CN110295225A - Primer combination and kit and method for instructing Lovastatin drug personalized medicine related gene to detect - Google Patents
Primer combination and kit and method for instructing Lovastatin drug personalized medicine related gene to detect Download PDFInfo
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Abstract
The primer combination that the invention discloses a kind of for instructing Lovastatin drug personalized medicine related gene to detect.The primer combination includes being directed to APOA5 (rs662799), CETP (rs708272), the specific primer and Taqman probe of SLCO1B1 (rs2291073) gene loci.The invention also discloses the kit combined comprising the primer and its analysis methods.The feature that operation of the present invention is simple, economic, detection is quick, accuracy is high, specificity is good and result interpretation is simple, it realizes to the quick and Accurate Determining for Lovastatin drug personalized medicine related gene polymorphism, to reduce the adverse reaction of the common medication of clinical Lovastatin drug, medical treatment cost is reduced, social resources are saved.
Description
Technical field
The present invention relates to vitro diagnostic techniques fields, and in particular to one kind is for instructing Lovastatin drug personalized medicine
The primer combination of related gene detection and kit and method.
Background technique
Hypercholesterolemia refers to that the cholesterol level in blood is exceeded and is greater than normal value (5.7mmol/L).Long-term gallbladder
Sterol is excessively high, will allow the artery of body is athero- to harden, and the disease of life can be jeopardized so as to cause coronary heart disease, cerebral infarction etc.
Disease.
Hyperlipidemia is primarily referred to as cholesterolemia and/or triglycerides increases.Hyperlipidemia is body fat metabolic disorder
Performance, be broadly divided into three classes: hypercholesterolemia, hypertriglyceridemia and combined hyperlipidemia.Hyperlipidemia is to face
Common metabolic disease on bed is because of fat metabolism or to turn the abnormal disease for causing the one or more lipids of blood plasma to be higher than normal index
Disease.Hyperlipemia is a kind of chronic disease that blood lipid level is higher, and pathogenic factor is complex, age, gender, heredity, ring
Border and undesirable living habit will lead to the generation of the disease.Clinical research shows that hyperlipidemia is luring for a variety of cardiovascular diseases
Hair factor, such as atherosclerosis, coronary heart disease.In recent years, with the continuous improvement of people 's material life, the disease incidence of the disease
Also increase year by year, at present the disease be generally acknowledged myocardial infarction, cerebral apoplexy etc. disable, lethal atherosclerotic lesion it is only
One of vertical risk factor, and it is related with diabetes, cardiovascular disease generation.
Lovastatin is most commonly used to treatment hypercholesterolemia, and especially with LDL increased perosn (II type), mixed type is high in fat
Mass formed by blood stasis also can be used, it can also be used to which nephrosis or diabetes are with hypercholesterolemia.
" drug metabolic enzyme and drug target technique of gene detection guide (tentative) " is pointed out: to drug metabolic enzyme and medicine
Object target gene is detected, and can be instructed clinical for the suitable drug of specific patient selection and dosage realization " individual
Change " medication, to improve the validity and safety of drug therapy.It is recommended that clinically selecting statin according to SLCO1B1 genotype
Class drug is treated.
Patient largely uses statins that the adverse reactions such as hepatic injury, musclar toxicity, renal dysfunction easily occur, hair
Raw rate is presented geometric multiple and increases.There are apparent individuation difference, the following poison is secondary to be made treatment effect
With the health for also seriously endangering sufferer.
APOA5 is the important determinant of blood plasma lipide concentration, is made a variation between the individual of serum triglyceride related, different
The variation of Genotyping influences internal low density lipoprotein cholesterol content, and Lovastatin is to APOA5 (rs662799) site AA
Type patient outcomes are good compared with AG and GG type patient.
The protein of CETP gene coding is present in blood plasma, participates in cholesteryl ester and turns from high-density lipoprotein (HDL)
Other lipoprotein are moved on to, the reason of defect of the gene is hyperlipoprotememia 1 (HALP1).Lovastatin is to CETP
(rs708272) site GG type patient outcomes are poor.
By the metabolism of detection Lovastatin, transhipment or action target spot gene, it can find that early Different Individual cuts down him to Lip river
The medicaments insensitive degree in spit of fland enhances the compliance of its dosage to realize the personalized medicine of Lovastatin, to reduce use
Risk caused by pharmaceutical quantities are improper improves therapeutic effect.
There are no the gene detecting kit for being directed to Lovastatin Resistance detection, clinically genetic tests currently on the market
Class kit takes Sanger PCR sequencing PCR, high-resolution solubility curve method, chip hybridization methods detection gene pleiomorphism more.But it is above-mentioned
Method has the following problems that wherein Sanger sequencing includes the operation such as PCR amplification, PCR product purifying, DNA sequencing, and step is numerous
Trivial, time-consuming for experiment, and professional technique requires height, and sequencing cost is high, is not suitable for clinical expansion；High-resolution solubility curve method pair
Equipment requirement is special, and detection sensitivity is not high, is also not suitable for clinical expansion；Chip hybridization methods detection sensitivity is low and specific
Difference is easy to appear false positive results.The detection method of quantitative fluorescent PCR compared with above method, possess at low cost, high sensitivity,
The advantages that high specificity, as a result reproducible, the 12-13 hours flux that could be completed are sequenced in achievable Sanger in 2 hours,
The clinical detection time is substantially reduced, is a kind of extraordinary genetic polymorphism detection means, Genotyping Genotyping dissipates
Point diagram is a kind of detection method of more simple and quick intuitive interpretation genotype based on quantitative fluorescent PCR, but currently without base
In this method for instructing that Lovastatin drug personalized medicine related gene detects such as the primer combination in claim
And reagent kit product.
Summary of the invention
In order to clinical application easily occur not when solving the diagnosis of traditional clinical experience medical mode and Lovastatin drug medication
The technical issues of good reaction and no suitable reagent kit product, the purpose of the present invention is to provide a kind of easy to operate, detections
Quickly, the primer combination for instructing Lovastatin drug personalized medicine related gene to detect that accuracy is high and specificity is good
And kit and its method.
One of in order to achieve the object of the present invention, used technical solution is:
A kind of primer combination for instructing Lovastatin drug personalized medicine related gene to detect, including be directed to
APOA5 (rs662799), CETP (rs708272), the specific primer and probe of SLCO1B1 (rs2291073) gene loci,
It is as follows:
APOA5 (rs662799) forward primer:
5'-GGGTGAAGATGAGATGGCAAG-3'(SEQ ID NO.1)；
APOA5 (rs662799) reverse primer:
5'-CTGCGAGTGGAGTTCAGCTTT-3'(SEQ ID NO.2)；
APOA5 (rs662799) wild-type probe:
5'FAM-CTGGAGCGAAAGTGAGAT-MGB 3'(SEQ ID NO.3)；
APOA5 (rs662799) saltant type probe:
5'VIC-CTGGAGCGAAAGTAAGAT-MGB 3'(SEQ ID NO.4)；
CETP (rs708272) forward primer:
5'-CCAACCTCCTAATCTTTACCCC-3'(SEQ ID NO.5)；
CETP (rs708272) reverse primer:
5'-TGAGAAGGTCCTAGCTGCATTG-3'(SEQ ID NO.6)；
CETP (rs708272) wild-type probe:
5'FAM-CTGAACCCTAACTCGAAC-MGB 3'(SEQ ID NO.7)；
CETP (rs708272) saltant type probe:
5'VIC-CTGAACCCTAACTTGAAC-MGB 3'(SEQ ID NO.8)；
SLCO1B1 (rs2291073) forward primer:
5'-TTGTTGGTTTTATTGACGGAAGC-3'(SEQ ID NO.9)；
SLCO1B1 (rs2291073) reverse primer:
5'-TTATTGCCAAATTGCCTGTGAG-3'(SEQ ID NO.10)；
SLCO1B1 (rs2291073) wild-type probe:
5'FAM-CAACTGGGGTAAATTTATCTCTCAC-TAMRA 3'(SEQ ID NO.11)；
SLCO1B1 (rs2291073) saltant type probe:
5'VIC-CAACTGGGGTAAATGTATCTCTCAC-TAMRA 3'(SEQ ID NO.12)。
In order to achieve the object of the present invention two, used technical solution is:
A kind of kit for instructing Lovastatin drug personalized medicine related gene to detect, including containing such as right
It is required that the PCR reaction solution of specific primer described in 1 and probe, the PCR reaction solution is respectively APOA5 (rs662799),
CETP (rs708272), SLCO1B1 (rs2291073) PCR reaction solution, respectively include:
APOA5 (rs662799) forward primer, APOA5 (rs662799) reverse primer, APOA5 (rs662799) wild type
Probe and APOA5 (rs662799) saltant type probe；
CETP (rs708272) forward primer, CETP (rs708272) reverse primer, CETP (rs708272) wild type are visited
Needle and CETP (rs708272) saltant type probe；
SLCO1B1 (rs2291073) forward primer, SLCO1B1 (rs2291073) reverse primer, SLCO1B1
(rs2291073) wild-type probe and SLCO1B1 (rs2291073) saltant type probe；
Above-mentioned PCR reaction solution further includes NuHi SNP Mix (2 ×) PCR premixed liquid and nuclease-free water.
In a preferred embodiment of the invention, the ingredient of the PCR reaction solution is final concentration of: 1 × NuHi SNP
Mix, 0.5 μM of each specific primer and 0.2 μM of probe.
It in a preferred embodiment of the invention, further include positive reference substance one, positive reference substance two and positive control
Product three；
The positive reference substance one is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1
(rs2291073) gene loci wild plasmid；
The positive reference substance two is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1
(rs2291073) plasmid of gene loci wild type and saltant type by 1:1 quantity than heterozygosis；
The positive reference substance three is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1
(rs2291073) gene point mutation type plasmid.
It in a preferred embodiment of the invention, further include blank control product, the blank control product are nuclease free
Water.
In order to achieve the object of the present invention three, used technical solution is:
A method of for instructing Lovastatin drug personalized medicine related gene to detect, include the following steps:
Step 1: the DNA of sample extracting to be detected is taken, as pcr template；
Step 2: providing kit as claimed in claim 5, takes out appropriate PCR reaction solution, by the PCR reaction solution
It is mixed with the appropriate pcr template；
Step 3: pcr amplification reaction: 95 DEG C of denaturation 5min is carried out；95 DEG C of 10sec, 60 DEG C of 40sec (acquisition fluorescence),
45cycles；
Step 4: PCR result judgement: the case where the positive reference substance and blank control product of the kit meet regulation
Under, result judgement is carried out according to fluorescent amplification curve and Genotyping Genotyping scatter plot, is obtained for Lovastatin medicine
The genotype of object personalized medicine related gene.
Provided by the present invention for the primer combination and examination for instructing Lovastatin drug personalized medicine related gene to detect
The beneficial effect of agent box and method is:
The feature that operation of the present invention is simple, economic, detection is quick, accuracy is high, specificity is good and result interpretation is simple, it is real
Now to the quick and Accurate Determining for Lovastatin drug personalized medicine related gene polymorphism, him is cut down to reduce clinical Lip river
The adverse reaction of the common medication of spit of fland drug reduces medical treatment cost, saves social resources.
Detailed description of the invention
Fig. 1 is the qPCR for the CC parting that clinical sample APOA5 (rs662799) protects primed probe using present patent application
Amplification curve diagram (FAM is upper)；
Fig. 2 is the qPCR for the CT parting that clinical sample APOA5 (rs662799) protects primed probe using present patent application
Amplification curve diagram (FAM is upper)；
Fig. 3 is the qPCR for the TT parting that clinical sample APOA5 (rs662799) protects primed probe using present patent application
Amplification curve diagram (FAM is under)；
Fig. 4 be clinical sample APOA5 (rs662799) using present patent application protect primed probe CC, tri- kinds of CT, TT
The Genotyping scatter plot of parting；
Fig. 5 is the qPCR expansion for the GG parting that clinical sample CETP (rs708272) protects primed probe using present patent application
Increase curve graph (FAM is upper)；
Fig. 6 is the qPCR expansion for the GA parting that clinical sample CETP (rs708272) protects primed probe using present patent application
Increase curve graph (FAM is under)；
Fig. 7 is the qPCR expansion for the AA parting that clinical sample CETP (rs708272) protects primed probe using present patent application
Increase curve graph (FAM is under)；
Fig. 8 is the GG, tri- kinds points of GA, AA that clinical sample CETP (rs708272) protects primed probe using present patent application
The Genotyping scatter plot of type；
Fig. 9 is the TT parting that clinical sample SLCO1B1 (rs2291073) protects primed probe using present patent application
QPCR amplification curve diagram (FAM is upper)；
Figure 10 is the TG parting that clinical sample SLCO1B1 (rs2291073) protects primed probe using present patent application
QPCR amplification curve diagram (FAM is under)；
Figure 11 is the GG parting that clinical sample SLCO1B1 (rs2291073) protects primed probe using present patent application
QPCR amplification curve diagram (FAM is under)；
Figure 12 is the TT, TG, GG that clinical sample SLCO1B1 (rs2291073) protects primed probe using present patent application
The Genotyping scatter plot of three kinds of partings.
Figure 13 is the qPCR amplification curve diagram of clinical sample APOA5 (rs662799) using the CC parting of comparison primed probe
(FAM is under)；
Figure 14 is the qPCR amplification curve diagram of clinical sample APOA5 (rs662799) using the CT parting of comparison primed probe
(FAM is under)；
Figure 15 is the qPCR amplification curve diagram of clinical sample APOA5 (rs662799) using the TT parting of comparison primed probe
(FAM is under)；
Figure 16 is the CC of clinical sample APOA5 (rs662799) using comparison primed probe, tri- kinds of partings of CT, TT
Genotyping scatter plot；
Figure 17 is clinical sample SLCO1B1 (rs2291073) bent using the qPCR amplification of the TT parting of comparison primed probe
Line chart (FAM is under)；
Figure 18 is clinical sample SLCO1B1 (rs2291073) bent using the qPCR amplification of the TG parting of comparison primed probe
Line chart (FAM is under)；
Figure 19 is clinical sample SLCO1B1 (rs2291073) bent using the qPCR amplification of the GG parting of comparison primed probe
Line chart (FAM is under)；
Figure 20 is the TT of clinical sample SLCO1B1 (rs2291073) using comparison primed probe, tri- kinds of partings of TG, GG
Genotyping scatter plot.
Specific embodiment
The principle of the invention lies in:
By design specificity high primer and Taqman fluorescent detection probe and pass through the release and intensity of detection fluorescence
To detect gene pleiomorphism.For design of primers in the upstream and downstream in probe-binding region domain, two probes respectively correspond two kinds of different genes
Type, during qPCR, after probe combination complementary with template strand, when probe is digested hydrolysis, FAM or the VIC report of 5 ' end connections
It accuses group and issues corresponding fluorescence signal, it can interpretation genotype by fluorescent amplification curve；With the positive of three kinds of corresponding genotype
Reference substance passes through the more intuitive quickly interpretation sample gene of Genotyping Genotyping scatter plot as polymorphism distribution standard
Type.Furthermore kit of the present invention is packed by the way of premixing, and when use only needs that genomic DNA is added, and is simplified
The response procedures of qPCR.
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is described in further detail.It should be appreciated that described herein, specific examples are only used to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1: the preparation of kit
One, the design and synthesis of primer and probe
For APOA5 in human genome (rs662799), CETP (rs708272), SLCO1B1 (rs2291073) gene position
Point (sequence is referring to mankind's whole genome sequence disclosed in ncbi database), uses Primer Premier 5.0 and Primer
3.0 software of Express separately designs specific primer and Taqman probe.
Specific primer and probe sequence, as shown in the table 1:
1 primer and probe sequence of table and length information
Two, reference substance selects
The positive reference substance one is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1
(rs2291073) gene loci wild plasmid；
The positive reference substance two is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1
(rs2291073) plasmid of gene loci wild type and saltant type by 1:1 quantity than heterozygosis；
The positive reference substance three is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1
(rs2291073) gene point mutation type plasmid.
Three, PCR reaction solution forms
Including the PCR reaction solution containing above-mentioned specific primer and probe, the PCR reaction solution is respectively APOA5
(rs662799), CETP (rs708272), SLCO1B1 (rs2291073) PCR reaction solution, respectively include:
APOA5 (rs662799) forward primer, APOA5 (rs662799) reverse primer, APOA5 (rs662799) wild type
Probe and APOA5 (rs662799) saltant type probe；
CETP (rs708272) forward primer, CETP (rs708272) reverse primer, CETP (rs708272) wild type are visited
Needle and CETP (rs708272) saltant type probe；
SLCO1B1 (rs2291073) forward primer, SLCO1B1 (rs2291073) reverse primer, SLCO1B1
(rs2291073) wild-type probe and SLCO1B1 (rs2291073) saltant type probe；
Above-mentioned PCR reaction solution further includes NuHi SNP Mix (2 ×) PCR premixed liquid and nuclease-free water.
Wherein, NuHi SNP Mix (2 ×) the PCR premixed liquid is purchased from the new marine growth Science and Technology Co., Ltd. in Suzhou；
The nuclease-free water is Ambion brand Nuclease-Free Water.
The PCR reaction solution ingredient it is final concentration of:
1×NuHi SNP Mix
0.5 μM of specific primer
0.2 μM of probe
System (is supplemented to 23ul) by nuclease free appropriate amount of water.
Embodiment 2: the side detected for Lovastatin drug personalized medicine related gene is carried out using mentioned reagent box
Method
One, biological sample
Biomaterial of the present invention is all from Pai Sennuo medical test institute.For during the 9-12 month in 2018 send it is gloomy
Promise medical test 500 detected remaining crowd's anticoagulations.
Two, the DNA for taking sample extracting to be detected, as pcr template:
DNA extraction kit be TIANGEN Biotech (Beijing) Co., Ltd. extracts kit " poba gene group DNA is mentioned
Take kit " extract (article No.: DP318).
1) whole blood 400ul is taken, 800ul cell pyrolysis liquid CL is added to mix, 10000rpm/11500 × g, 1min are centrifuged, in abandoning
Clearly, it can be repeated once if cracking is not thorough.
2) 200ul buffer GS is added in precipitating, mixes, adds 20ul Proteinase K, mixes.
3) 200ul buffer GB is added, mixes, 56 DEG C, 10min, during which overturns for several times, until solution becomes clarification (if not
Clarification can extend the time to clarification).
4) plus 200ul dehydrated alcohol mixes, of short duration centrifugation.
5) transfer sample mixed liquor is centrifuged to adsorption column CB3,13400 × g/12000rpm, 30s, abandons filtrate.
6) 500ul buffer GD is added to be centrifuged to adsorption column CB3,13400 × g/12000rpm, 30s, abandons filtrate.
7) 600ul buffer PW is added to be centrifuged to adsorption column CB3,13400 × g/12000rpm, 30s, abandons filtrate, repeat
Once.
8) adsorption column CB3 is put into clean centrifuge tube, 13400 × g/12000rpm, and filtrate is abandoned in 2min centrifugation, and room temperature is quiet
It sets 3 minutes, dries.
9) add 100ulddH2O, is incubated at room temperature 2-5min, and 13400 × g/12000rpm, 2min centrifugation are collected elution and produced
Object, -20 DEG C of preservations.
Three, the kit is provided, appropriate PCR reaction solution is taken out, by the PCR reaction solution and the appropriate pcr template
It mixes:
8 connecting leg of requirement PCR reaction solution (PCR pipe number=sample number+1 of every detection site is taken out from kit
+ 3 positive controls of blank control).
After PCR reaction solution room temperature is melted, brief centrifugation opens pipe lid.
2 μ L are taken to add in PCR reaction solution from sample to be examined DNA (or reference substance).
After oscillation mixes, brief centrifugation 10s moves it to amplification region.
Four, pcr amplification reaction: 95 DEG C of denaturation 5min is carried out；95 DEG C of 10sec, 60 DEG C of 40sec (acquisition fluorescence),
45cycles。
The PCR instrument used is ABI StepOnePlus, reaction system 25ul；
PCR reaction condition is as shown in table 2 below:
2 qPCR reaction condition of table
Five, PCR result judgement: in situation as defined in meeting in the positive reference substance and blank control product of the kit,
Result judgement is carried out according to fluorescent amplification curve and Genotyping Genotyping scatter plot, is obtained for Lovastatin drug
The genotype of body medication related gene.
As a result availability deciding, as shown in table 3 below:
Table 3: different detection site reference substance Genotypings
Blank control result is negative (value >=38 No Ct or Ct).
PCR result judgement distinguishes each gene loci according to fluorescent amplification curve and Genotyping Genotyping scatter plot
Wild type, heterozygous and saltant type, are detailed in Figure of description.
PCR result judgement distinguishes each gene loci according to fluorescent amplification curve and Genotyping Genotyping scatter plot
Wild type, heterozygous and saltant type, are detailed in Figure of description.
The interpretation result of 100 samples is as follows in the present embodiment:
APOA5 (rs662799) CC pattern sheet 55, wherein 1 testing result is as shown in Figure 1, APOA5 (rs662799)
CT pattern sheet 43, wherein 1 testing result is as shown in Fig. 2, APOA5 (rs662799) TT pattern sheet 2, wherein 1 detection
As a result as shown in Figure 3；Wherein 16 Genotyping Genotyping scatter plots (wherein AA type is the site positive reference substance 3)
As shown in Figure 4；
CETP (rs708272) GG pattern sheet 36, wherein 1 testing result is as shown in figure 5, CETP (rs708272) GA
Pattern sheet 62, wherein 1 testing result is as shown in fig. 6, CETP (rs708272) AA pattern sheet 2；Wherein 1 testing result
As shown in Figure 7；Wherein 19 Genotyping Genotyping scatter plots (wherein AA type is the site positive reference substance 3) are as schemed
Shown in 8；
SLCO1B1 (rs2291073) TT pattern sheet 64, wherein 1 testing result is as shown in figure 9, SLCO1B1
(rs2291073) TG pattern sheet 33, wherein 1 testing result is as shown in Figure 10, SLCO1B1 (rs2291073) GG pattern sheet 3
Example；Wherein 1 testing result is as shown in figure 11；Wherein 15 Genotyping Genotyping scatter plots are as shown in figure 12；
Six, kit test result of the invention is instructing the application in Lovastatin drug medication
It is obtained APOA5 (rs662799) according to the result judgement in quantitative fluorescent PCR, CETP (rs708272), SLCO1B1
(rs2291073) genotype in each site.Genotype table corresponding with Lovastatin drug personalized medicine relationship such as the following table 4 institute
Show:
4 different loci of table, different genotype and Lovastatin drug personalized medicine corresponding relationship
Comparative example 1
Change the Genotyping of primer combination sequence information detection people's anticoagulation tissue samples.
1 materials and methods
1.1 samples sources
Biomaterial of the present invention is all from Pai Sennuo medical test institute.For during the 9-12 month in 2018 send it is gloomy
Promise medical test 100 detected remaining crowd's anticoagulations.
1.2 take the DNA of sample extracting to be detected, as qPCR template:
DNA extraction kit be TIANGEN Biotech (Beijing) Co., Ltd. extracts kit " poba gene group DNA is mentioned
Take kit " it extracts (article No.: DP318), specific extraction steps are the same as people's DNA extraction steps in embodiment 2.
1.3 synthesis PCR amplification primers
It is gained knowledge and the relevant bioinformaticies softwares such as DNAstar using biological information, to institute's energy in Genbank database
The APOA5 (rs662799) retrieved, CETP (rs708272), SLCO1B1 (rs2291073) gene nucleic acid sequence carry out gene
Sequence alignment analysis, the specific sequence in selected target region design the corresponding specific gene segment for each region
PCR primer and probe (see the table below 5).
The specific primer probe and comparison primer probe sequence and length information of 5 different genes site the application of table protection
1.4 prepare qPCR reaction solution
The primer different to APOA5 (rs662799), SLCO1B1 (rs2291073), probe combinations are grouped, specifically
Primer and probe sequence be shown in Table 5:
First group of addition gene loci is the primed probe of APOA5 (rs662799), primer sequence be SEQ ID NO.1 and
SEQ ID NO.2, probe sequence are SEQ ID NO.3 and SEQ ID NO.4；
Second group of addition gene loci is the primed probe of APOA5 (rs662799), and primer sequence is SEQ ID NO.13
With SEQ ID NO.14, probe sequence is SEQ ID NO.15 and SEQ ID NO.16；
Third group adds the primed probe that gene loci is SLCO1B1 (rs2291073), and primer sequence is SEQ ID
NO.9 and SEQ ID NO.10, probe sequence are SEQ ID NO.11 and SEQ ID NO.12；
4th group of addition gene loci is the primed probe of SLCO1B1 (rs2291073), and primer sequence is SEQ ID
NO.17 and SEQ ID NO.18, probe sequence are SEQ ID NO.19 and SEQ ID NO.20；
Above-mentioned qPCR reaction solution further includes NuHi SNP Mix (2 ×) PCR premixed liquid and nuclease-free water；
The qPCR reaction solution ingredient it is final concentration of:
1×NuHi SNP Mix
0.5 μM of specific primer
0.2 μM of probe
System (is supplemented to 23ul) by nuclease free appropriate amount of water.
1.5 carry out qPCR amplified reaction: 95 DEG C of denaturation 5min；95 DEG C of 10sec, 60 DEG C of 40sec (acquisition fluorescence),
45cycles。
The qPCR instrument used is ABI StepOnePlus, reaction system 25ul；
QPCR reaction condition is as shown in table 2.
2 results
Parting figure such as Fig. 1 of 2.1 comparison primer combinations 1, shown in 2,3,4, Fig. 1,2,3 be 1 clinical sample APOA5 of combination
(rs662799) the qPCR amplification curve diagram of CC, CT, TT totally 3 kinds of partings, Fig. 4 are 1 clinical sample APOA5 (rs662799) of combination
3 kinds of Genotyping scatter plots, amplification curve and Genotyping scatter plot are distinguished well.
Parting figure such as Figure 13 of 2.2 comparison primer combinations 2, shown in 14,15,16, Figure 13,14,15, for the clinical sample of combination 2
The qPCR amplification curve diagram of this APOA5 (rs662799) CC, CT, TT totally 3 kinds of partings, Figure 16 are 2 clinical sample APOA5 of combination
(rs662799) 3 kinds of Genotyping scatter plots of CC, CT, TT, amplification curve and Genotyping scatter plot are distinguished ineffective；
Graftal such as Fig. 9 of 2.3 comparison primer combinations 3, shown in 10,11,12, Fig. 9,10,11 be 3 clinical samples of combination
The qPCR amplification curve diagram of SLCO1B1 (rs2291073) TT, TG, GG totally 3 kinds of partings, Figure 12 are 3 clinical samples of combination
3 kinds of Genotyping scatter plots of SLCO1B1 (rs2291073), amplification curve and Genotyping scatter plot are distinguished well.
Parting figure such as Figure 17 of 2.4 comparison primer combinations 4, shown in 18,19,20, Figure 17,18,19 be 4 clinical samples of combination
The qPCR amplification curve diagram of this SLCO1B1 (rs2291073) TT, TG, GG totally 3 kinds of partings, Figure 20 are 4 clinical samples of combination
3 kinds of Genotyping scatter plots of SLCO1B1 (rs2291073), amplification curve and Genotyping scatter plot are distinguished ineffective.
The working principle of the invention is: this method is directed to different genes site areas by design, comprising: APOA5
(rs662799), CETP (rs708272), SLCO1B1 (rs2291073) detect main influence Lovastatin curative effect gene position
The mutation type of point directly carries out qPCR amplification to target sequence by design primer combination, on the time and in testing cost all
It is effectively reduced, while in detection site, by 3 reaction systems, completes the inspection for influencing the gene loci of Lovastatin curative effect
It surveys.
Sequence table
<110>Nanjing Pai Sennuo Gene Tech. Company Limited
<120>the primer combination for instructing Lovastatin drug personalized medicine related gene to detect and kit and method
<130> 20190521
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> homo sapiens
<400> 1
gggtgaagat gagatggcaa g 21
<210> 2
<211> 21
<212> DNA
<213> homo sapiens
<400> 2
ctgcgagtgg agttcagctt t 21
<210> 3
<211> 18
<212> DNA
<213> homo sapiens
<400> 3
ctggagcgaa agtgagat 18
<210> 4
<211> 18
<212> DNA
<213> homo sapiens
<400> 4
ctggagcgaa agtaagat 18
<210> 5
<211> 22
<212> DNA
<213> homo sapiens
<400> 5
ccaacctcct aatctttacc cc 22
<210> 6
<211> 22
<212> DNA
<213> homo sapiens
<400> 6
tgagaaggtc ctagctgcat tg 22
<210> 7
<211> 18
<212> DNA
<213> homo sapiens
<400> 7
ctgaacccta actcgaac 18
<210> 8
<211> 18
<212> DNA
<213> homo sapiens
<400> 8
ctgaacccta acttgaac 18
<210> 9
<211> 23
<212> DNA
<213> homo sapiens
<400> 9
ttgttggttt tattgacgga agc 23
<210> 10
<211> 22
<212> DNA
<213> homo sapiens
<400> 10
ttattgccaa attgcctgtg ag 22
<210> 11
<211> 25
<212> DNA
<213> homo sapiens
<400> 11
caactggggt aaatttatct ctcac 25
<210> 12
<211> 25
<212> DNA
<213> homo sapiens
<400> 12
caactggggt aaatgtatct ctcac 25
<210> 13
<211> 22
<212> DNA
<213> homo sapiens
<400> 13
agggtgaaga tgagatggca ag 22
<210> 14
<211> 23
<212> DNA
<213> homo sapiens
<400> 14
gtagacggag tgggtgtgtc atc 23
<210> 15
<211> 22
<212> DNA
<213> homo sapiens
<400> 15
cgaaagtgag atttgcccca tg 22
<210> 16
<211> 22
<212> DNA
<213> homo sapiens
<400> 16
cgaaagtaag atttgcccca tg 22
<210> 17
<211> 20
<212> DNA
<213> homo sapiens
<400> 17
ttggttttat tgacggaagc 20
<210> 18
<211> 19
<212> DNA
<213> homo sapiens
<400> 18
ttgccaaatt gcctgtgag 19
<210> 19
<211> 19
<212> DNA
<213> homo sapiens
<400> 19
ctgtgagaga taaatttac 19
<210> 20
<211> 19
<212> DNA
<213> homo sapiens
<400> 20
ctgtgagaga tacatttac 19
Claims (6)
1. a kind of primer combination for instructing Lovastatin drug personalized medicine related gene to detect, which is characterized in that packet
Include for APOA5 (rs662799), CETP (rs708272), the specific primer of SLCO1B1 (rs2291073) gene loci and
Probe, as follows:
APOA5 (rs662799) forward primer:
5'-GGGTGAAGATGAGATGGCAAG-3'；
APOA5 (rs662799) reverse primer:
5'-CTGCGAGTGGAGTTCAGCTTT-3'；
APOA5 (rs662799) wild-type probe:
5'FAM-CTGGAGCGAAAGTGAGAT-MGB3'；
APOA5 (rs662799) saltant type probe:
5'VIC-CTGGAGCGAAAGTAAGAT-MGB3'；
CETP (rs708272) forward primer:
5'-CCAACCTCCTAATCTTTACCCC-3'；
CETP (rs708272) reverse primer:
5'-TGAGAAGGTCCTAGCTGCATTG-3'；
CETP (rs708272) wild-type probe:
5'FAM-CTGAACCCTAACTCGAAC-MGB3'；
CETP (rs708272) saltant type probe:
5'VIC-CTGAACCCTAACTTGAAC-MGB3'；
5'VIC-ATTGGTCTTTTCCAAACTCTTTGGTCATATCAG-TAMRA3'；
SLCO1B1 (rs2291073) forward primer:
5'-TTGTTGGTTTTATTGACGGAAGC-3'；
SLCO1B1 (rs2291073) reverse primer:
5'-TTATTGCCAAATTGCCTGTGAG-3'；
SLCO1B1 (rs2291073) wild-type probe:
5'FAM-CAACTGGGGTAAATTTATCTCTCAC-TAMRA3'；
SLCO1B1 (rs2291073) saltant type probe:
5'VIC-CAACTGGGGTAAATGTATCTCTCAC-TAMRA3'。
2. a kind of reagent for instructing Lovastatin drug personalized medicine related gene to detect as described in claim 1
Box, which is characterized in that including the PCR reaction solution containing specific primer as described in claim 1 and probe, the PCR is anti-
Answering liquid is respectively APOA5 (rs662799), CETP (rs708272), and SLCO1B1 (rs2291073) PCR reaction solution is wrapped respectively
It includes:
APOA5 (rs662799) forward primer, APOA5 (rs662799) reverse primer, APOA5 (rs662799) wild-type probe
And APOA5 (rs662799) saltant type probe；
CETP (rs708272) forward primer, CETP (rs708272) reverse primer, CETP (rs708272) wild-type probe and
CETP (rs708272) saltant type probe；
SLCO1B1 (rs2291073) forward primer, SLCO1B1 (rs2291073) reverse primer, SLCO1B1 (rs2291073)
Wild-type probe and SLCO1B1 (rs2291073) saltant type probe；
Above-mentioned PCR reaction solution further includes NuHi SNP Mix (2 ×) PCR premixed liquid and nuclease-free water.
3. a kind of reagent for instructing Lovastatin drug personalized medicine related gene to detect as claimed in claim 2
Box, which is characterized in that the ingredient of the PCR reaction solution is final concentration of: 1 × NuHi SNP Mix, 0.5 μM of each specific primer
And 0.2 μM of probe.
4. a kind of examination for instructing Lovastatin drug personalized medicine related gene to detect as claimed in claim 2 or claim 3
Agent box, which is characterized in that further include positive reference substance one, positive reference substance two and positive reference substance three；
The positive reference substance one is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1 (rs2291073) base
Because of site wild plasmid；
The positive reference substance two is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1 (rs2291073) base
Plasmid because of site wild type and saltant type by 1:1 quantity than heterozygosis；
The positive reference substance three is respectively APOA5 (rs662799), CETP (rs708272), SLCO1B1 (rs2291073) base
Because of site mutation type plasmid.
5. a kind of reagent for instructing Lovastatin drug personalized medicine related gene to detect as claimed in claim 2
Box, which is characterized in that further include blank control product, the blank control product are nuclease-free water.
6. one kind as described in claim 2-5 any one is for instructing Lovastatin drug personalized medicine related gene to examine
The method of survey, which comprises the steps of:
Step 1: the DNA of sample extracting to be detected is taken, as pcr template；
Step 2: providing kit as claimed in claim 5, takes out appropriate PCR reaction solution, by the PCR reaction solution and fits
The pcr template is measured to mix；
Step 3: pcr amplification reaction: 95 DEG C of denaturation 5min is carried out；95 DEG C of 10sec, 60 DEG C of 40sec, 45cycles；
Step 4: PCR result judgement: in situation as defined in meeting in the positive reference substance and blank control product of the kit,
Result judgement is carried out according to fluorescent amplification curve and Genotyping Genotyping scatter plot, is obtained for Lovastatin drug
The genotype of body medication related gene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110295224A (en) * | 2019-05-28 | 2019-10-01 | 南京派森诺基因科技有限公司 | Primer combination of probe and kit and application for instructing Rosiglitazone drug personalized medicine related gene to detect |
| CN111909996A (en) * | 2020-07-08 | 2020-11-10 | 广西医大睿谷医学检验有限公司 | Detection kit for polymorphism of gene related to individualized medication |
| CN116837089A (en) * | 2023-07-06 | 2023-10-03 | 重庆京因生物科技有限责任公司 | Gene polymorphism rapid detection primer set and kit for guiding drug administration of Parkinson's disease |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1566449A1 (en) * | 2004-02-18 | 2005-08-24 | Max-Delbrück-Centrum Für Molekulare Medizin | Use of haplotypes and SNPs in lipid-relevant genes for the analyses and diagnosis of cardiovascular diseases |
| CN105803099A (en) * | 2016-05-16 | 2016-07-27 | 钟诗龙 | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation |
-
2019
- 2019-05-28 CN CN201910452542.5A patent/CN110295225A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1566449A1 (en) * | 2004-02-18 | 2005-08-24 | Max-Delbrück-Centrum Für Molekulare Medizin | Use of haplotypes and SNPs in lipid-relevant genes for the analyses and diagnosis of cardiovascular diseases |
| CN105803099A (en) * | 2016-05-16 | 2016-07-27 | 钟诗龙 | Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110295224A (en) * | 2019-05-28 | 2019-10-01 | 南京派森诺基因科技有限公司 | Primer combination of probe and kit and application for instructing Rosiglitazone drug personalized medicine related gene to detect |
| CN111909996A (en) * | 2020-07-08 | 2020-11-10 | 广西医大睿谷医学检验有限公司 | Detection kit for polymorphism of gene related to individualized medication |
| CN116837089A (en) * | 2023-07-06 | 2023-10-03 | 重庆京因生物科技有限责任公司 | Gene polymorphism rapid detection primer set and kit for guiding drug administration of Parkinson's disease |
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