CN104152551B - A kind of compositions detecting pulmonary carcinoma hot spot mutation gene and using method thereof - Google Patents
A kind of compositions detecting pulmonary carcinoma hot spot mutation gene and using method thereof Download PDFInfo
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Abstract
The invention discloses a kind of compositions detecting pulmonary carcinoma hot spot mutation gene, described compositions comprises the primer sequence of SEQ NO:1 to SEQ NO:53, also includes the block sequence of SEQ NO:54 to SEQ NO:63.The present invention has the advantage such as high specificity, quick, safety little, simple to operate highly sensitive, contaminated to the detection method of pulmonary carcinoma related mutation, testing result has preferable accuracy and repeatability, it is especially suitable for from the body fluid such as blood plasma detecting pulmonary carcinoma and drives the hot spot mutation of gene, can diagnose in real time, non-invasively to patients with lung cancer, recurrence monitoring and curative effect evaluation, have important value.
Description
Technical field
The present invention relates to field of gene detection, particularly relate to detect pulmonary carcinoma and drive the compositions of hotspot mutation.
Background technology
Pulmonary carcinoma is a class high incidence, the malignant tumor of high mortality.Over nearly 10 years, the sickness rate of China's pulmonary carcinoma is with dead
Rate quickly increases.The site of pathological change of pulmonary carcinoma is different, according to differentiation degree and the morphological characteristic of various pulmonary carcinoma, at present by pulmonary carcinoma
Being divided into two big classes, i.e. small cell lung cancer and nonsmall-cell lung cancer, the latter includes scale cancer, adenocarcinoma and large cell carcinoma.
Research finds, the generation of pulmonary carcinoma has the biggest relation with the sudden change of EGFR, KRAS, PIK3CA focus gene.
EGFR gene is expressed in normal epithelium cell surface, and in some tumor cells normal process LAN, EGFR crosses table
Reach the transfer with tumor cell, to invade profit, poor prognosis relevant.The signal transduction pathway in EGFR downstream mainly has two: one
Ras/Raf/MEK/ERK-MAPK path, and another is PI3K/Akt/mTOR path.When after signal conduction to nucleus, draw
The increase of gene transcription level in nuclei of origin, makes cell proliferation, conversion.The exception of EGFR signal conduction is to cause kinds of tumors to occur
Reason.
K-ras gene is positioned on No. 12 the short arm of a chromosome, and 35kb belongs to a member of Ras gene family.In Ras gene,
K-Ras is on human cancer impact maximum, and it seems molecular switch: can control the path of regulating cell growth when normal；Occur
Time abnormal, then cause cell continued propagation, and stop cell self murder.It participates in the transmission of intracellular signal, when K-ras base
During because of sudden change, this gene forever activates, it is impossible to produce normal ras albumen, makes Cellular Signaling Transduction Mediated disorderly, and cell proliferation loses
Control and canceration.
The p110 catalytic subunit of PIK3CA gene code I class phosphatidylinositol-3-kinase, under normal circumstances at lung, breast
The organs such as gland, gastrointestinal and ovary all have expression, have regulation and control somatic cell proliferation, break up, many critical functions such as survival.Research
Find that PIK3CA gene produces the kinds cancer morbidities such as sudden change and pulmonary carcinoma and there is dependency, gene in the patients with lung cancer body of 4%
There is sudden change in PIK3CA.
The method of detection gene mutation at present mainly has following several:
(1) direct sequencing.Direct sequencing is to use probe carry out PCR amplification and check order amplified production and TA
Clone also checks order finally to determine the base positions of sudden change again.The method is comparatively laborious, the longest, and due to the limit of self
System causes its sensitivity the highest, can only detect the content gene mutation more than 20%.Therefore, this method is not suitable in clinic
In application and substantial amounts of clinical sample analysis.
(2) denaturing high-performance liquid chromatography (DHPLC).The principle of the method is based on the heteroduplex DNA that mispairing occurs
They are separated by the difference of feature of unwinding with the double-stranded DNA that isozygotys mated completely.Determine whether prominent by the difference of eluting peak
Become and exist.The heteroduplex fragment that DHPLC can detect that the sudden change containing single detection, inserts or lack.This method is to known
All can detect with unknown mutational site, sensitivity is about 5%, but needs to open reaction tube during detection, easily makes
Become to pollute, and positive findings not can confirm that it is the most unknown, the most also need order-checking to confirm.
(3) high-resolution melting curve (HRM).High-resolution melting curve is physical property based on nucleic acid, by full
It is incorporated into pcr amplification product, the mutation analysis gene mutation of monitoring product melting curve with dyestuff.Detection sensitivity is left 5%
The right side, can not determine its particular location for positive findings, finally also needs to be confirmed by order-checking.
(4) probe amplification retardance sudden change method (ARMS).3 ' the end end bit bases utilizing PCR primer must be with its template DNA
The complementary principle that could effectively expand, designs the specific PCR amplimer for mutational site, under strict conditions, only
There is the pcr amplification reaction when primer 3 ' base is matched with template just can be normally carried out, thus detect sudden change.By design two
5 ' end primers, one complementary with normal DNA, and one complementary with mutant DNA, suddenlys change for pure and mild property, is separately added into two kinds of primers
And 3 ' end primer carry out two parallel PCR, be combined with the primer with mutant DNA complete complementary and just can extend and obtain PCR
Amplified production, the sensitivity of this detection method is about 1%.
(5) allele specific oligonucleotide Taqman polymerase chain reaction (CAST-PCR).CAST-PCR method uses and optimizes
TaqMan probe, stops primer to be combined with wild type DNA by the MGB probe of one section of special design, selective preferential expansion
Increasing mutant DNA, the sensitivity of this detection method is 1～about 0.1%.
But the sensitivity of these technology is the highest, the poor specificity of detection, misdiagnosis rate is high, it is impossible to meet lung cancer detection
Demand.
Summary of the invention
It is an object of the invention to provide the compositions of a kind of pulmonary carcinoma focus gene noninvasive detection, said composition and user thereof
Method high specificity and highly sensitive.
For reaching above-mentioned purpose, the technical scheme is that a kind of compositions detecting pulmonary carcinoma hot spot mutation gene, described
Compositions comprises the primer sequence of SEQ NO:1 to SEQ NO:53, also includes the block sequence of SEQ NO:54 to SEQ NO:63
Row.
Wherein, the described compositions reaction system when expanding for PCR is: 1～10 × PCR buffer, 0.1～1mM
DNTPs, 0.1～1uM forward primer, 0.1～1 μM of reverse primer, 0.1～2 μM of block, 0.05～2ng/ μ L masterplate DNA,
Eva Green dyestuff 0.5～2 μ L, 0.01～0.10U/ μ l Taq DNA polymerase, 1～5mM magnesium chloride.
Wherein, described block sequence is used in pcr amplification reaction, is one section and mates completely with wild-type DNA-sequence
Can skewed popularity amplification mutant DNA sequences saltant type specific primer.
Wherein, described template DNA concentration can as little as 0.1～1ng/ μ L.
Wherein, described pulmonary carcinoma focus gene is EGFR, KRAS, PIK3CA gene wild type and EGFR, KRAS, PIK3CA base
Because of saltant type.
Wherein, described EGFR, KRAS, PIK3CA gene comprise G719A, G719S, G719C, E746_A750del (1),
E746_A750del(2)、L747_P753>S、E746_T751>I、E746_T751del、E746_T751>A、E746_S752>A、
E746_S752>V、E746_S752>D、L747_A750>P、L747_T751>Q、L747_E749del、L747_T751del、
L747_S752del、L747_A750>P、L747_P753>Q、L747_T751>S、L747_T751del、L747_T751>P、
T790M、S768I、H773_V774insH、D770_N771insG、V769_D770insASV、L858R、L861Q、G12R、
G12S、G12C、G12A、G12D、G12V、G13D、E542K、E545K、E545D、H1047L、H1047R、H1047。
Present invention also offers the using method of a kind of compositions detecting pulmonary carcinoma hot spot mutation gene, described using method
Step be:
The first step: by testing sample, negative quality-control product, positive quality control product and carry out pcr amplification reaction without masterplate comparison, its
Described in pcr amplification reaction with DNA to be measured as template, design for the specific primer in genes of interest mutational site, add simultaneously
Enter LNA lock nucleic acid to modify, by the genes of interest in DNA sample to be measured is carried out skewed popularity pcr amplification reaction；
Second step: according to Ct value in Instrumental results and the interpretation of MeltCure, it is judged that purpose in described testing sample
The catastrophe of gene.
Wherein, the specific sudden change of DNA is differentiated by amplification curve in pcr amplification reaction result and melting curve main peak.
The described interpretation according to Ct value and MeltCure judges the catastrophe of genes of interest in testing sample, and it is concrete
Step is:
1). running the calculating of CT value, no template control should be without specific amplified, or the fluorescence signal that only primer dimer causes
Strengthen；When Ct value >=45.0, showing that no template control has faint amplification, the impact on test is atomic, can continue to analyze
Test situation；
2.) run internal control, general Ct value<40.0, can continue to analyze, if Ct value>=40.0, then explanation sample DNA fall
Solve serious, be not suitable for testing needs；
3). running positive quality control product, < 45.0, the Tm value of melting curve summit is in corresponding sudden change for the Ct value of positive quality control
In the range of, illustrate that test system is normal, can continue to analyze result of the test；
4). meeting above-mentioned condition, run Tm calling program, by melting curve analysis and Ct value, interpretation sample is
No existence suddenlys change: run Tm calling program, if the Tm value of sample melting curve summit is in the range of corresponding sudden change, and
And Ct < 45, show that expanding section undergos mutation；If sample exists one of following three kinds of situations, then it is judged as feminine gender: a, without expanding
Increase；B, has amplification, Ct >=45；C, has amplification, Ct < 45, but runs Tm calling program, the Tm value of melting curve summit
Not in the range of corresponding sudden change；
5) if. do not meet above-mentioned 1st, 3 requirements, should reform this experiment；Do not meet the 2nd requirement, it is proposed that again
From sample, extract DNA, again detect.
Wherein, described DNA sample to be measured is human genome DNA, described positive quality control product be containing EGFR, KRAS,
The mixing plasmid of PIK3CA gene mutation or the plasmid that isozygotys, described negative quality-control product is not for containing EGFR, KRAS, PIK3CA base
Mixing plasmid or the plasmid that isozygotys because of sudden change.
Wherein, described DNA to be measured is mankind's normal DNA, cell DNA, tumor tissues genomic DNA, peripheral blood cells
DNA, peripheral blood serum or plasma dna, body fluid DNA or Excreta cell DNA.
Wherein, described pulmonary carcinoma focus gene is EGFR, KRAS, PIK3CA gene wild type and EGFR, KRAS, PIK3CA base
Because of saltant type.
Wherein, described EGFR, KRAS, PIK3CA gene comprise G719A, G719S, G719C, E746_A750del (1),
E746_A750del(2)、L747_P753>S、E746_T751>I、E746_T751del、E746_T751>A、E746_S752>A、
E746_S752>V、E746_S752>D、L747_A750>P、L747_T751>Q、L747_E749del、L747_T751del、
L747_S752del、L747_A750>P、L747_P753>Q、L747_T751>S、L747_T751del、L747_T751>P、
T790M、S768I、H773_V774insH、D770_N771insG、V769_D770insASV、L858R、L861Q、G12R、
G12S、G12C、G12A、G12D、G12V、G13D、E542K、E545K、E545D、H1047L、H1047R、H1047。
Wherein, the DNA concentration of described sample can as little as 0.1～1ng/ μ L.
Wherein, the detailed process of described pcr amplification reaction is divided into 2 stages:
(1) amplified reaction, 92～97 DEG C of denaturations 5～15 minutes, the polymerase chain reaction (PCR) amplification stage, 92～97 DEG C of changes
Property 10～20s, 50～65 DEG C of annealing 10～30s, 70～75 DEG C extend 10～35s, and carry out 35～55 circulations；
(2) melting curve is done, 92～97 DEG C of denaturations 0.2～5 minutes, anneal 0.2～5 minute for 20～55 DEG C, 60～97
DEG C gather fluorescence, collection fluorescence 10 per second～30 times.
The primer sequence of the present invention is as shown in Table 1 and Table 2.
Table 1 EGFR, KRAS, PIK3CA focus gene test primer sequence
Table 2 EGFR, KRAS, PIK3CA focus gene test block
Described block refers to a kind of oligonucleotide, and its 3 ' end did following modification: phosphorylation or an arm: Spacer 3,
One or more during Spacer 6 and Spacer 18 processes process, and in PCR course of reaction, do not have primer extension amplification
Function.Block as template, is designed sequence and synthesis with the wild type DNA after lock nucleic acid (LNA) modification.Block and template
The position of specific bond cover the binding site of primer, in the presence of not mutated DNA, block specificity is in combination, enters
One step improves mutant-specific primers and the specificity of mutant DNA combination.Described detection gene is EGFR, KRAS, PIK3CA
Gene wild type, EGFR, KRAS, PIK3CA genic mutation type, the mutational site that site to be detected may be contained is: G719A,
G719S、G719C、E746_A750del(1)、E746_A750del(2)、L747_P753>S、E746_T751>I、E746_
T751del、E746_T751>A、E746_S752>A、E746_S752>V、E746_S752>D、L747_A750>P、L747_T751
>Q、L747_E749del、L747_T751del、L747_S752del、L747_A750>P、L747_P753>Q、L747_T751>
S、L747_T751del、L747_T751>P、T790M、S768I、H773_V774insH、D770_N771insG、V769_
D770insASV、L858R、L861Q、G12R、G12S、G12C、G12A、G12D、G12V、G13D、E542K、E545K、E545D、
H1047L、H1047R、H1047。
The use step of the present invention and condition are as follows:
Material: plasma sample to be checked, positive quality control product, negative quality-control product.
Instrument: Lightcycler 480, nanodrop 1000, high speed centrifuge, water-bath, whirlpool concussion instrument, refrigerator,
Baking oven, autoclave.
Reagent: archaeal dna polymerase (Roche Holding Ag), 10 × PCR Buffer (Roche Holding Ag), MgCl2(Roche Holding Ag),
DNTP (TaKaRa), purified water.
Primer: all primer purity should reach electrophoresis level (PAGE) or HPLC level, without miscellaneous band.Offer combination mechanism is provided
The quality inspection of synthetic product prove, as PAGE electrophoresis result or HPLC analyze collection of illustrative plates, it was demonstrated that use PAGE or HPLC after purification
Should have obvious unimodal PAGE or HPLC purification collection of illustrative plates, concentration is that 10ng/ μ l is standby.
(1) PCR reaction system and reaction condition
The final concentration of described pcr amplification reaction system consists of: 1～10 × PCR buffer, 0.1～1mM dNTPs, 0.1
～1uM forward primer, 0.1～1 μM of reverse primer, 0.1～2 μM of block, 0.05～2ng/ μ L masterplate DNA, Eva Green dye
Material 0.5～2 μ L, 0.01～0.10U/ μ l Taq DNA polymerase, 1～5mM magnesium chloride.
(2) the sample-adding layout of PCR reaction
DNA sample to be measured, negative quality-control product, positive quality control product and all carry out 3 multiple holes detections without masterplate comparison.
After experiment terminates, carry out the analysis of testing result according to the following steps, judge (with Roche LightCyclerTM
Illustrate as a example by 480 instruments):
1. running the calculating of CT value, no template control (NTC) should be without specific amplified, or the fluorescence that only primer dimer causes
Signal strengthens (running Tm calling program, the Tm value of melting curve summit is not in the range of corresponding sudden change).When Ct value
When >=45.0, showing that no template control has faint amplification, the impact on test is atomic, can continue to analyze test situation.
2. operation internal control, general Ct value<40.0, can continue to analyze, if Ct value>=40.0, then explanation sample DNA fall
Solve serious, be not suitable for testing needs.
3. running positive quality control product (STD), < 45.0, the Tm value of melting curve summit is accordingly for the Ct value of positive quality control
In the range of sudden change, illustrate that test system is normal, can continue to analyze result of the test.
4. meeting above-mentioned condition, run Tm calling program, by melting curve analysis and Ct value, whether interpretation sample
There is sudden change.Run Tm calling program, if the Tm value of sample melting curve summit is (concrete in the range of corresponding sudden change
It is shown in Table 3), and Ct < 45, show that expanding section undergos mutation；If sample exists one of following three kinds of situations, then it is judged as the moon
Property: a, without amplification；B, has amplification, Ct >=45；C, has an amplification, Ct < 45, but runs Tm calling program, and melting curve is
The Tm value on peak is not in the range of corresponding sudden change.
If 5. not meeting above-mentioned 1st, 3 requirements, this experiment of should reforming；Do not meet the 2nd requirement, it is proposed that again from
Sample extracts DNA, again detects.
The Tm value scope of table 3 different loci sudden change
The present invention, based on real-time fluorescence quantitative PCR platform, utilizes deflection amplification round pcr (Asymmetric PCR) and melts
The specific sudden change of technology for detection DNA that solution curve analytical technology (Melting curve analysis) combines.In PCR system, with
Wild type DNA match block sequence specific containment wild type DNA cloning, deflection amplimer high specific expands specific sudden change
Template, differentiates the specific sudden change of DNA by amplification curve and melting curve main peak.The present invention can detect the gene containing detection and comprise
KRAS, BRAF, PIK3CA gene, minimum detectability is 2～5 copy mutant nucleotide sequences.The present invention detection to pulmonary carcinoma related mutation
Method has the advantage such as high specificity, quick, safety little, simple to operate highly sensitive, contaminated, and testing result has preferably
Accuracy and repeatability, be especially suitable for from the body fluid such as blood plasma detect pulmonary carcinoma drive gene hot spot mutation, can in real time, nothing
Invasively patients with lung cancer diagnosed, recur monitoring and curative effect evaluation.
The present invention is directed to, to pulmonary carcinoma, relevant focus gene design primer special and block sequence, high specificity, spirit occur
Sensitivity is high, it is possible to covers the pulmonary carcinoma mutant gene type of focus, disposably detects possible catastrophe point, precise and high efficiency.
Relative to prior art, present invention have the advantage that (1) high specificity: add and can tie with wild type specificity
The block closed；(2) highly sensitive, detection sensitivity reaches 0.005%；(3) detection process is stopped pipe reaction, reduces pollution
Probability；(4) simple to operate quickly, whole PCR course of reaction only has 90 minutes；And direct Sequencing rule needs the time of 2 days,
And operate for open pipe, it is greatly increased the probability of pollution；(5) result interpretation is the most objective, only need to be according to amplification data and melting
Curve has got final product the interpretation of paired samples gene type；(6) safety, whole system does not comprise poisonous and harmful substance, it is not necessary to PCR produces
The open pipe of thing, to testing crew and environment all non-hazardous.
Accompanying drawing explanation
Fig. 1 is pulmonary carcinoma focus detection in Gene Mutation amplification curve diagram.1 is positive curve, and 2 is negative graph.
Fig. 2 is pulmonary carcinoma focus detection in Gene Mutation melting curve figure.3 is positive curve, and 4 is negative graph.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read present disclosure, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within limited range of the present invention equally.
Embodiment 1, lung cancer patient focus gene mutation is screened
One, experiment material
1. collect 30 example pathology and be diagnosed as the tissue samples of patients with lung cancer and supporting plasma sample.Plasma sample is adopted
After collection, it is immediately placed in-80 degree refrigerators and preserves.
The most all primer purity should reach electrophoresis level (PAGE) or HPLC level, without miscellaneous band.Combination mechanism is provided to provide
The quality inspection of synthetic product proves, as PAGE electrophoresis result or HPLC analyze collection of illustrative plates, it was demonstrated that use PAGE or HPLC after purification should
Having obvious unimodal PAGE or HPLC purification collection of illustrative plates, concentration is that 10ng/ μ l is standby.
The most all reagent buy regular producer: archaeal dna polymerase (Roche Holding Ag), 10 × PCR Buffer (Roche Holding Ag),
MgCl2(Roche Holding Ag), dNTP (TaKaRa), purified water.
Two, key instrument: Roche Light CyclerTM 480, nanodrop 1000, high speed centrifuge, water-bath,
Whirlpool concussion instrument, refrigerator.
Three, experimental design
30 example tissue of patient samples and supporting preoperative blood sample is detected respectively, then to tissue sample by the present invention
Originally check order, determine the type of sudden change according to tissue samples sequencing result.
Four, reaction system and program
1. according to the existing experiment basis of laboratory and empirical studies, and reference molecule cloning experimentation guide third edition chapter 8
Polymerase chain reaction amplification in vitro DNA content and various raw-material use requirement determine basic research system.
The research system determined is as follows:
2. carry out upper machine according to above reaction system, after having tested, experimental result is carried out data process&analysis, instead
Answer program: 95 DEG C of denaturations 5 minutes, in the polymerase chain reaction (PCR) amplification stage, 95 DEG C of degeneration 10s, 60 DEG C of annealing 15s, 72 DEG C are prolonged
Stretch 25s, and carry out 50 circulations；(2) melting curve is done, 95 DEG C of denaturations 1 minute, anneal 1 minute for 40 DEG C, 65～95 DEG C of collections
Fluorescence, collection fluorescence per second 30 times.
Five, experimental result and analysis
Fig. 1 is the amplification curve obtained during pulmonary carcinoma focus detection in Gene Mutation, therein 1, i.e. and left-hand curve is positive sample
This, 2 i.e. dextrad curve is negative sample, it is seen that uses the present invention highly desirable to expand and obtains specific amplification song
Line.
Fig. 2 is the melting curve obtained during pulmonary carcinoma focus detection in Gene Mutation, therein 3, and the most single high peak profile is
Positive sample, 4 i.e. multi-peak curve is negative graph, and it is bent that the explanation of this curve uses the present invention can obtain the single melting of peak value
Line, it is possible to well distinguish negative and positive.
30 example clinical sample blood plasma abrupt climatic change results are as shown in table 4, illustrate that the present invention can effectively detect pulmonary carcinoma
The sudden change existed in the patient.
According to the pattern detection situation gathered, total sudden change in 42 mutational sites of EGFR, KRAS, PIK3CA gene in blood plasma
Rate is about 60%.
Table 4 30 example clinical sample abrupt climatic change result
The tissue samples of present invention detection is 100% with the sudden change coincidence rate of sequence measurement detection tissue samples.The present invention
The tissue samples that the plasma sample of detection detects with sequence measurement, coincidence rate more than 90%.Illustrate that the present invention may be used for tissue,
Especially in blood plasma, pulmonary carcinoma drives the detection of gene EGFR, KRAS, PIK3CA hot spot mutation.
Patients with lung cancer tissue samples and plasma sample detection comparative result are shown in Table 5.From table 5, use the present invention
Carry out blood plasma abrupt climatic change consistent with the result that tumor tissues carries out abrupt climatic change.Although the blood plasma of people is easier to obtain, but
Plasma dna content in human peripheral blood is considerably less, detects more difficult, but the present invention but highly effective can detect blood
The catastrophe of slurry DNA.
Table 5 patients with lung cancer tissue samples and plasma sample detection are compared
The present invention, based on real-time fluorescence quantitative PCR platform, utilizes deflection amplification round pcr (Asymmetric PCR) and melts
The specific sudden change of technology for detection DNA that solution curve analytical technology (Melting curve analysis) combines.In PCR system, with
Wild type DNA match block sequence specific containment wild type DNA cloning, deflection amplimer high specific expands specific sudden change
Template, differentiates the specific sudden change of DNA by amplification curve and melting curve main peak.The present invention can detect the gene containing detection and comprise
EGFR, KRAS, PIK3CA gene, minimum detectability is 2～5 copy mutant nucleotide sequences.The present invention detection to pulmonary carcinoma related mutation
Method has the advantage such as high specificity, quick, safety little, simple to operate highly sensitive, contaminated, and testing result has preferably
Accuracy and repeatability, be especially suitable for from the body fluid such as blood plasma detect pulmonary carcinoma drive gene hot spot mutation, can in real time, nothing
Invasively patients with lung cancer diagnosed, recur monitoring and curative effect evaluation.
The present invention is directed to, to pulmonary carcinoma, relevant focus gene design primer special and block sequence, high specificity, spirit occur
Sensitivity is high, it is possible to covers focus pulmonary carcinoma mutant gene type, disposably detects possible catastrophe point, precise and high efficiency.
Above-described is only the preferred embodiment of the present invention, it is noted that for those of ordinary skill in the art
For, without departing from the concept of the premise of the invention, it is also possible to make some deformation and improvement, these broadly fall into the present invention
Protection domain.
Claims (3)
1. the compositions detecting pulmonary carcinoma hot spot mutation gene, it is characterised in that described compositions comprises SEQ NO:1 to SEQ
The primer sequence of NO:53, also includes the block sequence of SEQ NO:54 to SEQ NO:63.
2. the as claimed in claim 1 compositions detecting pulmonary carcinoma hot spot mutation gene, it is characterised in that described compositions with
Reaction system when PCR expands is: 1～10 × PCR buffer, 0.1～1mM dNTPs, 0.1～1uM forward primer, 0.1
～1 μM of reverse primer, 0.1～2 μM of block, 0.05～2ng/ μ L template DNA, Eva Green dyestuff 0.5～2 μ L, 0.01～
0.10U/ μ l Taq DNA polymerase, 1～5mM magnesium chloride.
3. the compositions detecting pulmonary carcinoma hot spot mutation gene as claimed in claim 1 or 2, it is characterised in that described block sequence
Be classified as in pcr amplification reaction used, be one section mate completely with wild-type DNA-sequence can expand mutant DNA sequences by skewed popularity
Saltant type specific primer.
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| CN104774963A (en) * | 2015-04-29 | 2015-07-15 | 苏州工业园区为真生物医药科技有限公司 | Kit for detecting PIK3CA gene mutation and detecting method thereof |
| CN106834283A (en) * | 2015-11-04 | 2017-06-13 | 深圳市瀚海基因生物科技有限公司 | Primer, kit and the method for multiple target areas sequences are obtained and detected simultaneously |
| CN105713965A (en) * | 2015-11-23 | 2016-06-29 | 宋现让 | Kit for detecting PIK3CA gene mutation |
| CN108342480B (en) * | 2018-03-05 | 2022-03-01 | 北京医院 | Gene variation detection quality control substance and preparation method thereof |
| CN108795930B (en) * | 2018-07-09 | 2020-11-24 | 博奥生物集团有限公司 | Enrichment technology for low-abundance DNA mutation and application thereof |
| CN114277114B (en) * | 2021-12-30 | 2023-08-01 | 深圳海普洛斯医学检验实验室 | Method for adding unique identifier in amplicon sequencing and application |
| CN114891883B (en) * | 2022-02-24 | 2023-10-20 | 杭州联川生物技术股份有限公司 | Primer, probe and kit for detecting lung cancer polygenic variation and application thereof |
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