CN108728536A - Primer, probe, PCR reaction solution and the kit of Human epidermal growth factor receptor gene T790M site primers - Google Patents
Primer, probe, PCR reaction solution and the kit of Human epidermal growth factor receptor gene T790M site primers Download PDFInfo
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Abstract
The present invention relates to a kind of primer, probe, PCR reaction solution and the kits of Human epidermal growth factor receptor gene T790M site primers, belong to molecular biology field.The nucleotide sequence of primer of the present invention such as SEQ ID NO:1 and SEQ ID NO:Shown in 2；Probe of the present invention includes the saltant type probe and wild-type probe for the sites Human epidermal growth factor receptor gene T790M, the nucleotide sequence such as SEQ ID NO of the saltant type probe:Shown in 3, the nucleotide sequence such as SEQ ID NO of the wild-type probe:Shown in 4；5 ' ends of the saltant type probe and wild-type probe are marked with different fluorescent reporter groups, and 3 ' ends label has and is connected with MGB modification groups；At least one base of the saltant type probe and wild-type probe also carries out lock nucleic acid modification.Primer amplification specificity of the present invention and sensitivity degree are higher；Probe of the present invention is the TaqMan MGB probes of LNA modifications, and the Tm values of probe can be improved 10 DEG C or so by MGB, and LNA modifies probe, improved the stability and affinity of DNA molecular in PCR reactions, increased specificity and sensitivity.
Description
Technical field
The present invention relates to a kind of primer, probe, PCR reaction solution and the kits of Human epidermal growth factor receptor gene T790M site primers, belong to
In molecular biology field.
Background technology
Lung cancer is the general designation of various pulmonary malignant tumours, is divided into two class Small Cell Lung Cancer (small cell lung
Carcinoma, SCLC) and non-small cell lung cancer (non-small cell lung carcinoma, NSCLC), non-small cell lung
Cancer accounts for about 80% or more of all cases of lung cancer, including two major classes：1) non-squamous carcinoma (including gland cancer, large cell carcinoma and other are sub-
Type)；2) squamous cell (epiderm-like) cancer.Lung cancer is one of highest malignant tumour of morbidity and mortality, position in world wide
It occupies first of China's urban population Death Cause for Malignant Tumors.
The treatment of malignant tumour includes chemotherapy and targeted therapy, and traditional tumor chemical therapy has treatment targeting not
Clear, the features such as selectivity is not strong and is also easy to produce drug resistance, causes efficient low and damage while killing tumor cell at it
The shortcomings of normal tissue cell.Targeted therapy refer to antitumor drug targeting occur with the different characteristics target site of tumour
It acts on and kills tumour cell, targeted therapy normal tissue influences smaller.In recent years, ideas of cancer therapy generates revolutionary
Variation is the individualized treatment of tumour.Tumor individual therapy is according to each individual tumors biological property and genomics
It is targetedly treated with the difference in transcription group, curative effect is made to maximize and toxicity minimumization.Targeted therapy is undoubtedly at present
It is that lung cancer is effective and the lower treatment means of toxicity.
EGF-R ELISA (epidermal growth factor receptor, EGFR) is a kind of cross-film junket ammonia
Acid kinase receptor, this receptor kinase domain activate related with multi-signals conduction paths such as cancer cell multiplication, transfer and apoptosis.Lung gland
Cancer patient's EGFR gene sensitizing mutation asian ancestry crowd's positive rate is higher than Caucasia crowd.EGFR mutation occur mainly in intracellular junket
On the first four exon in the region histidine kinase (TK) (18~21), presently found TK region mutagenesis has more than 30 kinds.A generation
Epidermal growth factor recipient tyrosine kinase inhibitor (epidermal growth factor receptor tyrosine
Kinase inhibitors, EGFR-TKIs) Gefitinib, Tarceva, angstrom can replace Buddhist nun, the Afatinib in two generations, for
Advanced Non-small cell lung (non-small cell lung cancer, the NSCLC) patient of EGFR gene sensitizing mutation, in nothing
Progress existence (progressionfree survival, PFS), efficient aspect, are obviously prolonged compared with classic chemotherapy,
Life in patients and tolerance are preferable simultaneously.However, usually after targeted therapy, patient can inevitably occur acquired
Drug resistance.
Wherein, most commonly seen with the T790M mutation on 20 exons.For the quantitative inspection of EGFR gene T790M mutation
Survey can more precisely instruct clinic, and mutation abundance and the EGFR-TKI treatment of T790M is related with prognosis, therefore EGFR gene T790M
Abrupt climatic change has become the clear resistance mechanism of NSCLC patient and adjusts the important indicator that therapeutic scheme must refer to.
The detection technique of gene mutation at present mainly has sanger sequencings, high-flux sequence, ARMS-PCR detections, number
PCR is detected.It is qualitative detection that sanger sequencings, high-flux sequence, ARMS-PCR, which detect these three technologies, remolding sensitivity compared with
Difference.Digital pcr, independent of standard curve and sample for reference, directly detects copying for target sequence by the way of absolute quantitation
Shellfish number, this detection mode have higher sensitivity and specificity, accuracy.
Invention content
A species specificity is provided it is an object of the invention to overcome the deficiencies in the prior art place and sensitivity is higher
Human epidermal growth factor receptor gene T790M site primers primer, probe, PCR reaction solution and kit.
To achieve the above object, the technical solution that the present invention takes is：
In a first aspect, the present invention provides the primer of Human epidermal growth factor receptor gene T790M site primers, the nucleotides sequence of the primer
Row such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.
Above-mentioned primer is to carry out the special of specific amplified to destination region for the design of the sites Human epidermal growth factor receptor gene T790M to draw
Object.It, can for the primer that can be expanded to destination region gone out designed by the sites Human epidermal growth factor receptor gene T790M according to design of primers principle
Have multigroup, however it is not limited to specific primer of the present invention.But inventor has found in numerous primers, primer amplification of the invention
Specificity and sensitivity degree are higher, therefore the specific primer is selected from numerous primers.
Second aspect, the present invention provides the probes of Human epidermal growth factor receptor gene T790M site primers comprising is directed to Human epidermal growth factor receptor base
Because of the saltant type probe and wild-type probe in the sites T790M, the nucleotide sequence such as SEQ ID NO of the saltant type probe:3 institutes
Show, the nucleotide sequence such as SEQ ID NO of the wild-type probe:Shown in 4；The 5 ' of the saltant type probe and wild-type probe
End is marked with different fluorescent reporter groups, and 3 ' ends label has and is connected with MGB modification groups；It is described
At least one base of saltant type probe and wild-type probe also carries out lock nucleic acid (LNA) modification.
The quenching group that probe of the present invention uses is non-fluorescence quenching group (Non-Fluorescent Quencher),
Itself fluorescence is not generated, the intensity of background signal can be substantially reduced.
The probe of the present invention is the TaqMan MGB specific probes of lock nucleic acid (LNA) modification.Lock nucleic acid (LNA) is a kind of spy
Different double-ring nucleotide derivative contains one or more 2'-O, 4'-C- methylene -- D-RIBOSE nucleic acid list in structure
Body, the positions 2'-O of ribose act on forming Oxymethylene epithio methylene bridge or amine methylene by different shrinks with 4'-C
Bridge, and connect and circularize, this annular bridge has locked the N configurations of furanose C3'- inner mold, reduces the flexibility of ribose structure,
The stability for increasing phosphate backbone partial structurtes improves the stability and affinity of DNA molecular in PCR reactions, increases
Specificity and sensitivity.
Minor groove binders (minor groove binder, abbreviation MGB) are derived from the chemical base of certain antibiotic molecules
, the ditch in energy intercalation of DNA double-spiral structure forms Non-covalent binding, generally can the Tm values of few nucleotide be improved 10
DEG C or so, the length of primer or probe can be shortened, specificity is improved and brought convenience to design.MGB makes what probe designed
Success rate greatly improves, and also reduces cost.
The preferred embodiment of probe as Human epidermal growth factor receptor gene T790M site primers of the present invention, the fluorescence report
It is FAM, TET, VIC or HEX to accuse group；The non-fluorescence quenching group is NFQ.
The preferred embodiment of probe as Human epidermal growth factor receptor gene T790M site primers of the present invention, the saltant type
7th and the 11st base of probe carries out lock nucleic acid modification, and the 7th base of the wild-type probe carries out lock nucleic acid modification.
The third aspect, the present invention also provides above-mentioned primers and/or above-mentioned probe to prepare for detecting Human epidermal growth factor receptor gene
The kit in the sites T790M or the application in PCR reaction solution.
Fourth aspect, the present invention provides the PCR reaction solutions of Human epidermal growth factor receptor gene T790M site primers comprising above-mentioned spy
Needle and such as SEQ ID NO:1 and SEQ ID NO:Primer shown in 2.
The preferred embodiment of PCR reaction solution as Human epidermal growth factor receptor gene T790M site primers of the present invention, it is described
PCR reaction solution further includes the PCR premixed liquid Master Mix for droplet digital pcr.
The more preferable embodiment of PCR reaction solution as Human epidermal growth factor receptor gene T790M site primers of the present invention, it is described
In PCR reaction solution, such as SEQ ID NO:1 and SEQ ID NO:The concentration of primer shown in 2 is 20 μm/L, and the saltant type is visited
The concentration of needle and wild-type probe is 10 μm/L.
5th aspect, the present invention provides the kits of Human epidermal growth factor receptor gene T790M site primers comprising above-mentioned PCR is anti-
Answer liquid.
The preferred embodiment of kit as Human epidermal growth factor receptor gene T790M site primers of the present invention, the reagent
Box further includes DNA extracts reagents.
The preferred embodiment of kit as Human epidermal growth factor receptor gene T790M site primers of the present invention, the DNA are carried
It is Qiagen DNeasy Blood&Tissue Kit to take reagent.
The sites Human epidermal growth factor receptor gene T790M can be detected by the method for droplet digital pcr, the present inventor's EGFR gene
The method of the sites T790M droplet digital pcrs detection is：
(1) DNA is extracted from sample to be tested as template, template concentrations 20ng/ μ l~50ng/ μ l；
(2) PCR reaction solution of above-mentioned Human epidermal growth factor receptor gene T790M site primers is prepared；
(3) using the DNA of step (1) extraction as template, the droplet digital pcr detection of the sites Human epidermal growth factor receptor gene T790M is prepared
PCR specific amplified reaction systems；
(4) instrument is generated by Creator droplets and PCR specific amplified reaction systems is generated into droplet；
(5) digital pcr amplification is carried out, the response procedures of amplification are：95 DEG C, 10 minutes 1 cycles；It 94 DEG C, is denaturalized within 30 seconds,
60 DEG C, annealing in 60 seconds extends 40 cycles of progress altogether；98 DEG C, 10 minutes 1 cycles；4 DEG C of holdings；
(6) Detector droplet detectors are used to detect fluorescence signal, using MicroDropTMDigital pcr instrument matches sets of data
Analysis software (MD Plotter Data Analysis Software), which calculates, provides the copy number with inspection target molecule.
Droplet digital pcr is a kind of nucleic acid molecules absolute quantitation technology, and principle is：Standard PCR reaction systems are by micro-
After drop occurs, each droplet includes or not comprising the target molecule (DNA profiling) that one or more copies, realizes " unimolecule mould
Plate PCR amplification ", after PCR cycle after, the droplet containing nucleic acid templates can provide fluorescence signal, not template
Droplet is just generated without fluorescence signal.Finally according to Poisson distribution principle and the ratio of positive droplet, using MicroDropTM
The mating Data Analysis Software of digital pcr instrument (MD Plotter Data Analysis Software) can calculate the concentration for providing target molecule to be checked or
Copy number.Digital pcr can directly calculate the copy number of target sequence, and there is no need to depend on control sample and standard curve just
It can carry out accurate absolute quantitation detection；Further, since digital pcr only judges to expand with/without two kinds when carrying out result interpretation
Increasing state, therefore the intersection point of fluorescence signal and given threshold line need not be also detected, it is completely independent of the identification of Ct values, therefore
The reaction of digital pcr is influenced to substantially reduce by amplification efficiency, is greatly improved to the tolerance of PCR response inhabitation objects；Number
The process of PCR experiment Plays reaction system distribution can largely reduce the background with the competitive effect of target sequence
Sequence concentration.Calculate the concentration or copy number for providing target molecule to be checked.
Compared with prior art, beneficial effects of the present invention are：
(1) the TaqMan MGB probes that the present invention is modified using lock nucleic acid (LNA), the quenching group of probe is using non-fluorescence
Quenching group (Non-Fluorescent Quencher), itself does not generate fluorescence, can substantially reduce the intensity of background signal；
MGB modification groups are also associated on probe simultaneously, the Tm values of probe can be improved 10 DEG C or so so that the success of probe design
Rate greatly improves, and reduces cost；Also, lock nucleic acid (LNA) modify probe, improve PCR reaction in DNA molecular stability and
Affinity increases specificity and sensitivity.In short, using the specific probe of the present invention, specificity and sensitivity can be improved.
(2) with compared with the technology having, droplet digital pcr by the way of absolute quantitation, independent of standard curve and
Sample for reference, directly detects the copy number of target sequence, and this detection mode has higher sensitivity and specificity, accurate
Property, provide important reference frame for the accurate medication guide of lung cancer individuation, pharmacodynamic assessment and examination of curative effect.
(3) present invention is suitable for the free core of blood circulation of tumour Fresh Frozen tissue, paraffin-embedded tissue and low concentration
The detection of EGFR gene T790M mutation in acid；Ginseng is provided for the clear resistance mechanism of NSCLC patients with terminal and adjustment therapeutic scheme
It examines.
Description of the drawings
Fig. 1 is the range of linearity fitting that the present invention detects EGFR gene T790M mutant plasmids using droplet type digital pcr
Figure；
Fig. 2 is the range of linearity fitting that the present invention detects EGFR gene T790M wild plasmids using droplet type digital pcr
Figure；
Fig. 3 is EGFR gene T790M frequency of mutation Linear Fit Charts of the present invention.
Specific implementation mode
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment to the present invention
It is described further.
In following embodiments, MicroDropTMDigital pcr instrument comes from the bio tech ltd Shunde Yong Nuo.It is following
Experimental method used in embodiment is conventional method unless otherwise specified；Material as used in the following examples, reagent
Deng being commercially available unless otherwise specified.
1 primer of embodiment
A kind of embodiment of the primer of the present inventor's EGFR gene T790M site primers, Human epidermal growth factor receptor base described in the present embodiment
Because the primer of T790M site primers is as follows：
Forward primer：ATCTGCCTCACCTCCACCG (as shown in SEQ ID No.1)；
Reverse primer：AGGAGGCAGCCGAAGGG (as shown in SEQ ID No.2).
Design of primers：Human epidermal growth factor receptor (NM_005228.3) gene cds sequences are searched from NCBI, utilize primer softwares
Design can amplify the upstream and downstream primer for including the mutational sites T790M segment.According to design of primers principle, multipair draw is devised
Object.Synthesis includes such as SEQ ID NO:1 and SEQ ID NO:The multipair primer of primer shown in 2, and drawn using 6 pairs of round pcr pair
Object is tested, it is found that primer can influence the length of amplified fragments, different amplification lengths have the sensitivity of the experimental program
It influences.Also, the efficiency of different primers amplification is different.
The study found that SEQ ID NO:1 and SEQ ID NO:Specificity, the efficiency of primer amplification shown in 2 are higher, and adopt
With the sensitivity higher of the primer detection.
2 probe of embodiment
A kind of embodiment of the probe of the present inventor's EGFR gene T790M site primers, probe described in the present embodiment is by needle
To the saltant type probe (being combined with mutant DNA template) and wild-type probe in the sites Human epidermal growth factor receptor gene T790M (with wild type
DNA profiling combination) composition.The saltant type probe is Mut probe1：5'-FAM-AGCTGC+ATGA+TGAGC-MGB-NFQ-
3 ' ("+N " indicates that the base is modified for LNA) (such as SEQ ID NO:Shown in 3)；The wild-type probe is Wt probe1：5'-
VIC-AGCTGC+GTGATGAGC-MGB-NFQ-3 ' ("+N " indicates that the base is modified for LNA) (such as SEQ ID NO:Shown in 4).
I.e.：5 ' ends of saltant type probe are marked with FAM, and 3 ' ends are marked with non-fluorescence quenching group NFQ, while being also associated with MGB modifications
7th and the 11st base of group, saltant type probe carries out lock nucleic acid modification；5 ' ends of wild-type probe are marked with VIC, 3 ' ends
It is marked with MGB and NFQ, the 7th base of wild-type probe carries out lock nucleic acid modification.
The specific probe of the present embodiment and the specific primer of embodiment 1 are used cooperatively, for detecting Human epidermal growth factor receptor gene
T790M site mutations.
Embodiment 3PCR reaction solutions and kit
A kind of embodiment of the PCR reaction solution of the present inventor's EGFR gene T790M site primers, PCR described in the present embodiment
Reaction solution is as shown in table 1.Wherein, PCR premixed liquids are the MicroDrop of the bio tech ltd Shunde Yong NuoTMNumber
The mating PCR premixed liquid Master Mix of PCR instrument；Forward primer and reverse primer are as described in Example 1；Saltant type probe and open country
Raw type probe is as described in Example 2.
Table 1
| Premixed liquid Master Mix | 10ul |
| Forward primer (20 μm/L) | 1.8μl |
| Reverse primer (20 μm/L) | 1.8μl |
| Saltant type probe (10 μm/L) | 0.5μl |
| Wild-type probe (10 μm/L) | 0.5μl |
| ddH2O | 3.4μl |
| Total volume | 18μl |
In experimentation, the PCR reaction solution of table 1 is mixed with 2 μ lDNA templates, obtains the reaction of 20 μ l PCR specific amplifieds
System.
Kit described in the present embodiment includes PCR reaction solution and DNA extracts reagents described in the present embodiment, the DNA extractions
Reagent is Qiagen DNeasy Blood&Tissue Kit.
Embodiment 4
The present embodiment is using PCR reaction solution described in embodiment 3/sites kit detection Human epidermal growth factor receptor gene T790M, specifically
Detection method is：
(1) extracting genome DNA：Using Qiagen DNeasy Blood&Tissue Kit, with reference to kit specification
Extract normal peripheral blood genomic DNA and H1975 cell line dnas.The genomic DNA of acquisition is detected using NanoDrop 100
DNA purity, the integrality of detected through gel electrophoresis DNA, qubit3.0 fluorometric quantifications, DNA concentration range 20ng/ μ l~
50ng/μl。
(2) EGFR gene T790M is mutated being formulated as follows for the PCR reaction solution of droplet digital pcr detection：PCR premixed liquids
10 μ l of Master Mix, 20 μm/L sense primers, 1.8 μ l, 20 μm/L downstream primers, 1.8 μ l, 10 μm/L saltant types probe, 0.5 μ
L, 10 μm/L wild-type probes, 0.5 μ l, ddH2O 3.4 μ l, the generally 18 μ l of a reaction.
(3) EGFR gene T790M is mutated the preparation of the PCR amplification system of droplet digital pcr detection：Take 18 μ lPCR reactions
2 μ l DNA profilings are added in liquid, and gently vortex mixed is uniform.
(4) droplet generates：Prepared 20 μ l digital pcr amplification systems are added to the water phase of 8 droplet generation chip
Then Kong Zhong is added 50 μ l droplets and generates in oil to the oil phase hole of 8 droplet generation chip, it is micro- that chip is placed in Creator
Drop, which generates, prepares the micro- reaction drops of PCR in instrument (bio tech ltd Shunde Yong Nuo).
(5) the micro- reaction drops of the PCR prepared are carefully transferred on 96 hole reaction plates, are sealed with sealing plate film.
(6) 96 orifice plates sealed are put into PCR instrument, setting response procedures carry out PCR amplification, pcr amplification reaction journey
Sequence is：95 DEG C, 10 minutes 1 cycles；It 94 DEG C, is denaturalized within 30 seconds, 60 DEG C, annealing in 60 seconds extends 40 cycles of progress altogether；98 DEG C,
10 minutes 1 cycles；4 DEG C of holdings.
(7) 96 orifice plates are placed in Detector droplets detector after pcr amplification reaction (promise biotechnology has Shunde forever
Limit company) in be detected fluorescence signal, have the droplet signal detecting and measuring apparatus of nucleic acid templates that will provide fluorescence signal, do not have
The droplet signal detecting and measuring apparatus of template is just without fluorescence signal.Finally according to Poisson distribution principle and the ratio of positive droplet, use
MicroDropTMThe mating Data Analysis Software of digital pcr instrument (MD Plotter Data Analysis Software), which can calculate, provides band inspection target point
The copy number of son.
Embodiment 5
The present embodiment is using PCR reaction solution described in embodiment 3/sites kit detection Human epidermal growth factor receptor gene T790M, specifically
Detection method is：
(1) sample：Diluting EGFR gene T790M in proportion using normal human gene group DNA, to sport positive H1975 thin
Born of the same parents strain DNA, it is 50%, 10%, 1%, 0.1% frequency of mutation sample, a concentration of 50ng/ μ l to obtain the frequency of mutation.
(2) EGFR gene T790M is mutated being formulated as follows for the PCR reaction solution of droplet digital pcr detection：PCR premixed liquids
10 μ l of Master Mix, 20 μm/L sense primers, 1.8 μ l, 20 μm/L downstream primers, 1.8 μ l, 10 μm/L saltant types probe, 0.5 μ
L, 10 μm/L wild-type probes, 0.5 μ l, ddH2O 3.4 μ l, the generally 18 μ l of a reaction.
(3) EGFR gene T790M is mutated the preparation of the PCR amplification system of droplet digital pcr detection：Take 18 μ lPCR reactions
2 μ l DNA profilings are added in liquid, and gently vortex mixed is uniform.
(4) droplet generates：Prepared 20 μ l digital pcr amplification systems are added to the water phase of 8 droplet generation chip
Then Kong Zhong is added 50 μ l droplets and generates in oil to the oil phase hole of 8 droplet generation chip, it is micro- that chip is placed in Creator
Drop, which generates, prepares the micro- reaction drops of PCR in instrument (bio tech ltd Shunde Yong Nuo).
(5) the micro- reaction drops of the PCR prepared are carefully transferred on 96 hole reaction plates, are sealed with sealing plate film.
(6) 96 orifice plates sealed are put into PCR instrument, setting response procedures carry out PCR amplification, pcr amplification reaction journey
Sequence is：95 DEG C, 10 minutes 1 cycles；It 94 DEG C, is denaturalized within 30 seconds, 60 DEG C, annealing in 60 seconds extends 40 cycles of progress altogether；98 DEG C,
10 minutes 1 cycles；4 DEG C of holdings.
(7) 96 orifice plates are placed in Detector droplets detector after pcr amplification reaction (promise biotechnology has Shunde forever
Limit company) in be detected fluorescence signal, have the droplet signal detecting and measuring apparatus of nucleic acid templates that will provide fluorescence signal, do not have
The droplet signal detecting and measuring apparatus of template is just without fluorescence signal.Finally according to Poisson distribution principle and the ratio of positive droplet, use
MicroDropTMThe mating Data Analysis Software of digital pcr instrument (MD Plotter Data Analysis Software), which can calculate, provides band inspection target point
The copy number of son.
The linear model of EGFR gene T790M mutant plasmids is detected using droplet type digital pcr (ddPCR) according to the present invention
Enclose fitting result as shown in Figure 1, detection EGFR gene T790M wild plasmids range of linearity fitting result as shown in Fig. 2,
The results are shown in Figure 3 for EGFR gene T790M frequencies of mutation linear fit.As seen from Figure 1, the present invention detects EGFR gene
The range of linearity of T790M mutant plasmids and wild plasmid is 2 × 101~2 × 106copy。
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>The Guangzhou bio tech ltd Yong Nuo
The bio tech ltd Shunde Yong Nuo
<120>Primer, probe, PCR reaction solution and the kit of Human epidermal growth factor receptor gene T790M site primers
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
atctgcctca cctccaccg 19
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<400> 2
aggaggcagc cgaaggg 17
<210> 3
<211> 15
<212> DNA
<213>Artificial sequence
<400> 3
agctgcatga tgagc 15
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence
<400> 4
agctgcgtga tgagc 15
Claims (10)
1. the primer of Human epidermal growth factor receptor gene T790M site primers, which is characterized in that the nucleotide sequence of the primer such as SEQ ID
NO:1 and SEQ ID NO:Shown in 2.
2. the probe of Human epidermal growth factor receptor gene T790M site primers, which is characterized in that include for the sites Human epidermal growth factor receptor gene T790M
Saltant type probe and wild-type probe, the nucleotide sequence such as SEQ ID NO of the saltant type probe:Shown in 3, the wild type
The nucleotide sequence of probe such as SEQ ID NO:Shown in 4；5 ' ends of the saltant type probe and wild-type probe are marked with difference
Fluorescent reporter group, 3 ' end label have and be connected with MGB modification groups；The saltant type probe and
At least one base of wild-type probe also carries out lock nucleic acid modification.
3. the probe of Human epidermal growth factor receptor gene T790M site primers as claimed in claim 2, which is characterized in that the fluorescence report
Group is FAM, TET, VIC or HEX；The non-fluorescence quenching group is NFQ.
4. the probe of Human epidermal growth factor receptor gene T790M site primers as claimed in claim 2 or claim 3, which is characterized in that the saltant type
7th and the 11st base of probe carries out lock nucleic acid modification, and the 7th base of the wild-type probe carries out lock nucleic acid modification.
5. primer as described in claim 1 and/or as described in any one of claim 2~4 probe prepare for detecting people
The kit in the sites EGFR gene T790M or the application in PCR reaction solution.
6. the PCR reaction solution of Human epidermal growth factor receptor gene T790M site primers, which is characterized in that including any one of such as claim 2~4
The probe and such as SEQ ID NO:1 and SEQ ID NO:Primer shown in 2.
7. the PCR reaction solution of Human epidermal growth factor receptor gene T790M site primers as claimed in claim 6, which is characterized in that further include using
In the PCR premixed liquids of droplet digital pcr.
8. the PCR reaction solution of Human epidermal growth factor receptor gene T790M site primers as claimed in claim 7, which is characterized in that the PCR
In reaction solution, such as SEQ ID NO:1 and SEQ ID NO:The concentration of primer shown in 2 is 20 μm/L, the saltant type probe
Concentration with wild-type probe is 10 μm/L.
9. the kit of Human epidermal growth factor receptor gene T790M site primers, which is characterized in that including as described in any one of claim 6~8
PCR reaction solution.
10. the kit of Human epidermal growth factor receptor gene T790M site primers as claimed in claim 9, which is characterized in that further include DNA
Extracts reagent.
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