CN113088572A - Solid phase PCR kit for hepatitis B drug detection - Google Patents
Solid phase PCR kit for hepatitis B drug detection Download PDFInfo
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- CN113088572A CN113088572A CN202110569064.3A CN202110569064A CN113088572A CN 113088572 A CN113088572 A CN 113088572A CN 202110569064 A CN202110569064 A CN 202110569064A CN 113088572 A CN113088572 A CN 113088572A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a solid phase PCR kit for detecting hepatitis B medication, which comprises a PCR reaction buffer solution, a primer group and hot start DNA polymerase; the nucleotide sequences of the upstream primer and the downstream primer of the primer pair 03IL-28B x 1rs12979860 are respectively shown by SEQ ID NO.1 and SEQ ID NO. 2; detecting 04IL-28B 2rs8099917 primer pair, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown by SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the upstream primer and the downstream primer of the 116ITPA rs1127354 primer pair are respectively shown by SEQ ID NO.7 and SEQ ID NO.8, and the invention relates to the technical field of gene detection. According to the invention, the reliability of a detection result is ensured by adopting a specific primer sequence; the extracted nucleic acid can be directly detected; the requirements on a detection platform and detection personnel are low, and the method can be widely popularized in the clinical front-line.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a solid-phase PCR kit for detecting hepatitis B medication.
Background
The PEG-IFN alpha-2 b injection is also named as Pellent and a prescription drug, is produced by Ireland XinlingBaoya company and authorized by the American XinlingBaoya company, and is ranked first in the prescription amount in the global long-acting interferon. The PEG-IFN alpha-2 b injection is the first long-acting interferon approved by the FDA of the European Union and the United states in 2000 and 2001. Approval was obtained in china in month 5 of 2004 for the treatment of chronic hepatitis c, and in month 3 of 2007 for chronic hepatitis b indications. Pelargoni is an optimally designed pegylated interferon that maximizes antiviral activity while extending half-life. The pelargonii is the only long-acting interferon which is administrated according to the body weight, so that the incidence rate of adverse reactions of patients with different body weights is the same, and the defect of high incidence rate of bone marrow suppression of patients with small body weight when a single fixed dose is administrated is avoided. The 2009 american liver disease research association (AASLD) chronic hepatitis b guideline, the 2012 asia-pacif liver disease research association (APASL) chronic hepatitis b guideline, the 2012 european liver disease research association (EASL) chronic hepatitis b guideline, and the 2010 chinese chronic hepatitis b control guideline recommends peg interferon alpha-2 b as one of the first-line medications for the initial treatment of HBeAg-positive chronic hepatitis b patients.
Ribavirin, also known as ribavirin, nimesulide and the like, is a broad-spectrum powerful antiviral drug and is widely applied to prevention and treatment of viral diseases at present. The common dosage forms include injection, tablet, oral liquid, aerosol, etc. Ribavirin has few side effects and low adverse reaction incidence, and according to the pharmacological action, the application needs to pay attention to that the large dose of the medicine can cause leucopenia, anemia, serum transaminase and bilirubin increase after long-term use. Blind and overdose administration should be avoided. The increase of bilirubin and decrease of hemoglobin caused by the use of the medicine in large dose can interfere with clinical examination and diagnosis.
The asia-pacific liver disease research association (APASL) chronic hepatitis c guideline, the european liver disease research association (EASL) chronic hepatitis c guideline, and the american liver disease research association (AASLD) chronic hepatitis c guideline recommend peginterferon alfa-2 b in combination with oral ribavirin as the first line treatment option for chronic hepatitis c.
Disclosure of Invention
The invention aims to solve the technical problem of providing a convenient and quick 2-type hepatitis B drug gene detection kit with high flux, which can be used for detecting 03IL-28B 1rs12979860, 04IL-28B 2rs8099917, 05IL-28B 3rs12980275 and 116ITPA rs1127354 genes.
In order to achieve the purpose, the invention adopts the following technical scheme: a hepatitis B drug detection solid phase PCR kit, the said solid phase PCR kit includes PCR reaction buffer solution, primer group, hot start DNA polymerase to form;
the primer group consists of the following 4 pairs of primers:
03IL-28B*1rs12979860、04IL-28B*2rs8099917、05IL-28B*3rs12980275、116ITPA rs1127354；
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 03IL-28B x 1rs12979860 primer pair are respectively shown by SEQ ID NO.1 and SEQ ID NO. 2;
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 04IL-28B x 2rs8099917 primer pair are respectively shown by SEQ ID NO.3 and SEQ ID NO. 4;
detecting the nucleotide sequences of the upstream primer and the downstream primer of the 05IL-28B, 3rs12980275 primer pair respectively represented by SEQ ID NO.5 and SEQ ID NO. 6;
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair for detecting 116ITPA rs1127354 are respectively shown by SEQ ID NO.7 and SEQ ID NO. 8.
An amplification method of a solid phase PCR kit, wherein the solid phase PCR kit is fixed in a 96-hole PCR reaction plate or 12-row PCR reaction holes.
Preferably, the primer group, the hot-start DNA polymerase and the PCR reaction buffer solution are fixed in a 96-well PCR reaction plate or 12-row PCR reaction wells, and the upper-level amplification can be realized only by adding a nucleic acid template during sample adding.
Preferably, the concentration of the primer in the solid phase PCR kit is 100nM-1 uM.
A detection method for detecting 2 medicines for hepatitis B based on a solid phase PCR kit comprises the following steps: the method comprises the following steps: 2, producing a solid-phase PCR amplification kit for hepatitis B drug detection; step two: extracting nucleic acid from the oral swab; step three: solid phase PCR amplification reaction; step four: sequencing the PCR product after electrophoretic analysis.
Preferably, the primer concentration is 100nM-1000nM, preferably 500nM-800 nM.
Preferably, the solid phase PCR kit is characterized in that a layer of paraffin mixture is sealed on the surface of the mixed solution containing the PCR reaction, the melting point of the paraffin mixture is between 30 and 38 ℃, the paraffin mixture is used for isolating enzyme and other components in the PCR reaction solution, the melting point is moderate, and the sensitivity of the PCR reaction is not influenced. The paraffin mixture is prepared by mixing liquid paraffin and melted solid paraffin, wherein the solid paraffin is selected from solid paraffin with melting point higher than 50 ℃, and the ratio of the solid paraffin to the liquid paraffin is 1: 4-1: 12, preferably 1: 7-1: 9.
Collecting sample and extracting nucleic acid
Nucleic acid is extracted by adopting an oral swab DNA extraction kit according to the instruction, the concentration is not lower than 10ng/ul, and the purity is 1.6-1.8.
Solid phase PCR amplification reaction using nucleic acid sample as template
The 25ul PCR reaction system was: solid phase PCR reaction MIX, nucleic acid template 2 ul.
The PCR reaction MIX contains Buffer, Mg +, dNTPs, enzyme and a primer group in the table 1.
Compared with the prior art, the invention has the following beneficial effects: the method is simple and convenient to operate, can be directly amplified on a computer only by adding the nucleic acid template into the solid-phase PCR plate, is simple to operate, has good clinical detection specificity and sensitivity equivalent to that of quantitative PCR, and has stronger clinical application and popularization values;
the invention has high sensitivity and strong specificity, and the solid phase PCR kit adopts hot start DNA polymerase, thereby avoiding non-specific amplification and improving the detection sensitivity and specificity;
the invention has complete detection sites, is suitable for hepatitis B medication guidance, and the solid phase PCR kit can detect 4 target spots related to hepatitis B medication detection at one time;
in conclusion, the invention provides a solid phase PCR kit for detecting hepatitis B medication. The invention adopts specific primer sequences to ensure the reliability of the detection result; the detection method is simple to operate, and time and labor are saved; the detection flux is large, and the cost of reagent consumables is low; the extracted nucleic acid can be directly detected; the requirements on a detection platform and detection personnel are low, and the method can be widely popularized in the clinical front-line.
Drawings
The invention is described in further detail below with reference to the following figures and detailed description:
FIG. 1 is an electrophoretogram of a patient 1 detected by the kit;
FIG. 2 shows the sequencing result of the kit for detecting patient 1.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-2.
Example 1:
a hepatitis B drug detection solid phase PCR kit, the said solid phase PCR kit includes PCR reaction buffer solution, primer group, hot start DNA polymerase to form;
the primer group consists of the following 4 pairs of primers:
03IL-28B*1rs12979860、04IL-28B*2rs8099917、05IL-28B*3rs12980275、116ITPA rs1127354；
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 03IL-28B x 1rs12979860 primer pair are respectively shown by SEQ ID NO.1 and SEQ ID NO. 2;
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 04IL-28B x 2rs8099917 primer pair are respectively shown by SEQ ID NO.3 and SEQ ID NO. 4;
detecting the nucleotide sequences of the upstream primer and the downstream primer of the 05IL-28B, 3rs12980275 primer pair respectively represented by SEQ ID NO.5 and SEQ ID NO. 6;
the nucleotide sequences of the upstream primer and the downstream primer of the primer pair for detecting 116ITPA rs1127354 are respectively shown by SEQ ID NO.7 and SEQ ID NO. 8.
An amplification method of a solid phase PCR kit, wherein the solid phase PCR kit is fixed in a 96-hole PCR reaction plate or 12-row PCR reaction holes.
The primer group, the hot start DNA polymerase and the PCR reaction buffer solution are fixed in a 96-hole PCR reaction plate or 12-row PCR reaction holes, and the upper-level amplification can be realized only by adding a nucleic acid template during sample adding.
The concentration of the primer in the solid phase PCR kit is 100nM-1 uM.
Table 1 detection target and primer sequences of the kit:
example 2:
the composition of 4 hepatitis B solid phase PCR detection kits:
the kit consists of a PCR reaction liquid mixed solution and a 12-row tube or 96-hole PCR reaction plate, wherein the PCR reaction mixed solution consists of 5 XPCRBuffer (Tris-HCl pH8.5100mM, KCl 500nM, MgCl215nM), dNTPs (10mM each), Mg2+, 4 pairs of primers and hot start DNA polymerase.
The kit is characterized in that a layer of paraffin mixture is sealed on the surface of the PCR reaction mixed solution, and the paraffin mixture is formed by mixing solid paraffin and liquid paraffin according to the ratio of 1:8 (W/V).
The reaction system (25ul) of the kit is as follows: PCR reaction solution (23 ul) and template (2 ul).
Example 3:
operation and result determination of reagent kit
1. Extraction of genomic DNA
Human oral cavity inner surface cast-off cells were extracted using an oral swab, and DNA was extracted using an oral swab genomic DNA extraction kit (Tiangen Biochemical technology, cat # DP322) according to the kit instructions.
2. Preparation of the reaction System
2ul of extracted DNA is added into a 12-row tube or a 96-hole PCR reaction plate of the hepatitis B solid-phase PCR reaction kit.
3. PCR amplification
The PCR tube is put into a common PCR instrument and is programmed according to the instruction proposal for PCR amplification.
4. 1.0% agarose gel electrophoresis analysis of PCR products
5. Interpretation of results
The result interpretation standards of the kit are as follows:
example 4:
sequencing result interpretation and medication guidance
After 1 example of DNA extracted from the oral swab of a healthy person is taken as a template and amplified by using the kit, the following sequencing result is obtained, and the corresponding medication guidance is given according to the sequencing result as follows:
the foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (4)
1. The solid phase PCR kit for detecting hepatitis B is characterized by comprising a PCR reaction buffer solution, a primer group and hot start DNA polymerase;
the primer group consists of the following 4 pairs of primers:
03IL-28B*1rs12979860、04IL-28B*2rs8099917、05IL-28B*3rs12980275、116ITPA rs1127354；
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 03IL-28B x 1rs12979860 primer pair are respectively shown by SEQ ID NO.1 and SEQ ID NO. 2;
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 04IL-28B x 2rs8099917 primer pair are respectively shown by SEQ ID NO.3 and SEQ ID NO. 4;
detecting that the nucleotide sequences of the upstream primer and the downstream primer of the 05IL-28B x 3rs12980275 primer pair are respectively shown by SEQ ID NO.5 and SEQ ID NO. 6;
the nucleotide sequences of the upstream primer and the downstream primer for detecting the 116ITPA rs1127354 primer pair are respectively shown by SEQ ID NO.7 and SEQ ID NO. 8.
2. The amplification method of the solid phase PCR kit is characterized in that the solid phase PCR kit is fixed in a 96-hole PCR reaction plate or 12-row PCR reaction holes.
3. The method for amplifying a solid-phase PCR kit according to claim 2, wherein: the primer group, the hot start DNA polymerase and the PCR reaction buffer solution are fixed in a 96-hole PCR reaction plate or 12-row PCR reaction holes, and the upper-level amplification can be realized only by adding a nucleic acid template during sample adding.
4. The solid-phase PCR kit for detecting hepatitis B virus of claim 2, wherein: the concentration of the primer in the solid phase PCR kit is 100nM-1 uM.
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Citations (5)
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| CN102459647A (en) * | 2009-05-21 | 2012-05-16 | 先灵公司 | Genetic markers associated with interferon-alpha response |
| US20120282224A1 (en) * | 2009-11-09 | 2012-11-08 | Albrecht Janice K | Markers associated with ribavirin-induced anemia |
| CN107435076A (en) * | 2017-09-08 | 2017-12-05 | 银川安龙基因科技有限公司 | A kind of hypertension medication genetic test Solid phase PCR kit |
| CN108070646A (en) * | 2017-06-23 | 2018-05-25 | 安徽安龙基因医学检验所有限公司 | A kind of accurate medication genetic test Solid phase PCR kit of cardiovascular and cerebrovascular disease |
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2021
- 2021-05-25 CN CN202110569064.3A patent/CN113088572A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102459647A (en) * | 2009-05-21 | 2012-05-16 | 先灵公司 | Genetic markers associated with interferon-alpha response |
| US20120282224A1 (en) * | 2009-11-09 | 2012-11-08 | Albrecht Janice K | Markers associated with ribavirin-induced anemia |
| CN108070646A (en) * | 2017-06-23 | 2018-05-25 | 安徽安龙基因医学检验所有限公司 | A kind of accurate medication genetic test Solid phase PCR kit of cardiovascular and cerebrovascular disease |
| CN107435076A (en) * | 2017-09-08 | 2017-12-05 | 银川安龙基因科技有限公司 | A kind of hypertension medication genetic test Solid phase PCR kit |
| CN109097459A (en) * | 2018-08-16 | 2018-12-28 | 深圳道医学检验实验室 | A kind of adult based on SNP site commonly uses the detection method and its application of quo of anti-infective medicine analysis |
Non-Patent Citations (2)
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