CN103468814A - Kit and method for detecting polymorphism of CYP3A4 gene - Google Patents
Kit and method for detecting polymorphism of CYP3A4 gene Download PDFInfo
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Abstract
The invention relates to a kit and a method for detecting the polymorphism of a CYP3A4 gene and belongs to the technical field of gene sequencing. The kit comprises primers for detection, wherein the primers for the detection comprise at least one pair in long-fragment amplification specific forward and reverse primers for a first exon to a third exon of the CYP3A4 gene, long-fragment amplification specific forward and reverse primers for a fourth exon to a seventh exon of the CYP3A4 gene, long-fragment amplification specific forward and reverse primers for an eighth exon to an eleventh exon of the CYP3A4 gene and long-fragment amplification specific forward and reverse primers for a twelfth exon and a thirteenth exon of the CYP3A4 gene and at least one pair in forward and reverse sequencing primers for each exon of the CYP3A4 gene corresponding to long-fragment deoxyribose nucleic acid (DNA) of the first exon to the third exon, long-fragment DNA of the fourth exon to the seventh exon, long-fragment DNA of the eighth exon to the eleventh exon and long-fragment DNA of the twelfth exon and the thirteenth exon. According to the kit and the method, the detection time is short and the workload is low.
Description
Technical field
The present invention relates to the gene sequencing technical field, be specifically related to a kind of test kit and method of the CYP3A4 of detection gene pleiomorphism.
Background technology
Cytochrome P 450 enzymes (Cytochrome P450, CYP), claim again mixed-function oxidase (Mixed function oxcidase), monooxygenase (Monooxygenase) or drug metabolism enzyme, it is distributed widely in animal, plant and microbe.Have been found that more than 20 kind of isozyme in mammiferous tissue, be divided into 4 families of CYP1～4.CYP3A type enzyme accounts for 70% of CYP450 total amount in vivo, accounts for 30% of the interior CYP450 of liver, participates in 60% above common drug metabolic conversion in vivo.At present, in human body, oneself finds 4 kinds of CYP3A～genes, be CYP3A4, CYF3A5, CYP3A7 and CYP3A43, and wherein CYP3A4 is one of most important P450 enzyme clinically, participating in the oxidative metabolism of 150 multi-medicaments of 38 classifications of metabolism, account for 50% of whole medicines, is that the root of untoward reaction occurs because interacting many medicines.CYP3A4 is P450 metabolic enzyme main in liver and gi tract, and its activity shows as singlet and distributes, and in the metabolism of many medicines and environmental pollutant, plays an important role.Research shows, CYP3A4 not only has significant individual difference on expression level, and the internal metabolism of its substrate also can differ more than 10 times.The gene of coding CYP3A4 albumen is positioned at karyomit(e) 7q21.3-22.1, coding region comprises 13 exons and 12 introns, main its structural area of transcribing and expressing of regulation and control is positioned at 5 ends, be about 27kb, the CYP3A4 gene pleiomorphism specifically refers to CYP allelotrope database (http://www.cypalleles.ki.se).Up to the present existing 24 allelotrope of CYP3A4 are determined, the occurrence frequency difference of not agnate CYP3A4SNPs, as CYP3A4*3 and * 17 only find in white people, CYP3A4*15 only finds in black race, and CYP3A4*16 exists only in Japanese and Mexican, in 39 SNPs that found, CYP3A4*4, * 5, and * 6, * 18, * 19 is definite in Chinese, and their occurrence frequency is respectively 1.5%, 0.98%, 0.5%, 2% and 2%.Many common drugs are strong inductor or inhibitor of CYP3A4, as CYP3A4 during as inductor, itself and metabolism substrate drug combination, can greatly improve the latter's metabolic rate, thereby the Plasma Concentration in the time of may making the latter can't reach the performance drug effect, reduce the latter's availability or can't reach result for the treatment of.On the contrary, as CYP3A4 during as inhibitor, they can reduce the latter's metabolic rate during with medicine drug combination through the CYP3A4 metabolism, cause the latter's Plasma Concentration to raise, and may cause untoward reaction.Therefore, by detecting the CYP3A4 gene pleiomorphism, the activity of judgement CYP3A4 enzyme, for the clinical application individuation provides the guidance foundation.
The available technology adopting direct sequencing detects the CYP3A4 gene pleiomorphism, after at first adopting the different purpose fragment of pcr amplification, reclaims purified pcr product, is then checked order.
Because the detection time of direct sequencing is long, generally need more than 48 hours, make the Efficiency Decreasing of this detection, therefore, for detecting the CYP3A4 gene pleiomorphism, make troubles.
Summary of the invention
For time length that detects the CYP3A4 gene pleiomorphism in prior art, complex steps, the needs problem of PCR repeatedly, the object of the present invention is to provide a kind of test kit of the CYP3A4 of detection gene pleiomorphism.
Another object of the present invention is to provide a kind of method of the CYP3A4 of detection gene pleiomorphism.
Detect CYP3A4 gene pleiomorphism time length, complex steps, need the repeatedly problem of PCR in order to solve prior art, the present invention has adopted following technical scheme:
A kind of test kit that detects the CYP3A4 gene pleiomorphism, comprise detecting and use primer, described detection comprises with primer: the long segment specific amplification upstream and downstream primer of CYP3A4 gene 1st～3 exons, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 4th～7 exons, at least one pair of in the long segment specific amplification upstream and downstream primer of the long segment specific amplification upstream and downstream primer of CYP3A4 gene 8th～11 exons and CYP3A4 gene 12nd～13 exons, and with the DNA long fragment of described 1st～3 exons, the DNA long fragment of 4th～7 exons, at least one pair of in the upstream and downstream sequencing primer of each exon of the CYP3A4 gene that the DNA long fragment of the DNA long fragment of 8th～11 exons and 12nd～13 exons is corresponding, wherein:
Described CYP3A4 gene 1st～3 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:1 in sequence table;
Described CYP3A4 gene 1st～3 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:2 in sequence table;
Described CYP3A4 gene 4th～7 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:3 in sequence table;
Described CYP3A4 gene 4th～7 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:4 in sequence table;
Described CYP3A4 gene 8th～11 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:5 in sequence table;
Described CYP3A4 gene 8th～11 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:6 in sequence table;
Described CYP3A4 gene 12nd～13 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:7 in sequence table;
Described CYP3A4 gene 12nd～13 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:8 in sequence table;
Described CYP3A4 exon 1 upstream sequencing primer sequence is as shown in SEQ ID NO:9 in sequence table;
Described CYP3A4 exon 1 downstream sequencing primer sequence is as shown in SEQ ID NO:10 in sequence table;
Described CYP3A4 Exon 2 upstream sequencing primer sequence is as shown in SEQ ID NO:11 in sequence table;
Described CYP3A4 Exon 2 downstream sequencing primer sequence is as shown in SEQ ID NO:12 in sequence table;
Described CYP3A4 gene the 3rd exon upstream sequencing primer sequence is as shown in SEQ ID NO:13 in sequence table;
Described CYP3A4 gene the 3rd exon downstream sequencing primer sequence is as shown in SEQ ID NO:14 in sequence table;
Described CYP3A4 gene the 4th exon upstream sequencing primer sequence is as shown in SEQ ID NO:15 in sequence table;
Described CYP3A4 gene the 4th exon downstream sequencing primer sequence is as shown in SEQ ID NO:16 in sequence table;
Described CYP3A4 gene 5th～6 exon upstream sequencing primer sequences are as shown in SEQ IDNO:17 in sequence table;
Described CYP3A4 gene 5th～6 exon downstream sequencing primer sequences are as shown in SEQ IDNO:18 in sequence table;
Described CYP3A4 gene the 7th exon upstream sequencing primer sequence is as shown in SEQ ID NO:19 in sequence table;
Described CYP3A4 gene the 7th exon downstream sequencing primer sequence is as shown in SEQ ID NO:20 in sequence table;
Described CYP3A4 gene the 8th exon upstream sequencing primer sequence is as shown in SEQ ID NO:21 in sequence table;
Described CYP3A4 gene the 8th exon downstream sequencing primer sequence is as shown in SEQ ID NO:22 in sequence table;
Described CYP3A4 gene the 9th exon upstream sequencing primer sequence is as shown in SEQ ID NO:23 in sequence table;
Described CYP3A4 gene the 9th exon downstream sequencing primer sequence is as shown in SEQ ID NO:24 in sequence table;
Described CYP3A4 gene exon10 upstream sequencing primer sequence is as shown in SEQ IDNO:25 in sequence table;
Described CYP3A4 gene exon10 downstream sequencing primer sequence is as shown in SEQ IDNO:26 in sequence table;
Described CYP3A4 gene the 11st exon upstream sequencing primer sequence is as shown in SEQ IDNO:27 in sequence table;
Described CYP3A4 gene the 11st exon downstream sequencing primer sequence is as shown in SEQ IDNO:28 in sequence table;
Described CYP3A4 gene the 12nd exon upstream sequencing primer sequence is as shown in SEQ IDNO:29 in sequence table;
Described CYP3A4 gene the 12nd exon downstream sequencing primer sequence is as shown in SEQ IDNO:30 in sequence table;
Described CYP3A4 gene the 13rd exon upstream sequencing primer sequence is as shown in SEQ IDNO:31 in sequence table;
Described CYP3A4 gene the 13rd exon downstream sequencing primer sequence is as shown in SEQ IDNO:32 in sequence table.
In the mentioned reagent box, as a kind of preferred implementation, described test kit also comprises reference gene ACTB and reference gene ACTB amplimer, wherein,
The upstream primer sequence of described reference gene ACTB amplimer is as shown in SEQ ID NO:33 in sequence table;
The downstream primer sequence of described reference gene ACTB amplimer is as shown in SEQ ID NO:34 in sequence table;
The sequence of described reference gene ACTB is as shown in SEQ ID NO:35 in sequence table.
In the mentioned reagent box, as a kind of preferred implementation, described test kit also comprises the order-checking damping fluid.More electedly, described order-checking damping fluid is the Buffer damping fluid, and the pH of described Buffer damping fluid is 8.3.
In the mentioned reagent box, as a kind of preferred implementation, described test kit also comprises negative control and positive control, and wherein, described negative control is deionized water; In the CYP3A4 gene pleiomorphism that described positive control is the CYP3A4*8 type containing 389G the gene order DNA sample in A mutational site.
In the mentioned reagent box, as a kind of preferred implementation, described test kit also comprises the mixture of various PCR reaction reagents or various PCR reaction reagents; More preferably, described various PCR reaction reagent comprises: PCR premix, Mg
2+, dNTPs dUTP, Taq enzyme, BSA, T4 phage gene 32 proteins encoded and without the RNase deionized water.
In the mentioned reagent box, as a kind of preferred implementation, described test kit also comprises the amplimer of inner positive control sequence and inner positive control sequence, and wherein: the positive control sequence in described inside is as shown in SEQ ID NO:36 in sequence table; The upstream primer of the amplimer of the positive control sequence in described inside is as shown in SEQ ID NO:37 in sequence table; The downstream primer of the amplimer of the positive control sequence in described inside is as shown in SEQ ID NO:38 in sequence table.
In the mentioned reagent box, various preferred implementations can arbitrary combination.
A kind of method that detects the CYP3A4 gene pleiomorphism, comprise the steps:
Step 1, extract person under inspection's genomic dna;
Step 2, the genomic dna that the step 1 of take obtains is template, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 1st～3 exons of employing as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 4th～7 exons as shown in SEQ ID NO:3 in sequence table and SEQ ID NO:4, the long segment specific amplification upstream and downstream primer of the long segment specific amplification upstream and downstream primer of CYP3A4 gene 8th～11 exons as shown in SEQ ID NO:5 in sequence table and SEQ ID NO:6 or CYP3A4 gene 12nd～13 exons as shown in SEQ ID NO:7 in sequence table and SEQ ID NO:8, the DNA long fragment of pcr amplification CYP3A4 gene 1st～3 exons, the DNA long fragment of CYP3A4 gene 4th～7 exons, the DNA long fragment of the DNA long fragment of CYP3A4 gene 8th～11 exons or CYP3A4 gene 12nd～13 exons,
Step 3, pcr amplification product to step 2 carries out gel electrophoresis, and reclaims DNA long fragment, the DNA long fragment of CYP3A4 gene 4th～7 exons, the DNA long fragment of CYP3A4 gene 8th～11 exons or the DNA long fragment of CYP3A4 gene 12nd～13 exons of CYP3A4 gene 1st～3 exons that purifying obtains;
Step 4, it is template that the step 3 of take reclaims the product obtained after purifying, the upstream and downstream primer of employing as shown in SEQ IDNO:9 in sequence table and SEQ ID NO:10, upstream and downstream primer shown in SEQ ID NO:11 and SEQ ID NO:12, upstream and downstream primer shown in SEQ ID NO:13 and SEQ ID NO:14, upstream and downstream primer shown in SEQ IDNO:15 and SEQ ID NO:16, upstream and downstream primer shown in SEQ ID NO:17 and SEQ ID NO:18, upstream and downstream primer shown in SEQ ID NO:19 and SEQ ID NO:20, upstream and downstream primer shown in SEQ IDNO:21 and SEQ ID NO:22, upstream and downstream primer shown in SEQ ID NO:23 and SEQ ID NO:24, upstream and downstream primer shown in SEQ ID NO:25 and SEQ ID NO:26, upstream and downstream primer shown in SEQ IDNO:27 and SEQ ID NO:28, at least one pair of corresponding upstream and downstream sequencing primer in upstream and downstream primer shown in upstream and downstream primer shown in SEQ ID NO:29 and SEQ ID NO:30 and SEQ ID NO:31 and SEQ ID NO:32 carries out sequencing reaction,
Step 5, after the sequencing reaction product purification that step 4 is obtained, upper sequenator is checked order, and will record sequence and compare to obtain the CYP2D6 gene pleiomorphism in the NCBI nucleic acid database.
In aforesaid method, as a kind of preferred implementation, in described step 2, the reaction conditions of described PCR is as follows: first through 95 ℃ of 3min; Then enter the PCR circulation, described PCR circulation is 95 ℃ of 30s, 57 ℃ of 3.6min, 72 ℃ of 3min, totally 35 circulations; Last 72 ℃ of 10min.More preferably, in the reaction solution of described PCR, part component and concentration are as follows: 1 * PCR premix, Mg
2+: 2.5-4.0mM, dNTPs:0.2-0.4mM, dUTP:0.3-0.6mM, Taq enzyme: gene 32 proteins encoded of 0.2U/ μ L, BSA:1.25wt%, T4 phage: 1nmol/L.
In aforesaid method, as a kind of preferred implementation, in described step 4, the condition of described sequencing reaction is as follows: first 98 ℃ of sex change 2min, then carry out the PCR circulation, and the PCR loop parameter is 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 4min, totally 25 circulations.
In aforesaid method, as a kind of preferred implementation, in described step 2, also added the downstream primer of amplimer of the positive control sequence of upstream primer and the inside as shown in SEQ ID NO:38 in sequence table of the amplimer of the positive control sequence in inside as shown in SEQ ID NO:36 in sequence table, the positive control sequence in the inside as shown in SEQ ID NO:37 in sequence table in described PCR reaction.
Advantage and the effect of test kit of the present invention and detection method are as follows:
(1) susceptibility is high: can detect 24 kinds of CYP3A4 gene pleiomorphisms of current report simultaneously, specifically refer to CYP allelotrope database (http://www.cypalleles.ki.se).
(2) high specificity: use primer amplified CYP3A4 gene length fragment sequence, then checked order, specific molecule is identified, accuracy is high, specificity is good, false positive is low, can reach the accuracy of direct sequencing, even more accurate than direct sequencing.
(3) handy and safe: simple to operate, safety, only need can carry out a plurality of exon order-checking detection polymorphisms after a pcr amplification simultaneously, greatly reduced detection, reduced the possibility of polluting.
(4) complete monitoring: the test kit that the embodiment of the present invention provides has been introduced the inner positive quality control system of controlling, and testing process is carried out to Complete Quality Supervision, effectively avoids false positive or false negative.
(5) quick: speed is fast, high-throughput, only needs disposable amplification CYP3A4 gene length fragment sequence, and, without each exon of CYP3A4 gene is increased, has greatly reduced labour intensity, has saved detection time, can in 8 hours, complete.
Test kit of the present invention and method, utilize the LA-PCR sequencing to detect fast and accurately 24 kinds of CYP3A4 gene pleiomorphisms of current report, effectively stop false positive and false-negative generation, for the prediction of the using dosage of the medicine through the CYP3A4 metabolism and for the selection of medicine, provide important means.
The accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, in below describing embodiment, the accompanying drawing of required use is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is that the long segment amplimer of test kit of the employing embodiment 1 that provides of the embodiment of the present invention 2 is to amplification person under inspection peripheral blood DNA PCR product gel electrophoresis result, the nucleic acid fragment in the same size that its clip size is estimated, amplification corresponds to: the first band is Marker, the second band is CYP3A4 1st～3 exon primer amplification long segment, the 3rd band is CYP3A4 4th～7 exon primer amplification long segment, the 4th band is CYP3A4 8th～11 exon primer amplification long segment, the 6th band is CYP3A4 12nd～13 exon primer amplification long segment, the positive contrast of the 7th band, the negative contrast of the 8th band,
Fig. 2 is the checked order result of rear gained of CYP3A4 gene the 7th exon upstream sequencing primer of employing embodiment 1 test kit that provides of the embodiment of the present invention 2, at the 7th exon nucleotide sequence 566 base places, 566T has occurred > C, prompt for CYP3A4 gene pleiomorphism * 17;
Fig. 3 is the checked order result of rear gained of CYP3A4 gene the 11st exon upstream sequencing primer of employing embodiment 1 test kit that provides of the embodiment of the present invention 2, at the 11st exon nucleotide sequence 1088 base places, 1088C has occurred > T, prompt for CYP3A4 gene pleiomorphism * 11.
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.The experimental technique of unreceipted actual conditions in following embodiment, be generally this area ordinary method, as according to the normal experiment condition as people such as Sambrook, molecular cloning, laboratory manual (third edition) (Science Press, 2002) condition described in, or the condition of advising according to reagent manufacturing firm.
The preparation of embodiment 1. test kits
1, the design of primers of reference gene ACTB and CYP3A4 gene
Derive from American National biotechnology information center nucleic acid database (NCBI) according to gene order ACTB gene order and CYP3A4 gene order, wherein the ACTB gene I/D is 60, reference sequences number is NG_007992.1, also can be referring to SEQ ID NO:35 in sequence table of the present invention, the CYP3A4 gene I/D is 1574, reference sequences number is NG_008421.1, adopt the Primer5.0 primer-design software to design respectively the special primer of reference gene ACTB and CYP3A4 gene, comprise: the long segment specific amplification upstream and downstream primer of CYP3A4 gene 1st～3 exons, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 4th～7 exons, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 8th～11 exons, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 12nd～13 exons, and CYP3A4 exon 1 upstream and downstream sequencing primer, CYP3A4 Exon 2 upstream and downstream sequencing primer, CYP3A4 gene the 3rd exon upstream and downstream sequencing primer, CYP3A4 gene the 4th exon upstream and downstream sequencing primer, CYP3A4 gene 5th～6 exon upstream and downstream sequencing primers, CYP3A4 gene the 7th exon upstream and downstream sequencing primer, CYP3A4 gene the 8th exon upstream and downstream sequencing primer, CYP3A4 gene the 9th exon upstream and downstream sequencing primer, CYP3A4 gene exon10 upstream and downstream sequencing primer, CYP3A4 gene the 11st exon upstream and downstream sequencing primer, CYP3A4 gene the 12nd exon upstream and downstream sequencing primer and CYP3A4 gene the 13rd exon upstream and downstream sequencing primer, wherein, as the long segment specific amplification upstream and downstream primer of CYP3A4 gene 1st～3 exons is respectively used to differentiate the CYP3A4*14 type of CYP3A4 gene, CYP3A4 gene the 7th exon upstream and downstream primer is for differentiating the CYP3A4*17 gene pleiomorphism, CYP3A4 gene the 11st exon upstream and downstream primer is one-to-one relationship for differentiating CYP2D6*11 etc., can detect 24 kinds of CYP3A4 gene pleiomorphisms of current report simultaneously, wherein, 24 kinds of CYP3A4 gene pleiomorphisms specifically refer to CYP allelotrope database (http://www.cypalleles.ki.se).
By above-mentioned design of primers, obtain:
CYP3A4 gene 1st～3 exon long segment specific amplification upstream primer sequence: 5 '-TGGACTTGGGGTGCTAATCA-3 ', as shown in SEQ ID NO:1 in sequence table;
CYP3A4 gene 1st～3 exon long segment specific amplification downstream primer sequence: 5 '-TGTGTGTGTGTAAAAACCCTGAC-3 ', as shown in SEQ ID NO:2 in sequence table;
CYP3A4 gene 4th～7 exon long segment specific amplification upstream primer sequence: 5 '-GGCTGTTGATGTTTAATCAACTCTG-3 ', as shown in SEQ ID NO:3 in sequence table;
CYP3A4 gene 4th～7 exon long segment specific amplification downstream primer sequence: 5 '-CAAATGTACTACAAATCACTGAACTG-3 ', as shown in SEQ ID NO:4 in sequence table;
CYP3A4 gene 8th～11 exon long segment specific amplification upstream primer sequence 5 '-AGCAAATAATTATACAACCACATGAC-3 ', as shown in SEQ ID NO:5 in sequence table;
CYP3A4 gene 8th～11 exon long segment specific amplification downstream primer sequence: 5 '-GCTCCAGGTAAATTTTGCACA-3 ', as shown in SEQ ID NO:6 in sequence table;
CYP3A4 gene 12nd～13 exon long segment specific amplification upstream primer sequence: 5 '-GTAAGAAACCCTAACATGTAACT-3 ', as shown in SEQ ID NO:7 in sequence table;
CYP3A4 gene 12nd～13 exon long segment specific amplification downstream primer sequence: 5 '-AGGCCAGGATGTCTCTCCA-3 ', as shown in SEQ ID NO:8 in sequence table;
CYP3A4 exon 1 upstream sequencing primer sequence: 5 '-GCCCTGCTACTGGCTGCA-3 ', as shown in SEQ ID NO:9 in sequence table;
CYP3A4 exon 1 downstream sequencing primer sequence: 5 '-CGGCCTGAACATCTTTTTTG-3 ', as shown in SEQ ID NO:10 in sequence table;
CYP3A4 Exon 2 upstream sequencing primer sequence: 5 '-TCGCTGTGACTTGATTTCTGT-3 ', as shown in SEQ ID NO:11 in sequence table;
CYP3A4 Exon 2 downstream sequencing primer sequence: 5 '-AAAACTTCAGACCTTCCCCCT-3 ', as shown in SEQ ID NO:12 in sequence table;
CYP3A4 gene the 3rd exon upstream sequencing primer sequence: 5 '-TGACAAGAGCTTCATCCCAA-3 ', as shown in SEQ ID NO:13 in sequence table;
CYP3A4 gene the 3rd exon downstream sequencing primer sequence: 5 '-TCCCAATTAGAGGCAGGGTTA-3 ', as shown in SEQ ID NO:14 in sequence table;
CYP3A4 gene the 4th exon upstream sequencing primer sequence: 5 '-TTGGGCTCCAGCTGTAGAATA-3 ', as shown in SEQ ID NO:15 in sequence table;
CYP3A4 gene the 4th exon downstream sequencing primer sequence: 5 '-CCTGGACATTTTTTAGGCAAC-3 ', as shown in SEQ ID NO:16 in sequence table;
CYP3A4 gene 5th～6 exon upstream sequencing primer sequence: 5 '-AAGTGTTCCTGTTTAACACATTTTC-3 ', as shown in SEQ ID NO:17 in sequence table;
CYP3A4 gene 5th～6 exon downstream sequencing primer sequence: 5 '-TCACTGGAATAACCCAACAGCA-3 ', as shown in SEQ ID NO:18 in sequence table;
CYP3A4 gene the 7th exon upstream sequencing primer sequence: 5 '-GTGGATGAATTACATGGTGATTT-3 ', as shown in SEQ ID NO:19 in sequence table;
CYP3A4 gene the 7th exon downstream sequencing primer sequence: 5 '-GGAAGGATGGTAAAAAGGTGCT-3 ', as shown in SEQ ID NO:20 in sequence table;
CYP3A4 gene the 8th exon upstream sequencing primer sequence: 5 '-GCTCCAGGTAAATTTTGCACA-3 ', as shown in SEQ ID NO:21 in sequence table;
CYP3A4 gene the 8th exon downstream sequencing primer sequence: 5 '-AAAACATACAGGTAACTGCACTGA-3 ', as shown in SEQ ID NO:22 in sequence table;
CYP3A4 gene the 9th exon upstream sequencing primer sequence: 5 '-TCAGGAGCCACTTTCTGTCA-3 ', as shown in SEQ ID NO:23 in sequence table;
CYP3A4 gene the 9th exon downstream sequencing primer sequence: 5 '-TATGCCTGCATGCCTCTAGAA-3 ', as shown in SEQ ID NO:24 in sequence table;
CYP3A4 gene exon10 upstream sequencing primer sequence: 5 '-CTCTTCATCTAAACTGTGATGCCC-3 ', as shown in SEQ ID NO:25 in sequence table;
CYP3A4 gene exon10 downstream sequencing primer sequence: 5 '-AGAGTCAGTGAAAGAATCAGTGATT-3 ', as shown in SEQ ID NO:26 in sequence table;
CYP3A4 gene the 11st exon upstream sequencing primer sequence: 5 '-CTTCATTAGTACTGCATGGACTGAG-3 ', as shown in SEQ ID NO:27 in sequence table;
CYP3A4 gene the 11st exon downstream sequencing primer sequence: 5 '-AGCAAATAATTATACAACCACATGACTG-3 ', as shown in SEQ ID NO:28 in sequence table;
CYP3A4 gene the 12nd exon upstream sequencing primer sequence: 5 '-TCTCAACAAGACTGAAAGCTCC-3 ', as shown in SEQ ID NO:29 in sequence table;
CYP3A4 gene the 12nd exon downstream sequencing primer sequence: 5 '-GGGCTTTACATGATGTTTTTGATG-3 ', as shown in SEQ ID NO:30 in sequence table;
CYP3A4 gene the 13rd exon upstream sequencing primer sequence: 5 '-TCCCCTCAACACTGAAGGAGT-3 ', as shown in SEQ ID NO:31 in sequence table;
CYP3A4 gene the 13rd exon downstream sequencing primer sequence: 5 '-CAATGCATGTACAGAATCCCC-3 ', as shown in SEQ ID NO:32 in sequence table;
Reference gene ACTB amplification upstream sequencing primer sequence: 5 '-CGATTTCTCGCAGCTCACCAT-3 ', as shown in SEQ ID NO:33 in sequence table;
Reference gene ACTB amplification downstream sequencing primer sequence: 5 '-AATACACACTCCAAGGCCGC-3 ', as shown in SEQ ID NO:34 in sequence table.
2, the design of inner positive control sequence and primer thereof
The positive control sequence in this inside comprises CYP3A4 Gene Partial sequence and the artificial composition sequence of a part, as shown in SEQ ID NO:36 in sequence table.
Adopt the Primer5.0 primer-design software and be respectively according to the upstream and downstream primer that above-mentioned design of primers principle is designed the positive control sequence in above-mentioned inside: 5 '-GCGCGTGACACTGCTACATG-3 ' (as shown in SEQ ID NO:37 in sequence table), 5 '-TCGAGTAGCTGAGACTGCGT-3 ' (as shown in SEQ ID NO:38 in sequence table).
3, the composition of test kit and preparation
Test kit of the present invention is composed as follows:
1. extracting genome DNA reagent: this extraction reagent is this area common agents, and the present embodiment adopts tissue gene group DNA extraction test kit (Qiagen company, article No.: the reagent 69504).
2. primer: long segment amplification upstream and downstream primer, above-mentioned CYP3A4 gene the 1st, 2,3,4,5～6,7,8,9,10,11,12 and the 13 exons order-checking upstream and downstream primer, the positive control sequence amplification upstream and downstream primer in above-mentioned inside and the reference gene ACTB amplification upstream and downstream primer that comprise above-mentioned can increase respectively CYP3A4 gene 1st～3 exons, 4～7 exons, 8～11 exons and 12～13 exons.
3. above-mentioned reference gene ACTB and inner positive control sequence.
Above-mentioned primer sequence, inner positive control sequence and reference gene ACTB sequence are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
4. negative control and positive control: with the negative contrast of deionized water, with in the CYP3A4 gene pleiomorphism that contains the CYP3A4*8 type containing 389G the positive contrast of gene order DNA sample in A mutational site;
The preparation of positive control: adopt tissue gene group DNA extraction test kit (Qiagen company, article No.: in the CYP3A4 gene pleiomorphism that contains the CYP3A4*8 type that 69504) rapid extraction has been made a definite diagnosis containing 389G the 0.5ml person under inspection peripheral blood genomic dna of the gene order in A mutational site, as positive control.
5. various PCR reaction reagents: the PCR premix, select 2 * PCR Premix(Qiagen company in the present embodiment, product article No.: 210212); Mg
2+, dNTPs, dUTP, Taq enzyme, BSA(Albumin from bovine serum, bovine serum albumin), gene 32 proteins encoded of T4 phage, without the RNase deionized water.
6. the damping fluid that checks order, the Buffer damping fluid that pH is 8.3, the preparation of its Buffer damping fluid: 1 μ lBigdye and 3 μ l5x are containing 10mM MgCl
2400mM Tris solution.
The test kit of embodiment 2. use embodiment 1 preparations detects the CYP3A4 gene pleiomorphism
Take with the routine person under inspection's peripheral blood of machine testing 30 sample result is example.
The testing process that detects crowd CYP3A4 gene pleiomorphism with test kit of the present invention is: at first obtain clinical person under inspection's peripheral blood sample, the rapid extraction genomic dna; Secondly, first prepare the PCR reaction solution of reference gene ACTB, then add concentration to be 2ng/ μ l reference gene ACTB sequence and each 2 μ l of inner positive control sequence, carry out pcr amplification, carry out electrophoresis in the agarose DNA gel that PCR is 2% in concentration after finishing, check the pcr amplification situation.If there are two bands of 390bp and 800bp left and right in the pcr amplification product of reference gene ACTB, point out whole testing process effective, as shown in Figure 1, if lack one or two bands, prompting detects unsuccessfully, needs to re-start detection.Again, if the PCR result proof testing process by reference gene ACTB is effective, prepare CYP3A4 gene length fragment amplification PCR reaction solution and negative, positive reaction liquid, the person under inspection's peripheral blood genomic dna that adds 2 μ l to extract in the former, the latter adds respectively 2 μ l deionized waters and positive DNA, and respectively to add concentration be the inner positive control sequence 2 μ l of 2ng/ μ l, carry out pcr amplification, imaging on the agarose DNA gel electrophoresis that pcr amplification is 2% in concentration after finishing, check the pcr amplification situation.Person under inspection's peripheral blood sample CYP3A4 gene 1st～3 exons, 4～7 exons, 8～11 exons and 12～13 exon amplification clip size should be respectively 3780bp, 4500bp, 5210bp, 4281bp left and right, and one of 390bp left and right band all arranged, positive controls is each of 800bp and 390bp left and right band, negative control group is 1 of 390bp band, as shown in Figure 1.
Finally, reclaim purified pcr product with one in the long segment that obtains person under inspection CYP3A4 1st～3 exons, 4～7 exons, 8～11 exons and 12～13 exons, template by this DNA fragmentation for sequencing reaction, the laggard performing PCR sequencing reaction of preparation PCR sequencing reaction liquid, checked order upper sequenator after PCR sequencing reaction thing purifying.
The concrete detecting step of CYP3A4 gene pleiomorphism is as follows:
1. the extracting of person under inspection's peripheral blood genomic dna: the extracting genome DNA reagent in employing embodiment 1 test kit is pressed the method rapid extraction 0.5ml person under inspection peripheral blood genomic dna of DNA extracting and purifying.
2. person under inspection's peripheral blood genomic dna of said extracted is identified to its integrity through agarose gel electrophoresis, measure purity and the concentration of 260nm and 280nm optical density value calculating DNA by ultraviolet spectrophotometer, the DNA that regulates extracting with aseptic deionized water, to same concentrations, puts refrigerator-20 ℃ preservation.
3. the positive control sequence standard substance in the inside in embodiment 1 test kit and reference gene ACTB standard substance being diluted to respectively to concentration is 2ng/ μ l.
4. the pcr amplification of reference gene ACTB:
The PCR reaction system is 20 μ L, and in this reaction system, each composition and final concentration are as follows: 1 * PCR premix (Qiagen company, product article No.: 210212,2 * PCR Premix, 10.0 μ L), Mg
2+: 3mM; DNTPs:0.3mM; DUTP:0.5mM; Taq enzyme: 0.2U/ μ L; BSA:1.25wt%; Gene 32 proteins encoded of T4 phage: 1nmol/L; Reference gene ACTB upstream and downstream primer is: 0.25pmol/ μ L; Inner positive control sequence upstream and downstream primer is: 0.25pmol/ μ L; Inner positive control sequence: 0.3pmol/ μ L; Reference gene ACTB sequence: 0.2ng/ μ l; All the other are without the RNase deionized water.
In the amplification of the enterprising performing PCR of ABI9700PCR instrument, the PCR response procedures is: first through 95 ° of C3min, and 95 ℃ of 30s then, 57 ℃ of 3.6min, 72 ℃ of 3min, 35 circulations, last 72 ℃ of 10min.
Imaging on the agarose DNA gel electrophoresis that pcr amplification is 2% in concentration after finishing can be seen two bands that have 390bp and 800bp left and right from electrophorogram, points out whole testing process effective, therefore can carry out following operation.
5. CYP3A4 gene 1st～3 exons, 4～7 exons, 8～11 exons and the amplification of 12～13 exon LA-PCRs:
The PCR reaction system of above-mentioned four CYP3A4 gene length fragments is 20 μ L, and in this reaction system, each composition and final concentration are as follows: 1 * PCR premix (Qiagen company, product article No.: 210212,2 * PCR Premix, 10.0 μ L); Mg
2+: 3mM; DNTPs:0.3mM; DUTP:0.5mM; Taq enzyme: 0.2U/ μ L; BSA:1.25wt%; Gene 32 proteins encoded of T4 phage: 1nmol/L; CYP3A4 gene 1st～3 exons, 4th～7 exons, 8th～11 exons or 12nd～13 exon long segment specific amplification upstream and downstream primers are 0.25pmol/ μ L; Inner positive control sequence upstream and downstream primer is 0.25pmol/ μ L; Inner positive control sequence: 0.3pmol/ μ L; The consumption of person under inspection's peripheral blood genomic dna is 2 μ L; All the other are without the RNase deionized water.
In CYP3A4 gene 1st～3 exons, 4～7 exons, 8～11 exons and the amplification of 12～13 exon LA-PCRs, positive control and negative control are set, the reaction system of positive control and negative control is 20 μ L, in this reaction system, each composition and final concentration are as follows: 1 * PCR premix (Qiagen company, product article No.: 210212,2 * PCR Premix, 10.0 μ L); Mg
2+: 3mM; DNTPs:0.3mM; DUTP:0.5mM; Taq enzyme: 0.2U/ μ L; BSA:1.25wt%; Gene 32 proteins encoded of T4 phage: 1nmol/L; Reference gene ACTB amplification upstream and downstream primer is: 0.25pmol/ μ L; Inner positive control sequence upstream and downstream primer is 0.25pmol/ μ L; Inner positive control sequence: 0.3pmol/ μ L; The consumption of positive or negative sample is 2 μ L, and all the other are without the RNase deionized water.
In the amplification of the enterprising performing PCR of ABI9700PCR instrument, the PCR response procedures is: first through 95 ℃ of 3min, and 95 ℃ of 30s then, 57 ℃ of 3.6min, 72 ℃ of 3min, 35 circulations, last 72 ℃ of 10min.
Imaging on the agarose DNA gel electrophoresis that pcr amplification is 2% in concentration after finishing.As shown in Figure 1.
6. cut respectively CYP3A4 gene 1st～3 exons, 4th～7 exons, 8th～11 exons and 12nd～13 exon long segment amplification PCR product and inner positive control sequence PCR product, utilize Axy company DNA gel to reclaim the DNA fragmentation that test kit reclaims the purifying amplification.
7. the preparation of sequencing reaction liquid: get 4 μ l Buffer order-checking damping fluids, 6. the step that concentration is 2ng/ μ l reclaims each 1 μ l of four DNA long fragments that purifying obtains, then gets accordingly CYP3A4 gene the 1st, 2,3,4,5～6,7,8,9,10,11,12 and each 1 μ l of 13 exon upstream and downstream sequencing primers that concentration is 1pmol/ml.
On the ABI9700 instrument, first 98 ℃ of sex change 2min, then carry out the PCR circulation, and the PCR loop parameter is 96 ℃ of 10s, 50 ℃ of 5s, and 60 ℃ of 4min, 25 circulations, amplification arranges 4 ℃ of insulations after finishing.
8. adopt sodium-acetate/Ethanol Method purification step sequencing reaction product 7., checked order according to the operation instructions of ABI3130 sequenator.
9. data collection process and analysis: measured sequence is compared in the NCBI nucleic acid database, analyze the CYP3A4 gene pleiomorphism, result is referring to table 1.
Table 1 is analyzed CYP3A4 gene pleiomorphism in person under inspection's peripheral blood genome for the LA-PCR sequencing
| Sample | The nucleic acid variation | Amino acid variation | Gene pleiomorphism |
| Person under inspection's peripheral blood 1 | 508G>A | V170I | CYP3A4*9 |
| Person under inspection's peripheral blood 2 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 3 | - | — | CYP3A4*1 |
| Person under inspection's peripheral blood 4 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 5 | 600A>T | Q200H | CYP3A4*24 |
| Person under inspection's peripheral blood 6 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 7 | 664T>C | S222P | CYP3A4*2 |
| Person under inspection's peripheral blood 8 | 1247C>T | P416L | CYP3A4*13 |
| Person under inspection's peripheral blood 9 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 10 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 11 | 485G>A | R162Q | CYP3A4*15 |
| Person under inspection's peripheral blood 12 | 653C>G | P218R | CYP3A4*5 |
| Person under inspection's peripheral blood 13 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 14 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 15 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 16 | 1247C>T | P416L | CYP3A4*13 |
| Person under inspection's peripheral blood 17 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 18 | 352A>G | I118V | CYP3A4*4 |
| Person under inspection's peripheral blood 19 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 20 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 21 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 22 | 352A>G | I118V | CYP3A4*4 |
| Person under inspection's peripheral blood 23 | 1247C>T | P416L | CYP3A4*13 |
| Person under inspection's peripheral blood 24 | 664T>C | S222P | CYP3A4*2 |
| Person under inspection's peripheral blood 25 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 26 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 27 | — | — | CYP3A4*1 |
| Person under inspection's peripheral blood 28 | 352A>G | I118V | CYP3A4*4 |
| Person under inspection's peripheral blood 29 | 1088C>T | T363M | CYP3A4*11 |
| Person under inspection's peripheral blood 30 | — | — | CYP3A4*1 |
Test kit detectivity of the present invention is estimated:
Using direct sequencing as comparison and detection method of the present invention, above-mentioned 30 routine person under inspection's peripheral blood samples are detected simultaneously, analytical results is identical with table 1, therefore the susceptibility, specificity and the sensitivity that adopt this test kit of the present invention to be detected are consistent with direct sequencing, but obviously shorten detection time, labour intensity obviously descends, and meets real requirement (specifically referring to table 2) fully.
Table 2 is the comparison that two kinds of different methods detect CYP3A4 gene pleiomorphism in person under inspection's peripheral blood
As seen from the above table, by the LA-PCR sequencing check CYP3A4 gene pleiomorphism that increases, using direct sequencing as reference, LA-PCR amplification sequencing and direct sequencing detect 13 routine saltant types and 17 routine wild-types simultaneously, the two detected result is consistent, draw thus, the positive predictive value of LA-PCR amplification sequencing is 100%; LA-PCR amplification sequencing and direct sequencing detect 17 routine wild-types simultaneously, all do not detect saltant type, draw thus, the negative predictive value of LA-PCR amplification sequencing is 100%, simultaneously, specificity and sensitivity are 100%, positive predictive value and negative predictive value also reach 100%, and repeatedly the repeated experiments result is consistent, are about 10h the detection time of a clinical samples, consuming time short, and direct sequencing method about 48h consuming time.
Above-mentioned experiment can illustrate, adopt all higher test kits of the present invention utilize the LA-PCR sequencing to detect the CYP3A4 gene pleiomorphism of susceptibility and specificity, under the condition that the specificity of its detected result and susceptibility do not reduce, detection time obviously shortens and labour intensity obviously reduces, the somatotype that this test kit is person under inspection's peripheral blood CYP3A4 gene pleiomorphism provides a kind of brand-new fast and convenient gene diagnosis technology, simultaneously, have and detect fast and reduced the characteristics such as pollution, and not yet there is the LA-PCR sequencing to detect the relevant report of CYP3A4 gene pleiomorphism test kit at present, test kit provided by the invention is conducive to predict curative effect of medication, instruct the selectivity medication of medicine.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. a test kit that detects the CYP3A4 gene pleiomorphism, comprise detecting and use primer, it is characterized in that, described detection comprises with primer: the long segment specific amplification upstream and downstream primer of CYP3A4 gene 1st～3 exons, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 4th～7 exons, at least one pair of in the long segment specific amplification upstream and downstream primer of the long segment specific amplification upstream and downstream primer of CYP3A4 gene 8th～11 exons and CYP3A4 gene 12nd～13 exons, and with the DNA long fragment of described 1st～3 exons, the DNA long fragment of 4th～7 exons, at least one pair of in the upstream and downstream sequencing primer of each exon of the CYP3A4 gene that the DNA long fragment of the DNA long fragment of 8th～11 exons and 12nd～13 exons is corresponding, wherein:
Described CYP3A4 gene 1st～3 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:1 in sequence table;
Described CYP3A4 gene 1st～3 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:2 in sequence table;
Described CYP3A4 gene 4th～7 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:3 in sequence table;
Described CYP3A4 gene 4th～7 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:4 in sequence table;
Described CYP3A4 gene 8th～11 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:5 in sequence table;
Described CYP3A4 gene 8th～11 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:6 in sequence table;
Described CYP3A4 gene 12nd～13 exon long segment specific amplification upstream primer sequences are as shown in SEQ ID NO:7 in sequence table;
Described CYP3A4 gene 12nd～13 exon long segment specific amplification downstream primer sequences are as shown in SEQ ID NO:8 in sequence table;
Described CYP3A4 exon 1 upstream sequencing primer sequence is as shown in SEQ ID NO:9 in sequence table;
Described CYP3A4 exon 1 downstream sequencing primer sequence is as shown in SEQ ID NO:10 in sequence table;
Described CYP3A4 Exon 2 upstream sequencing primer sequence is as shown in SEQ ID NO:11 in sequence table;
Described CYP3A4 Exon 2 downstream sequencing primer sequence is as shown in SEQ ID NO:12 in sequence table;
Described CYP3A4 gene the 3rd exon upstream sequencing primer sequence is as shown in SEQ ID NO:13 in sequence table;
Described CYP3A4 gene the 3rd exon downstream sequencing primer sequence is as shown in SEQ ID NO:14 in sequence table;
Described CYP3A4 gene the 4th exon upstream sequencing primer sequence is as shown in SEQ ID NO:15 in sequence table;
Described CYP3A4 gene the 4th exon downstream sequencing primer sequence is as shown in SEQ ID NO:16 in sequence table;
Described CYP3A4 gene 5th～6 exon upstream sequencing primer sequences are as shown in SEQ IDNO:17 in sequence table;
Described CYP3A4 gene 5th～6 exon downstream sequencing primer sequences are as shown in SEQ IDNO:18 in sequence table;
Described CYP3A4 gene the 7th exon upstream sequencing primer sequence is as shown in SEQ ID NO:19 in sequence table;
Described CYP3A4 gene the 7th exon downstream sequencing primer sequence is as shown in SEQ ID NO:20 in sequence table;
Described CYP3A4 gene the 8th exon upstream sequencing primer sequence is as shown in SEQ ID NO:21 in sequence table;
Described CYP3A4 gene the 8th exon downstream sequencing primer sequence is as shown in SEQ ID NO:22 in sequence table;
Described CYP3A4 gene the 9th exon upstream sequencing primer sequence is as shown in SEQ ID NO:23 in sequence table;
Described CYP3A4 gene the 9th exon downstream sequencing primer sequence is as shown in SEQ ID NO:24 in sequence table;
Described CYP3A4 gene exon10 upstream sequencing primer sequence is as shown in SEQ IDNO:25 in sequence table;
Described CYP3A4 gene exon10 downstream sequencing primer sequence is as shown in SEQ IDNO:26 in sequence table;
Described CYP3A4 gene the 11st exon upstream sequencing primer sequence is as shown in SEQ IDNO:27 in sequence table;
Described CYP3A4 gene the 11st exon downstream sequencing primer sequence is as shown in SEQ IDNO:28 in sequence table;
Described CYP3A4 gene the 12nd exon upstream sequencing primer sequence is as shown in SEQ IDNO:29 in sequence table;
Described CYP3A4 gene the 12nd exon downstream sequencing primer sequence is as shown in SEQ IDNO:30 in sequence table;
Described CYP3A4 gene the 13rd exon upstream sequencing primer sequence is as shown in SEQ IDNO:31 in sequence table;
Described CYP3A4 gene the 13rd exon downstream sequencing primer sequence is as shown in SEQ IDNO:32 in sequence table.
2. test kit according to claim 1, is characterized in that, described test kit also comprises reference gene ACTB and reference gene ACTB amplimer, wherein,
The upstream primer sequence of described reference gene ACTB amplimer is as shown in SEQ ID NO:33 in sequence table;
The downstream primer sequence of described reference gene ACTB amplimer is as shown in SEQ ID NO:34 in sequence table;
The sequence of described reference gene ACTB is as shown in SEQ ID NO:35 in sequence table.
3. test kit according to claim 1, is characterized in that, described test kit also comprises the order-checking damping fluid, and preferably, described order-checking damping fluid is the Buffer damping fluid, and the pH of described Buffer damping fluid is 8.3.
4. test kit according to claim 1, is characterized in that, described test kit also comprises negative control and positive control, and wherein, described negative control is deionized water; In the CYP3A4 gene pleiomorphism that described positive control is the CYP3A4*8 type containing 389G the gene order DNA sample in A mutational site.
5. test kit according to claim 1, is characterized in that, described test kit also comprises the mixture of various PCR reaction reagents or various PCR reaction reagents, and preferably, described various PCR reaction reagents comprise: PCR premix, Mg
2+gene 32 proteins encoded of (such as magnesium chloride), dNTPs, dUTP, Taq enzyme, bovine serum albumin, T4 phage and without the RNase deionized water.
6. according to the described test kit of claim 1-5 any one, it is characterized in that, described test kit also comprises the amplimer of inner positive control sequence and inner positive control sequence, wherein:
The positive control sequence in described inside is as shown in SEQ ID NO:36 in sequence table;
The upstream primer of the amplimer of the positive control sequence in described inside is as shown in SEQ ID NO:37 in sequence table; The downstream primer of the amplimer of the positive control sequence in described inside is as shown in SEQ ID NO:38 in sequence table.
7. a method that detects the CYP3A4 gene pleiomorphism, is characterized in that, comprises the steps:
Step 1, extract person under inspection's genomic dna;
Step 2, the genomic dna that the step 1 of take obtains is template, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 1st～3 exons of employing as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2, the long segment specific amplification upstream and downstream primer of CYP3A4 gene 4th～7 exons as shown in SEQ ID NO:3 in sequence table and SEQ ID NO:4, the long segment specific amplification upstream and downstream primer of the long segment specific amplification upstream and downstream primer of CYP3A4 gene 8th～11 exons as shown in SEQ ID NO:5 in sequence table and SEQ ID NO:6 or CYP3A4 gene 12nd～13 exons as shown in SEQ ID NO:7 in sequence table and SEQ ID NO:8, the DNA long fragment of pcr amplification CYP3A4 gene 1st～3 exons, the DNA long fragment of CYP3A4 gene 4th～7 exons, the DNA long fragment of the DNA long fragment of CYP3A4 gene 8th～11 exons or CYP3A4 gene 12nd～13 exons,
Step 3, pcr amplification product to step 2 carries out gel electrophoresis, and reclaims DNA long fragment, the DNA long fragment of CYP3A4 gene 4th～7 exons, the DNA long fragment of CYP3A4 gene 8th～11 exons or the DNA long fragment of CYP3A4 gene 12nd～13 exons of CYP3A4 gene 1st～3 exons that purifying obtains;
Step 4, it is template that the step 3 of take reclaims the product obtained after purifying, the upstream and downstream primer of employing as shown in SEQ IDNO:9 in sequence table and SEQ ID NO:10, upstream and downstream primer shown in SEQ ID NO:11 and SEQ ID NO:12, upstream and downstream primer shown in SEQ ID NO:13 and SEQ ID NO:14, upstream and downstream primer shown in SEQ IDNO:15 and SEQ ID NO:16, upstream and downstream primer shown in SEQ ID NO:17 and SEQ ID NO:18, upstream and downstream primer shown in SEQ ID NO:19 and SEQ ID NO:20, upstream and downstream primer shown in SEQ IDNO:21 and SEQ ID NO:22, upstream and downstream primer shown in SEQ ID NO:23 and SEQ ID NO:24, upstream and downstream primer shown in SEQ ID NO:25 and SEQ ID NO:26, upstream and downstream primer shown in SEQ IDNO:27 and SEQ ID NO:28, at least one pair of corresponding upstream and downstream sequencing primer in upstream and downstream primer shown in upstream and downstream primer shown in SEQ ID NO:29 and SEQ ID NO:30 and SEQ ID NO:31 and SEQ ID NO:32 carries out sequencing reaction,
Step 5, after the sequencing reaction product purification that step 4 is obtained, upper sequenator is checked order, and will record sequence and compare to obtain the CYP2D6 gene pleiomorphism in the NCBI nucleic acid database.
8. method according to claim 7, is characterized in that, in described step 2, the reaction conditions of described PCR is as follows: first through 95 ℃ of 3min; Then enter the PCR circulation, described PCR circulation is 95 ℃ of 30s, 57 ℃ of 3.6min, 72 ℃ of 3min, totally 35 circulations; Last 72 ℃ of 10min.
9. method according to claim 7, is characterized in that, in described step 4, the condition of described sequencing reaction is as follows: first 98 ℃ of sex change 2min, then carry out the PCR circulation, and the PCR loop parameter is 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 4min, totally 25 circulations.
10. method according to claim 7, it is characterized in that, in described step 2, also added the downstream primer of amplimer of the positive control sequence of upstream primer and the inside as shown in SEQ ID NO:38 in sequence table of the amplimer of the positive control sequence in inside as shown in SEQ ID NO:36 in sequence table, the positive control sequence in the inside as shown in SEQ ID NO:37 in sequence table in described PCR reaction.
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| CN106754991B (en) * | 2016-12-28 | 2020-11-10 | 温州医科大学 | CYP3A4 gene segment containing 972C > A mutation, protein segment coded by same and application thereof |
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