CN103627789A - Kit for detecting warfarin sensitivity gene by using electrochemical gene sensor method - Google Patents
Kit for detecting warfarin sensitivity gene by using electrochemical gene sensor method Download PDFInfo
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Abstract
The invention provides a warfarin sensitivity gene detection kit (an electrochemical gene sensor method) which is a kit used for rapid detection of a warfarin sensitivity gene in a human genome by using electrochemical gene sensor technology. The kit is applicable to qualitative detection of *2, *3 and *5 allele types of the cytochrome CYP2C9 gene and 1639G>A and 1173C>T allele types of vitamin K epoxide reductase complex 1 (VKORC 1) in clinical specimens like human peripheral blood and a variety of tissue and cells; the above-mentioned genotypes are helpful for clinical discrimination of patients with increased drug susceptibility to warfarin and enable the dosage range of warfarin to be rapidly determined, curative effects to be guaranteed and the risk of hemorrhage to be reduced. Combination of warfarin gene detection results with INR monitoring enables the maintenance dose of warfarin to be effectively and rapidly adjusted, thereby lowering down the risk of hemorrhage while achieving curative effects.
Description
Technical field
The present invention relates to the test kit that a kind of electrochemical gene sensor method detects warfarin sensitivity genes, particularly relate to the test kit with warfarin sensitivity genes in a kind of electrochemical gene sensor technology rapid detection human genome.This test kit has very high sensitivity and specificity.By test kit of the present invention, realize the method woods sensitivity genes in the human genome in the samples such as human body cell, anticoagulated whole blood or tissue is carried out to rapid detection and analysis.
Background technology
1998 the end of the year AAAS classify electrochemical gene sensor technology one of as large progress in 1998 annual natural science fields ten, it serves to show the meaning in the Qi history of science.It can be analyzed the ability of thousands of genomic informations simultaneously, quickly and accurately and demonstrate huge power with it.These application mainly comprise the aspects such as genetic expression detection, sudden change detection, genome polymorphism analysis and gene library mapping and sequencing by hybridization.
Biochip is applied to bio-science field more and more with the detecting pattern of its high-throughput, many target position.The micro-array chip that the printed circuit board (PCB) of take is carrier, the features such as more efficient with it, high-throughput, low price, have been widely used in fundamental research and Clinical screening, auxiliary diagnosis.
Micro-array chip comprises oligonucleotide chip, cDNA chip, antigen chip, antibody chip etc., the printed circuit board (PCB) that electrochemical gene sensor chip be take after the processing of special chemical method is carrier, various probes or target fragment are fixed on to printed circuit board surface in the mode of chemical bonds, and utilize ferrocene deriv as electrochemistry indicator, form the micro-array chip that can be used for hybridization or antigen antibody reaction.
Warfarin (warfarin) is coumarins oral anticoagulation, is at present domestic and international the most frequently used long-acting anticoagulation, is used for the oral anticoagulation therapy of artificial valve replacement, venous thrombosis (pulmonary infarction), atrial fibrillation etc.Its anti-freezing effect is often usingd prothrombin time (prothrombin time, PT) and INR (international normalized ratio, INR) as monitoring index.First the common employing of dosage regimen of warfarin gives certain standard dosage clinically at present, and then clinician, according to the situation of each patient INR value, increases or reduce dosage until INR reaches target.But in such anticoagulant therapy, the cycle of adjusting dosage is longer, patient there is thrombus or hemorrhage possibility higher.
Cause the reason of warfarin consumption individual difference a lot, can be divided into non-genetic factor and inherited genetic factors.Non-genetic factor mainly contains interaction, food habits and the morbid state etc. of age, sex, body surface area, medicine.Yet the influence degree of non-genetic factor is comparatively limited, it is not the major cause of warfarin consumption individual difference.In recent years, along with the progress of pharmacogenomics and illustrating of warfarin pharmacological action molecular mechanism, the effect of inherited genetic factors in warfarin consumption individual difference is more and more subject to people's attention.At present, pharmacodynamics known and warfarin the gene relevant with pharmacokinetics reaches more than 30 plants, and wherein the gene pleiomorphism of vitamin K epoxide reductase complex body subunit 1 gene (VKORC1) and cytochrome P450 2C9 gene (CYP2C9) are to affect topmost two inherited genetic factorss of warfarin consumption individual difference.
VKORC1 and CYP2C9 affect the topmost inherited genetic factors of warfarin consumption individual difference known at present, and existing scholar is studied the contribution of these two genes in warfarin individual difference.These studies show that the contribution proportion difference 5～22% and 6～37% of the polymorphism of CYP2C9 and VKORC1 to warfarin consumption individual difference.By the polymorphism of VKORC1 and CYP2C9 and non-genetic factor, as age, sex, body weight, combination with medication and treatment disease etc. combine, 33.3%～60.8% of soluble warfarin consumption individual difference reason.In Chinese population, 60.8% of Veenstra etc. report VKORC1, CYP2C9, age, the soluble Hong Kong of sex Chinese's warfarin consumption individual difference reason, wherein the contribution of VKORC1 and CYP2C9 is 31% and 7.9%.In another group Chinese of the report such as Miao, VKORC1 and CYP2C9 are respectively 49.4% and 1.7% to the contribution of warfarin consumption individual difference.
Summary of the invention
The present invention relates to the test kit that a kind of electrochemical gene sensor method detects warfarin sensitivity genes, particularly relate to the test kit with method woods sensitivity genes in a kind of electrochemical gene sensor technology rapid detection human genome.Method woods sensitivity genes in human genome in the samples such as this test kit can human body cell, anticoagulated whole blood or tissue.
The present invention is applicable to cytochrome C YP2C9 gene * 2 in the clinical samples such as qualitative detection human peripheral, various tissue and cell, * 3, * 5 allelotypes and vitamin K epoxide reductase complex body 1 (VKORC1) gene-1 639G > A, 1173C > T allelotype be totally 5 warfarin sensitive gene SNP sites.
The object of this invention is to provide a kind of test kit that carrys out warfarin sensitivity genes in qualitative detection sample by electrochemical gene sensor technology, particularly relate to the judgement application of doctor to patient's warfarin dosage.Its ultimate principle is to utilize the Auele Specific Primer of four pairs of oligonucleotide at UNG enzyme, hot resistant DNA polymerase (Taq enzyme), high-quality deoxyribonucleoside triphosphate (dNTPs), Mg
2+in PCR reaction buffer, by commercially available qualitative PCR amplification instruments such as ABI2700, ABI9700, realize the cyclic amplification of target polynucleotide, obtain multiplex amplification product; The specificity capture probe of design is separately fixed at special printed circuit board (PCB) gold electrode surfaces, is prepared into electrochemical sensor (chip), for catching PCR multiplex amplification product; Specific signals probe and the PCR product specific combination of having caught.Because signal probe is marked with ferrocene molecule, by the variation of sandwich hybridization generation current, Applied Electrochemistry gene sensor analytical system detects current value (signal value), and the base in each SNP site is judged, thereby reach the object of rapid detection target polynucleotide, in order to instruct clinical application.
In detection sample provided by the present invention, the test kit of warfarin sensitivity genes comprises: a plurality of reagent bottles or pipe that (1) is equipped with respectively pcr amplification reaction liquid, pcr amplification reaction enzyme system, electrochemistry hybridization solution, blank, warfarin sensitivity genes quality control product and positive reference material and is sealed, and the packing box of these reagent bottles of packing or pipe is separated and concentrated in (2).It is characterized in that in pcr amplification reaction liquid, 24 primers for target polynucleotide amplification comprise:
The sequence of forward primer W*2-F1 is: 5 '-GGATCTCCCTCCTAGTTTCG-3 ' (SEQ ID NO:1),
The sequence of forward primer W*2-F2 is: 5 '-ATCTCCCTCCTAGTTTCGTT-3 ' (SEQ ID NO:2),
The sequence of forward primer W*2-F3 is: 5 '-ATCTCCCTCCTAGTTTCGTTTCTCT-3 ' (SEQ ID NO:3),
The sequence of reverse primer W*2-R1 is: 5 '-GTAAGGTCAGTGATATGGAGT-3 ' (SEQ ID NO:4),
The sequence of reverse primer W*2-R2 is: 5 '-CCTTGGTTTTTCTCAACTCCTCC-3 ' (SEQ ID NO:5),
The sequence of reverse primer W*2-R3 is: 5 '-ATCTCCCTCCTAGTTTCGTT-3 ' (SEQ ID NO:6),
The sequence of forward primer W*3/5-F1 is: 5 '-CAGGAAGAGATTGAACGTG-3 ' (SEQ ID NO:7),
The sequence of forward primer W*3/5-F2 is: 5 '-AGTTTTTACTTGTGTCTTATCAG-3 ' (SEQ ID NO:8),
The sequence of forward primer W*3/5-F3 is: 5 '-TAAAGTCCAGGAAGAGATTGAACG-3 ' (SEQ ID NO:9),
The sequence of reverse primer W*3/5-R1 is: 5 '-AAACAAACTTACCTTGGGAAT-3 ' (SEQ ID NO:10),
The sequence of reverse primer W*3/5-R2 is: 5 '-CGAAAACATGGAGTTGCAGTGTAG-3 ' (SEQ ID NO:11),
The sequence of reverse primer W*3/5-R3 is: 5 '-GTTGCAGTGTAGGAGAAACAAACTT-3 ' (SEQ ID NO:12),
The sequence of forward primer W1173F1 is: 5 '-TGACATGGAATCCTGACGT-3 ' (SEQ ID NO:13),
The sequence of forward primer W1173F2 is: 5 '-ACATGGAATCCTGACGTGGC-3 ' (SEQ ID NO:14),
The sequence of forward primer W1173F3 is: 5 '-GTATGACATGGAATCCTGACGTG-3 ' (SEQ ID NO:15),
The sequence of reverse primer W1173R1 is: 5 '-GAACCAGGTTAGGACTGTCAAC-3 ' (SEQ ID NO:16),
The sequence of reverse primer W1173R2 is: 5 '-GTGGAACCAGGTTAGGACTGTC-3 ' (SEQ ID NO:17),
The sequence of reverse primer W1173R3 is: 5 '-CTGTCAACCCAGTGCCTTGG-3 ' (SEQ ID NO:18),
The sequence of forward primer W1639F1 is: 5 '-AGGGTAGGTGCAACAGTAA-3 ' (SEQ ID NO:19),
The sequence of forward primer W1639F2 is: 5 '-AGAAGGGTAGGTGCAACAGTAA-3 ' (SEQ ID NO:20),
The sequence of forward primer W1639F3 is: 5 '-CAGAAGGGTAGGTGCAACAGTAAG-3 ' (SEQ ID NO:21),
The sequence of reverse primer W1639R1 is: 5 '-CAAGACGCTAGACCCAATGGTTATT-3 ' (SEQ ID NO:22),
The sequence of reverse primer W1639R2 is: 5 '-CCTGACCTCAAGTGATCCACCC-3 ' (SEQ ID NO:23),
The sequence of reverse primer W1639R3 is: 5 '-CACCAAGACGCTAGACCCAATG-3 ' (SEQ ID NO:24).
According to a preferred embodiment of the invention, in electrochemistry hybridization solution for specific binding PCR product and indicate the oligonucleotide signal probe of the base in each SNP site to have 27 to comprise:
The sequence of 2C9star2.WT.SP1 is: 5 '-GAACACGGTCCT-3 ' (SEQ ID NO:25),
The sequence of 2C9star2.WT.SP2 is: 5 '-TGAACACGGTCCT-3 ' (SEQ ID NO:26),
The sequence of 2C9star2.WT.SP3 is: 5 '-AACACGGTCCT-3 ' (SEQ ID NO:27),
The sequence of 2C9star2.MUT.SP1 is: 5 '-GAACACAGTCCTCAA-3 ' (SEQ ID NO:28),
The sequence of 2C9star2.MUT.SP2 is: 5 '-TGAACACAGTCCTCAA-3 ' (SEQ ID NO:29),
The sequence of 2C9star2.MUT.SP3 is: 5 '-TTGAACACAGTCCTCAA-3 ' (SEQ ID NO:30),
2C9star3/5.WT.SP1 sequence be: 5 '-GAAGGTCAATGTAT-3 ' (SEQ ID NO:31),
2C9star3/5.WT.SP2 sequence be: 5 '-GAAGGTCAATGTATC-3 ' (SEQ ID NO:32),
2C9star3/5.WT.SP3 sequence be: 5 '-GAGAAGGTCAATGTATC-3 ' (SEQ ID NO:33),
The sequence of 2C9star3.MUT.SP1 is: 5 '-GAAGGTCAAGGTAT-3 ' (SEQ ID NO:34),
The sequence of 2C9star3.MUT.SP2 is: 5 '-GAGAAGGTCAAGGTATC-3 ' (SEQ ID NO:35),
The sequence of 2C9star3.MUT.SP3 is: 5 '-GAAGGTCAAGGTATC-3 ' (SEQ ID NO:36),
The sequence of VKORC1 1173C > T.WT.SP1 is: 5 '-TCCAAGGGTCGAT-3 ' (SEQ ID NO:37),
The sequence of VKORC1 1173C > T.WT.SP2 is: 5 '-AGTCCAAGGGTCG-3 ' (SEQ ID NO:38),
The sequence of VKORC1 1173C > T.WT.SP3 is: 5 '-TCCAAGGGTCG-3 ' (SEQ ID NO:39),
The sequence of VKORC1 1173C > T.MT.SP1 is: 5 '-TCCAAGAGTCG-3 ' (SEQ ID NO:40),
The sequence of VKORC1 1173C > T.MT.SP2 is: 5 '-AGTCCAAGAGTCGATG-3 ' (SEQ ID NO:41),
The sequence of VKORC1 1173C > T.MT.SP3 is: 5 '-TCCAAGAGTCGATG-3 ' (SEQ ID NO:42),
The sequence of VKORC1-1639G > A.WT.SP1 is: 5 '-GCACCCGGCCA-3 ' (SEQ ID NO:43),
The sequence of VKORC1-1639G > A.WT.SP2 is: 5 '-CGCACCCGICCAAT-3 ' (SEQ ID NO:44),
The sequence of VKORC1-1639G > A.WT.SP3 is: 5 '-GCACCCGACCAATG-3 ' (SEQ ID NO:45),
The sequence of VKORC1-1639G > A.MT.SP1 is: 5 '-GCACCTGGCCA-3 ' (SEQ ID NO:46),
The sequence of VKORC1-1639G > A.MT.SP2 is: 5 '-GCACCTGGCCAA-3 ' (SEQ ID NO:47),
The sequence of VKORC1-1639G > A.MT.SP3 is: 5 '-GCACCTGGACAATGG-3 ' (SEQ ID NO:48),
The sequence of 2C9star5.MUT.SP1 is: 5 '-GAAGCTCAATGTAT-3 ' (SEQ ID NO:49),
The sequence of 2C9star5.MUT.SP2 is: 5 '-GAGAAGCTCAATGTATCTCT-3 ' (SEQ ID NO:50),
2C9star5.MUT.SP3 sequence be: 5 '-GAGAAGCTCAATGTATCT-3 ' (SEQ ID NO:51).
According to another preferred embodiment of the present invention, wherein pcr amplification reaction liquid can be respectively and 4 forward primers of article one chain combination of 4 double-stranded target polynucleotides by (a) hot resistant DNA polymerase (Taq enzyme), (b) UNG enzyme, (c) deoxyribonucleoside triphosphate (dNTPs), (d), (e) can be respectively and 4 reverse primers of the second chain combination of double-stranded target polynucleotide.
According to another preferred embodiment of the present invention, wherein the working concentration for each primer of pcr amplification reaction liquid is respectively 0.1 μ mol/L W*2-F1, 0.1 μ mol/L W*2-F2, 0.1 μ mol/L W*2-F3, 0.1 μ mol/L W*3/5-F1, 0.1 μ mol/L W*3/5-F2, 0.1 μ mol/L W*3/5-F3, 0.1 μ mol/L W1173F1, 0.1 μ mol/L W1173F2, 0.1 μ mol/L W1173F3, 0.1 μ mol/L W1639F1, 0.1 μ mol/L W1639F2, 0.1 μ mol/L W1639F3, 0.01 μ mol/L W*2-R1, 0.01 μ mol/L W*2-R2, 0.01 μ mol/L W*2-R3, 0.02 μ mol/L W*3/5-R1, 0.02 μ mol/L W*3/5-R2, 0.02 μ mol/L W*3/5-R3, 0.01 μ mol/L W1173R1, 0.01 μ mol/L W1173R2, 0.01 μ mol/L W1173R3, 0.01 μ mol/L W1639R1, 0.01 μ mol/L W1639R2, 0.01 μ mol/L W1639R3.
According to another preferred embodiment of the present invention, wherein pcr amplification reaction enzyme system is comprised of (a) hot resistant DNA polymerase (Taq enzyme), (b) UNG enzyme, (c) deoxyribonucleoside triphosphate (dNTPs).
According to another preferred embodiment of the present invention, wherein the magnesium ion optimum concn for pcr amplification reaction liquid and enzyme system is that 5mmol/L, heat-resisting Taq enzyme optimum amount are that 8U/ reaction, UNG enzyme optimum amount are that 0.1U/ reaction, deoxyribonucleoside triphosphate (dNTPs) optimum concn are 0.24mmol/L.
According to another preferred embodiment of the present invention, wherein optimal reaction temperature and the time for pcr amplification is: 50 ℃ of 3min; Then 95 ℃ of denaturation 15min; Last 94 ℃ of 30s, 56 ℃ of 35s, 72 ℃ 40s45 circulation; 72 ℃ of 7min.
According to another preferred embodiment of the present invention, wherein electrochemistry hybridization solution comprises tri-kinds of hybridization solution I, hybridization solution II, hybridization solution III, the main component of hybridization solution I is above-mentioned 9 kinds of signal probes (SEQ ID NO:25～SEQ ID NO:51), and the concentration of every kind of signal probe is 0.175pmol/ μ L; The main component of hybridization solution II is protein; The main component of hybridization solution III is sodium perchlorate, and its concentration is 0.8mol/L.
According to another preferred embodiment of the present invention, wherein blank is sterilizing purified water.
According to another preferred embodiment of the present invention, wherein warfarin sensitivity genes quality control product is the DNA that contains the common type fragment of warfarin sensitivity genes having increased, and double-stranded DNA concentration is 30ng/ μ L.
The minimum concentration that detection kit provided by the invention can detect human gene group DNA is 10ng/ μ L, illustrates that this test kit has extraordinary sensitivity.
The present invention is directed to method woods sensitivity genes fragment design special primer and probe in human genome, can detect the method woods sensitivity genes in human genome, but can not detect the illegal woods sensitivity genes fragment in human genome, illustrate that this test kit has good specificity.
Detection kit provided by the invention can be sensitive, the type of the warfarin sensitivity genes in the human genome in the sample such as human body cell, anticoagulated whole blood or tissue rapidly; The clinical application amount that can be warfarin provides reliable experimental evidence, effectively reduces the patient's bleeding risk that warfarin inappropriate medication causes, and also can effectively monitor curative effect.
Accompanying drawing explanation
Fig. 1 shows warfarin sensitivity genes detected result
Fig. 2 shows warfarin sensitivity genes detected result
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: the development of warfarin sensitivity genes detection reagent
1, the design of primer and probe: by the warfarin sensitivity genes sequence on the existing human genome of Genebank database and both at home and abroad delivered the associated nucleic acid sequences of reporting in document and carried out sequence alignment analysis, the conservative fragments of warfarin sensitivity genes of take is amplified target site, selection is without the section of secondary structure and high conservative, according to the fundamental principle of primer probe design, utilize software, manually design multipair primer and probe.
2, the selection of sample: show according to domestic and international pertinent literature report, can select the samples such as human body cell, anticoagulated whole blood or tissue.
3, the Establishment and optimization of reaction system
The preparation of sample: the empirical tests of clinical collection of usining has anticoagulated whole blood sample 6 examples of sudden change and people's cell sample 2 examples of vitro culture as positive reference material as warfarin drug susceptibility gene test site, by absorption method, extract the human gene group DNA of above-mentioned positive reference material, stand-by.
The screening of primer probe: the many groups primer probe with design in above-mentioned 1 detects respectively the above-mentioned human gene group DNA who is extracted by positive reference material, through repetition test, filter out specificity, sensitivity and reproducible best primer probe combinations: SEQ ID NO:1～SEQ ID NO:51 in sequence table.
The optimization of primer concentration: the in the situation that other components being constant in reaction system, upstream and downstream primer amount ratio is by 4/1 to 20/1 gradient, use respectively from the upstream primer of 0.05 μ mol/L to 0.30 μ mol/L concentration gradient and carry out PCR reaction, through revision test repeatedly, finally determine that the working concentration of each best primer is respectively 0.1 μ mol/L W*2-F1, 0.1 μ mol/L W*2-F2, 0.1 μ mol/L W*2-F3, 0.1 μ mol/L W*3/5-F1, 0.1 μ mol/L W*3/5-F2, 0.1 μ mol/L W*3/5-F3, 0.1 μ mol/LW1173F1, 0.1 μ mol/L W1173F2, 0.1 μ mol/L W1173F3, 0.1 μ mol/L W1639F1, 0.1 μ mol/LW1639F2, 0.1, μ mol/L W1639F3, 0.01 μ mol/L W*2-R1, 0.01 μ mol/L W*2-R2, 0.01 μ mol/LW*2-R3, 0.02 μ mol/L W*3/5-R1, 0.02 μ mol/L W*3/5-R2, 0.02 μ mol/L W*3/5-R3, 0.01 μ mol/LW1173R1, 0.01 μ mol/L W1173R2, 0.01 μ mol/L W1173R3, 0.01 μ mol/L W1639R1, 0.01 μ mol/LW1639R2, 0.01 μ mol/L W1639R3.
The optimization of warm start Taq enzyme dosage: the in the situation that other components being constant in 50 μ L reaction systems, use respectively the enzyme dosage/reaction from 4U (unit of enzyme) to 10U concentration gradient to carry out PCR reaction, through revision test repeatedly, finally determine that best Taq enzyme dosage is 6U/ reaction.
The optimization of UNG enzyme dosage: the in the situation that other components being constant in 50 μ L reaction systems, use respectively the enzyme dosage/reaction from 0.05U (unit of enzyme) to 0.2U concentration gradient to carry out PCR reaction, through revision test repeatedly, finally determine that best UNG enzyme dosage is 0.1U/ reaction.
The optimization of dNTPs concentration: the in the situation that other components being constant in reaction system, use respectively from the dNTPs of 0.1mmol/L to 0.28mmol/L concentration gradient and carry out PCR reaction, through revision test repeatedly, finally determine that best dNTPs concentration is 0.24mmol/L.
The optimization of application of sample amount: the in the situation that other components being constant in reaction system, test respectively from the application of sample amount of 5 μ L to 15 μ L gradients, carry out PCR reaction, through revision test repeatedly, finally determine that best application of sample amount is 10 μ L.
The optimization of temperature of reaction: according to the length of the activity of enzyme and few polynucleotide, mainly annealing temperature and extension time are optimized, through revision test repeatedly, finally determine that best temperature of reaction and the time is: 50 ℃ of 3min, 95 ℃ of denaturation 15min; Then 94 ℃ of 30s, 56 ℃ of 35s, 72 ℃ of 40s, 40 circulations; Last 72 ℃ are extended 7min.
The optimization of signal probe concentration in hybridization solution I: the in the situation that other components being constant in hybridization system, use respectively the signal probe from 0.10pmol/ person-portion to 0.20pmol/ person-portion concentration gradient to carry out hybridization, through revision test repeatedly, finally determine that the concentration of every kind of best signal probe is 0.175pmol/ μ L.
The optimization of hybridization solution II amount: the in the situation that other components being constant in hybridization system, using respectively from the hybridization solution II of 5 μ L to 15 μ L gradients and carry out hybridization, through revision test repeatedly, finally determine that best hybridization solution II amount is 10 μ L.
The optimization of hybridization solution III concentration: the in the situation that other components being constant in hybridization system, use respectively from the hybridization solution III of 0.4mol/L to 1.5mol/L gradient and carry out hybridization, through revision test repeatedly, finally determine that best hybridization solution III concentration is 0.8mol/L.
4, limit of identification experiment: the DNA ladder degree being extracted by people's anticoagulated whole blood that is 20ng/ μ L by human genome concentration is diluted to 10ng/ μ L, 5ng/ μ L, 2ng/ μ L, 1ng/ μ L, 0.5ng/ μ L, as linear and limit of identification reference material, carries out PCR and detects analysis, when DNA concentration is 1ng/ μ L, detecting the amount of sample in reaction system is 10ng, and each site still can be detected normal signal value, and limit of identification can reach 10ng.
5, Samples detection: using the samples such as human body cell, anticoagulated whole blood or tissue as sample to be checked, by absorption method, extract after the DNA of sample respectively, the nucleic acid amplification system of setting up through above-mentioned optimization detects, and result shows: this test kit can be very sensitive detects the warfarin drug susceptibility gene locus in clinical samples.
Embodiment 2: warfarin sensitivity genes detection kit (electrochemical gene sensor method) and use thereof
1, preparation comprises the test kit of following moiety: warfarin PCR reaction solution A (888 μ L/ pipe) 1 pipe, warfarin PCR reaction solution B (72 μ L/ pipe) 1 pipe, warfarin sensitivity genes quality control product (50 μ L/ pipe) 1 pipe, blank (50 μ L/ pipe) 1 pipe, hybridization solution I (1680 μ L/ pipe) 1 pipe, hybridization solution II (240 μ L/ pipe) 1 pipe, hybridization solution III (480 μ L/ pipe) 1 pipe, electrochemical sensor (chip) (8/bag) 3 bags.
2, collection of specimens, transport and preserve
2.1 applicable sample types: anticoagulated whole blood etc.
2.2 sample collecting and pre-treatment (attention aseptic technique): with disposable sterilized injector, extract person under inspection's venous blood 2mL, inject the Glass tubing containing EDTA or sodium citrate anticoagulant, put upside down gently immediately Glass tubing and mix 5～10 times, antithrombotics and venous blood are fully mixed, airtight censorship.
2.3 preserve and transport: sample can be immediately for test;-20 ± 5 ℃ of preservation perives, it is 9 months; If want prolonged preservation, need be stored in-80 ± 5 ℃; Sample should be avoided multigelation.Sample transports and adopts the on the rocks or bubble chamber sealing transportation on the rocks of curling stone, and haulage time should be over 7 days.
3, detecting step
(1) extracting genome DNA
Suggestion is got 200 μ L samples and is carried out nucleic acid extraction.Can adopt the whole blood genome that Da'an Gene Company, Zhongshan University produces to extract test kit, this test kit can be used for the extraction to people's Whole Blood Genomic DNA, please according to test kit specification sheets, operates; Also can select other suitable commercially produced products.
(2) pcr amplification
Get appropriate PCR reaction tubes, every pipe adds warfarin PCR reaction solution A37 μ L, warfarin PCR reaction solution B3 μ L (also can react consumption according to PCR, calculate the required total amount of each component, mix rear packing 40 μ L in single PCR reaction tubes); In above-mentioned PCR reaction tubes, add respectively the sample nucleic acid 10 μ L after extraction, separately get two pipes and add respectively warfarin sensitivity genes quality control product and each 10 μ L of blank, the centrifugal several seconds of 8,000g, put into pcr amplification instrument.On applicable instrument, according to following condition, carry out the setting of PCR cycling program: 50 ℃ 3 minutes, 95 ℃ of denaturations 15 minutes, then by 94 ℃ 30 seconds → 56 ℃ 35 seconds → 72 ℃ amplifications in 40 seconds, 45 circulations, last 72 ℃ are extended 7 minutes.
(3) crossover operation
Get the centrifuge tube of a 0.2mL, add successively 70 μ L hybridization solution I, 10 μ L hybridization solution II, 20 μ L hybridization solution III and 25 μ LPCR products, fully mix (attention tried not bubble), the liquid mixing is moved in the pipe of electrochemical sensor (chip), compress pipe lid.Before sensor inserts the detection module of DA9000 instrument (by the special-purpose hybridization detecting instrument mating with this test kit of Da'an Gene Company, Zhongshan University's independent research), correctly whether inspection mark, and whether the sequence number of confirmatory sample pipe mates with sensor.Then selected special-purpose hybridization check program of mating with this test kit, brings into operation.Approximately, after 30min, hybridization completes, output report list.
(4) interpretation of result
Illustrate: above form has been listed in 2 target genes that this test kit detects the gene type of 5 allelotrope site corresponding wild-types and saltant type, take 430C > T (* 2) as example explanation, what C represented is the base of wild-type, and what T represented is the base of saltant type." genotype result " hurdle in examining report can provide the genotype in above each allelotrope site.Examining report can be printed, and also can directly check the information of electronic version, and detailed information can be read electrochemical gene sensor system specification.
Effective result: the high signal control point of electrochemical sensor chips (HiS Control) and chip low signal reference mark (LoS Control) are by test, and corresponding gene type result is all reported out in all SNP sites.
Invalid result: the high signal control point of electrochemical sensor chips (HiS Control) or chip low signal reference mark (LoS Control) be not by test, or in all SNP sites, the genotype result of any one or several site report is " low signal " " error " " contradictory score " " indeterminate score " etc., it is invalid that this experiment is considered as, and suggestion retests.
Embodiment 3: application warfarin sensitivity genes detection kit (electrochemical gene sensor method) detects clinical sample
All experimentations complete in the Chinese People's Liberation Army the 3rd O seven hospital inspection chambers, clinical sample is provided by the Chinese People's Liberation Army the 3rd O seven hospital inspection chambers, comprise anticoagulated whole blood, the sample types such as DNA sample that extracted, specimen dna extracts, PCR reaction is carried out with reference to embodiment 2 with interpretation of result.
The detected result of clinical sample is as shown in drawings: the gene type (seeing Figure of description Fig. 1, Fig. 2) of the warfarin drug susceptibility gene locus from the examining report Dan Zhongjun of 2 clinical samples can intuitive judgment clinical samples.
Fig. 1 explanation: the genotype in 5 warfarin sensitive gene SNP sites of this routine sample is respectively: 430C > T (* 2) loci gene type is C/C, and wild-type is isozygotied; 1075A > C (* 3) loci gene type is A/A, and wild-type is isozygotied; 1080C > G (* 5) loci gene type is C/C, and wild-type is isozygotied; 1173C > T loci gene type is C/T, i.e. heterozygosis;-1639G > A loci gene type is G/A, i.e. heterozygosis.Contrast with sequencing result, result is consistent.
Fig. 2 explanation: the genotype in 5 warfarin sensitive gene SNP sites of this routine sample is respectively: 430C > T (* 2) loci gene type is C/C, and wild-type is isozygotied; 1075A > C (* 3) loci gene type is A/C, i.e. heterozygosis; 1080C > G (* 5) loci gene type is C/C, and wild-type is isozygotied; 1173C > T loci gene type is T/T, and saltant type is isozygotied;-1639G > A loci gene type is A/A, and saltant type is isozygotied.Contrast with sequencing result, result is consistent.
Warfarin sensitivity genes detection kit of the present invention (electrochemical gene sensor method) is easy and simple to handle, and can obtain detected result accurately half an hour; And a chip can detect the allelotype in 5 SNP sites of a patient simultaneously, with low cost.
Claims (9)
1. a test kit that detects warfarin sensitivity genes, this test kit comprises: a plurality of reagent bottles or pipe that (1) is equipped with respectively pcr amplification reaction liquid, pcr amplification reaction enzyme system, electrochemistry hybridization solution, blank, warfarin sensitivity genes quality control product and positive reference material and is sealed, and the packing box of these reagent bottles of packing or pipe is separated and concentrated in (2).It is characterized in that in pcr amplification reaction liquid, 24 primers for target polynucleotide amplification comprise:
The sequence of forward primer W*2-F1 is: 5 '-GGATCTCCCTCCTAGTTTCG-3 ',
The sequence of forward primer W*2-F2 is: 5 '-ATCTCCCTCCTAGTTTCGTT-3 ',
The sequence of forward primer W*2-F3 is: 5 '-ATCTCCCTCCTAGTTTCGTTTCTCT-3 ',
The sequence of reverse primer W*2-R1 is: 5 '-GTAAGGTCAGTGATATGGAGT-3 ',
The sequence of reverse primer W*2-R2 is: 5 '-CCTTGGTTTTTCTCAACTCCTCC-3 ',
The sequence of reverse primer W*2-R3 is: 5 '-ATCTCCCTCCTAGTTTCGTT-3 ',
The sequence of forward primer W*3/5-F1 is: 5 '-CAGGAAGAGATTGAACGTG-3 ',
The sequence of forward primer W*3/5-F2 is: 5 '-AGTTTTTACTTGTGTCTTATCAG-3 ',
The sequence of forward primer W*3/5-F3 is: 5 '-TAAAGTCCAGGAAGAGATTGAACG-3 ',
The sequence of reverse primer W*3/5-R1 is: 5 '-AAACAAACTTACCTTGGGAAT-3 ',
The sequence of reverse primer W*3/5-R2 is: 5 '-CGAAAACATGGAGTTGCAGTGTAG-3 ',
The sequence of reverse primer W*3/5-R3 is: 5 '-GTTGCAGTGTAGGAGAAACAAACTT-3 ',
The sequence of forward primer W1173F1 is: 5 '-TGACATGGAATCCTGACGT-3 ',
The sequence of forward primer W1173F2 is: 5 '-ACATGGAATCCTGACGTGGC-3 ',
The sequence of forward primer W1173F3 is: 5 '-GTATGACATGGAATCCTGACGTG-3 ',
The sequence of reverse primer W1173R1 is: 5 '-GAACCAGGTTAGGACTGTCAAC-3 ',
The sequence of reverse primer W1173R2 is: 5 '-GTGGAACCAGGTTAGGACTGTC-3 ',
The sequence of reverse primer W1173R3 is: 5 '-CTGTCAACCCAGTGCCTTGG-3 ',
The sequence of forward primer W1639F1 is: 5 '-AGGGTAGGTGCAACAGTAA-3 ',
The sequence of forward primer W1639F2 is: 5 '-AGAAGGGTAGGTGCAACAGTAA-3 ',
The sequence of forward primer W1639F3 is: 5 '-CAGAAGGGTAGGTGCAACAGTAAG-3 ',
The sequence of reverse primer W1639R1 is: 5 '-CAAGACGCTAGACCCAATGGTTATT-3 ',
The sequence of reverse primer W1639R2 is: 5 '-CCTGACCTCAAGTGATCCACCC-3 ',
The sequence of reverse primer W1639R3 is: 5 '-CACCAAGACGCTAGACCCAATG-3 '.
2. test kit according to claim 1, the working concentration that is further characterized in that each primer in pcr amplification reaction liquid is respectively 0.1 μ mol/L W*2-F1, 0.1 μ mol/L W*2-F2, 0.1 μ mol/L W*2-F3, 0.1 μ mol/L W*3/5-F1, 0.1 μ mol/L W*3/5-F2, 0.1 μ mol/L W*3/5-F3, 0.1 μ mol/L W1173F1, 0.1 μ mol/L W1173F2, 0.1 μ mol/L W1173F3, 0.1 μ mol/L W1639F1, 0.1 μ mol/L W1639F2, 0.1 μ mol/L W1639F3, 0.01 μ mol/L W*2-R1, 0.01 μ mol/L W*2-R2, 0.01 μ mol/L W*2-R3, 0.02 μ mol/L W*3/5-R1, 0.02 μ mol/L W*3/5-R2, 0.02 μ mol/L W*3/5-R3, 0.01 μ mol/L W1173R1, 0.01 μ mol/L W1173R2, 0.01 μ mol/LW1173R3, 0.01 μ mol/LW1639R1, 0.01 μ mol/LW1639R2, 0.01 μ mol/LW1639R3.
3. test kit according to claim 1, the concentration that is further characterized in that hot resistant DNA polymerase in pcr amplification reaction liquid is 8U/ reaction.
4. test kit according to claim 1, the concentration that is further characterized in that UNG enzyme in pcr amplification reaction liquid is 0.1U/ reaction.
5. test kit according to claim 1, the concentration that is further characterized in that dNTPs in pcr amplification reaction liquid is 0.24mmol/L.
6. test kit according to claim 1, is further characterized in that for pcr amplification reaction condition to be: 50 ℃ of 3min; 95 ℃ of 15min; 94 ℃ of 30s, 56 ℃ of 35s, 72 ℃ of 40s, 45 circulations; 72 ℃ of 7min.
7. according to the test kit of claim 1, be further characterized in that electrochemistry hybridization solution comprises tri-kinds of hybridization solution I, hybridization solution II, hybridization solution III, the main component of hybridization solution I is 9 kinds of signal probes; The main component of hybridization solution II is protein; The main component of hybridization solution III is sodium perchlorate.
8. electrochemistry hybridization solution according to claim 7, is further characterized in that in electrochemistry hybridization solution I for specific binding PCR product and indicates the oligonucleotide signal probe of the base in each SNP site to have 27 to comprise:
The sequence of 2C9star2.WT.SP1 is: 5 '-GAACACGGTCCT-3 ',
The sequence of 2C9star2.WT.SP2 is: 5 '-TGAACACGGTCCT-3 ',
The sequence of 2C9star2.WT.SP3 is: 5 '-AACACGGTCCT-3 ',
The sequence of 2C9star2.MUT.SP1 is: 5 '-GAACACAGTCCTCAA-3 ',
The sequence of 2C9star2.MUT.SP2 is: 5 '-TGAACACAGTCCTCAA-3 ',
The sequence of 2C9star2.MUT.SP3 is: 5 '-TTGAACACAGTCCTCAA-3 ',
The sequence of 2C9star3/5.WT.SP1 is: 5 '-GAAGGTCAATGTAT-3 ',
The sequence of 2C9star3/5.WT.SP2 is: 5 '-GAAGGTCAATGTATC-3 ',
The sequence of 2C9star3/5.WT.SP3 is: 5 '-GAGAAGGTCAATGTATC-3 ',
The sequence of 2C9star3.MUT.SP1 is: 5 '-GAAGGTCAAGGTAT-3 ',
The sequence of 2C9star3.MUT.SP2 is: 5 '-GAGAAGGTCAAGGTATC-3 ',
The sequence of 2C9star3.MUT.SP3 is: 5 '-GAAGGTCAAGGTATC-3 ',
The sequence of VKORC1 1173C > T.WT.SP1 is: 5 '-TCCAAGGGTCGAT-3 ',
The sequence of VKORC1 1173C > T.WT.SP2 is: 5 '-AGTCCAAGGGTCG-3 ',
The sequence of VKORC1 1173C > T.WT.SP3 is: 5 '-TCCAAGGGTCG-3 ',
The sequence of VKORC1 1173C > T.MT.SP1 is: 5 '-TCCAAGAGTCG-3 ',
The sequence of VKORC1 1173C > T.MT.SP2 is: 5 '-AGTCCAAGAGTCGATG-3 ',
The sequence of VKORC1 1173C > T.MT.SP3 is: 5 '-TCCAAGAGTCGATG-3 ',
The sequence of VKORC1-1639G > A.WT.SP1 is: 5 '-GCACCCGGCCA-3 ',
The sequence of VKORC1-1639G > A.W T.SP2 is: 5 '-CGCACCCGICCAAT-3 ',
The sequence of VKORC1-1639G > A.WT.SP3 is: 5 '-GCACCCGACCAATG-3 ',
The sequence of VKORC1-1639G > A.MT.SP1 is: 5 '-GCACCTGGCCA-3 ',
The sequence of VKORC1-1639G > A.MT.SP2 is: 5 '-GCACCTGGCCAA-3 ',
The sequence of VKORC1-1639G > A.MT.SP3 is: 5 '-GCACCTGGACAATGG-3 ',
The sequence of 2C9star5.MUT.SP1 is: 5 '-GAAGCTCAATGTAT-3 ',
The sequence of 2C9star5.MUT.SP2 is: 5 '-GAGAAGCTCAATGTATCTCT-3 ',
The sequence of 2C9star5.MUT.SP3 is: 5 '-GAGAAGCTCAATGTATCT-3 '.
9. test kit according to claim 1, is further characterized in that detected sample can be selected from but be not limited to the samples such as human body cell, anticoagulated whole blood or tissue.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107292110A (en) * | 2017-06-28 | 2017-10-24 | 贵州省人民医院 | Anti-freezing for medical treatment auxiliary terminal device and rear end equipment manages method and device |
| WO2018036881A1 (en) * | 2016-08-24 | 2018-03-01 | Quantumdx Group Limited | Warfarin sensitivity assay |
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| 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 |
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| 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 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018036881A1 (en) * | 2016-08-24 | 2018-03-01 | Quantumdx Group Limited | Warfarin sensitivity assay |
| CN107292110A (en) * | 2017-06-28 | 2017-10-24 | 贵州省人民医院 | Anti-freezing for medical treatment auxiliary terminal device and rear end equipment manages method and device |
| CN107292110B (en) * | 2017-06-28 | 2021-01-22 | 贵州省人民医院 | Anticoagulation management method and device for medical auxiliary terminal equipment and rear-end equipment |
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