CN102108390A - Entecavir resistant mutation test kit - Google Patents
Entecavir resistant mutation test kit Download PDFInfo
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- CN102108390A CN102108390A CN2009102004951A CN200910200495A CN102108390A CN 102108390 A CN102108390 A CN 102108390A CN 2009102004951 A CN2009102004951 A CN 2009102004951A CN 200910200495 A CN200910200495 A CN 200910200495A CN 102108390 A CN102108390 A CN 102108390A
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- Prior art keywords
- entecavir
- test kit
- resistant mutation
- testing
- kit
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Abstract
The invention discloses a kit for testing entecavir resistant mutation heredity in an individual. The kit comprises a specific primer pair and a specific fluorescent probe pair for testing the polymorphism of entecavir resistant mutation loci rtM204V, rtL180M, rtT184, rtS202 and rtM250, as well as a fluorescence quantitative rolymerase chain reaction (PCR) normal component and the like. The kit disclosed by the invention estimates the entecavir resistance in the individual by testing the gene polymorphism of an entecavir administration target locus.
Description
Technical field
The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects individual Entecavir resistance mutation genetic, assess individual Entecavir resistance by detecting Entecavir medication target site gene pleiomorphism.
Background technology
Entecavir is the guanosine-analogue, and is inhibited to hepatitis B virus (HBV) polymerase.It can become by phosphorylation and has active triphosphate, and triphosphate is 15 hours in the intracellular transformation period.Be applicable to that virus replication is active, lasting rising of serum transaminase ALT or liver histological show the treatment of the chronic one-tenth human hepatitis B of reactivity pathology.The oral administration biaavailability height, serum protein bonding force low (13%) is not inductor, inhibitor and the substrate of cytopigment isozyme.It appears in the urine with female medicine form by renal excretion, and the transformation period is 0.5-15h in the cell, through renal excretion.
The covariation of Entecavir resistance is on rtM204V+rtL180M variation basis, unites the amino acid replacement variation at least one site in three sites of rtT184, rtS202 or rtM250 again.
Summary of the invention
The invention provides a kind of test kit that detects individual Entecavir resistance mutation genetic target site gene pleiomorphism.
This test kit comprises: it is right to reaching the specificity fluorescent probe to detect the Auele Specific Primer that draws rtM204V, rtL180M, rtT184, rtS202 and rtM250 loci polymorphism, Taq enzyme, dNTP mixed solution, MgCl
2Solution, reaction buffer, deionized water.
Auele Specific Primer described in this test kit and fluorescent probe can be finished design for the person of ordinary skill of the art easily, and Auele Specific Primer and fluorescent probe synthesize the DNA synthetic technology of available routine.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The use of embodiment detection kit
The extraction of step 1:DNA template
Genomic dna with silica gel adsorption extracting mouth epithelial cells.
Step 2: quantitative fluorescent PCR reaction
Use the quantitative fluorescent PCR suit in the detection kit, carry out the quantitative fluorescent PCR reaction, the system of reaction is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mMMgCl of 20ng/ μ l
2The band VIC fluorescent probe that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.
React on the pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on quantitative real time PCR Instrument.
Step 4: gene type assay
According to the gene type diagram that the test kit working instructions indicate gene type assay is carried out in the SNP site.
The those skilled in the art that are familiar with fluorescent quantitative PCR technique can be by the final sample fluorescence volume that shows on the identification quantitative real time PCR Instrument, can determine the genotype in the SNP site detected according to the power of different sequence fluorescence probe signals.
Claims (1)
1. a test kit that detects individual Entecavir resistance mutation genetic comprises that Auele Specific Primer is right to reaching the specificity fluorescent probe, Taq enzyme, dNTP mixed solution, MgCl
2Solution, quantitative fluorescent PCR reaction buffer and deionized water is characterized in that: described Auele Specific Primer is to reaching the specificity fluorescent probe to designing at rtM204V, rtL180M, rtT184, rtS202 and rtM250 loci polymorphism.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102004951A CN102108390A (en) | 2009-12-23 | 2009-12-23 | Entecavir resistant mutation test kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102004951A CN102108390A (en) | 2009-12-23 | 2009-12-23 | Entecavir resistant mutation test kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102108390A true CN102108390A (en) | 2011-06-29 |
Family
ID=44172675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102004951A Pending CN102108390A (en) | 2009-12-23 | 2009-12-23 | Entecavir resistant mutation test kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102108390A (en) |
-
2009
- 2009-12-23 CN CN2009102004951A patent/CN102108390A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| DANIEL J. TENNEY ET AL.: "Two-Year Assessment of Entecavir Resistance in Lamivudine-Refractory Hepatitis B Virus Patients Reveals Different Clinical Outcomes Depending on the Resistance Substitutions Present", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| 薛瑞霞等: "恩替卡韦治疗失败的慢性乙型肝炎患者耐药分析20例", 《世界华人消化杂志》 * |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: Ginza wealth International Plaza No. 43 Shanghai Guoquan road 200433 4 floor Applicant after: XINBAXIANG (SHANGHAI) MOLECULAR MEDICAL TECHNOLOGY (SHANGHAI) CO., LTD. Address before: 200433 No. 335, building 2, No. 6, National Road, Shanghai Applicant before: Shanghai Zhujian Bioengineering Co., Ltd. |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: SHANGHAI ZHUJIAN BIOENGINEERING CO., LTD. TO: INSTITUTE OF TARGETED THERAPY AND MOLECULAR MEDICINE OF SHANGHAI |
|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110629 |