 parts: a target region complementary sequence, a random sequence of varying lengths, and a universal sequence linked to a FAM fluorescent label.
19 related SNP sites of 16 genes related to cardiovascular disease drug metabolism are specifically as follows: rs4149056, rs12248560, rs4986893, rs10306114, rs4343, rs5186, rs4244285, rs1057910, rs776746, rs2108622, rs429358, rs671, rs5918, rs7412, rs9923231, rs5065, rs1801253, rs1065852 and rs12041331. The primer sequence in the PCR reaction system is shown as SEQ ID NO. 1-38;
the sequence of a specific probe in the LDR reaction system is shown in SEQ ID NO. 58-95.
The sequence of the modified probe is shown in SEQ ID NO. 39-57.
The PCR reaction system also comprises Taq enzyme, UNG enzyme and dNTP, PCR buffer solution and primers; the reaction system of LDR also includes: ligase, UNG enzyme, dNTP, probe and buffer solution.
The operation instruction book of the gene detection kit for cardiovascular disease individualized medication comprises the following steps:
(1) Extracting genome DNA from a sample to be detected, adding the genome DNA serving as a template into a PCR system, and performing multiple PCR reaction; the PCR reaction system comprises Taq enzyme, UNG enzyme and dNTP, PCR buffer solution and primers;
(2) Taking the product of the multiple PCR reaction as a template, adding LDR reaction, and performing multiple LDR reaction; wherein the LDR reaction system comprises ligase, UNG enzyme, dNTP, a probe and a buffer solution;
(3) Capillary electrophoresis is carried out on the LDR reaction products, and the allele type of each SNP locus is judged according to the size of the LDR product fragment shown in an ABI3730XL electrophoresis peak chart, and particularly, the following corresponding data are shown.
The cardiovascular disease individualized medication gene detection kit provided by the invention detects 19 related SNP loci of 11 common drug metabolism related 16 genes for 5 types of cardiovascular diseases.
The invention relates to common medicines, medicine metabolic genes and SNP loci which are respectively as follows:
the invention provides a gene detection kit for cardiovascular disease individualized medication, 19 SNP loci of 16 genes, and corresponding multiple PCR primer sequences SEQ ID NO.1-38 are as follows:
the invention provides a gene detection kit for cardiovascular disease individualized medication, which comprises 16 genes, LDR identification probe sequences of 19 SNP sites and universal probe sequences (SEQ ID NO. 39-95), wherein the lengths and product lengths are as follows:
reagents required for the PCR reaction are MgCl 2 dNTP, primer mix and Taq enzyme.
Reagents required for the LDR reaction include Tris-HCl (pH 8.5), mgCl 2 KCl, DTT, NAD +, taq DNA ligase.
According to the embodiment provided by the invention, the sample to be detected of the kit is whole blood, plasma, serum, saliva sample and the like obtained by various ways.
According to the embodiment provided by the invention, the standard of the kit for judging the gene detection result is based on a peak image of an electrophoresis result, the genotype of a sample is judged, the genotype of a corresponding drug of a patient from which the sample is derived is judged according to the genotype type and by referring to technical guidelines (trial) for detecting genes of drug metabolism enzymes and drug action targets proposed by national defense committee, and a doctor judges whether the type and dosage of the drug are suitable for the corresponding patient according to the description and calculation formula of the guidelines on the characteristics of the metabolism, so that the doctor and the patient are guided to select a more reasonable drug administration scheme.
Advantageous effects
The present invention relates to 11 kinds of common medicines with 5 kinds of use, and the present invention has comprehensive coverage range and makes it possible for cardiovascular disease patient to have complicated disease.
The invention designs probes with different lengths to distinguish different alleles of the same SNP locus and different SNP loci. Both the modify probe and the discrimination probe contain a random sequence, which widens the range of variation in probe detection. Only 1 probe, to which a fluorescent label is attached, can integrate all LDR products and label the discrimination probe for each SNP site.
The kit disclosed by the invention adopts a multiple PCR technology and a multiple LDR technology, combines the two technologies, is based on hybridization reaction and ligation reaction, ensures double guarantee, does not need DNA purification in the detection process, reduces the possibility of pollution, and has more accurate result. The accuracy of PCR-LDR detection can be compared favorably with Sanger sequencing, but the diversity of the PCR-LDR detection is higher and the cost is lower.
Therefore, the gene detection kit for the individual medication of the cardiovascular disease is an efficient clinical gene detection kit, and is used for gene detection before the medication is selected for treatment, so that doctors and patients are helped to select correct medication for treatment, wrong medication is avoided, and adverse reactions are reduced.
Drawings
FIG. 1 is a diagram of the effect of ABI3730 electrophoresis of LDR product;
FIG. 2 shows the results of Sanger's method for verifying typing of rs1057910, rs776746and rs4244285 8 samples;
FIG. 3 shows the 19 SNP allele distribution in the 19 SNP237 samples.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Sample Collection and DNA extraction
200 mul of blood of a cardiovascular disease patient is collected, and genome DNA is extracted by using an Axygen DNA extraction kit, wherein the specific method refers to the kit specification. DNA was quality controlled using 0.8% agarose gel electrophoresis and OD 260/280.
Multiplex PCR
The invention discloses a primer sequence for 19 SNP locus multiplex PCR primer sequences related to cardiovascular disease treatment drug metabolic pathways. The 19 pairs of primers were divided into 2 groups and subjected to multiplex PCR amplification. Group 1 included 10 SNP sites: rs4149056, rs12248560, rs4986893, rs10306114, rs4343, rs5186, rs4244285, rs1057910, rs776746, rs2108622; group 2 included 9 SNP sites: rs429358, rs671, rs5918, rs7412, rs9923231, rs5065, rs1801253, rs1065852, rs12041331.PCR system a 15 μ l system was used: 1.5mM MgCl2,0.2mM dNTP, 2. Mu.l primer mix, 2. Mu.l DNA template, 1U Takara Hot Start Taq enzyme (Takara Co. LTD). The PCR reaction conditions were set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 90 seconds, extension at 74 ℃ for 1 minute, and repeating 40 cycles; extension at 72 ℃ for 10 min; taq enzyme was denatured at 98 ℃ for 10 minutes.
Multiple LDR
The sequence information of the LDR probe is disclosed in the corresponding invention (specifically, see SEQ ID NO. 39-95).
The invention designs 3 LDR probes, one probe is a modification probe (modified probe), and the other two probes are two allele-specific probes of an SNP locus. The universal probe comprises 3 parts: a target region complementary sequence, a random sequence of varying length, and a universal sequence linked to a fluorescent label of FAM. The allele-specific probes have different lengths, and the last base is complementary to both alleles of the SNP. The probes for each SNP site were grouped according to the grouping scheme of multiplex PCR in the previous step. LDR reaction oligonucleotide allele 1, allele 2, modification probe (complementary and random sequences), FAM fluorescence label ligated universal sequence and template were expressed as follows 1:1:2: n: n (n refers to the number of SNPs in one reaction, e.g., set 1, n = 10).
LDR reaction was performed using a 5. Mu.l system comprising 20mM Tris-HCl (pH 8.5), 5mM MgCl 2 100mM KCl,10mM DTT, 10mM NAD +,1U Taq DNA ligase (New England Biolabs, USA), 2. Mu.l probe mix and 2. Mu.l PCR product. The PCR program was set to: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 15 sec, annealing at 50 ℃ for 25 sec, setting 30 cycles of denaturation and annealing. The LDR product was detected by capillary electrophoresis on an ABI3730XL sequencer at 3KV for 30 min. Off-line data were analyzed using GeneMapper 3.3 software.
Example 2
Clinical gene detection application
Sample Collection and DNA extraction
237 blood samples are from patients with cardiovascular diseases including hypertension, coronary heart disease, chronic heart failure, hyperlipidemia or myocardial infarction in Songjiang district Central Hospital of Shanghai city. The patient information is shown in attached table 1. 200. Mu.l of blood was taken and genomic DNA was extracted using Axygen DNA extraction kit, and the specific method was described in the kit instructions. DNA was quality controlled using 0.8% agarose gel electrophoresis and OD260/280 values.
TABLE 1 age and sex profiles of patients from whom blood samples were obtained
| Information | Male/female | Upper limit of age | Lower limit of age | 50～59(y) | 60～69(y) | 70～79(y) | 80～89(y) | 90～100(y) |
| Number of | 106/131 | 98 | 53 | 10 | 105 | 71 | 48 | 3 |
Method evaluation
In order to simultaneously type 19 SNPs, the probe for LDR detection was designed to have a difference in length. The length difference of the probe at the same site is 2 nucleotides, and the length of the probe at different sites is more than 3 nucleotides. The results showed that 19 SNP sites were clearly distinguishable in one lane (as shown in FIG. 1). Of the 19 SNPs, 16 were in harmony with the harmony balance, and 3 were not in harmony with the harmony balance. To further verify the reliability of the method, 8 samples of 3 SNP loci (rs 1057910, rs776746and rs 4244285) are sampled and detected by adopting a Sanger method, and the results show that the typing results of the two methods are consistent (as shown in figure 2).
PCR-LDR genotyping of 19 SNP loci in clinical samples
237 blood samples used in this study were from patients treated in the Songjiang Central Hospital, shanghai, and aged between 53 and 98 years. All patients received antithrombotic therapy.
The genotype distribution, allele frequencies of the 19 SNP sites in the patient samples were analyzed and compared with the data of the public database (Table 2).
Table 2 shows the genotype frequency statistics for 19 SNP sites in 237 patients:
*:Ensemble waston
**:CASAgilent
the results show that the distribution of genotypes across the large cohort is very similar in the cases. However, there were no drug metabolism pathway variations in only 22%, at least 1 variation in 78%, only one in 99 individuals (42%), 2 in 25%, and 4 in 3 patients (fig. 3).
The kit provided by the invention detects 19 SNP sites of metabolic pathways of cardiovascular disease treatment drugs of 237 cases of patients with cardiovascular diseases. 78% of the patients carry at least 1 variation, which indicates that most patients are in urgent need of such genetic testing results to guide them to the correct drugs and doses for treatment. Before selecting the medicine for treatment, personalized medicine safety gene detection is carried out to assist in selecting the medicine, adverse medicine reaction is reduced, and wrong medicine taking is avoided, which is very helpful for cardiovascular disease patients and doctors.
SEQUENCE LISTING
<110> university of east China
SHANGHAI SONGJIANG DISTRICT CENTRAL Hospital
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<211> 59
<212> DNA
<213> Artificial sequence
<400> 48
tgtggccgga ccctgaggtg tttttttttt tttttttttt ttttcgcaaa cctgtactc 59
<210> 49
<211> 59
<212> DNA
<213> Artificial sequence
<400> 49
cacgtcctcc atgtccgcgc ccagcttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 50
<211> 59
<212> DNA
<213> Artificial sequence
<400> 50
aagtgaaaac tgtgagtgtg ggaccttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 51
<211> 59
<212> DNA
<213> Artificial sequence
<400> 51
gggctcacct cgctgtgacc tgaagttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 52
<211> 59
<212> DNA
<213> Artificial sequence
<400> 52
gcctggcagt gtaccaggcc ggggcttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 53
<211> 59
<212> DNA
<213> Artificial sequence
<400> 53
ggtgcggtgg ctcacgccta taatcttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 54
<211> 59
<212> DNA
<213> Artificial sequence
<400> 54
gaagataaca gccagggagg acaagttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 55
<211> 59
<212> DNA
<213> Artificial sequence
<400> 55
ctggaaggcc ttgcggaagt cggggttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 56
<211> 59
<212> DNA
<213> Artificial sequence
<400> 56
gtagcgtgca gcccagcgtt ggcgcttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 57
<211> 59
<212> DNA
<213> Artificial sequence
<400> 57
ggaagtgaga cagcagaagg gacttttttt ttttcgcaaa cctgtactcg catcactca 59
<210> 58
<211> 24
<212> DNA
<213> Artificial sequence
<400> 58
tacgaagcat attacccatg aacg 24
<210> 59
<211> 26
<212> DNA
<213> Artificial sequence
<400> 59
tttacgaagc atattaccca tgaaca 26
<210> 60
<211> 26
<212> DNA
<213> Artificial sequence
<400> 60
tttcattatc tcttacatca gagatg 26
<210> 61
<211> 28
<212> DNA
<213> Artificial sequence
<400> 61
tttttcatta tctcttacat cagagata 28
<210> 62
<211> 28
<212> DNA
<213> Artificial sequence
<400> 62
tttttaaaaa cttggcctta cctggatt 28
<210> 63
<211> 30
<212> DNA
<213> Artificial sequence
<400> 63
tttttttaaa aacttggcct tacctggatc 30
<210> 64
<211> 30
<212> DNA
<213> Artificial sequence
<400> 64
tttttttaca caaatctcct ggtgcagtgt 30
<210> 65
<211> 32
<212> DNA
<213> Artificial sequence
<400> 65
ttttttttta cacaaatctc ctggtgcagt gc 32
<210> 66
<211> 32
<212> DNA
<213> Artificial sequence
<400> 66
ttttttttta tctgacgaat gtgatggcca cg 32
<210> 67
<211> 34
<212> DNA
<213> Artificial sequence
<400> 67
tttttttttt tatctgacga atgtgatggc caca 34
<210> 68
<211> 34
<212> DNA
<213> Artificial sequence
<400> 68
tttttttttt tttcaattct gaaaagtagc taat 34
<210> 69
<211> 36
<212> DNA
<213> Artificial sequence
<400> 69
tttttttttt tttttcaatt ctgaaaagta gctaag 36
<210> 70
<211> 36
<212> DNA
<213> Artificial sequence
<400> 70
tttttttttt ttttaagtaa tttgttatgg gttcct 36
<210> 71
<211> 38
<212> DNA
<213> Artificial sequence
<400> 71
tttttttttt ttttttaagt aatttgttat gggttccc 38
<210> 72
<211> 38
<212> DNA
<213> Artificial sequence
<400> 72
tttttttttt tttttaggct ggtggggaga aggtcaat 38
<210> 73
<211> 40
<212> DNA
<213> Artificial sequence
<400> 73
tttttttttt tttttttagg ctggtgggga gaaggtcaag 40
<210> 74
<211> 43
<212> DNA
<213> Artificial sequence
<400> 74
tttttttttt tttttttttt tggtccaaac agggaagaga tat 43
<210> 75
<211> 45
<212> DNA
<213> Artificial sequence
<400> 75
tttttttttt tttttttttt tttggtccaa acagggaaga gatac 45
<210> 76
<211> 46
<212> DNA
<213> Artificial sequence
<400> 76
tttttttttt tttttttttt tttcggaacc catcacaacc cagctg 46
<210> 77
<211> 48
<212> DNA
<213> Artificial sequence
<400> 77
tttttttttt tttttttttt tttttcggaa cccatcacaa cccagcta 48
<210> 78
<211> 21
<212> DNA
<213> Artificial sequence
<400> 78
gtactgcacc aggcggccgc g 21
<210> 79
<211> 23
<212> DNA
<213> Artificial sequence
<400> 79
ttgtactgca ccaggcggcc gca 23
<210> 80
<211> 25
<212> DNA
<213> Artificial sequence
<400> 80
ttttacgggc tgcaggcata cactg 25
<210> 81
<211> 27
<212> DNA
<213> Artificial sequence
<400> 81
ttttttacgg gctgcaggca tacacta 27
<210> 82
<211> 29
<212> DNA
<213> Artificial sequence
<400> 82
ttttctcctg tcttacaggc cctgcctct 29
<210> 83
<211> 31
<212> DNA
<213> Artificial sequence
<400> 83
ttttttctcc tgtcttacag gccctgcctc c 31
<210> 84
<211> 33
<212> DNA
<213> Artificial sequence
<400> 84
tttttttttt ttatgccgat gacctgcaga agt 33
<210> 85
<211> 35
<212> DNA
<213> Artificial sequence
<400> 85
tttttttttt ttttatgccg atgacctgca gaagc 35
<210> 86
<211> 37
<212> DNA
<213> Artificial sequence
<400> 86
tttttttttt ttagacctga aaaacaacca ttggccg 37
<210> 87
<211> 39
<212> DNA
<213> Artificial sequence
<400> 87
tttttttttt ttttagacct gaaaaacaac cattggcca 39
<210> 88
<211> 41
<212> DNA
<213> Artificial sequence
<400> 88
tttttttttt ttttttatgt ctgtgttctc tttgcagtac t 41
<210> 89
<211> 43
<212> DNA
<213> Artificial sequence
<400> 89
tttttttttt ttttttttat gtctgtgttc tctttgcagt acc 43
<210> 90
<211> 45
<212> DNA
<213> Artificial sequence
<400> 90
tttttttttt tttttttttt ccctgcgcgc gcagcagagc agtcg 45
<210> 91
<211> 47
<212> DNA
<213> Artificial sequence
<400> 91
tttttttttt tttttttttt ttccctgcgc gcgcagcaga gcagtcc 47
<210> 92
<211> 49
<212> DNA
<213> Artificial sequence
<400> 92
tttttttttt tttttttttt ttttcgggca gtggcagggg gcctggtgg 49
<210> 93
<211> 51
<212> DNA
<213> Artificial sequence
<400> 93
tttttttttt tttttttttt ttttttcggg cagtggcagg gggcctggtg a 51
<210> 94
<211> 53
<212> DNA
<213> Artificial sequence
<400> 94
tttttttttt tttttttttt ttttttttat agaaagcaga gagtaagggt gat 53
<210> 95
<211> 55
<212> DNA
<213> Artificial sequence
<400> 95
tttttttttt tttttttttt tttttttttt atagaaagca gagagtaagg gtgac 55
Claims (4)
1. A gene detection kit for cardiovascular disease individualized medication comprises a PCR reaction system and a LDR reaction system, and is characterized in that: aiming at 19 related SNP sites of 16 genes related to cardiovascular disease drug metabolism, the PCR reaction system comprises 19 pairs of primers; the LDR reaction system comprises 19 groups of probes, wherein each group of probes comprises 3 LDR probes, one of the probes is a modification probe, and the other two probes are two allele specific probes of the SNP locus; wherein the primer sequence in the PCR reaction system is shown as SEQ ID NO. 1-38; the sequence of a specific probe in the LDR reaction system is shown as SEQ ID NO. 58-95; the sequence of the modified probe is shown in SEQ ID NO. 39-57.
2. The kit of claim 1, wherein the modified probe comprises 3 parts: a target region complementary sequence, a random sequence of varying lengths, and a universal sequence linked to a FAM fluorescent label.
3. The kit as claimed in claim 1, wherein the 19 related SNP sites of the 16 genes related to cardiovascular disease drug metabolism are rs4149056, rs12248560, rs4986893, rs10306114, rs4343, rs5186, rs4244285, rs1057910, rs776746, rs2108622, rs429358, rs671, rs5918, rs7412, rs9923231, rs5065, rs1801253, rs1065852 and rs12041331.
4. The kit of claim 1, wherein the PCR reaction system further comprises MgCl 2 dNTP, primer mix and Taq enzyme; the reaction system of LDR also includes: tris-HCl, mgCl, pH 8.5 2 KCl, DTT, NAD +, taq DNA ligase.
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| CN110846399B true CN110846399B (en) | 2022-11-04 |
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| CN112080563B (en) * | 2020-09-01 | 2021-04-30 | 美思睿(北京)生物科技有限公司 | Kit for detecting accurate medication genes of chronic diseases |
| CN113981068B (en) * | 2021-11-04 | 2024-03-01 | 武汉菲思特生物科技有限公司 | Primer probe system for simultaneously detecting cardiovascular and cerebrovascular drug metabolism ability genes and detection kit |
| CN116515994A (en) * | 2023-06-26 | 2023-08-01 | 广州凯普医药科技有限公司 | Primer group and kit for detecting cardiovascular disease drug genes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013014069A1 (en) * | 2011-07-22 | 2013-01-31 | Universite Pierre Et Marie Curie (Paris 6) | Method for predicting the risk of early stent thrombosis in a subject with a clopidogrel treatment |
| CN103184271A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Warfarin drug gene VKORC1 and CYP2C9 mutation detection kit |
| CN109468372A (en) * | 2018-11-21 | 2019-03-15 | 中国人民解放军陆军军医大学第附属医院 | Primer combination, method and the kit in a variety of hereditary metabolic disorders hepatopathy targetings library are constructed based on high-flux sequence |
| CN109868314A (en) * | 2018-12-29 | 2019-06-11 | 浙江迪谱诊断技术有限公司 | A kind of kit and detection method detecting Drugs for Cardiovascular Diseases gene |
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2019
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013014069A1 (en) * | 2011-07-22 | 2013-01-31 | Universite Pierre Et Marie Curie (Paris 6) | Method for predicting the risk of early stent thrombosis in a subject with a clopidogrel treatment |
| CN103184271A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Warfarin drug gene VKORC1 and CYP2C9 mutation detection kit |
| CN109468372A (en) * | 2018-11-21 | 2019-03-15 | 中国人民解放军陆军军医大学第附属医院 | Primer combination, method and the kit in a variety of hereditary metabolic disorders hepatopathy targetings library are constructed based on high-flux sequence |
| CN109868314A (en) * | 2018-12-29 | 2019-06-11 | 浙江迪谱诊断技术有限公司 | A kind of kit and detection method detecting Drugs for Cardiovascular Diseases gene |
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