CN102690881B - For the polymerase chain reaction method of fast constant temperature detection in Gene Mutation - Google Patents
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Abstract
Provide a kind of polymerase chain reaction method for fast constant temperature detection in Gene Mutation in the present invention, described method comprises: A) provide specific primer group for target gene DNA sequence dna to be detected; B) providing package is containing the reaction system of this primer sets, and adds DNA sample to be measured; C) polymerase chain reaction is carried out at a constant temperature; D) gene test result is obtained.Described specific primer group, test kit and their purposes is additionally provided in the present invention.Method of the present invention and product can be used for simply, responsive and gene amplification fast and detection (especially mutator gene detection), have broad prospects clinically.
Description
Technical field
The present invention relates to biotechnology and medical field, be specifically related to a kind of simple, responsive and polymerase chain reaction constant temperature gene amplification method that is real-time nucleic acid amplification fast and for the special primer sets of the method and test kit.
The method of this aspect, primer and test kit can be widely used in the detection (such as including but not limited to wild-type or the mutator gene such as EGFR, KRAS, BRAF, PI3K, ALK, C-Kit, PDGFR, ABL or other oncogene) of specific DNA gene wild-type or sudden change sample in the various biological sample such as blood, cell tissue in fundamental research and clinical diagnosis, also can be used for the constant-temperature amplification of specific gene.
Background technology
A. the clinical meaning of gene test
Gene test is mainly used for the diagnosis of disease, the prevention of disease and instruct personalized medicine.Such as, to the diagnosis of mycobacterium tuberculosis infection, mainly relied on phlegm, ight soil or blood cultivation in the past, whole inspection process needed more than two weeks, and adopt now the method for gene diagnosis, not only susceptibility improves greatly, and just can obtain a result in 1 hour.The prevention of disease, detects the genotype of healthy population exactly, the risk that prediction individual is ill, and proposes guidance in life to person under inspection, avoids the generation of disease.Such as colorectal carcinoma, the defect of apc gene, DCC gene and P53 gene and intestinal cancer have close relationship.The people of these gene existing defects has the excessive risk suffering from colorectal carcinoma.By gene test, when epithelial hyperplasia is not also really evolved into colorectal carcinoma, just can find the symptom of a trend of colorectal carcinoma, take measures early to prevent, just likely prevent the generation of colorectal carcinoma.Research finds that the polymorphism of gene UGT1A1 promoter region and the toxic side effect of medicine irinotecan have dependency clinically.If blindly medication can cause the side effect of Neutrophilic granulocytopenia and diarrhoea.U.S. FDA built view patient will carry out the genotypic detection of UGT1A1 before use irinotecan.The China national Ministry of Health has also added " pharmaceutical chemicals personalized medicine gene test project " in new Clinical laboratory test catalogue.UGT1A1 genotype detection, for clinical correctly taking drugs, reduces toxic side effect, improves curative effect and has clear and definite clinical meaning.
Therefore, say from Point of View of Clinical, in the urgent need to developing more easy, sensitive and gene tester and be applicable to primer and the test kit of the method fast in this area.
B. the clinical meaning of detection in Gene Mutation
Along with modern molecular biology research deepen continuously, to disease incidence mechanism, diagnosis and control research reach molecular level, become the important topic of modern medicine from gene level Diagnosis and treatment disease.
Mutator gene changes original structure and fuction, causes original inherited character to change, and wherein a part of transgenation can cause inherited disease or have the disease even tumour of genetic predisposition.As hemophilia be the sudden change of coagulation factor gene, thalassemia is the transgenation etc. of globin.Having the essential hypertension of genetic predisposition, diabetes, Peptic Ulcers etc. is the coefficient result of polygenic variation and environmental factors.Tumour is the result of somatic cell gene sudden change.
Gene diagnosis (gene diagnosis) is exactly the technological method utilizing modern molecular biology and molecular genetics, and whether normally direct gene detection structure and expression level thereof, thus make the method for diagnosis to disease.The testing goal thing of gene diagnosis is DNA or RNA, and the former reflects the existence of gene, and the latter reflects the expression status of gene.Traditional diagnostic method mainly with the phenotypic alternation of disease for foundation, the phenotypic character of biont is gene embodiment under certain condition, and the change of gene can cause the change of various phenotype and occur disease.In recent years along with the develop rapidly of Protocols in Molecular Biology, making to carry out gene diagnosis becomes possibility.
Therefore, the detection of transgenation for the study of pathogenesis of transgenation relative disease, diagnosis, susceptibility assessment, patient medication selects and/or prognosis evaluation has very important significance.
For EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) and KRAS gene.At present, the treatment of EGFR and KRAS gene target has become advanced Non-small cell lung (Non-smallcell lung cancer, NSCLC) focus of the research of tyrosine kinase inhibitor (tyrosine kinase inhibitor, TKI) Iressa and Erlotinib pharmacological agent.Research shows, in Iressa and the significant patient of Erlotinib result for the treatment of, the incidence of EGFR genetic mutation is apparently higher than the person of failing to respond to any medical treatment.In the patient having EGFR genetic mutation, the tumour objective remission rate of Iressa and Erlotinib is apparently higher than the patient without sudden change.EGFR genetic mutation rate is 33%, and mutable site about 90% is positioned at exons 19 and the exon 21 (as L858R) in encoding tyrosine kinases district, is heterozygous mutant.The mutation expression of EGFR has important directive significance to selection treatment with tyrosine kinase inhibitors, the tumor tissues sample of excision mostly is at present for the sample detecting EGFR genetic mutation, but end-stage patients are difficult to obtain tumor tissues, the tumor tissues finding a kind of easy acquisition substitutes the detection that sample carries out EGFR genetic mutation, therefrom filter out the patient of applicable treatment with tyrosine kinase inhibitors, realize individualized treatment, extremely important to clinical position.
KRAS gene is one of important molecule in EGFR signal path, and it is the albumen of the 21kD size be positioned on No. 12 karyomit(e) p12.1 positions.Saltant type KRAS does not rely on the activation of stimulus signal, namely saltant type KRAS gene is not by the impact of upstream EGFR gene state, all the time state of activation is in, only has wild-type KRAS gene by the impact of upstream EGFR token stimulus, this is also have saltant type KRAS gene patient to resist the theoretical basis that EGFR medicine (such as Erbitux (Erbitux), dimension gram are for than (Vectibix, Panitumumab) etc.) fails to respond to any medical treatment.The point mutation of KRAS gene mainly concentrates on specific amino acid codes (the 12nd, 13,61 codons), accounts for more than 90% of all sudden changes.KRAS gene mutation can cause cell evasion apoptosis, and the incidence of this exception in the tumours such as carcinoma of the pancreas, large bowel cancer, lung cancer is higher.In carcinoma of the pancreas, the point mutation incidence of KRAS gene is up to more than 90%.
Detect KRAS gene mutation, to judging the developing of these tumours, the result for the treatment of of prognosis and understanding tumour has positive meaning.In normal human blood, detect KRAS gene mutation then point out and there is tumor susceptibility; If innocent tumour patient detects KRAS gene and to dash forward the possibility may pointed out and cancerate; The KRAS gene mutation positive then imply that the possibility of cancer return is also very high, poor prognosis; Without the tumour patient such as lung cancer, colorectal cancer of KRAS gene mutation, obvious through anti-EGFR targeted drug treatment curative effect.Current KRAS gene mutation detects and has been written into U.S.'s latest edition " NCCN colorectal cancer clinical practice guideline ".Therefore, can be filtered out by detection KRAS gene mutation state and treat effective colorectal cancer patients for anti-EGFR targeted drug, help clinician to select to formulate the most effective methods for the treatment of of tumour patient.Under normal conditions, the KRAS gene of about 60% colorectal cancer patients is all wild-type, detects, by the comprehensive therapeutic plan of individuation, efficiently can significantly to increase if all receive KRAS gene mutation.
Therefore, say from Point of View of Clinical, in the urgent need to developing more easy, sensitive and detection method of gene mutation and be applicable to primer and the test kit of the method fast in this area.
C. the detection technique of transgenation
Transgenation, mainly refers to heritable variation that DNA molecular occurs, and is the change that gene base pair composition structurally occurs or puts in order.The form of transgenation can be that replacement nucleic acid, insertion, disappearance and overlap etc. are multiple.
In current life science, the dependency of transgenation and various disease (such as tumour) becomes one of study hotspot, and detection method also develops rapidly thereupon.The application of PCR amplification technology (PCR) makes detection in Gene Mutation technology have significant progress, the molecular diagnostic techniques of detection in Gene Mutation nearly all be at present all build on PCR basis on, and the novel method derived due to PCR constantly occurs, precision of analysis, susceptibility are also greatly improved.Method representative in these methods comprises: the sequential analysis of PCR combined nucleic acid, Taqman probe real-time PCR methodology, Mutant-enriched PCR, DNA chip technology, amplification refractory mutation system etc.
the sequential analysis of PCR combined nucleic acid: this is a kind of method for determining nucleic acid sequence of PCR-based, is the basis of other fast and convenient detection mutations all, is also that check point suddenlys change the most directly, a kind of the most basic method.The template of sequencing is mainly derived from traditional round pcr, finally all needs could determine mutation type and mutated site with sequential analysis, and its efficiency can reach 100%.The ultimate principle of sequence measurement is the dideoxy dna chain end cessation method of Sanger invention.This direct Sequencing is the classical way detecting transgenation, but susceptibility is low, only has and just can be detected when mutant gene accounts for more than 25% of total gene.
mutant-enriched PCR: the method utilizes oncogene or certain sub-position of encoding of cancer suppressor gene to there is known restriction endonuclease sites (the BstNI site as KRAS gene the 12nd codon), increase with the nest-type PRC of continuous quadratic the DNA fragmentation (such as comprising the DNA fragmentation of KRAS the 12nd codon) comprising and there is enzyme site coding, with the DNA fragmentation that corresponding endonuclease digestion increases between twice amplified reaction, wild-type can not enter second time pcr amplification because of digested, saltant type then completely can enter second time pcr amplification and obtain the enrichment of product.The susceptibility of the method is about 10%.
dNA chip technology: the method is the DNA analysis new technology developed after the nineties, and it has gathered the modern techniquies such as IC computer, laser confocal scanning, fluorescence labeling probe and DNA synthesis.Its ultimate principle is be arranged in by the oligonucleotide DNA of many known arrays on 1 piece of surface-mounted integrated circuit, overlapping 1 base each other, and cover all required genes detected, fluorescently-labeled normal DNA and mutant DNA are hybridized with 2 pieces of DNA chip respectively, owing at least there is the difference of 1 base, DNA that is normal and sudden change will obtain different hybridization collection of illustrative plates, detects the fluorescent signal of two kinds of DNA moleculars generations respectively, can determine whether there is sudden change through aggregation microscope.The method is quick simply, level of automation is high.
amplification refractory mutation system (Amplification Refractory Mutation System, ARMS): for detecting known mutations gene.The method is the method for holding error-prone PCR based on primer 3 ', as primer 3 ' holds base and template mispairing that Taq enzyme amplification efficiency can be caused to decline.This method is by design two 5 ' end primer, one complementary with normal DNA, and one complementary with mutant DNA, for homozygous mutant, add these two kinds of primers respectively and 3 ' end primer carries out two parallel PCR, just extensible and obtain pcr amplification product with the primer of the complete complementation of mutant DNA.If mispairing is positioned at 3 ' end of primer, causes PCR not extend, ARMS technological borrowing multiplex PCR principle, two or more allelic mutation sites can be detected in same system simultaneously, but advantage is that reaction is by selective amplification strand gene.The susceptibility of this technology brings up to about 1%.
Above-mentioned diverse ways in specificity on all increase compared with traditional PCR, but the aspects such as sensitivity, cost performance, clinical application high-throughput await further reinforcement.Thus, in this area still in the urgent need to developing more easy, sensitive and detection method of gene mutation fast.
Summary of the invention
Main purpose of the present invention is just to provide a kind of easy, sensitive and gene tester fast.Another object of the present invention is to provide the primer being applicable to the method, and comprise and be applicable to the primer of the inventive method and the test kit of other reagent.
In a first aspect of the present invention, provide a kind of constant temperature polymerase chain reaction method for gene test, described method comprises:
A) for target gene DNA sequence dna to be detected, following 5 kinds of primers are provided around 5 different zones around its amplification gene fragment:
A external primers that () holds based on target gene 3 ';
Turnover primer inside b external primers that () holds based on target gene 3 ';
C () is based on the excitation amplimer of target gene middle part;
D () is based on the folding primer inside target gene external primers; With
E external primers that () holds based on target gene 5 ';
B) providing package is containing A) described in the polymerase chain reaction system of primer, and DNA sample to be measured is added in described reaction system, if wherein this sample is contained is double-stranded DNA, then, before being added described reaction system, sex change is carried out to this sample;
C) under the constant temperature of 50 ~ 70 DEG C, polymerase chain reaction is carried out;
D) by the constant temperature gene amplification response data of DNA to be detected or collection of illustrative plates being compared with positive control, gene test result is obtained.
In an embodiment of the invention, described DNA sample to be measured is from blood, tissue or tissue slice, culturing cell.
In a preference, described DNA sample to be measured derives from: the cell of people and peripheral blood (as whole blood, serum or blood plasma).In another preference, described DNA sample to be measured can be available from: the fresh acquisition of people or fixing surgical samples, endoscopic biopsy sample or puncture ascites pleural fluid sample.In another preference, described DNA sample to be measured derives from: people, animal or pathogenic bacteria.In another preference, described target gene is selected from: EGFR, KRAS, BRAF, PI3K, ALK, C-Kit, PDGFR and abl gene or their mutant.In another preference, described target gene is wild type gene or mutator gene.In another preference, described mutator gene is homozygous mutation or heterozygous mutant, and saltation zone is arranged in amplification gene fragment.
In yet another embodiment of the present invention, described primer is the nucleic acid primer of synthesis or the primer of peptide nucleic acid(PNA) synthesis.
In yet another embodiment of the present invention, described reaction system comprises: DNA, water, MgCl
2, 4 kinds of dNTP, damping fluid and fatty bacillus polysaccharases.
In a preference, the consumption of described fatty bacillus polysaccharase is 2 ~ 12U, preferably 4 ~ 10U, more preferably 4 ~ 6U.In another preference, described fatty bacillus polysaccharase is purchased from NEB.In another preference, described reaction system also comprises: nucleic acid dye (preferred SYBR Green I) or tagged molecule.In another preference, the sex change of described double-stranded DNA is at 90 ~ 110 DEG C, preferably at the temperature of 98 ± 0.5 DEG C, processes 3 ~ 10 minutes, preferably 5 minutes.
In yet another embodiment of the present invention, described reaction carries out 20 ~ 60 minutes at the temperature of 55 ~ 65 DEG C.In a preference, described reaction is carried out at the temperature of 60 ± 0.5 DEG C, and the reaction times is 20 ~ 50 minutes.
In yet another embodiment of the present invention, described response data or collection of illustrative plates are that method by being selected from lower group obtains: the quantitative analysis of biomarker or the quantitative analysis of fluorescent marker.
In a preference, described gene test result is selected from: determine whether, exist sudden change or sport heterozygous mutant or homozygous mutation in target gene whether the existence of sample target gene.In another preference, described positive control is: known target-gene sequence and/or comprise the sequence of known mutations, and described sequence is obtained by synthetic, genetic engineering methods is obtained or is isolated from biological sample by this area ordinary method.
In another preference, whether method of the present invention for judging the existence of target-gene sequence, positive control is target-gene sequence, if result continues positive signals (namely occurring and the same or analogous signal of positive control), then shows to there is target-gene sequence in sample.
In another preference, when method of the present invention is for judging whether there is specific sudden change in target gene, positive control is the target-gene sequence comprising this specific sudden change, if when sudden change target spot primer reaction result continues positive signals, show to there is transgenation in target gene; If during the non-positive signals of result, shown in target gene without transgenation.
In another preference, method of the present invention sports homozygous mutation or heterozygous mutant for judging in target gene, positive control is respectively the target-gene sequence comprising this specific sudden change and the normal target gene sequence not comprising sudden change, when detected result display to only have without normal gene target spot primer reaction signal suddenly change target spot primer reaction result time, what show to exist in target gene is homozygous mutation; When result display normal gene target spot primer reaction result and sudden change target spot primer reaction result all time exist time, show that transgenation existing in target gene is heterozygous mutant.
In a second aspect of the present invention, provide a kind of primer sets, it comprises:
A () holds the external primers of DNA sequence dna based on target gene 3 ';
B () holds the turnover primer inside the external primers of DNA sequence dna based on target gene 3 ';
C () is based on the excitation amplimer of target gene DNA sequence dna middle part;
D () is based on the folding primer inside target gene DNA sequence dna external primers; With
E external primers that () holds based on target gene DNA sequence dna 5 '.
In a third aspect of the present invention, provide a kind of test kit, it comprises: each member separately or in the primer sets of the present invention of mixing; One or more container.
In a preference, described test kit also optionally comprise be selected from lower group of material one or more: DNA, water, MgCl
2, 4 kinds of dNTP, nucleic acid dye, PCR damping fluid and fatty bacillus polysaccharases.In another preference, the amount of described fatty bacillus polysaccharase is 2 ~ 12U, preferably 4 ~ 10U, more preferably 4 ~ 6U.In another preference, described nucleic acid dye is any can combination with dsDNA, preferred SYBRGreen.
In a fourth aspect of the present invention, provide the purposes of primer sets of the present invention in the test kit of the polymerase chain reaction for the preparation of constant temperature gene amplification or detection.
In a preference, described test kit as previously mentioned.In another preference, described test kit for detect the existence of sample target gene whether, whether exist in target gene and sport homozygous mutation or heterozygous mutant in sudden change or target gene.
In another preference, described test kit be used for heredity or transgenation relative disease diagnosis, susceptibility assessment, patient medication select and/or prognosis evaluation.In another preference, described heredity or transgenation relative disease are selected from: cancer, hemopathy or congenital hereditary disease, and described cancer is preferred: the rectum cancer, gastrointestinal cancer, nonsmall-cell lung cancer, gastrointestinal stromal tumors (GISTs), leukemia (preferred chronic myelocytic leukemia), gland cancer etc.
In a fifth aspect of the present invention, provide the purposes of primer sets of the present invention in polymerase chain reaction, described reaction is selected from: polymerase chain reaction (PCR) amplification, loop-mediated isothermal polymerase chain reaction (PCR) amplification, symmetry or asymmetric strand polymerase chain reaction (PCR) amplification.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1: the Representative fluorescence peak figure of the positive findings adopting nucleic acid gene constant-temperature amplification method of the present invention to obtain, wherein: X-coordinate is reaction times (unit: minute), and ordinate zou is standardized fluorescence intensity level (Fluorescence (norm)).
Fig. 2: the Representative fluorescence peak figure of the negative findings adopting nucleic acid gene constant-temperature amplification method of the present invention to obtain, wherein: X-coordinate is reaction times (unit: minute), and ordinate zou is standardized fluorescence intensity level.
Fig. 3: adopt the result that nucleic acid gene constant-temperature amplification method of the present invention detects different copy number DNA, wherein: A:3000 copies; B:600 copies; C:300 copies; D:60 copies; E:30 copies; F:6 copies; G:3 copies; H:0 copies; X-coordinate is reaction times (unit: minute), and ordinate zou is standardized fluorescence intensity level.
Fig. 4: adopt the result that the instant PCR amplification method of fluorescent probe of prior art detects different copy number DNA, wherein: A:3000 copies; B:600 copies; C:300 copies; D:60 copies; E:30 copies; F:6 copies; G:3 copies; H:0 copies; X-coordinate is reaction cycle number, and ordinate zou is standardized fluorescence intensity level.
Embodiment
The present inventor researchs and develops a kind of simple, sensitive and carry out the polymerase chain reaction constant temperature gene amplification technological method of nucleic acid amplification fast through long-term and deep, and the method can be widely used in detecting normal gene and/or mutator gene in blood, cell, tissue in fundamental research and clinical diagnosis.
As used herein, term " nucleic acid gene constant-temperature amplification method ", " nucleic acid polymerase reaction ", " polymerase chain reaction of the present invention " are used interchangeably, and all refer in the present invention the nucleic acid amplification reaction adopting specific primer group, carry out at a constant temperature.
Polymerase chain reaction method for constant temperature gene amplification of the present invention comprises the steps:
A) for target gene DNA sequence dna to be detected, following primer is provided around 5 different zones around its amplification gene fragment:
A external primers that () holds based on target gene 3 ';
Turnover primer inside b external primers that () holds based on target gene 3 ';
C () is based on the excitation amplimer of target gene middle part;
D () is based on the folding primer inside target gene external primers; With
E external primers that () holds based on target gene 5 ';
B) providing package is containing A) described in the polymerase chain reaction system of primer, and DNA sample to be measured is added in described reaction system, if wherein this sample is contained is double-stranded DNA, then, before being added described reaction system, sex change is carried out to this sample;
C) at 50 ~ 70 DEG C, preferably polymerase chain reaction is carried out under the constant temperature of 55 ~ 65 DEG C.
When method of the present invention is used for constant temperature gene test, it also comprises step further on the basis of above-mentioned amplification:
D) by the constant temperature gene amplification response data of DNA to be detected or collection of illustrative plates being compared with positive control, gene test result is obtained.
Of the present invention one exemplary but nonrestrictive embodiment is as follows:
Step 1. sample collection: obtain sample, as the source of DNA detection from tissues such as human whole blood, the tissue gathered through the tissue, operation of skin penetrating collection and freezing and paraffin-embedded tissues.
Step 2. extracting genome DNA: according to methods known in the art or employing commercial reagent box, extracts genomic dna (such as adopting commercially available centrifugal column or solution-type whole blood to extract test kit to extract whole blood DNA).
Step 3.DNA purity and Concentration Testing: detect DNA purity and concentration (such as adopting spectrophotometry), and carry out denaturing treatment.
Step 4. design of primers: mainly for target gene around the different zone design 5 kinds of different primers of amplification point around 5 and probe: the folding primer inside the external primers that the turnover primer, 5 ' inside the external primers held based on target gene 3 ' and external primers is held and external primers and the excitation amplimer of middle part.
Step 5. polymerase chain amplification reaction system is prepared: in polymerase chain amplified reaction pipe, preparation comprises the reaction system of the reagent such as 5 kinds of different primers and probe, dNTP, damping fluid, fluorescent marker reagent, sample DNA, polysaccharase (preferred fat bacillus polysaccharase).
Step 6. real time aggregation enzyme chain amplified reaction: carry out real time aggregation enzyme chain amplified reaction to reaction system, reaction conditions is 60 ~ 65 DEG C, 20 ~ 60 minutes.
Step 7. interpretation of result and evaluation: by each testing sample of observation and comparison and the real-time fluorescence curves figure result of positive control adopting same detection method gained, analyze to judge whether to exist in target gene or gene and whether there is sudden change or existing sport homozygous mutation or heterozygous mutant.
Should be understood that those of ordinary skill in the art after reading this disclosure, necessary improvement or change can be carried out based on prior art and/or practical situation to this embodiment.
The preparation of DNA sample
For the DNA sample to be measured in the inventive method, by Method and Technology preparation well known to those skilled in the art and acquisition.
Sample can be obtained from such as blood sample (as whole blood, serum or blood plasma), the tissue gathered through the tissue, operation of skin penetrating collection, culture, freezing, fixing (such as formalin is fixed) or paraffin-embedded tissue, as the source of DNA, preferably from blood sample, obtain DNA.In an embodiment of the invention, DNA sample derives from: people, animal or pathogenic bacteria.
Methods known in the art can be adopted (as the people such as Sambrook " molecular cloning: lab guide " (NewYork:Cold Spring Harbor Laboratory Press,) or adopt commercial reagent box to extract genomic dna (such as adopting commercially available centrifugal column or solution-type whole blood to extract test kit to extract whole blood DNA) in sample, and the purity of gained DNA and concentration are detected (such as adopting spectrophotometry) 1989).
Design of primers
Have employed specific primer sets in the present invention, comprising: the external primers that (a) holds based on target gene 3 '; Turnover primer inside b external primers that () holds based on target gene 3 '; C () is based on the excitation amplification probe of target gene middle part; D () is based on the folding primer inside target gene external primers; (e) based on the external primers that target gene 5 ' is held.
As used herein, term " target gene " refers to target gene to be detected, this gene can be wild type gene or wherein comprises the specific gene of sudden change, the existence of this target gene whether or wherein whether exist sudden change can to the generation of the susceptibility of disease, disease, to select for the medication of disease and/or the prognosis etc. of disease relevant.The wild-type of described target gene can be selected from (but being not limited to): EGFR, KRAS, BRAF, PI3K, ALK, C-Kit, PDGFR, abl gene or other oncogene or saltant type.
As used herein, term " external primers " refers to: for the Auele Specific Primer of gene fragment end.
As used herein, term " turnover primer " refers to: between two external primers, and the sequence of its end itself is complementary, can the primer of folded in half upon itself.
As used herein, term " folding primer " refers to: between two external primers, with sequence target fragment having corresponding complementation, can with the primer of complementary.
As used herein, term " excitation amplimer " refers to: between turnover primer and folding primer, and is the primer of upstream-downstream relationship with turnover primer or folding primer.
Such as; when method of the present invention is for detecting Urogastron (EGF) acceptor gene (NM_001982) exon 21 region mutation; the primer sets of SEQ ID NOs:1-5 can be adopted to detect mutator gene, and adopt the primer sets of SEQ ID NOs:6-10 to detect normal gene.
Again such as; when method of the present invention is for detecting KRAS (NM_033360) 12 exons mutation gene; the primer sets of SEQ ID NOs:11-15 can be adopted to detect mutator gene, and adopt the primer sets of SEQ ID NOs:16-20 to detect normal gene.
Those of ordinary skill in the art, after having read specification sheets of the present invention, according to primer design method as known in the art, can design for various target gene and obtain these primers.
Reaction system and reaction process
The template DNA of conventional amount used, water, MgCl is comprised in reaction system of the present invention
2, 4 kinds of dNTP, PCR damping fluid and archaeal dna polymerases.Those of ordinary skill in the art can select the consumption of these components according to general knowledge.
In the method for the invention, preferably adopt fatty bacillus polysaccharase, the consumption of described fatty bacillus polysaccharase is 2 ~ 12U, preferably 4 ~ 10U, more preferably 4 ~ 6U.By the commercially available fatty bacillus polysaccharase of commercially available acquisition, such as, purchased from NEB company.
Also can comprise in PCR reaction system of the present invention: any nucleic acid dye, tagged molecule that can be combined with dsDNA.Described nucleic acid dye includes but not limited to: SYBR Green I.
Before DNA to be measured is added PCR system of the present invention, denaturing treatment can be carried out to DNA, such as, at the temperature of 90 ~ 110 DEG C (preferably 98 ± 0.5 DEG C), process 3 ~ 10 minutes (preferably 5 minutes).
Isothermal amplification reactions of the present invention can adopt the PCR instrument of this area routine that the Other Instruments of constant temperature or equipment maybe can be provided to carry out.Temperature of reaction is generally 50 ~ 70 DEG C, preferably 55 ~ 65 DEG C, more preferably 60 ± 0.5 DEG C.The time of reaction is generally 20 ~ 60 minutes, preferably 30 ~ 40 minutes.
Reaction result and analysis
Experimental result (as data or collection of illustrative plates) is obtained, such as (but being not limited to): the quantitative analysis of biomarker or the quantitative analysis of fluorescent marker by method conventional in this area.
After acquisition experimental result, by testing sample is compared with adopting the detected result of the positive that detects of same procedure and/or negative control DNA sample, analyze testing sample target gene situation (as quantitatively, whether exist, whether have sudden change, sport the still heterozygous mutant that isozygotys).
Described positive control can be such as: known target-gene sequence and/or comprise the sequence of known mutations, and described sequence is obtained by synthetic, genetic engineering methods is obtained or is isolated from biological sample by this area ordinary method.
Negative control can adopt without DNA sample and/or the known sample not containing sudden change.
When the existence for judging target-gene sequence whether time, positive control is target-gene sequence, if result continues positive signals (namely occurring and the same or analogous signal of positive control), then shows to there is target-gene sequence in sample.
When for judging whether there is specific sudden change in target gene, positive control is the target-gene sequence comprising this specific sudden change, if when sudden change target spot primer reaction result continues positive signals, show to there is transgenation in target gene; If during the non-positive signals of result, shown in target gene without transgenation.
When for judge to exist in target gene sport heterozygous mutant or homozygous mutation time, positive control is respectively the target-gene sequence comprising this specific sudden change and the normal target gene sequence not comprising sudden change, when result display to only have without normal gene target spot primer reaction signal suddenly change target spot primer reaction result time, what show to exist in target gene is homozygous mutation; When result display normal gene target spot primer reaction result and sudden change target spot primer reaction result all time exist time, show that transgenation existing in target gene is heterozygous mutant.
Primer sets and test kit
Additionally provide the primer sets for the inventive method in the present invention, described primer sets comprises for particular target gene design: the external primers that (a) holds based on target gene 3 '; Turnover primer inside b external primers that () holds based on target gene 3 '; C () is based on the excitation amplification probe of target gene middle part; D () is based on the folding primer inside target gene external primers; (e) based on the external primers that target gene 5 ' is held.
These primers can Individual existence or with 2,3,4 or 5 kind mixing mode exist.Mode that can be conventional in this area is prepared (as synthetic), is provided primer as described in (as solution or solid) and preservation (as deepfreeze).With suitable solvent or damping fluid, primer can be mixed with required working concentration before use.
Additionally provide a kind of test kit in the present invention, it at least comprises primer sets of the present invention and one or more container.Described test kit also optionally comprise in the reagent being selected from lower group one or more: solvent (as water), MgCl
2, dNTP, nucleic acid dye (as SYBR Green I), molecular marked compound, PCR damping fluid, archaeal dna polymerase (preferred fat bacillus polysaccharase).
The purposes of the inventive method, primer sets and test kit
Method of the present invention, primer sets and test kit by the detection to target gene or transgenation, for heredity or transgenation relative disease diagnosis, susceptibility assessment, patient medication select and/or prognosis evaluation intermediate information is provided.In the present invention, term " heredity or transgenation relative disease " includes but not limited to: cancer, hemopathy, congenital hereditary type disease.
Such as, method of the present invention can be used for cancer patients medication selection, susceptibility assessment and/or prognosis evaluation, described cancer includes but not limited to: the rectum cancer, gastrointestinal cancer, nonsmall-cell lung cancer, gastrointestinal stromal tumors (GISTs), leukemia (preferred chronic myelocytic leukemia), gland cancer etc.
In addition, primer sets of the present invention and test kit also can be used for target gene amplified reaction or in the polymerase chain reaction of alternate manner, such as polymerase chain reaction (PCR) amplification, loop-mediated isothermal polymerase chain reaction (PCR) amplification, symmetry or asymmetric strand polymerase chain reaction (PCR) amplification.
Advantage of the present invention
Method tool of the present invention has the following advantages:
1. detection sensitivity obviously strengthens, and clearly can judge whether there is target gene or mutator gene in sample according to reaction result, can be widely used in clinical, for heredity or transgenation relative disease diagnosis, susceptibility assessment, patient medication select and/or prognosis evaluation relevant information is provided;
2. testing process is simple, and step is few, and without the need to frequent variations temperature, also reduces thus the requirement of operator and reaction kit;
3. 1 to a couple of days more of the prior art in reaction times and even one week shorten greatly, and whole process (comprising DNA separation and purification) only needs 2 hours, achieves rapid detection;
4. method of the present invention can detect blood sample, thus without the need to carrying out the complex operations such as sample of tissue, reduces the requirement of sampling personnel and improves the conformability of detected object, having application prospect more widely clinically.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as the people such as Sambrook " molecular cloning: lab guide " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
the real-time detection of embodiment 1.EFGR mutator gene
Provide the detection method of a kind of Urogastron (EGF) acceptor gene (NM_001982) exon 21 region mutation in the present embodiment, it can as the method selecting treatment with tyrosine kinase inhibitors clinically.
A. detection method
step 1. sample collecting
In " technical regulation of the collection of specimens of mankind's kinds of tumor flesh tissue, process and preservation " of promulgating according to the Ministry of Health, the method for defined carries out whole blood, surgical tissue collection of specimens.
Gather 200 μ l to 1ml fresh whole bloods, be placed in the heparin tube containing EDTA antithrombotics, be stored in-20 DEG C to-80 DEG C, all can extract in general 2 ~ 3 years, the shelf time is shorter, and the quality of extraction is higher, preferably extracts in 2 months.
The sample of positive control can adopt 1970 cell strains (purchased from Shanghai Sheng Ke institute of the Chinese Academy of Sciences) containing E21 site mutation; The sample of negative control is without DNA sample.
the separation of step 2. genomic dna
Adopt the DNA extraction kit of Tiangen Inc company.Get the anticoagulation 200 μ l in step 1, add 20 μ l Proteinase K mixings.Then add 200 μ l damping fluid GB (Tiangen Inc test kit), place at 56 DEG C and become limpid to solution in 10 minutes.Add 200 μ l dehydrated alcohols to occurring flocks.(Tiangen Inc test kit) in adsorption column CB3 is added to containing the overall solution of flocks, and with 12000rpm centrifugal 30 seconds, outwell the waste liquid in collection tube, adsorption column CB3 is put into collection tube.500 μ l damping fluid GD (Tiangen Inc test kit) are added, with 12000rpm centrifugal 30 seconds in adsorption column CB3.In adsorption column CB3, add three 700 μ l rinsing liquid PW respectively wash DNA (Tiangen Inc test kit), with 12000rpm centrifugal 30 seconds.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
Proceeded to by adsorption column CB3 in 1.5ml centrifuge tube, to the unsettled dropping 50 in adsorption film mid-way μ l elutriant TB, room temperature places 2-5 minute, with 12000rpm centrifugal 2 minutes, by solution collection in centrifuge tube.Added in same adsorption column CB3 by the centrifugal solution obtained, room temperature places 2 minutes again, with 12000rpm centrifugal 2 minutes, namely obtains Genomic DNA solution.
This DNA can leave 2 ~ 8 DEG C in, if want long storage time, can be placed on-20 DEG C.
The extracting genome DNA operating process of positive control and negative control sample is the same.
step 3.DNA purity detecting
Utilize spectrophotometry (Nanodrop2000, Thermo Scientific) to detect DNA purity and concentration to measure DNA concentration, require OD
260/ OD
280≈ 1.8, each sample is diluted to 25ng/ μ l by unification.
step 4.DNA sex change
The DNA getting 100 μ g is put in PCR pipe, is heated to 98 DEG C and within 3 minutes, makes DNA sex change, for follow-up polymerase chain reaction.
the preparation of step 5.PCR system
Prepare in the PCR pipe for detecting mutator gene: 100 μM of 3 ' end external primers (each primer is purchased from the raw work in Shanghai) of 0.5 μ l, 100 μM of 3 ' end turnover primer of 0.5 μ l, 50 μM of 5 ' end external primers of 0.5 μ l, the folding primer of 50 μM of 5 ' end of 0.5 μ l, 12.5 μMs of excitation primers of 0.5 μ l.The sequence of these primers is as shown in table 1:
Table 1. mutator gene detects primer
3 ' end external primers, 3 ' end turnover primer, 5 ' end external primers, the folding primer of 5 ' end, excitation primer (each primer concentration is the same) is prepared in the PCR pipe for detecting normal gene.The sequence of these primers is as shown in table 2:
Table 2. normal gene detects primer
In the detection system of 10 μ l that primer is housed, add respectively in PCR pipe: the water of 10ng DNA, 1.125 μ l, the 25mM MgCl of 2.5 μ l
2, the 10mM dNTPs of 1.25 μ l, 200 × SYBR GreenI of 0.125 μ l, 10 × PCR damping fluid of 1.5 μ l, and add the fatty bacillus polysaccharase (purchased from NEB, 8U/ μ l) of 1 μ l.
step 6. real time aggregation enzyme chain amplified reaction
The PCR pipe accommodating PCR system is put into real time PCR instrument, and the position in record hole, each sample place 96, carry out polymerase chain amplified reaction, reaction parameter is: 60 DEG C, 45 minutes.
step 7. sequence verification
Amplified production is checked order, to verify the exactness of acquired results.
b. interpretation of result and evaluation
Judged result is carried out by the real time aggregation enzyme chain amplified reaction fluorescence curve figure observing each sample, wherein the Representative fluorescence peak figure of positive findings (namely there is sudden change) as shown in Figure 1, as shown in Figure 2, two curves in figure are respectively multiple hole detected result to the Representative fluorescence peak figure of negative findings (namely without sudden change).
Sequence verification result confirms: the sample of display positive findings is contained is really mutant nucleotide sequence, and the sample showing negative findings is contained really for without mutant nucleotide sequence.
The above results show method of the present invention can easy, accurately and effectively for the detection of EGFR mutator gene.
the real-time detection of embodiment 2.KRAS (NM_033360) 12 exons mutation gene
A. detection method
Detecting step is with embodiment 1, and positive sample is SW480 cell strain, and the sample of negative control is without DNA sample.Distinguish as shown in Table 3 and Table 4 for the primer sequence that mutator gene detects and normal gene detects:
Table 3. mutator gene detects primer
Table 4. normal gene detects primer
b. interpretation of result and evaluation
Carry out to exist in judgement sample KRAS 12 by the real time aggregation enzyme chain amplified reaction fluorescence curve figure comparing each sample and control sample whether to suddenly change, and through sequence verification.Result confirm the sample of display positive findings contained be really mutant nucleotide sequence, and the sample showing negative findings is contained really for without mutant nucleotide sequence.
The above results show method of the present invention can easy, accurately and effectively for the detection of KRAS12 mutator gene.
the methods comparison of embodiment 3. nucleic acid gene isothermal amplification technology and the instant pcr amplification technology of fluorescent probe
A. nucleic acid gene isothermal amplification technology
step 1. sample collecting
As described in Example 1, collecting whole blood is carried out.
the separation of step 2. genomic dna
As described in Example 1, the genomic dna in separating whole blood sample.
step 3.DNA purity detecting
As described in Example 1, the purity of institute's isolation of genomic DNA is detected, and dilutes.
step 4.DNA sex change
As described in Example 1, sex change is carried out to DNA, difference be add respectively in each PCR pipe different copy number DNA:3000,600,300,60,30,6,3,0 copy number, totally eight kinds of different copy numbers (as shown in Figure 3).
the PCR system preparation of step 5. real time aggregation enzyme chain amplified reaction
The each mutator gene adding concentration shown in embodiment 1 step 5 in the PCR pipe of nucleic acid gene isothermal amplification technology detects primer (as shown in table 1).
In the detection system that primer 10 μ l is housed, in PCR pipe, add 10ng DNA, the water of 1.125 μ l, the 25mM MgCl of 2.5 μ l respectively
2, the 10mM dNTPs of 1.25 μ l, the 200X SYBR Green I of 0.125 μ l, 10 × PCR damping fluid of 1.5 μ l, and add the fatty bacillus polysaccharase (purchased from NEB, 8U/ μ l) of 1 μ l.
step 6. real time aggregation enzyme chain amplified reaction
Real time aggregation enzyme chain amplified reaction is carried out in the PCR pipe of nucleic acid gene isothermal amplification technology.
The PCR pipe accommodating PCR system is put into real time PCR instrument, and the position in record hole, each sample place 96, carries out PCR reaction.Polymerase chain amplified reaction parameter is: 60 DEG C, 45 minutes.
B. the instant PCR of fluorescent probe
step 1 ~ 4
With " A. nucleic acid gene isothermal amplification technology ", except DNA denaturing step carries out when PCR.
the preparation of the instant PCR system of step 5. fluorescent probe
Be furnished with in the pipe of the instant PCR of fluorescent probe (cumulative volume is 10 μ l): 3 ' end external primers (sequence GCCAGTTAACGTCTTCCT (SEQ ID NO.:1)) of 2.5 μMs/L and 5 ' of 2.5 μMs/L holds the MgCl of DNA, 1.25mM/L of external primers (sequence C CACACAGCAAAGCAG (SEQ ID NO.:3)), 1 μ g
2, dNTPs, 1X SYBR Green I of 200 μMs/L, 1 μ l 10 × PCR damping fluid and 1.0U Taqman polysaccharase (purchased from ABI), and the water of surplus.
the instant PCR reaction of step 6. fluorescent probe
Real time aggregation enzyme chain amplified reaction is carried out in the instant PCR pipe of fluorescent probe.PCR pipe is put into real time PCR instrument, the position in record hole, each sample place 96.Polymerase chain amplified reaction parameter be sex change 95 DEG C, 10 minutes, and 30 circulation 95 DEG C, 30 seconds, annealing 56 DEG C, 30 seconds, extend 72 DEG C, 30 seconds, then continue 72 DEG C, 10 minutes.
C. interpretation of result and comparing
As shown in Figure 3, the result of the instant PCR reaction of fluorescent probe as shown in Figure 4 for the result of real time aggregation enzyme chain amplified reaction of the present invention.
The result display of Fig. 3: 1) real time aggregation enzyme chain amplification reaction method of the present invention is stablized, and it is low to 3 copy numbers to detect DNA amount; 2) owing to being added with the accurate primer of height for catastrophe point in this reaction primer, therefore reaction result is variants.
The result display of Fig. 4: 1) adopt fluorescent probe instant pcr amplification EGFR gene to be merely able to detect that DNA measures low copy number between 300 to 60; 2) due to the accurate primer of the height of needleless to catastrophe point in this reaction primer, thus reaction result uncertain be normal gene or mutator gene.
Above result proves: the sensitivity of polymerase chain reaction constant temperature gene amplification technology of the present invention is obviously better than the instant pcr amplification technology of fluorescent probe, and whether can make judgement directly perceived and clear and definite to transgenation.
Further, more known by reaction process parameter: the reaction times of method of the present invention is short (only 45 minutes), and temperature of reaction keeps constant (65 DEG C); And the long reaction time of the instant PCR of fluorescent probe, and frequently need change temperature.Demonstrate thus: method of the present invention has easy, efficient advantage.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (14)
1., for a constant temperature polymerase chain reaction method for gene test, described method comprises:
A) for target gene DNA sequence dna to be detected, following 5 kinds of primers are provided around 5 different zones around its amplification gene fragment:
A external primers that () holds based on target gene 3 ';
Turnover primer inside b external primers that () holds based on target gene 3 ';
C () is based on the excitation amplimer of target gene middle part;
D () is based on the folding primer inside target gene external primers; With
E external primers that () holds based on target gene 5 ';
B) providing package is containing A) described in the polymerase chain reaction system of primer, and DNA sample to be measured is added in described reaction system, if wherein this sample is contained is double-stranded DNA, then before being added described reaction system, carry out sex change to this sample, the enzyme in the system of wherein said polymerase chain reaction is fatty bacillus polysaccharase;
C) under the constant temperature of 50 ~ 70 DEG C, polymerase chain reaction is carried out;
D) by the constant temperature gene amplification response data of DNA to be detected or collection of illustrative plates being compared with positive control, gene test result is obtained,
Wherein, described method is used for non-diseases diagnostic purpose.
2. the method for claim 1, is characterized in that, described DNA sample to be measured is from blood, tissue or tissue slice, culturing cell.
3. the method for claim 1, is characterized in that, described primer is the nucleic acid primer of synthesis or the primer of peptide nucleic acid(PNA) synthesis.
4. the method for claim 1, is characterized in that, described reaction system comprises: DNA, water, MgCl
2, 4 kinds of dNTP, damping fluid and fatty bacillus polysaccharases.
5. the method for claim 1, is characterized in that, described reaction carries out 20 ~ 60 minutes at the temperature of 55 ~ 65 DEG C.
6. the method for claim 1, is characterized in that, described response data or collection of illustrative plates are that the method by being selected from lower group obtains: the quantitative analysis of biomarker or the quantitative analysis of fluorescent marker.
7. a test kit, it comprises:
Each member separately or in the following primer sets of mixing:
A external primers that () holds based on target gene 3 ';
Turnover primer inside b external primers that () holds based on target gene 3 ';
C () is based on the excitation amplimer of target gene middle part;
D () is based on the folding primer inside target gene external primers; With
E external primers that () holds based on target gene 5 ';
Fat bacillus polysaccharase; With
One or more container.
8. fatty bacillus polysaccharase and the application of primer in the product of the constant temperature polymerase chain reaction method for the preparation of gene test for target gene DNA sequence dna to be detected in DNA sample to be measured, wherein said primer around 5 different zones around its amplification gene fragment, and is:
A external primers that () holds based on target gene 3 ';
Turnover primer inside b external primers that () holds based on target gene 3 ';
C () is based on the excitation amplimer of target gene middle part;
D () is based on the folding primer inside target gene external primers; With
E external primers that () holds based on target gene 5 '.
9. apply as claimed in claim 8, it is characterized in that, the constant temperature polymerase chain reaction method of described product for comprising the steps:
A) primer (a) ~ (e) is provided;
B) providing package is containing A) described in the polymerase chain reaction system of primer, and DNA sample to be measured is added in described reaction system, if wherein this sample is contained is double-stranded DNA, then before being added described reaction system, carry out sex change to this sample, the enzyme in the system of wherein said polymerase chain reaction is described fatty bacillus polysaccharase;
C) under the constant temperature of 50 ~ 70 DEG C, polymerase chain reaction is carried out;
D) by the constant temperature gene amplification response data of DNA to be detected or collection of illustrative plates being compared with positive control, gene test result is obtained.
10. apply as claimed in claim 8, it is characterized in that, described DNA sample to be measured is from blood, tissue or tissue slice, culturing cell.
11. apply as claimed in claim 8, it is characterized in that, described primer is the nucleic acid primer of synthesis or the primer of peptide nucleic acid(PNA) synthesis.
12. apply as claimed in claim 9, it is characterized in that, described reaction system comprises: DNA, water, MgCl
2, 4 kinds of dNTP, damping fluid and fatty bacillus polysaccharases.
13. apply as claimed in claim 9, it is characterized in that, described reaction carries out 20 ~ 60 minutes at the temperature of 55 ~ 65 DEG C.
14. apply as claimed in claim 9, it is characterized in that, described response data or collection of illustrative plates are that the method by being selected from lower group obtains: the quantitative analysis of biomarker or the quantitative analysis of fluorescent marker.
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| PCT/CN2012/072929 WO2012130102A1 (en) | 2011-03-25 | 2012-03-23 | Polymerase chain reaction method for detecting genes and gene mutation rapidly at constant temperature |
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