CN109355368A - A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism - Google Patents
A kind of kit and method of quick detection hypertension individuation medication gene pleiomorphism Download PDFInfo
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Abstract
The invention discloses the kits and method of a kind of quickly detection hypertension individuation medication gene pleiomorphism, belong to technical field of gene detection, with clinical antihypertensive β1receptorblocker, AngiotensinⅡreceptors type1, angiotensin converting enzyme inhibitors drug metabolism, kit includes the sample processing reagent quickly handled for people's whole blood sample, gene detection reagent PCR reaction solution 1, PCR reaction solution 2, PCR reaction solution 3, positive reference substance and negative controls through premixing single part packing.The present invention is guaranteeing on the specific and sensitive basis of detection that realization is convenient, fast and efficiently detects, and reduction detection operating procedure, reduction reduce production cost and testing cost while the detection reaction time, is conducive to clinical detection assays application.
Description
Technical field
The present invention relates to a kind of kit of genetic test and methods, more particularly to a kind of quickly detection Hypertensive subjects
The kit and method for changing medication gene pleiomorphism, belong to technical field of gene detection.
Background technique
CYP2D6 only accounts for 1-2%, but up to more than 80 kinds of drug through its catalysis metabolism, Different Individual in the total CYP of liver
The active maximum of CYP2D6 can differ 1000 times, and the most common polymorphism is CYP2D6*10 in Chinese population, and about 51.6%
Chinese carry this hereditary variation, which causes the enzymatic activity of CYP2D6 to reduce, and extremely unstable, to influence drug generation
It thanks.
CYP2C9 is the important member in Cytochrome P450 family, accounts for the 20% of hepatomicrosome P450 total amount, which urges
Change about 12% clinical commonly used drug, CYP2C9 gene there are polymorphism, predominantly CYP2C9*1 (Arg144/Ile359),
CYP2C9*2 (Cys144/Ile359), CYP2C9*3 (Arg144/Leu359), wherein CYP2C9*1 is normal enzymatic activity, China
The hereditary variation being primarily present in crowd is CYP2C9*3, causes enzymatic activity to reduce, influences drug metabolism.
1 adrenocepter of β belongs to g protein coupled receptor superfamily, is encoded by ADRB1 gene, and there are polymorphic for the gene
Property, research shows that ADRB1 gene G1165C polymorphism is related to condition of medicine treatment for hypertension, use metoprolol, Carvedilol etc.
After treatment, C1165 homozygotic individual effective percentage is higher.
AngiotensinⅡ is the important humoral factor of renin-angiotensin system, and 90% or more effect passes through
1 receptor (AGTR1) of angiotensinⅡ mediates, and the AGTR1 assignment of genes gene mapping encodes 359 ammonia in 3q21-q25, overall length 55kb
The receptor protein of base acid residue, there are polymorphisms for the gene, and wherein the site A1166C has weight in the drug therapy of hypertension
Meaning is wanted, research shows that the effective percentage of AA genotype individuals is apparently higher than AC and CC genotype individuals after treating using Losartan.
Angiotensin-Converting is the key enzyme of renin-angiotensin system, is encoded by ACE gene, and the gene is fixed
Positioned at No. 17 2nd area of chromosome long arm, 3 bands, the long 21kb of sequence.There are a variety of genetic polymorphisms for ACE gene, wherein the 16th introne
Present in 287bp Alu Insert Fragment (I) and deletion fragment (D) polymorphism it is related to condition of medicine treatment for hypertension, study table
The homozygous individual of bright DD is significant in efficacy using benazepil and fosinopril, and the homozygous individual of II is reached using enalapril and miaow
Puli's is significant in efficacy, and ID heterozygous individual uses hypertensin inhibitor class drug therapy, effectively.
Summary of the invention
The present invention for the prior art detection CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C,
Require height, detection cycle long, complicated for operation, at high cost and specific not strong sample process when ACE I/D type gene pleiomorphism
The deficiencies of, the kit and method of a kind of quickly detection hypertension individuation medication gene pleiomorphism are provided, anti-hypertension is related to
Medicine β1receptorblocker, AngiotensinⅡreceptors type1, angiotensin converting enzyme inhibitors drug metabolism, receptor are quick
Perceptual correlation CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C, ACE I/D genetic polymorphism detection
Kit and method.
The purpose of the present invention can reach by using following technical solution:
A kind of kit of quick detection hypertension individuation medication gene pleiomorphism, with clinical antihypertensive β1receptor
Retarding agent, AngiotensinⅡreceptors type1, angiotensin converting enzyme inhibitors drug metabolism, gene pleiomorphism include
Receptor sensitivity correlation CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C, ACE I/D gene polymorphic
Property, kit includes sample processing reagent, gene detection reagent, positive reference substance and negative controls.
Further, gene detection reagent is using the single part premix packing of PCR8 union, comprising: PCR reaction solution 1, PCR are anti-
Answer liquid 2 and PCR reaction solution 3.
Further, PCR reaction solution 1 includes:
Detect CYP2D6*10 primer pair: SEQ ID NO.1 and SEQ ID NO.2；
SEQ ID NO.1 is CYP2D6*10 upstream primer:
5'-AGTGAGGCAGGTATGGGGC-3'；
SEQ ID NO.2 is CYP2D6*10 downstream primer:
5'-GTCCACATGCAGCAGGTTG-3'；
Detect CYP2D6*10 specific probe: SEQ ID NO.3 and SEQ ID NO.4；
SEQ ID NO.3 is CYP2D6*10 wild type fluorescence probe:
5 '-fluorophor-GCTACCCACCAGGC-MGB-NFQ-3 '；
SEQ ID NO.4 is CYP2D6*10 saltant type fluorescence probe:
5 '-fluorophor-ACGCTACTCACCAT-MGB-NFQ-3 '；
Detect ADRB1 1165G > C primer pair: SEQ ID NO.5 and SEQ ID NO.6；
SEQ ID NO.5 is ADRB1 1165G > C upstream primer:
5'-CGCAGCCCCGACTTC-3'；
SEQ ID NO.6 is ADRB1 1165G > C downstream primer:
5'-CCGGTCTCCGTGGGT-3'；
Detect ADRB1 1165G > C specific probe: SEQ ID NO.7 and SEQ ID NO.8；
SEQ ID NO.7 is ADRB1 1165G > C wild type fluorescence probe:
5 '-fluorophor-ATTCCAGGGACTGC-MGB-NFQ-3 '；
SEQ ID NO.8 is ADRB1 1165G > C saltant type fluorescence probe:
5 '-fluorophor-TCCAGCGACTGCT-MGB-NFQ-3 '；
Further, PCR reaction solution 2 includes:
Detect CYP2C9*3 primer pair: SEQ ID NO.9 and SEQ ID NO.10；
SEQ ID NO.9 is CYP2C9*3 upstream primer:
5'-AGGAAGAGATTGAACGTGTGAT-3'；
SEQ ID NO.10 is CYP2C9*3 downstream primer:
5'-ATGAATTTGGGGACTTCGAA-3'；
Detect CYP2C9*3 specific probe: SEQ ID NO.11 and SEQ ID NO.12；
SEQ ID NO.11 is CYP2C9*3 wild type fluorescence probe:
5 '-fluorophor-GAGATACATTGACCTTCA-MGB-NFQ-3 '；
SEQ ID NO.12 is CYP2C9*3 saltant type fluorescence probe:
5 '-fluorophor-AGATACCTTGACCTTCA-MGB-NFQ-3 '；
Detect AGTR1 1166A > C primer pair: SEQ ID NO.13 and SEQ ID NO.14；
SEQ ID NO.13 is AGTR1 1166A > C upstream primer:
5'-GAGAACATTCCTCTGCAGCA-3'；
SEQ ID NO.14 is AGTR1 1166A > C downstream primer:
5'-CGGTTCAGTCCACATAATGC-3'；
Detect AGTR1 1166A > C specific probe: SEQ ID NO.15 and SEQ ID NO.16；
SEQ ID NO.15 is AGTR1 1166A > C wild type fluorescence probe:
5 '-fluorophor-CAAATGAGCATTAGCT-MGB-NFQ-3 '
SEQ ID NO.16 is AGTR1 1166A > C saltant type fluorescence probe:
5 '-fluorophor-ACATGAGCCTTAGCT-MGB-NFQ-3 '.
Further, PCR reaction solution 3 includes:
Detect ACE I/D primer pair: SEQ ID NO.17 and SEQ ID NO.18；
SEQ ID NO.17 is ACE I/D upstream primer:
5'-TGGAGAGCCACTCCCATCCTTTC-3'；
SEQ ID NO.18 is ACE I/D downstream primer:
5'-CGTGGCCATCACATTCGTCAG-3'；
Detect ACE I/D specific probe: SEQ ID NO.19 and SEQ ID NO.20；
SEQ ID NO.19 is ACE insert type fluorescence probe:
5 '-fluorophor-GGACCACAGGCGCCGCCACTAC-MGB-NFQ-3 '；
SEQ ID NO.20 is ACE deletion form fluorescence probe:
5 '-fluorophor-AGACCTGCTGCCTATA-MGB-NFQ-3 '；
Probe nucleic acid sequence 5 ' is used using one of FAM, JOE, VIC, NED and ROX modification, probe nucleic acid sequence 3 '
MGB-NFQ base group modification.
Further, gene detection reagent further include: the internal control primer pair for inner quality control: SEQ ID NO.21-SEQ
ID NO.22 and internal reference probe SEQ ID NO.23, probe use TaqMan probe；
SEQ ID NO.21 is internal reference upstream primer:
5'-CCTTCCTGCCACCGCTGAT-3'；
SEQ ID NO.22 is internal reference downstream primer:
5'-TGCCTTCGCAACCATTCCCTTA-3'；
SEQ ID NO.23 is internal reference probe:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ-3 '.
Further, gene detection reagent further include:
TaqPath ProAmp Master Mix premixed liquid with anti-PCR enzyme inhibitor；Premixed liquid includes after modifying
Hotstart-Taq polymerase, UNG enzyme, ROX fluorescent calibration dyestuff, dNTP, MgCl2、PCR buffer。
Further, positive reference substance is mixing CYP2D6100C, 100T, CYP2C91075A, 1075C, ADRB1
1165G, 1165C, AGTR11166A, 1166C, ACE Insertion (I), ACE Deletion (D) gene plasmid DNA and interior
Join Plasmid DNA；Negative controls are the ultrapure water without target gene and reference gene.
A kind of method of quick detection hypertension individuation medication gene pleiomorphism, includes the following steps:
Step 1: taking EDTA anticoagulated whole blood sample, take 2 μ L, 20 μ L sample processing reagents are added, are incubated under the conditions of 37 DEG C
5min。
Step 2: by 2 μ L of step 1 gained sample, being added separately to the gene detection reagent PCR reaction of 18 μ L premix packing
Liquid 1, PCR reaction solution 2 in PCR reaction solution 3, carry out PCR amplification after mixing；
Step 3: setting reaction process and response parameter, response parameter are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations open fluorescence channel in the 3rd stage and collect fluorescent value；
Step 4: sample is determined according to the difference △ Ct. value of same gene site wild type and saltant type fluorescence channel Ct. value
Genotyping, if detection positive findings have typical S type amplification curve to rise, and Ct. value is less than decision content, and NoCT indicates no expansion
Increase, Ct. value is based on 40.
Further, in step 4, Genotyping interpretation result is as follows:
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, Ct. >=3 △, gene type are as follows: Wild homozygous or
ACE insert type (CYP2D6*1/*1, CYP2C9*1/*1, ADRB1 1165G/G, AGTR1 1166A/A, ACE I/I)；
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, -3 < △ Ct. < 3, gene type are as follows: heterozygous
(CYP2D6*1/*10,CYP2C9*1/*3,ADRB1 1165G/C,AGTR1 1166A/C,ACE I/D)；
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, △ Ct.≤- 3, gene type are as follows: mutated homozygous or
ACE deletion form (CYP2D6*10/*10, CYP2C9*3/*3, ADRB1 1165C/C, AGTR1 1166C/C, ACE D/D).
Advantageous effects of the invention:
1, the kit and method of quick detection hypertension individuation medication gene pleiomorphism provided by the invention, for sample
This need to carry out the time-consumings such as gDNA extraction purification, effort, consuming operation, the mating quick reagent treatment of sample greatly drops
Low operating procedure and operating time, specifically processing method are as follows: take 2 μ LEDTA anticoagulated whole blood samples, be added to 20 μ L samples
Reagent treatment is incubated for 5min, that is, completion processing under the conditions of 37 DEG C.
2, the kit and method of quick detection hypertension individuation medication gene pleiomorphism provided by the invention, for sample
The change of present treatment mode and the requirement of multi-PRC reaction system, using containing modified Hotstart-Taq polymerase and excellent
The PCR premixed liquid TaqPath ProAmp Master Mix of the buffer system of change, enables anti-inhibition of the detection architecture for sample
Power greatly promotes, and gene detection reagent is made to have high-efficiency multiple PCR respond, and premixed liquid ingredient containing stable reagent is conducive to
Reagent under admixture saves steadily in the long term, enhances the stability of reagent.
3, the kit and method of quick detection hypertension individuation medication gene pleiomorphism provided by the invention, for every
Secondary detection, each detection architecture can only carry out substance PCR reaction, and each reaction system can only detect a gene loci polymorphism
Deficiency, gene detection reagent by multipair primer and a plurality of specificity fluorescent probe modified through different fluorescent dye groups with
The preparation of PCR premixed liquid blendes together a reaction system in advance, utilizes TaqPath ProAmp Master Mix premixed liquid multiple reaction energy
Power expands different target genes high-efficiency multiple PCR under the combination of multipair specific primer, and utilizes the different fluorescent dye bases of label
MGB-NFQ the and TaqMan-BHQ fluorescence probe of group is measured in real time analysis to target product.
4, the kit and method of quick detection hypertension individuation medication gene pleiomorphism provided by the invention, dependency basis
Because detection primer and specific probe are configured to a detection reagent, premix single tube packaging facilitates detection, realizes a reaction
The polygenic variation of system is analyzed, and improves detection efficiency while lowering production and testing cost.
Detailed description of the invention
Fig. 1 is that testing result of the invention is CYP2D6*1/*1, ADRB11165GG；
Fig. 2 is that testing result of the invention is CYP2D6*1/*10, ADRB11165GG；
Fig. 3 is that testing result of the invention is CYP2D6*10/*10, ADRB11165GG；
Fig. 4 is that testing result of the invention is CYP2D6*1/*1, ADRB11165GC；
Fig. 5 is that testing result of the invention is CYP2D6*1/*1, ADRB11165CC；
Fig. 6 is that testing result of the invention is CYP2D6*1/*10, ADRB11165GC；
Fig. 7 is that testing result of the invention is CYP2D6*1/*10, ADRB11165CC；
Fig. 8 is that testing result of the invention is CYP2D6*10/*10, ADRB11165GC；
Fig. 9 is that testing result of the invention is CYP2D6*10/*10, ADRB11165CC；
Figure 10 is that testing result of the invention is CYP2C9*1/*1, AGTR11166AA；
Figure 11 is that testing result of the invention is CYP2C9*1/*3, AGTR11166AA；
Figure 12 is that testing result of the invention is CYP2C9*3/*3, AGTR11166AA；
Figure 13 is that testing result of the invention is CYP2C9*1/*1, AGTR11166AC；
Figure 14 is that testing result of the invention is CYP2C9*1/*1, AGTR11166CC；
Figure 15 is that testing result of the invention is CYP2C9*1/*3, AGTR11166AC；
Figure 16 is that testing result of the invention is CYP2C9*1/*3, AGTR11166CC；
Figure 17 is that testing result of the invention is CYP2C9*3/*3, AGTR11166AC；
Figure 18 is that testing result of the invention is CYP2C9*3/*3, AGTR11166CC；
Figure 19 is that testing result of the invention is ACE I/I；
Figure 20 is that testing result of the invention is ACE I/D；
Figure 21 is that testing result of the invention is ACE D/D.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, below with reference to examples and drawings
The present invention is described in further detail, and embodiments of the present invention are not limited thereto.
In the present embodiment, be according to graphic result interpretation genotype, Fig. 1 be the present embodiment CYP2D6*1/*1,
ADRB11165GG result；Fig. 2 is CYP2D6*1/*10, ADRB11165GG result of the present embodiment；Fig. 3 is the present embodiment
CYP2D6*10/*10, ADRB11165GG result；Fig. 4 is CYP2D6*1/*1, ADRB11165GC result of the present embodiment；Fig. 5
For CYP2D6*1/*1, ADRB11165CC result of the present embodiment.
In the present embodiment, be according to graphic result interpretation genotype, Fig. 6 be the present embodiment CYP2D6*1/*10,
ADRB11165GC result；Fig. 7 is CYP2D6*1/*10, ADRB11165CC result of the present embodiment；Fig. 8 is the present embodiment
CYP2D6*10/*10, ADRB11165GC result；Fig. 9 is CYP2D6*10/*10, ADRB11165CC result of the present embodiment.
In the present embodiment, be according to graphic result interpretation genotype, Figure 10 be the present embodiment CYP2C9*1/*1,
AGTR11166AA result；Figure 11 is CYP2C9*1/*3, AGTR11166AA result of the present embodiment；Figure 12 is the present embodiment
CYP2C9*3/*3, AGTR11166AA result；Figure 13 is CYP2C9*1/*1, AGTR11166AC result of the present embodiment；Figure 14
For CYP2C9*1/*1, AGTR11166CC result of the present embodiment.
In the present embodiment, be according to graphic result interpretation genotype, Figure 15 be the present embodiment CYP2C9*1/*3,
AGTR11166AC result；Figure 16 is CYP2C9*1/*3, AGTR11166CC result of the present embodiment；Figure 17 is the present embodiment
CYP2C9*3/*3, AGTR11166AC result；Figure 18 is CYP2C9*3/*3, AGTR11166CC result of the present embodiment；Figure 19
For the ACEI/I result of the present embodiment；Figure 20 is the ACEI/D result of the present embodiment；Figure 21 is the ACED/D result of the present embodiment.
The kit of quick detection hypertension individuation medication gene pleiomorphism provided in this embodiment, with clinical anti-high blood
Pressing β1receptorblocker, AngiotensinⅡreceptors type1, angiotensin converting enzyme inhibitors drug metabolism, gene
Polymorphism includes receptor sensitivity correlation CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C, ACE I/
D gene pleiomorphism, kit include the sample processing reagent quickly handled for people's whole blood sample, are premixed single part and dispense
Gene detection reagent PCR reaction solution 1, PCR reaction solution 2, PCR reaction solution 3, positive reference substance and negative controls.
In the present embodiment, PCR reaction solution 1 includes:
Detect CYP2D6*10 primer pair: SEQ ID NO.1 and SEQ ID NO.2；
Detect CYP2D6*10 specific probe: SEQ ID NO.3 and SEQ ID NO.4；
Detect ADRB1 1165G > C primer pair: SEQ ID NO.5 and SEQ ID NO.6；
Detect ADRB1 1165G > C specific probe: SEQ ID NO.7 and SEQ ID NO.8.
In the present embodiment, PCR reaction solution 2 includes:
Detect CYP2C9*3 primer pair: SEQ ID NO.9 and SEQ ID NO.10；
Detect CYP2C9*3 specific probe: SEQ ID NO.11 and SEQ ID NO.12；
Detect AGTR1 1166A > C primer pair: SEQ ID NO.13 and SEQ ID NO.14；
Detect AGTR1 1166A > C specific probe: SEQ ID NO.15 and SEQ ID NO.16.
In the present embodiment, PCR reaction solution 3 includes:
Detect ACE I/D primer pair: SEQ ID NO.17 and SEQ ID NO.18；
Detect ACE I/D specific probe: SEQ ID NO.19 and SEQ ID NO.20；
TaqPath ProAmp Master Mix premixed liquid with anti-PCR enzyme inhibitor；Premixed liquid includes after modifying
Hotstart-Taq polymerase, UNG enzyme, ROX fluorescent calibration dyestuff, dNTP, MgCl2, PCR buffer etc..
In the present embodiment, SEQ ID NO.1 is CYP2D6*10 upstream primer:
5'-AGTGAGGCAGGTATGGGGC-3'；
SEQ ID NO.2 is CYP2D6*10 downstream primer:
5'-GTCCACATGCAGCAGGTTG-3'；
SEQ ID NO.3 is CYP2D6*10 wild type fluorescence probe:
5 '-fluorophor-GCTACCCACCAGGC-MGB-NFQ-3 '；
SEQ ID NO.4 is CYP2D6*10 saltant type fluorescence probe:
5 '-fluorophor-ACGCTACTCACCAT-MGB-NFQ-3 '；
SEQ ID NO.5 is ADRB1 1165G > C upstream primer:
5'-CGCAGCCCCGACTTC-3'；
SEQ ID NO.6 is ADRB1 1165G > C downstream primer:
5'-CCGGTCTCCGTGGGT-3'；
SEQ ID NO.7 is ADRB1 1165G > C wild type fluorescence probe:
5 '-fluorophor-ATTCCAGGGACTGC-MGB-NFQ-3 '；
SEQ ID NO.8 is ADRB1 1165G > C saltant type fluorescence probe:
5 '-fluorophor-TCCAGCGACTGCT-MGB-NFQ-3 '；
SEQ ID NO.9 is CYP2C9*3 upstream primer:
5'-AGGAAGAGATTGAACGTGTGAT-3'；
SEQ ID NO.10 is CYP2C9*3 downstream primer:
5'-ATGAATTTGGGGACTTCGAA-3'；
SEQ ID NO.11 is CYP2C9*3 wild type fluorescence probe:
5 '-fluorophor-GAGATACATTGACCTTCA-MGB-NFQ-3 '；
SEQ ID NO.12 is CYP2C9*3 saltant type fluorescence probe:
5 '-fluorophor-AGATACCTTGACCTTCA-MGB-NFQ-3 '；
SEQ ID NO.13 is AGTR1 1166A > C upstream primer:
5'-GAGAACATTCCTCTGCAGCA-3'；
SEQ ID NO.14 is AGTR1 1166A > C downstream primer:
5'-CGGTTCAGTCCACATAATGC-3'；
SEQ ID NO.15 is AGTR1 1166A > C wild type fluorescence probe:
5 '-fluorophor-CAAATGAGCATTAGCT-MGB-NFQ-3 '
SEQ ID NO.16 is AGTR1 1166A > C saltant type fluorescence probe:
5 '-fluorophor-ACATGAGCCTTAGCT-MGB-NFQ-3 '；
SEQ ID NO.17 is ACE I/D upstream primer:
5'-TGGAGAGCCACTCCCATCCTTTC-3'；
SEQ ID NO.18 is ACE I/D downstream primer:
5'-CGTGGCCATCACATTCGTCAG-3'；
SEQ ID NO.19 is ACE insert type fluorescence probe:
5 '-fluorophor-GGACCACAGGCGCCGCCACTAC-MGB-NFQ-3 '；
SEQ ID NO.20 is ACE deletion form fluorescence probe:
5 '-fluorophor-AGACCTGCTGCCTATA-MGB-NFQ-3 '.
In the present embodiment, gene detection reagent further include: the internal control primer pair for inner quality control: SEQ ID
NO.21-SEQ ID NO.22 and internal reference probe SEQ ID NO.23, probe use TaqMan probe；
SEQ ID NO.21 is internal reference upstream primer:
5'-CCTTCCTGCCACCGCTGAT-3'；
SEQ ID NO.22 is internal reference downstream primer:
5'-TGCCTTCGCAACCATTCCCTTA-3'；
SEQ ID NO.23 is internal reference probe:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ-3 '.
In the present embodiment, gene detection reagent further include:
TaqPath ProAmp Master Mix premixed liquid with anti-PCR enzyme inhibitor；Premixed liquid includes after modifying
Hotstart-Taq polymerase, UNG enzyme, ROX fluorescent calibration dyestuff, dNTP, MgCl2, PCR buffer etc..
In the present embodiment, positive reference substance is mixing CYP2D6100C, 100T, CYP2C91075A, 1075C, ADRB1
1165G, 1165C, AGTR11166A, 1166C, ACE Insertion (I), ACE Deletion (D) gene plasmid DNA and interior
Join Plasmid DNA；Negative controls are the ultrapure water without target gene and reference gene.
The present embodiment additionally provides the method for quickly detection hypertension individuation medication gene pleiomorphism, including walks as follows
It is rapid:
Step 1: taking EDTA anticoagulated whole blood sample, take 2 μ L, 20 μ L sample processing reagents are added, are incubated under the conditions of 37 DEG C
5min。
Step 2: by 2 μ L of step 1 gained sample, being added separately to the gene detection reagent PCR reaction of 18 μ L premix packing
Liquid 1, PCR reaction solution 2 in PCR reaction solution 3, carry out PCR amplification after mixing；
Step 3: setting reaction process and response parameter, response parameter are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations open fluorescence channel in the 3rd stage and collect fluorescent value；
Step 4: sample is determined according to the difference △ Ct. value of same gene site wild type and saltant type fluorescence channel Ct. value
Genotyping, if detection positive findings have typical S type amplification curve to rise, and Ct. value is less than decision content, and NoCT indicates no expansion
Increase, Ct. value is based on 40.
Genotyping interpretation result is as follows:
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, Ct. >=3 △, gene type are as follows: Wild homozygous or
ACE insert type (CYP2D6*1/*1, CYP2C9*1/*1, ADRB1 1165G/G, AGTR1 1166A/A, ACE I/I)；
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, -3 < △ Ct. < 3, gene type are as follows: heterozygous
(CYP2D6*1/*10,CYP2C9*1/*3,ADRB1 1165G/C,AGTR1 1166A/C,ACE I/D)；
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, △ Ct.≤- 3, gene type are as follows: mutated homozygous or
ACE deletion form (CYP2D6*10/*10, CYP2C9*3/*3, ADRB1 1165C/C, AGTR1 1166C/C, ACE D/D).
In the present embodiment, the kit of quick detection hypertension individuation medication gene pleiomorphism provided in this embodiment
And method, for sample need to carry out the time-consumings such as gDNA extraction purification, effort, consuming operation, mating sample quickly handles
Reagent significantly reduces operating procedure and operating time, specifically processing method are as follows: takes 2 μ LEDTA anticoagulated whole blood samples, adds
Enter to 20 μ L sample processing reagents, 5min, that is, completion processing is incubated under the conditions of 37 DEG C.
In the present embodiment, the kit of quick detection hypertension individuation medication gene pleiomorphism provided in this embodiment
And method, the requirement of change and multi-PRC reaction system for sample process mode, using containing modified Hotstart-
The PCR premixed liquid TaqPath ProAmp Master Mix of Taq polymerase and the buffer system of optimization, make detection architecture for
The anti-rejection ability of sample greatly promotes, and so that gene detection reagent is had high-efficiency multiple PCR respond, premixed liquid is containing stable examination
Agent ingredient, the reagent be conducive under admixture save steadily in the long term, enhance the stability of reagent.
In the present embodiment, the kit of quick detection hypertension individuation medication gene pleiomorphism provided in this embodiment
And method, for each detection, each detection architecture can only carry out substance PCR reaction, and each reaction system can only detect one
The deficiency of gene loci polymorphism, gene detection reagent by multipair primer and it is a plurality of modify through different fluorescent dye groups it is special
Property fluorescence probe and PCR premixed liquid preparation blend together a reaction system in advance, utilize TaqPath ProAmp Master Mix premix
Liquid multiple reaction ability expands different target genes high-efficiency multiple PCR under the combination of multipair specific primer, and not using label
MGB-NFQ and TaqMan-BHQ fluorescence probe with fluorescent dye groups is measured in real time analysis to target product.
In the present embodiment, the kit of quick detection hypertension individuation medication gene pleiomorphism provided in this embodiment
And method, related gene detection primer and specific probe are configured to a detection reagent, premix single tube packaging facilitates detection,
The polygenic variation analysis for realizing a reaction system, improves detection efficiency while lowering production and testing cost.
In the present embodiment, the kit of quick detection hypertension individuation medication gene pleiomorphism provided in this embodiment
And method, inhibit with hypertension drug β1receptorblocker, AngiotensinⅡreceptors type1, angiotensin converting enzyme
Agent drug metabolism, the kit of receptor sensitivity correlation CYP2D6, CYP2C9, ADRB1, AGTR1, ACE gene pleiomorphism and side
Method, detection gene pleiomorphism includes CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C, ACE I/D
Type, kit forms of the present invention include the gene detection reagent PCR reaction solution of the reagent quickly handled for sample, premix completion
1, gene detection reagent PCR reaction solution 2, gene detection reagent PCR reaction solution 3.
In the present embodiment, the positive reference substance of hybrid detection target gene Plasmid DNA, the negative control without target gene
Product, gene detection reagent have the function of stronger anti-PCR mortifier ability and multiplex PCR, when detection without gDNA extraction purification,
The gene detection reagent of premix, different tested target bases need to will can be only separately added into after the processing of whole blood sample cell rapid cleavage
Gene magnification is carried out because different primer pairs is respectively adopted, and amplification is produced using the probe that different fluorescent dye groups are modified
Object is measured in real time analysis, realizes detection of the reaction system simultaneously to polygenic variation.
In the present embodiment, guaranteeing on the specific and sensitive basis of detection, realization is convenient, fast and efficiently examines
It surveys, reduces production cost and testing cost while reducing detection operating procedure, reduce the detection reaction time, be conducive to clinic
Test and analyze application.
In conclusion in the present embodiment, quick detection hypertension individuation medication gene polymorphic provided in this embodiment
Property kit and method, for promoted detection specificity and sensitivity, using according to target-gene sequence design specificity expand
Increase primer pair and MGB-NFQ fluorescence probe, carries out specific amplified and real-time detection analysis, it can specificity detection
CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C, ACE I/D gene mutation.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Claims (10)
1. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism, hinders with clinical antihypertensive β1receptor
Stagnant dose, AngiotensinⅡreceptors type1, angiotensin converting enzyme inhibitors drug metabolism, which is characterized in that gene is more
State property includes receptor sensitivity correlation CYP2D6*10, CYP2C9*3, ADRB1 1165G > C, AGTR1 1166A > C, ACE I/D
Gene pleiomorphism, kit include sample processing reagent, gene detection reagent, positive reference substance and negative controls.
2. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as described in claim 1, feature
It is, gene detection reagent is using the single part premix packing of PCR8 union, comprising: PCR reaction solution 1, PCR reaction solution 2 and PCR are anti-
Answer liquid 3.
3. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as claimed in claim 2, feature
It is, PCR reaction solution 1 includes:
Detect CYP2D6*10 primer pair: SEQ ID NO.1 and SEQ ID NO.2；
SEQ ID NO.1 is CYP2D6*10 upstream primer:
5'-AGTGAGGCAGGTATGGGGC-3'；
SEQ ID NO.2 is CYP2D6*10 downstream primer:
5'-GTCCACATGCAGCAGGTTG-3'；
Detect CYP2D6*10 specific probe: SEQ ID NO.3 and SEQ ID NO.4；
SEQ ID NO.3 is CYP2D6*10 wild type fluorescence probe:
5 '-fluorophor-GCTACCCACCAGGC-MGB-NFQ-3 '；
SEQ ID NO.4 is CYP2D6*10 saltant type fluorescence probe:
5 '-fluorophor-ACGCTACTCACCAT-MGB-NFQ-3 '；
Detect ADRB1 1165G > C primer pair: SEQ ID NO.5 and SEQ ID NO.6；
SEQ ID NO.5 is ADRB1 1165G > C upstream primer:
5'-CGCAGCCCCGACTTC-3'；
SEQ ID NO.6 is ADRB1 1165G > C downstream primer:
5'-CCGGTCTCCGTGGGT-3'；
Detect ADRB1 1165G > C specific probe: SEQ ID NO.7 and SEQ ID NO.8；
SEQ ID NO.7 is ADRB1 1165G > C wild type fluorescence probe:
5 '-fluorophor-ATTCCAGGGACTGC-MGB-NFQ-3 '；
SEQ ID NO.8 is ADRB1 1165G > C saltant type fluorescence probe:
5 '-fluorophor-TCCAGCGACTGCT-MGB-NFQ-3 '.
4. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as claimed in claim 2, feature
It is, PCR reaction solution 2 includes:
Detect CYP2C9*3 primer pair: SEQ ID NO.9 and SEQ ID NO.10；
SEQ ID NO.9 is CYP2C9*3 upstream primer:
5'-AGGAAGAGATTGAACGTGTGAT-3'；
SEQ ID NO.10 is CYP2C9*3 downstream primer:
5'-ATGAATTTGGGGACTTCGAA-3'；
Detect CYP2C9*3 specific probe: SEQ ID NO.11 and SEQ ID NO.12；
SEQ ID NO.11 is CYP2C9*3 wild type fluorescence probe:
5 '-fluorophor-GAGATACATTGACCTTCA-MGB-NFQ-3 '；
SEQ ID NO.12 is CYP2C9*3 saltant type fluorescence probe:
5 '-fluorophor-AGATACCTTGACCTTCA-MGB-NFQ-3 '；
Detect AGTR1 1166A > C primer pair: SEQ ID NO.13 and SEQ ID NO.14；
SEQ ID NO.13 is AGTR1 1166A > C upstream primer:
5'-GAGAACATTCCTCTGCAGCA-3'；
SEQ ID NO.14 is AGTR1 1166A > C downstream primer:
5'-CGGTTCAGTCCACATAATGC-3'；
Detect AGTR1 1166A > C specific probe: SEQ ID NO.15 and SEQ ID NO.16；
SEQ ID NO.15 is AGTR1 1166A > C wild type fluorescence probe:
5 '-fluorophor-CAAATGAGCATTAGCT-MGB-NFQ-3 '
SEQ ID NO.16 is AGTR1 1166A > C saltant type fluorescence probe:
5 '-fluorophor-ACATGAGCCTTAGCT-MGB-NFQ-3 '.
5. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as claimed in claim 2, feature
It is, PCR reaction solution 3 includes:
Detect ACE I/D primer pair: SEQ ID NO.17 and SEQ ID NO.18；
SEQ ID NO.17 is ACE I/D upstream primer:
5'-TGGAGAGCCACTCCCATCCTTTC-3'；
SEQ ID NO.18 is ACE I/D downstream primer:
5'-CGTGGCCATCACATTCGTCAG-3'；
Detect ACE I/D specific probe: SEQ ID NO.19 and SEQ ID NO.20；
SEQ ID NO.19 is ACE insert type fluorescence probe:
5 '-fluorophor-GGACCACAGGCGCCGCCACTAC-MGB-NFQ-3 '；
SEQ ID NO.20 is ACE deletion form fluorescence probe:
5 '-fluorophor-AGACCTGCTGCCTATA-MGB-NFQ-3 '；
Probe nucleic acid sequence 5 ' uses MGB- using one of FAM, JOE, VIC, NED and ROX modification, probe nucleic acid sequence 3 '
NFQ base group modification.
6. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as described in claim 1, feature
Be, gene detection reagent further include: the internal control primer pair for inner quality control: SEQ ID NO.21-SEQ ID NO.22 and
Internal reference probe SEQ ID NO.23, probe use TaqMan probe；
SEQ ID NO.21 is internal reference upstream primer:
5'-CCTTCCTGCCACCGCTGAT-3'；
SEQ ID NO.22 is internal reference downstream primer:
5'-TGCCTTCGCAACCATTCCCTTA-3'；
SEQ ID NO.23 is internal reference probe:
5 '-fluorophor-CGCTATGCTCCGCGCGCATCGTCTGCTC-BHQ-3 '.
7. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as described in claim 1, feature
It is, gene detection reagent further include:
TaqPath ProAmp Master Mix premixed liquid with anti-PCR enzyme inhibitor；Premixed liquid includes after modifying
Hotstart-Taq polymerase, UNG enzyme, ROX fluorescent calibration dyestuff, dNTP, MgCl2、PCR buffer。
8. a kind of kit of quickly detection hypertension individuation medication gene pleiomorphism as described in claim 1, feature
Be, positive reference substance be mixing CYP2D6100C, 100T, CYP2C91075A, 1075C, ADRB1 1165G, 1165C,
AGTR11166A, 1166C, ACE Insertion (I), ACE Deletion (D) gene plasmid DNA and internal reference Plasmid DNA；Yin
Property reference substance be the ultrapure water without target gene and reference gene.
9. a kind of method of quickly detection hypertension individuation medication gene pleiomorphism, which comprises the steps of:
Step 1: taking EDTA anticoagulated whole blood sample, take 2 μ L, 20 μ L sample processing reagents are added, are incubated for 5min under the conditions of 37 DEG C.
Step 2: by 2 μ L of step 1 gained sample, be added separately to 18 μ L premix packing gene detection reagent PCR reaction solution 1,
In PCR reaction solution 2, PCR reaction solution 3, PCR amplification is carried out after mixing；
Step 3: setting reaction process and response parameter, response parameter are as follows:
1st stage: 95 DEG C, 10min, 1 circulation；
2nd stage: 95 DEG C, 10S, 40 circulations；
3rd stage: 60 DEG C, 30S, 40 circulations open fluorescence channel in the 3rd stage and collect fluorescent value；
Step 4: sample gene is determined according to the difference △ Ct. value of same gene site wild type and saltant type fluorescence channel Ct. value
Parting, if detection positive findings have typical S type amplification curve to rise, and Ct. value is less than decision content, and NoCT indicates no amplification, Ct.
Value is based on 40.
10. a kind of method of quickly detection hypertension individuation medication gene pleiomorphism as claimed in claim 9, feature exist
In in step 4, Genotyping interpretation result is as follows:
When internal reference Ct value≤35, △ Ct is that Ct mutation-Ct is wild, Ct. >=3 △, gene type are as follows: Wild homozygous or ACE are inserted
Enter type (CYP2D6*1/*1, CYP2C9*1/*1, ADRB1 1165G/G, AGTR1 1166A/A, ACE I/I)；
When internal reference Ct value≤35, △ Ct be Ct mutation-Ct it is wild, -3 < △ Ct. < 3, gene type are as follows: heterozygous (CYP2D6*
1/*10,CYP2C9*1/*3,ADRB1 1165G/C,AGTR1 1166A/C,ACE I/D)；
When internal reference Ct value≤35, △ Ct is that Ct mutation-Ct is wild, △ Ct.≤- 3, gene type are as follows: mutated homozygous or ACE are lacked
Mistake type (CYP2D6*10/*10, CYP2C9*3/*3, ADRB1 1165C/C, AGTR1 1166C/C, ACE D/D).
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| CN111172276A (en) * | 2020-03-09 | 2020-05-19 | 东莞博奥木华基因科技有限公司 | Primer for detecting ACE gene polymorphism and application thereof |
| CN112080562A (en) * | 2020-08-18 | 2020-12-15 | 珠海赛乐奇生物技术股份有限公司 | Gene chip and kit for detecting hypertension drug treatment related gene |
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