CN108315420A - A kind of kit for detecting hepatitis B canceration polymorphism - Google Patents
A kind of kit for detecting hepatitis B canceration polymorphism Download PDFInfo
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- CN108315420A CN108315420A CN201810299579.4A CN201810299579A CN108315420A CN 108315420 A CN108315420 A CN 108315420A CN 201810299579 A CN201810299579 A CN 201810299579A CN 108315420 A CN108315420 A CN 108315420A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- 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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The invention discloses a kind of kits for detecting hepatitis B canceration polymorphism, the polymorphic site of the hepatitis B canceration tumor susceptibility gene detection is HLA DQrs9275319 and STAT4rs2242480, and the kit includes PCR reaction solution 1, PCR reaction solution 2, the reversed amplimers of HLA DQ, HLA DQ sequencing primers, glass slide, coverslip, eluent and buffer solution.The present invention facilitates observation as a result, having the advantages that simple and practical, specific height, sensitivity are strong, being suitable for grass-roots unit and field quick detection.
Description
Technical field
The present invention relates to nucleic acid detection technique field, specifically a kind of kit for detecting hepatitis B canceration polymorphism.
Background technology
Liver cancer is the malignant tumour that whole world lethality is in third, and a kind of malignant disease that China is common.The whole world
There are about 700,000 people to die of liver cancer every year.Hepatocellular carcinoma (Hepato Cellular Carcinoma, HCC) is most normal in liver cancer
See, international cancer research institution's investigation in 2000 is shown, in HCC patient 80% with anisotropic hepatitis virus and hepatitis C virus
Poison is related.History questionnaire is bright, and 80% or more has hepatitis B medical history in Chinese hepatocarcinoma patient.The number issued according to the Ministry of Public Health
According to Chinese current total has 93,000,000 Hepatitis B patients, accounts for the one third of whole world hepatitis B patient sum or more.
After China's universal hepatitis B vaccine injection in 1992, onset of liver cancer rate is declined.But in the people being born before, about
8%~9% is positive in hepatitis B surface antigen (HBsAg).Therefore, China's Mainland will be in following 40~50 years hepatocellular carcinomas
One important public health problem.Often belong to the terminal stage of a disease since clinical manifestation occurs in primary carcinoma of liver (HCC) patient, it is dead
Rate is high, and therefore, risk of the screening high risk factor for the concurrent HCC of patient of dlinial prediction hepatitis B virus infection is conducive to disease
Early detection and treatment.
Major histocompatibility complex (MHC) participates in the cell that the adjusting of immune response, especially T cell mediate and exempts from
Epidemic disease is reacted.Human MHC is mainly human leucocyte antigen (HLA) (Human Leukocyte Antigen, HLA), is located at No. 6 dyes of people
Colour solid is divided into I class and II class.MHC class Ⅱmolecules are only expressed in a few cell, such as Dendritic Cells, bone-marrow-derived lymphocyte,
Wherein most representative is HLA-DR and HLA-DQ antigens.The T cell pair that HLA-DR ,-DQ are limited by MHC class Ⅱmolecules
The identification of exotic antigen plays in the immune responses such as mediated lymphocytes activation, antigen submission and process of immune regulation
Important role.
Genome-wide association study shows the SNPs and HCC caused by hepatic sclerosis, HBV and HCV in HLA-DQ gene clusters
Risk is related, is especially located between the HLA-DQB1 genes and HLA-DQA2 genes for having biological associations with HCC
The sites rs9275319.Signal transduction and activity factor (STAT) family are a kind of transcription factors of discovered in recent years, including
STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6.STAT families are mediating cytokine, growth factor
Start the important transcription factor of protein transcription on signal path, they all rise in the cellular elements event of many signal paths
To important function, such as cell differentiation, cell Proliferation and survival.
STAT4 is located at chromosome 2q32.2-2q32.3, is T cell, NK cells, Dendritic Cells, core macrophage etc.
The crucial constituent element of the intracellular IL-12/STAT4/IFN- γ signal transduction paths of immunoregulation, which is that IL-12 inductions are immune, to be adjusted
The key regulator that cell generates IFN-γ is controlled, the immune response of IL-12 and the differentiation of regulatory T-cell are mediated.Foreign study
It confirms, STAT4 gene pleiomorphisms participate in the generation of various autoimmune disease.STAT4 makes leucocyte by signal transduction
The induction of interleukin 12 generates IFN-γ.IFN-γ is a kind of pleiotropic cytokines, is played an important role in host defense.
Due to STAT4 gene pleiomorphisms, the IFN-γ of activation may reduce its antiviral and antitumor activity, be demonstrate,proved by studying
It is real：The single nucleotide polymorphism of G allele in STAT4 is related with the high risk of hepatitis B canceration.
HLA-DQ genes and STAT4 genes are the key that hepatitis B patient suffering from hepatic cancer tumor susceptibility genes, are further ground for the mankind
How study carefully, which reduces onset of liver cancer risk, treatment hepatitis B and liver cancer, indicates new direction.By detect HLA-DQ genes and
The parting of STAT4 genes, the Susceptible population of screening liver cancer, thus in advance to the corresponding pai n nursing of Susceptible population's progress and in advance
Anti- measure reduces onset of liver cancer risk.Therefore the Genotyping detection in the sites HLA-DQ and STAT4 is carried out for preventing hepatitis B
Canceration has very important meaning.
" goldstandard " of the hospital for Genotyping detection is PCR- direct sequencings, but the sensibility of direct sequencing is not
Height, can only detect mutant cell ratio 10-20% or more tumor tissues, for for ＆lt；10% mutant cell swells
Tumor tissue and peripheral blood, Standard PCR-direct sequencing are almost helpless.Compared to direct sequencing, pyrosequencing
Sensitivity higher, cost it is lower.
Kit in the prior art tends not to meet clinical hepatitis B canceration susceptible two gene parting when being used alone
Demand needs that two kits are used in combination, there is many operational issues and system cross-cutting issue in clinical practice；It will
Two tumor susceptibility gene kits, which are combined and optimize Primer redesign and correspond to PCR system stability, still larger improvement
Space.
Invention content
The purpose of the present invention is to provide a kind of kits for detecting hepatitis B canceration polymorphism, to solve above-mentioned background
The problem of being proposed in technology.
To achieve the above object, the present invention provides the following technical solutions：
A kind of kit for detecting hepatitis B canceration polymorphism, the polymorphic position of the hepatitis B canceration tumor susceptibility gene detection
Point is HLA-DQrs9275319 and STAT4rs2242480, and the kit includes PCR reaction solution 1, PCR reaction solution 2, HLA-
The reversed amplimers of DQ, HLA-DQ sequencing primers, glass slide, coverslip, eluent and buffer solution.
Preferably, the PCR reaction solution 1 contains HLA-DQ forward direction amplimers；The PCR reaction solution 2 contains STAT4
Positive amplimer.
Preferably, the eluent includes that eluent I, II, III, IV is as follows：
20×SSC
3mmol/L Nacl
0.3mol/L trisodium citrates
Eluent I：6 × SSC, 0.5%SDS；
Eluent II：5 × SSC, 0.1%SDS；
Eluent III：1 × SSC, 0.1%SDS；
Eluent IV：0.1 × SSC, 0.1%SDS.
Preferably, described includes buffer solution I, II, III：
The buffer solution I is as follows：0.15mol/L Nacl, 100mmol/L Tris, pH7.5；
The buffer solution II is as follows：0.15mol/L Nacl, 0.1mol/L Tris, 3% bovine serum albumin(BSA),
pH7.5；
The buffer solution III is as follows：0.1mol/L Nacl, 0.1mol/L Tris, 3% bovine serum albumin(BSA),
50mmol/LMgcl2, pH9.5.
Preferably, the reaction condition of PCR is 95 DEG C of pre-degeneration 5min, and 95 DEG C of denaturation 10s, 55 DEG C of annealing 20s, 72 DEG C are prolonged
1min is stretched, after 30 recycle, 72 DEG C extend polymerization 10min, 4 DEG C of preservations.
Preferably, the kit further includes HLA-DQ positive reference substances 1, HLA-DQ positive reference substances 2 and HLA-DQ sun
Property reference substance 3.
Preferably, the kit further includes STAT4 positive reference substances 1, STAT4 positive reference substances 2 and STAT4 positive
Reference substance 3.
Preferably, HLA-DQ no mutant homozygote plasmid and the HLA-DQ described in the HLA-DQ plasmid mixtures are wild
The quantity ratio of homozygote plasmid is 1:1.
Preferably, STAT4 no mutant homozygote plasmid and the STAT4 described in the STAT4 plasmid mixtures are wild pure
The quantity ratio of zygote plasmid is 1:1.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention can detect HLA-DQ and STAT4 gene pleiomorphisms simultaneously, and it is qualitative accurate to have, high sensitivity and specificity
Strong advantage；In addition, also having, sample treatment is simple, sequencing steps are simple, sequencing speed is fast, half an hour completes on primary
Machine reaction directly gives the advantages of detection site frequency analysis and visual result；When can monitor reaction process, reaction in real time
Between short, PCR product simple process can go up pyrophosphoric acid sequencer, easy to operate and high-throughput sample detection, and than gold mark
Quasi- method, i.e. Capillary Electrophoresis PCR sequencing method sensitivity higher, are particularly suited for the requirement of clinical examination.In conclusion of the invention
Facilitate observation as a result, with it is simple and practical, specific it is high, sensitivity is strong, suitable for the excellent of grass-roots unit and field quick detection
Point.
Specific implementation mode
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is general
The every other embodiment that logical technical staff is obtained without making creative work, belongs to protection of the present invention
Range.
In the embodiment of the present invention, a kind of kit for detecting hepatitis B canceration polymorphism, the easy sensillary base of hepatitis B canceration
Because the polymorphic site of detection is HLA-DQrs9275319 and STAT4rs2242480, the kit includes PCR reaction solution
1, the reversed amplimer of PCR reaction solution 2, HLA-DQ, HLA-DQ sequencing primers, glass slide, coverslip, eluent and buffering
Liquid；The PCR reaction solution 1 contains HLA-DQ forward direction amplimers；The PCR reaction solution 2 contains STAT4 forward direction amplimers；
The reaction condition of PCR is 95 DEG C of pre-degeneration 5min, and 95 DEG C of denaturation 10s, 55 DEG C of annealing 20s, 72 DEG C of extension 1min, 30 recycle
Afterwards, extend polymerization 10min, 4 DEG C of preservations for 72 DEG C.
The eluent includes that eluent I, II, III, IV is as follows：
20×SSC
3mmol/L Nacl
0.3mol/L trisodium citrates
Eluent I：6 × SSC, 0.5%SDS；
Eluent II：5 × SSC, 0.1%SDS；
Eluent III：1 × SSC, 0.1%SDS；
Eluent IV：0.1 × SSC, 0.1%SDS.
Described includes buffer solution I, II, III；The buffer solution I is as follows：0.15mol/L Nacl, 100mmol/L
Tris, pH7.5；The buffer solution II is as follows：0.15mol/L Nacl, 0.1mol/L Tris, 3% bovine serum albumin(BSA),
pH7.5；The buffer solution III is as follows：0.1mol/L Nacl, 0.1mol/L Tris, 3% bovine serum albumin(BSA), 50mmol/
LMgcl2, pH9.5.
The kit further includes HLA-DQ positive reference substances 1, HLA-DQ positive reference substances 2 and HLA-DQ positive reference substances
3；The kit further includes STAT4 positive reference substances 1, STAT4 positive reference substances 2 and STAT4 positive reference substances 3；It is described
The quantity ratio of HLA-DQ no mutant homozygote plasmid described in HLA-DQ plasmid mixtures and the wild homozygote plasmids of the HLA-DQ
It is 1:1；STAT4 no mutant homozygote plasmid described in the STAT4 plasmid mixtures and the wild homozygote plasmids of the STAT4
Quantity ratio be 1:1；The kit further includes blank control product, and the blank control product are sterilizing purified water.
Kit provided by the present invention for detecting hepatitis B canceration polymorphism has the advantages that：
One, the present invention can detect HLA-DQ and STAT4 gene pleiomorphisms simultaneously, and it is qualitative accurate to have, high sensitivity and spy
Anisotropic strong advantage；In addition, also having, sample treatment is simple, sequencing steps are simple, sequencing speed is fast, half an hour completes one
Secondary upper machine reaction directly gives the advantages of detection site frequency analysis and visual result；
Two, the present invention can monitor that reaction process, reaction time is short, PCR product simple process can go up pyrophosphoric acid in real time
Sequencer, easy to operate and high-throughput sample detection, and than goldstandard method, i.e. Capillary Electrophoresis PCR sequencing method sensitivity
Higher is particularly suited for the requirement of clinical examination.
Finally it should be noted that：Example preferably is applied the foregoing is merely the present invention, is not intended to restrict the invention, to the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, for those skilled in the art, still can be with
Technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features, all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in the guarantor of the present invention
Within the scope of shield.
Claims (9)
1. a kind of kit for detecting hepatitis B canceration polymorphism, which is characterized in that the hepatitis B canceration tumor susceptibility gene detection
Polymorphic site be HLA-DQrs9275319 and STAT4rs2242480, the kit includes PCR reaction solution 1, PCR anti-
Answer the reversed amplimer of liquid 2, HLA-DQ, HLA-DQ sequencing primers, glass slide, coverslip, eluent and buffer solution.
2. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 1, characterized in that the PCR
Reaction solution 1 contains HLA-DQ forward direction amplimers；The PCR reaction solution 2 contains STAT4 forward direction amplimers.
3. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 1, characterized in that described washes
De- liquid includes that eluent I, II, III, IV is as follows：
20×SSC
3mmol/L Nacl
0.3mol/L trisodium citrates
Eluent I：6 × SSC, 0.5%SDS；
Eluent II：5 × SSC, 0.1%SDS；
Eluent III：1 × SSC, 0.1%SDS；
Eluent IV：0.1 × SSC, 0.1%SDS.
4. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 1, characterized in that the packet
Include buffer solution I, II, III：
The buffer solution I is as follows：0.15mol/L Nacl, 100mmol/L Tris, pH7.5；
The buffer solution II is as follows：0.15mol/L Nacl, 0.1mol/L Tris, 3% bovine serum albumin(BSA), pH7.5；
The buffer solution III is as follows：0.1mol/L Nacl, 0.1mol/L Tris, 3% bovine serum albumin(BSA), 50mmol/
LMgcl2, pH9.5.
5. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 1, characterized in that PCR's is anti-
It is 95 DEG C of pre-degeneration 5min to answer condition, and 95 DEG C of denaturation 10s, 55 DEG C of annealing 20s, 72 DEG C of extension 1min, after 30 recycle, 72 DEG C are prolonged
Long polymerization 10min, 4 DEG C of preservations.
6. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 1, characterized in that the reagent
Box further includes HLA-DQ positive reference substances 1, HLA-DQ positive reference substances 2 and HLA-DQ positive reference substances 3.
7. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 1, characterized in that the reagent
Box further includes STAT4 positive reference substances 1, STAT4 positive reference substances 2 and STAT4 positive reference substances 3.
8. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 5, characterized in that the HLA-
The quantity ratio of HLA-DQ no mutant homozygote plasmid described in DQ plasmid mixtures and the wild homozygote plasmids of the HLA-DQ is 1:
1。
9. a kind of kit for detecting hepatitis B canceration polymorphism according to claim 6, characterized in that described
The quantity ratio of STAT4 no mutant homozygote plasmid described in STAT4 plasmid mixtures and the wild homozygote plasmids of the STAT4 is 1:
1。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810299579.4A CN108315420A (en) | 2018-04-04 | 2018-04-04 | A kind of kit for detecting hepatitis B canceration polymorphism |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810299579.4A CN108315420A (en) | 2018-04-04 | 2018-04-04 | A kind of kit for detecting hepatitis B canceration polymorphism |
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| CN108315420A true CN108315420A (en) | 2018-07-24 |
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| CN201810299579.4A Pending CN108315420A (en) | 2018-04-04 | 2018-04-04 | A kind of kit for detecting hepatitis B canceration polymorphism |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002006523A3 (en) * | 2000-07-14 | 2003-04-17 | Hoffmann La Roche | Method for detecting pre-disposition to hepatotoxicity |
| CN105821145A (en) * | 2016-05-26 | 2016-08-03 | 成都中创清科医学检验所有限公司 | Primer and method for detecting HLA-DQ gene rs9275319 site polymorphism |
| CN106086231A (en) * | 2016-08-30 | 2016-11-09 | 长沙三济生物科技有限公司 | The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit |
| CN107326102A (en) * | 2017-08-01 | 2017-11-07 | 广西中医药大学附属瑞康医院（广西中西医结合医院） | A kind of AIDS kit |
| CN107586840A (en) * | 2017-10-25 | 2018-01-16 | 长沙三济生物科技有限公司 | For detecting the primer pair and kit of hepatitis B canceration tumor susceptibility gene polymorphism |
-
2018
- 2018-04-04 CN CN201810299579.4A patent/CN108315420A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002006523A3 (en) * | 2000-07-14 | 2003-04-17 | Hoffmann La Roche | Method for detecting pre-disposition to hepatotoxicity |
| CN105821145A (en) * | 2016-05-26 | 2016-08-03 | 成都中创清科医学检验所有限公司 | Primer and method for detecting HLA-DQ gene rs9275319 site polymorphism |
| CN106086231A (en) * | 2016-08-30 | 2016-11-09 | 长沙三济生物科技有限公司 | The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit |
| CN107326102A (en) * | 2017-08-01 | 2017-11-07 | 广西中医药大学附属瑞康医院（广西中西医结合医院） | A kind of AIDS kit |
| CN107586840A (en) * | 2017-10-25 | 2018-01-16 | 长沙三济生物科技有限公司 | For detecting the primer pair and kit of hepatitis B canceration tumor susceptibility gene polymorphism |
Non-Patent Citations (1)
| Title |
|---|
| 徐望红 等: "《肿瘤流行病学》", 30 June 2017, 复旦大学出版社 * |
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Application publication date: 20180724 |