CN105219857A - A kind of Abacavir personalized medicine detection kit and method thereof - Google Patents

A kind of Abacavir personalized medicine detection kit and method thereof Download PDF

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CN105219857A
CN105219857A CN201510670708.2A CN201510670708A CN105219857A CN 105219857 A CN105219857 A CN 105219857A CN 201510670708 A CN201510670708 A CN 201510670708A CN 105219857 A CN105219857 A CN 105219857A
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abacavir
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汪大为
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Beijing Jinqi Biotechnology Co Ltd
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    • C12Q1/6858—Allele-specific amplification

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Abstract

The invention provides a kind of Abacavir personalized medicine detection kit and method thereof, belong to Abacavir medication detection technique field.Does the present invention adopt Allele-Specific? LAMP technology, the G allelotrope for rs2395029 devises special primer, utilizes the G allelotrope of rs2395029 and the cognation of HLA-B*5701 to judge whether patient carries HLA-B*5701 allelotrope.After test, the change of state (whether having the change of precipitation or color) of observing response system can judge whether positive reaction occurs, and naked eyes can judge, without the need to extra appliance arrangement, simple and easy to do.

Description

A kind of Abacavir personalized medicine detection kit and method thereof
Technical field
The present invention relates to Abacavir medication detection technique field, particularly relate to a kind of Abacavir personalized medicine detection kit and method thereof.
Background technology
Abacavir (abacavir is called for short ABC, trade(brand)name: Ziagen) is a kind of anti-AIDS new drug, and be new carbocyclic ring 2'-pancreatic desoxyribonuclease nucleoside medicine, its oral administration biaavailability is high, easily infiltrates central nervous system.The same with other efabirenzs, Abacavir is the prodrug of a non-activity, become the triguaiacyl phosphate of tool activity in vivo through 4 step metabolism, and play the effect of HIV inhibiting (HIV) reversed transcriptive enzyme by following 2 approach: 1. suppress 2'-deoxyguanosine triphosphate ester (dGTP) (DNA synthesizes one of fragment) to combine competitively and enter nucleic acid chains; 2. the synthesis of DNA chain is effectively stopped by stoping new adding of base.
Research in recent years shows, the generation of HLA-B gene polynorphisms and adverse drug reaction is closely related.2008 U.S. food Drug Administration (FDA) issue about Abacavir with containing the security information of Abacavir medicine, the HIV person pointing out to carry HLA-B*5701 gene uses Abacavir that Abacavir allergy (ABCHSR) more easily occurs.Abacavir allergy is a kind of multiple organ syndrome, there will be two or more S or S, comprises heating, fash, gastrointestinal reaction, respiratory response and systemic reaction.Allergy appears at usually treats the first six week that be early stage or treatment beginning, but, because symptom is more and the impact of drug combination, be difficult to make a definite diagnosis in early days.
CFDA points out in " drug metabolism enzyme and the drug target technique of gene detection guide (trying) " of issue in 2015: " carry HLA-B*5701 allelotrope person and be cautious use of Abacavir, in order to avoid cause medicamentous liver lesion." therefore, HIV person is before use abc drug, and necessary examination HLA-B*5701 gene, when detected result is negative, namely patient does not carry HLA-B*5701 gene and can use abc drug.
The detection means of HLA-B*5701 mainly contains serological detection method, PCR-direct sequencing (PCR-SBT) and fluorescent PCR method, but carries out HLA somatotype by Serologic detection, result out of true.Is then now generally the genotype being analyzed HLA by order-checking, although this method can obtain gene sequence information the most accurately, elapsed time is longer and cost is higher, large-scale promotion when not being adapted at detecting, so clinically and inapplicable; Although fluorescent PCR method relatively processing ease, be consuming timely also less than direct sequencing, still depend on test set costly and probe, be difficult in regional penetration and promotion remote or underdeveloped.Therefore, be necessary to develop the method that quick and precisely can must detect HLA-B*5701 with extremely low cost.
Recently discovery is studied, the G allelotrope of the SNP site rs2395029 near HLA-B locus and HLA-B*5701 are highly chain, the susceptibility of the two is 100%, this detection being HLA-B*5701 provides a new visual angle, namely we can by avoiding the sequential analysis directly to HLA-B, and the base situation just detecting rs2395029 learns whether detect sample exists HLA-B*5701 indirectly.
Ring mediated isothermal amplification (Loop-mediatedIsothermalAmplification, LAMP) technology is a kind of isothermal amplification technology that Japanese Scientists developed in 2000, for six zone design, four Auele Specific Primers of target gene, constant-temperature amplification under the effect of strand displacement archaeal dna polymerase, can produce 10 within tens minutes to one hour 9-10 10the product of the order of magnitude, judged the genotype of sample to be tested by the change of direct visual perception system status after reaction terminates, without the need to open pipe, therefore enormously simplify operation steps, effectively can avoid sample cross contamination again simultaneously, and, only need ortho-water bath to carry out detection, again save a large amount of testing costs.Therefore, it is a kind of novel and be suitable for the extensive method used for adopting LAMP method to detect the genotype of HLA-B*5701.
Summary of the invention
The problem to be solved in the present invention is that the method for existing detection HLA-B*5701 is undesirable, is unsuitable for large-scale promotion and uses.
For solving the problem, the present invention adopts allele-specific ring mediated isothermal amplification (Allele-SpecificLAMP) technology on the basis of LAMP, Tag SNP for HLA-B*5701 detects, provide a kind of be suitable for promoting the use of test kit and detection method.
First object of the present invention is to provide a kind of Abacavir personalized medicine to detect primer, for detecting the Tag SNP of HLA-B*5701---and the G allelotrope of rs2395029, comprises following primer:
5 '-GTGTGACAGCAGCCATGC-3 ' (SEQIDNo.1, hereinafter referred to as F3)
5 '-TCTCCCAAAACCACACTCTG-3 ' (SEQIDNo.2, hereinafter referred to as B3)
5′-ACCAGCGGGTGAGAAGAGGACCTCCTGGGGATCAGGATC-3′
(SEQIDNo.3, hereinafter referred to as FIP)
5′-GGACAGCTGTAATGTGTAGTTCAATGGAGGGTAGAAGGTCCT
GGATT-3 ' (SEQIDNo.4, hereinafter referred to as BIP).
In primer provided by the invention, BIP is for the allelic Auele Specific Primer of rs2395029G.In order to avoid potential non-specific interference, in BIP, introduce a catastrophe point of knowing clearly artificially.This catastrophe point can add the specificity of primer, reduces the possibility of non-specific amplification, improves the accuracy detected.
Preferably, described F3, B3, FIP, BIP are the primer after HPLC method of purification purifying.
Second object of the present invention is to provide a kind of Abacavir personalized medicine detection kit.Described test kit comprises the composition detecting rs2395029 loci gene type and then detect HLA-B*5701.Described composition comprises above-mentioned primer, also comprises dNTP, damping fluid, Bst polysaccharase and ultrapure water.
Further, described composition also comprises the indicator for showing composition color.
Preferably, described indicator is the composition of fluorexon and Manganous chloride tetrahydrate or is hydroxynaphthol blue.
Preferably, if indicator is the composition of fluorexon and Manganous chloride tetrahydrate, fluorexon is 25 μMs, and Manganous chloride tetrahydrate is 0.5mM.If indicator is hydroxynaphthol blue, it is 120 μMs.
Further, described composition also comprises additive.Described additive is the one in trimethyl-glycine or dimethyl sulfoxide (DMSO).If additive is trimethyl-glycine, its concentration is more than or equal to 0, is less than or equal to 1M.If additive is dimethyl sulfoxide (DMSO), its concentration is more than or equal to 0, is less than or equal to 10% (volume fraction).
Preferably, in described composition, F3 and B3 is 0.4 μM, FIP and BIP is 1.6 μMs.
Further, described damping fluid comprises: Tris-HCl20mM, KCl50mM, (NH 4) 2sO 410mM, MgSO 44mM, Tween-200.1% (volume fraction).
Preferably, in described composition, dNTP is 1.4mM, Bst polysaccharase is 8000U/mL.
3rd object of the present invention is to provide a kind of Abacavir personalized medicine detection method.Described detection method, use above-mentioned Abacavir personalized medicine detection kit, be specially: joined in described composition by nucleic acid to be detected, the reaction system obtained is reacted at 55 ~ 70 DEG C, and reaction terminates the change of rear observe system.
Further, the concentration of described nucleic acid to be detected in reaction system is 0.8 ~ 4ng/ μ L.
Preferably, after reaction terminates, reaction system is carried out inactivation treatment.Inactivation treatment makes the enzyme deactivation in system, when preventing system storage period long, non-specific amplification occurs.
Preferably, temperature of reaction is 63 DEG C.
Preferably, the described reaction times is 40 ~ 120min.
In reaction system of the present invention, reacting precursor is clear state, and when there is positive reaction, magnesium ion and by product tetra-sodium are formed and precipitate, and system produces macroscopic precipitation, become muddy state.The change of observe system state can directly determine whether amplified reaction occurs.
When there is indicator in system: (1) if indicator is hydroxynaphthol blue, the magnesium ion in reacting precursor system and dNTP chelating, system presents pansy; When there being positive reaction, magnesium ion and by product tetra-sodium are formed and precipitate, and pH value of solution changes, and color becomes sky blue from pansy gradually; (2) if when indicator is the composition of fluorexon and Manganous chloride tetrahydrate, before reaction, the mn ion of fluorexon in Manganous chloride tetrahydrate is combined, and system presents yellow; When there being positive reaction, by product tetra-sodium is combined with mn ion to generate and precipitates, and the magnesium ion of fluorexon in system is combined, and color becomes fluorescent green from yellow gradually.The change of observing response system color and/or state can directly determine whether amplified reaction occurs.
The advantage that the present invention has and positively effect are: (1) the present invention adopts Allele-SpecificLAMP technology, G allelotrope for rs2395029 devises special primer, the G allelotrope of rs2395029 and the cognation of HLA-B*5701 is utilized to judge whether patient carries HLA-B*5701 allelotrope, thus instruct Abacavir personalized medicine, avoid directly testing HLA-B*5701.(2) the present invention adopts Allele-SpecificLAMP technology, and the primer specificity of design is good.During detection, could follow-up amplification be caused when only having these 4 primers to match with template to be detected completely, improve the specificity of detection, accuracy.(3) the present invention uses Allele-SpecificLAMP technology, devise special primer, after test, the change of state (whether having the change of precipitation or color) of observing response system can judge whether positive reaction occurs, naked eyes can judge, without the need to extra appliance arrangement, simple and easy to do.(4) use detection primer provided by the invention, test kit and method to detect, required equipment is only water-bath or metal bath or thermal cycler, and testing cost is low, it is consuming time shorter to detect.And this detection method does not need extra open pipe to detect, and therefore effectively prevent the generation of crossed contamination, improves the accuracy of detection.
Test kit provided by the invention is with low cost, detection time is short, accuracy is high, is easy to promote.
Accompanying drawing explanation
Fig. 1 is the electrophorogram detecting the product that sample 1-5 is obtained by reacting through LAMP.
Fig. 2-Fig. 6 is the Sequencing chromatogram detecting the rs2395029 site that sample 1-5 PCR-sequencing obtains respectively.
Embodiment
For a better understanding of the present invention, below in conjunction with specific embodiments and the drawings, the present invention is conducted further description.
A kind of Abacavir personalized medicine detects primer, for detecting the G allelotrope of rs2395029, comprises following primer:
5 '-GTGTGACAGCAGCCATGC-3 ' (SEQIDNo.1, hereinafter referred to as F3)
5 '-TCTCCCAAAACCACACTCTG-3 ' (SEQIDNo.2, hereinafter referred to as B3)
5′-ACCAGCGGGTGAGAAGAGGACCTCCTGGGGATCAGGATC-3′
(SEQIDNo.3, hereinafter referred to as FIP)
5′-GGACAGCTGTAATGTGTAGTTCAATGGAGGGTAGAAGGTCCT
GGATT-3 ' (SEQIDNo.4, hereinafter referred to as BIP).
Above-mentioned F3, B3, FIP, BIP are the primer after HPLC method of purification purifying.
A kind of Abacavir personalized medicine detection kit, comprises the composition detecting rs2395029 loci gene type and then detect HLA-B*5701.Described composition comprises above-mentioned primer, also comprises dNTP, damping fluid, Bst polysaccharase, ultrapure water, indicator, additive.
Described indicator is the composition of fluorexon and Manganous chloride tetrahydrate or is hydroxynaphthol blue.
Described additive is the one in dimethyl sulfoxide (DMSO) or trimethyl-glycine.
The concentration that in composition, the concentration of each component can be reacted by normal LAMP is arranged.As preferably, F3 and B3 is 0.4 μM, FIP and BIP is 1.6 μMs, and dNTP is 1.4mM, Bst polysaccharase is 8U.Damping fluid comprises: Tris-HCl20mM, KCl50mM, (NH 4) 2sO 410mM, MgSO 44mM, Tween-200.1% (volume fraction).
If indicator is the composition of fluorexon and Manganous chloride tetrahydrate, fluorexon is 25 μMs, and Manganous chloride tetrahydrate is 0.5mM.If indicator is hydroxynaphthol blue, it is 120 μMs.
If additive is for there being trimethyl-glycine, its concentration is more than or equal to 0, is less than or equal to 1M; If additive is dimethyl sulfoxide (DMSO), its concentration is more than or equal to 0, is less than or equal to 10% (volume fraction).
A kind of Abacavir personalized medicine detection method, uses above-mentioned Abacavir personalized medicine detection kit.Be specially: nucleic acid to be detected is joined in above-mentioned composition, the reaction system obtained.Reaction system reacts 40 ~ 120min at 55 ~ 70 DEG C.After reaction terminates, reaction system is carried out inactivation treatment.Inactivation treatment makes the enzyme deactivation in system, when preventing system storage period long, non-specific amplification occurs.Reaction terminates the change of rear observe system.
As preferably, nucleic acid to be detected concentration in reaction system is 0.8 ~ 4ng/ μ L.Temperature of reaction is 63 DEG C, isothermal reaction.
During concrete enforcement, extract 4 people 1ml venous blood and preserve with EDTA anticoagulant tube, often 200uL blood got by pipe, and by the DNA extraction kit of full formula gold or other company, reference reagent box specification sheets extracts complete genome DNA as the sample detected, and sample is designated as template T.Above-mentioned 4 samples serial number 1-4 respectively, except above-mentioned 4 groups, also adds the plasmid of the G allelotrope positive of known rs2395029 as positive control, numbering 5.
Allele-SpecificLAMP technology is adopted to template T, makes nucleic acid to be detected carry out LAMP reaction, detect the genotype in rs2395029 site.By template T n(n is group numbering, 1-5) joins in composition respectively, obtains reaction system.Reaction system is isothermal reaction 60min at 63 DEG C, is warming up to 80 DEG C afterwards and carries out inactivation treatment 20min, lower the temperature afterwards, observe system colour-change.
As a kind of embodiment, in test kit, reaction system cubic capacity is 25 μ L, and each component is as shown in the table with its specification:
Reaction is carried out according to above-mentioned detection method, and before reaction, system color is pansy, and after reaction, color and the genotype of each group reaction system judge as shown in the table:
As seen from the above table, the sample reaction system being numbered 1-4 all keeps pansy, proves that positive reaction does not occur for it, points out the genotype of these 4 samples in rs2395029 site to be that T/T is homozygous.According to the allelic cognation of the G of HLA-B*5701 and rs2395029, detect sample 1-4 and do not carry HLA-B*5701 allelotrope, be suitable for Abacavir.
For the accuracy of further confirmatory reaction, electrophoresis test is carried out to the LAMP product of above-mentioned group.Condition is: with the sepharose of 2%, electrophoresis 30min under 6v/cm voltage.The electrophorogram obtained as shown in Figure 1.The positive reaction of the 5th group presents typical scalariform band, and negative reaction does not then have band.With coming to the same thing of detection method of the present invention.
Above-mentioned 5 other templates of experimental group are carried out somatotype by PCR-sequencing to rs2395029 respectively, the Sequencing chromatogram in the rs2395029 site obtained is as shown in Fig. 2-Fig. 6, contrast gene sequencing collection of illustrative plates and the detected result of test kit provided by the invention known, the two detection coincide rate 100%.Can verify that result of the present invention is accurate.
Above embodiments of the invention have been described in detail, but described content being only preferred embodiment of the present invention, can not being considered to for limiting practical range of the present invention.All equalizations done according to the scope of the invention change and improve, and all should still belong within this patent covering scope.

Claims (15)

1. Abacavir personalized medicine detects a primer, it is characterized in that: comprise following primer:
5 '-GTGTGACAGCAGCCATGC-3 ' (SEQIDNo.1, hereinafter referred to as F3)
5 '-TCTCCCAAAACCACACTCTG-3 ' (SEQIDNo.2, hereinafter referred to as B3)
5′-ACCAGCGGGTGAGAAGAGGACCTCCTGGGGATCAGGATC-3′
(SEQIDNo.3, hereinafter referred to as FIP)
5′-GGACAGCTGTAATGTGTAGTTCAATGGAGGGTAGAAGGTCCT
GGATT-3 ' (SEQIDNo.4, hereinafter referred to as BIP).
2. Abacavir personalized medicine according to claim 1 detects primer, it is characterized in that: described F3, B3, FIP, BIP are the primer after HPLC method of purification purifying.
3. an Abacavir personalized medicine detection kit, comprise Abacavir personalized medicine detection composition, described composition comprises the primer described in claim 1 or 2, it is characterized in that: described composition also comprises dNTP, damping fluid, Bst polysaccharase and ultrapure water.
4. Abacavir personalized medicine detection kit according to claim 3, is characterized in that: described composition also comprises the indicator for showing composition colour-change.
5. Abacavir personalized medicine detection kit according to claim 4, is characterized in that: described indicator is the composition of fluorexon and Manganous chloride tetrahydrate or is hydroxynaphthol blue.
6. Abacavir personalized medicine detection kit according to claim 5, is characterized in that: if indicator is the composition of fluorexon and Manganous chloride tetrahydrate, fluorexon is 25 μMs, and Manganous chloride tetrahydrate is 0.5mM;
If indicator is hydroxynaphthol blue, it is 120 μMs.
7. Abacavir personalized medicine detection kit according to claim 3, is characterized in that: described composition also comprises additive; Described additive is the one in trimethyl-glycine or dimethyl sulfoxide (DMSO);
If additive is trimethyl-glycine, its concentration is more than or equal to 0, is less than or equal to 1M;
If additive is dimethyl sulfoxide (DMSO), its concentration is more than or equal to 0, is less than or equal to 10% (volume fraction).
8., according to the arbitrary described Abacavir personalized medicine detection kit of claim 3-7, it is characterized in that: in described composition, F3 and B3 is 0.4 μM, FIP and BIP is 1.6 μMs.
9., according to the arbitrary described Abacavir personalized medicine detection kit of claim 3-7, it is characterized in that: described damping fluid comprises: Tris-HCl20mM, KCl50mM, (NH4) 2sO 410mM, MgSO 44mM, Tween-200.1% (volume fraction).
10., according to the arbitrary described Abacavir personalized medicine detection kit of claim 3-7, it is characterized in that: in described composition, dNTP is 1.4mM, Bst polysaccharase is 320U/mL.
11. an Abacavir personalized medicine detection method, use the arbitrary described Abacavir personalized medicine detection kit of claim 3-10, it is characterized in that: nucleic acid to be detected is joined in described composition, the reaction system obtained is reacted at 55 ~ 70 DEG C, and reaction terminates the change of rear observe system.
12. Abacavir personalized medicine detection methods according to claim 11, is characterized in that: the concentration of described nucleic acid to be detected in reaction system is 0.8 ~ 4ng/ μ L.
13. Abacavir personalized medicine detection methods according to claim 11 or 12, is characterized in that: after reaction terminates, reaction system is carried out inactivation treatment.
14. Abacavir personalized medicine detection methods according to claim 11 or 12, is characterized in that: temperature of reaction is 63 DEG C.
15. Abacavir personalized medicine detection methods according to claim 11 or 12, is characterized in that: the described reaction times is 40 ~ 120min.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102209791A (en) * 2008-11-12 2011-10-05 香港中文大学 Detection of hla genotype

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102209791A (en) * 2008-11-12 2011-10-05 香港中文大学 Detection of hla genotype

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BADOLO等: "Development of an alle-specific,loop-mediated,isothermal amplification method(AS-LAMP) to detect the L1014F kdr-w mutation in Anopheles gambiae s.l.", 《MALARIA》 *
IWASAKI等: "Validation of the Loop-mediated Isothermal Amplification Method for Single Nucleotide Polymorphism Genotyping with Whole Blood", 《GENOME LETTERS》 *
MINNUCCI等: "A novel,high sensitive and rapid allele-specific loop-mediated amplification assay for the detection of the JAK2V617F mutation in chronic myeloproliferative neoplasms", 《HAEMATOLOGICA-THE HEMATOLOGY JOURNAL》 *
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