CN107299131A - Intelligent constant-temperature amplification primer group, detection kit and method for detecting 21L858R point mutation of human EGFR gene - Google Patents
Intelligent constant-temperature amplification primer group, detection kit and method for detecting 21L858R point mutation of human EGFR gene Download PDFInfo
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Abstract
An intelligent constant-temperature amplification detection primer group, a detection kit and a method for detecting point mutation of exon 21L858R of human EGFR gene. The primer group comprises a TP primer, an FP primer, a BP primer, an OP1 primer and an OP2 primer; the detection kit comprises primer solution, reaction solution, Bst DNA polymerase, mismatch binding protein, color developing agent and contrast. The detection method is that the DNA polymerase with strand displacement activity is used, four specific primers and one mismatching binding protein are adopted to amplify the sample DNA template at 63 ℃, the pg grade of the DNA can be detected, and the identification mode is that the amplified product is added intoGreen I is detected and judged whether to be amplified or not in an ESE-tube scanner instrument, and whether the sample to be detected contains the human EGFR gene exon 21L858R point mutation DNA or not is determined. The scheme of the invention has the advantages of low cost, easy operation, rapidness, sensitivity and the like, can overcome the limitation that the traditional constant temperature amplification method can not detect gene polymorphism, accurately detects the gene polymorphism, has strong specificity and is suitable for popularization and application.
Description
Technical field
The invention belongs to technical field of molecular biology, it is related to a kind of based on intelligent constant-temperature amplification technique detection Human epidermal growth factor receptor
Primer sets, kit and the method for gene 21L858R point mutation.
Background technology
In recent years, the incidence of disease of non-small cell lung cancer (non-small cell lung cancer, NSCLC) increases year by year
Height, seriously threatens the health of the mankind.EGF-R ELISA (epidermal growth factor receptor,
EGFR) tyrosine kinase inhibitor changes the therapeutic strategy of NSCLC patient.EGFR mutation are the predictive factors of such medicine,
Played an important role in NSCLC Treatment decsion.Human epidermal growth factor acceptor (EGFR) gene extron 21L858R occurs prominent
The Patients with Non-small-cell Lung of change, to tyrosine kinase inhibitor class medicaments insensitive, i.e. receptor tyrosine kinase inhibitors medicine
It is effective in cure to these patients.L858R point mutation on exon 21 is typically to substitute mutation, and the T of 2573 is replaced by G, caused
The 858th framework amino acid of EGFR is changed into arginine (CTG-CGG) from leucine.Therefore EGFR exon 2s 1L858R points are dashed forward
The detection of change, can provide reference for screening targeted therapy patient；It can be used for the height of cancer patient, particularly patients with lung cancer simultaneously
Sensitivity early stage recurrence monitoring, and resistance mutation is monitored during medication.With the development of molecular biology, occur in that numerous
EGFR mutation detection methods based on Protocols in Molecular Biology, such as quantitative fluorescent PCR, DNA probe, DNA chip, DNA are straight
Connect sequencing etc., with its it is efficient, special the advantages of and favored；But these detection techniques are to the requirement of testing staff's technical merit
Height, and the detecting instrument that these technologies need is expensive, detection cycle is long, it is difficult to realize the mesh of quick detection and clinical expansion
's.So, a kind of reduction operating technology and expense are required in clinical detection and scientific research practice, fast and convenient, easy
Popularization, safe and reliable detection method.
The content of the invention
In view of this, technical problem solved by the invention is that providing a kind of detection Human epidermal growth factor receptor gene 21L858R points dashes forward
The intelligent constant-temperature amplimer group of change.
Technical problem solved by the invention also resides in a kind of detection Human epidermal growth factor receptor gene 21L858R point mutation intelligent constants of offer
Warm amplification detection kit.
Technical problem solved by the invention, which is also resided in, provides a kind of intelligence for detecting Human epidermal growth factor receptor gene 21L858R point mutation
Constant-temperature amplification detection method.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is as follows：
On the one hand, the invention discloses a kind of intelligent constant-temperature augmentation detection for detecting Human epidermal growth factor receptor gene 21L858R point mutation
Primer sets, including TP primers, FP primers, BP primers, OP1 primers and OP2 primers, its nucleotide sequence difference are as follows：
TP primers：CCTATCGGCATAGGATCACAGATTTGGGCG；
FP primers：GCTGGGTGCGCTCCTTACTTTGCCTCCTTC；
BP primers：AAGAGAAAGAATACCATGC；
OP1 primers：AAACACCGCAGCATGTC；
OP2 primers：CTGGCTGACCTAAAGCC.
Preferably, during amplified reaction, TP primers, FP primers, BP primers, the mol ratio of OP1 primers and OP2 primers are：8：
8：4：1：1.
On the other hand, the invention also discloses a kind of intelligent constant-temperature amplification for detecting Human epidermal growth factor receptor gene 21L858R point mutation
Contain intelligent constant-temperature amplimer group listed in such scheme in detection kit, the detection kit.
Preferably, the intelligent constant-temperature amplification detection kit is also some or all of including following component：(1) DNA gathers
Synthase；(2) mispairing associated proteins；(3) intelligent constant-temperature amplification reaction solution；(4) fluorescent color-developing agent；(5) positive control and feminine gender are right
According to.
Preferably, in the intelligent constant-temperature amplification detection kit, TP primers, FP primers, BP primers, OP1 draw in its primer
The mol ratio of thing and OP2 primers is：8：8：4：1：1.
Preferably, the archaeal dna polymerase is Bst archaeal dna polymerases；The mispairing associated proteins are Tap Muts mispairing knots
Hop protein.
Preferably, the intelligent constant-temperature amplification reaction solution contains：28mM dNTPs, 10%DMSO, 40mM Tris-HCl
(pH8.8), 20mM KCl, 20mM (NH4) 2SO4,16mM MgSO4,0.2%20。
Preferably, positive control is the carrier T containing detection Human epidermal growth factor receptor gene extron 21L858R catastrophe point genetic fragments
Clone, negative control is water or other solvents without Human epidermal growth factor receptor gene extron 21L858R catastrophe point genetic fragments.It is more excellent
Ground, negative control is deionized water.
Preferably, the developer is what 1/50000 stoste dilutedGreen I。
Further, the invention also discloses a kind of intelligent constant-temperature augmentation detection for detecting Human epidermal growth factor receptor gene 21L858R point mutation
Method, comprises the following steps：
(1) measuring samples DNA extraction；
(2) intelligent constant-temperature amplified reaction：The measuring samples DNA extracted in step (1) is added into intelligent constant-temperature amplified reaction
In system, centrifuged after mixing, and 40min is reacted in 63 DEG C；Contain institute in such scheme in the intelligent constant-temperature amplification reaction system
The intelligent constant-temperature amplimer group stated；
(3) result judges：Amplification is judged by the amplification curve for observing ESE-tubescanner amplification instruments.
Preferably, the system of intelligent constant-temperature amplified reaction used is：It is each containing TP and EP primers in 25 μ L reaction systems
Each 0.5 μm of ol/L, dNTP 0.5 mmol/L of 1.6 μm of ol/L, BP primers, 0.8 μm of ol/L, OP1 and OP2 primer, glycine betaine
0.6mol/L、(NH4)2SO4mmol/L、MgCl2 2.5mmol/L、KCl 10 mmol/L、MgSO4 2mmol/L、
Green I 0.5 μ L, pH for 8.8 the mmol/L of Tris-HCl 20,0.2%20th, 6U Bst archaeal dna polymerases, 1 μ
The measuring samples DNA extracted in g Tap Muts, 1 μ L DNA steps (1), with deionized water polishing to 25 μ L.
The intelligent constant-temperature amplification detection method of the detection Human epidermal growth factor receptor gene 21L858R point mutation of the present invention can be by this hair
Disclosed intelligent constant-temperature amplification detection kit in bright scheme is carried out, it would however also be possible to employ based on the present invention principle and
Mentality of designing is achieved using other similar intelligent constant-temperature amplification detection kits.
It is, in principle, that the present invention using intelligent constant-temperature amplification technique for rely on, develop one kind can quick detection go out Human epidermal growth factor receptor
Primer sets, kit and the method for gene 21L858R point mutation, intelligent constant-temperature TRAP (Smart amplification
Process) it is a kind of detection in gene mutation system that albumen is repaired based on strand displacement enzyme and DNA mismatch.Intelligent constant-temperature expands skill
The general principle of art, that is, it is repeated under isothermal conditions amplification after obtaining target sequence, and is monitored in real time by fluorescence,
To detect the presence of target sequence.
Therefore, understood with reference to the technique effect in above-mentioned principle and the embodiment of the present invention, the present invention is comprised at least to be had as follows
Beneficial effect：
In 5 primers designed by the present invention, the nucleotides that TP can be complementary with target sequence by anneal sequence and one section
Sequence composition, FP is with a loop-stem structure, and their annealing site is located at the two ends of target sequence respectively, can be respectively from two ends
With the complementary single-stranded annealing of 2 DNA, extend to offside.OP1 and OP2 annealing site is located on the outside of TP and FP respectively, Xiang Zhong
Between anneal extension while the obtained double-strand of TP and FP extensions is peeled off.Single-stranded with TP or FP is set to after changing and can made
For template, proceed as described above, can so form 2 kinds of crucial single-stranded intermediate products, this 2 kinds of intermediate products are then respectively with TP
Special construction with FP is starting point, carries out the DNA synthesis of self-priming.So move in circles, in the presence of archaeal dna polymerase
Cause self-loopa strand replacement reaction, realize a large amount of amplifications of target sequence.In the process, wherein combining one kind on a primer
Hybridize sensitive fluorescent primer, once with complementary series anneal, you can send the fluorescence of some strength, it is then low by cost
Honest and clean constant-temperature amplification instrument (such as ESE-Tube scanner) is detected.With the progress of amplified reaction, fluorescence intensity increases,
When the presence more than the realization that amplified reaction is can determine that when detecting minimum I values, namely target sequence.Whole process is in constant temperature bar
30-40min can be completed under part.Intelligent constant-temperature amplification technique has unique mispairing suppression technology, can recognize single nucleosides
The difference of acid, the powerful approach detected as SNP.This is also and other isothermal amplification techniques, such as ring mediation perseverance
Warm amplification, strand displacement amplification, amplification of nucleic acid sequences, transcription enzymatic amplification, rolling circle amplification, the enzymatic amplification that unwinds etc. are compared, intelligent constant-temperature
Expand the catastrophe point in clear advantage the most, these traditional constant-temperature amplification None- identified DNA fragmentations and be difficult to gene
The detection of polymorphism, and hinder popularization of the isothermal amplification technology in genetic polymorphism detection field.In addition, intelligent constant-temperature is expanded
Technology and the method for traditional detection gene pleiomorphism, such as DNA chip, RFLP PCR, allele are special
The many PCR of the opposite sex, DNA sequencing, TaqMan probe, Invader etc. are compared, and its main advantage is that this characteristic of isothermal reaction is determined
Intelligent constant-temperature TRAP has been determined without expensive operating instrument, with the clear superiority such as simple, quick, cost is low, sensitivity is high.Phase
Than in other detection techniques, the technology of the present invention has that quick, accurate, sensitive, special, background is low, low cost and other advantages, and breaks away from
Accurate requirement of the common nucleic acid detection technique to temperature control is fettered, and cheap with detecting instrument, detection time is short, reagent expense
It is low, the low advantage of technical requirements, specifically：
(1) detection time is short：It is can be achieved in 20-40min by the detection to gene polymorphism sites；
(2) Constant Temperature Detection, common mutation detection techniques need alternating temperature amplification procedure, it is therefore desirable to containing costly
The detector of alternating temperature module；This technology only need under constant temperature by special enzyme be can be achieved Constant Temperature Detection, without costliness
Instrument, it is with low cost；
(3) high specificity：Intelligent constant-temperature amplification technique has unique mispairing suppression technology, can recognize single nucleotide acid
Difference.First, reaction has used 5 asymmetric primers, when DNA and any primer mismatch, after may be prevented from
Mispairing associated proteins Taq MutS are also added into continuous amplified reaction, outer reaction system, if single base occurs in primer
Mispairing, Taq MutS are to be attached to mismatched regions, stop amplified reaction.Therefore, this technology can overcome traditional constant-temperature amplification method
The limitation of genetic polymorphism detection can not be carried out, gene pleiomorphism, high specificity can be detected exactly.
Brief description of the drawings
Fig. 1 is the sensitivity experiment result figure carried out using the kit and method of the present invention in the embodiment of the present invention 3.
Fig. 2 is the sensitivity experiment result figure carried out using quantitative fluorescent PCR in the embodiment of the present invention 3.
Fig. 3 is the result figure one of the test sample of the embodiment of the present invention 4 experiment.
Fig. 4 is the result figure two of the test sample of the embodiment of the present invention 4 experiment.
Embodiment
The present invention is further illustrated with reference to embodiments, but is not limited thereto.
The foundation of the intelligent constant-temperature amplification detection kit of embodiment 1
The intelligent constant-temperature amplification detection kit of Human epidermal growth factor receptor gene extron 21L858R point mutation, including intelligent constant-temperature expand
Increase primer sets, intelligent constant-temperature amplification reaction solution, Bst archaeal dna polymerases, mispairing associated proteins Tap Muts, positive control and feminine gender
Control, fluorescent color-developing agent.
(1) intelligent constant-temperature amplimer is designed：Drawn using Human epidermal growth factor receptor gene extron 21L858R catastrophe points as target gene
The design of thing.Primer sequence is shown in Table 1.
The primer sequence table of table 1
(2) intelligent constant-temperature amplification reaction solution contains：28mM dNTPs, 10%DMSO, 40mM Tris-HCl (pH8.8),
20mM KCl, 20mM (NH4) 2SO4,16mM MgSO4,0.2%20。
(3) positive control is the T carrier clonings containing Human epidermal growth factor receptor gene extron 21L858R catastrophe point genetic fragments, its
Preparation method is：Mutant DNA template source is utilized in the cell line H1975 for carrying L858R point mutation on EGFR exon 2s 1
OP1 and OP2 primers (SEQ ID NO in table 1：4 and SEQ ID NO：5) pcr amplification reaction is carried out to masterplate DNA, contained
The DNA of target-gene sequence, is reclaimed the amplified fragments, is connected to using conventional method in carrier T, as positive control.
(4) negative control is deionized water.
Embodiment 2 sets up Human epidermal growth factor receptor gene extron 21L858R point mutation using ESE-Quant tube scanner instrument
Detection method：
The method for detecting Human epidermal growth factor receptor gene extron 21L858R point mutation using the kit of embodiment 1, including following step
Suddenly：
(1) measuring samples DNA extraction；
(2) constant temperature gene amplification reacts：Contain each 1.6 μm of ol/L, BP primers of TP and FP primers in 25 μ L reaction systems
Each 0.5 μm of ol/L, dNTP 0.5mmol/L of 0.8 μm of ol/L, OP1 and OP2 primer, glycine betaine 0.6mol/L, (NH4)2SO4mmol/L、MgCl2 2.5mmol/L、KCl 10mmol/L、MgSO4 2 mmol/L、Green I 0.5μL、pH
For 8.8 Tris-HCl 20mmol/L, 0.2%20th, 6U Bst archaeal dna polymerases, 1 μ g Tap Muts, 1 μ L
DNA profiling, with deionized water polishing to 25 μ L；Positive control and negative control are set；By the PCR pipe prepared mix after from
The heart, and react 40min in 63 DEG C.
(3) result judges：Reaction tube is placed in ESE-Tube Scanner and reacted, ESE-Tube is observed
Scanner softwares judge amplification, are then the positive if there is " S " type curve, are then feminine gender without " S " type curve.
The detection method of embodiment 3 and the comparison of fluorescence quantitative PCR method (Taqman-MGB sonde methods) sensitivity
(1) detected using the kit of embodiment 1 and the method for embodiment 2
Prepare sample：
The DNA of the gene containing Wild type EGFR, DNA source can be serum, blood plasma, peripheral blood, oral cavity strength film, thoracic cavity product
Liquid, body fluid or tissue etc..
EGFR gene exon 2 1L858R is mutated positive DNA, and mutant DNA template source is on carrying EGFR exon 2s 1
The cell line H1975 cells of L858R point mutation.
The content ratio respectively 50% of wild type DNA and mutant DNA mixing sample, wherein mutant and wild type,
10%th, 5%, 1%, 0.5%, 0.25%.
Detected that testing result is shown in Fig. 1 using the kit of embodiment 1 and the method for embodiment 2.Experimental result table
Face, the sensitivity of the inventive method detection is up to 0.5%.
(2) fluorescence quantitative PCR method (Taqman-MGB sonde methods)
The sequence of TaqMan probe is：
5’-VIC-TTGGGCTGGCCAA-MGB-3’
5’-FAM-TTGGGCGGGCCAA-MGB-3’
5’-CCGCAGCATGTCAAGATCAC-3’
5’-TCCTTCTGCATGGTATTCTTTCTCT-3’
Quantitative fluorescent PCR reaction is carried out using ABI PCR kit for fluorescence quantitative.
Response procedures are：94 DEG C of pre-degenerations 5 minutes；94 DEG C are denatured 10 seconds；65 DEG C are annealed 10 seconds；56 DEG C extend 45 seconds；Altogether
Carry out 35 circulations；10 DEG C of terminating reactions.
Testing result is shown in Fig. 2, test result indicates that, there is detection result when sudden change sample content is 1%, sudden change sample contains
Measure for 0.5% when can not detect.
By two methods compare it can be seen from the present invention kit sensitivity result can reach 0.5% it is dense
Degree, and the sensitivity of fluorescence quantitative PCR method (Taqman-MGB sonde methods) method is 1%, and 1% or shown below as feminine gender
As a result；Through comparing, kit of the invention and method sensitivity are apparently higher than fluorescence quantitative PCR method (Taqman-MGB sonde methods)
The susceptibility of method, can detect the sample of lower loading.
Detection of the detection side of the invention of embodiment 4 to actual blood sample
(1) sample explanation：Fetch from three, Guangzhou three, 200 samples of hospital's oncology, be non-small cell lung cancer and
To the patient of tyrosine kinase inhibitor class medicaments insensitive, wherein blood sample 125,37, pleural effusion sample organizes sample
This 38.
(2) detected that specific steps and agents useful for same are referred to using the kit of embodiment 1 and the method for embodiment 2
Embodiment 1 and embodiment 2.
(3) testing result：
Table 2, pattern detection experimental result
Experimental result shows, 126 are positive sample that EGFR gene exon 2 1L858R undergos mutation in 200 samples,
Remaining is negative sample.Fig. 3 and Fig. 4 are two positive target test experiments result figures of any selection.Sent out by sequence verification
It is existing, it is that actually to have 127 be positive model in 200 samples, 73 are negative sample.Except a tissue samples are to detect sun
Outside property, the result and the result of sequence verification that other samples are detected by the solution of the present invention are corresponded, therefore, from detection
Accuracy rate from the point of view of, primer, kit and method accuracy of the invention is higher than 99%.In addition, although sample comes autoblood, group
Knit or the different sample acquisition positions such as pleural effusion, but the positive for using the kit and method of the present invention to be detected
Proportion is also very approximate in the type sample, can also absolutely prove primer sets, kit and the method for the present invention
The detection of the sample of various samples sources can be applied to exactly.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Sequence table
<110>Ji'nan University
<120>Detect intelligent constant-temperature amplimer group, detection kit and the method for Human epidermal growth factor receptor gene 21L858R point mutation
<160> 5
<210> 1
<211> 30
<212>Artificial sequence
<400> 1
CCTATCGGCATAGGATCACAGATTTGGGCG
<210> 2
<211> 30
<212>Artificial sequence
<400> 2
GCTGGGTGCGCTCCTTACTTTGCCTCCTTC
<210> 3
<211> 19
<212>Artificial sequence
<400> 3
AAGAGAAAGAATACCATGC
<210> 4
<211> 17
<212>Artificial sequence
<400> 4
AAACACCGCAGCATGTC
<210> 5
<211> 17
<212>Artificial sequence
<400> 5
CTGGCTGACCTAAAGCC
Claims (10)
1. it is a kind of detect Human epidermal growth factor receptor gene 21L858R point mutation intelligent constant-temperature amplimer group, including TP primers, FP primers,
BP primers, OP1 primers and OP2 primers, its nucleotide sequence difference are as follows：
TP primers：CCTATCGGCATAGGATCACAGATTTGGGCG；
FP primers：GCTGGGTGCGCTCCTTACTTTGCCTCCTTC；
BP primers：AAGAGAAAGAATACCATGC；
OP1 primers：AAACACCGCAGCATGTC；
OP2 primers：CTGGCTGACCTAAAGCC.
2. the intelligent constant-temperature amplimer group of Human epidermal growth factor receptor gene 21L858R point mutation, its feature are detected as claimed in claim 1
It is：During amplified reaction, TP primers, FP primers, BP primers, the mol ratio of OP1 primers and OP2 primers are：8：8：4：1：1.
3. a kind of intelligent constant-temperature amplification detection kit for detecting Human epidermal growth factor receptor gene 21L858R point mutation, it is characterised in that the inspection
Contain intelligent constant-temperature amplimer group as claimed in claim 1 or 2 in test agent box.
4. the intelligent constant-temperature amplification detection kit of detection Human epidermal growth factor receptor gene 21L858R point mutation according to claim 3,
Characterized in that, also some or all of including following component：(1) archaeal dna polymerase；(2) mispairing associated proteins；(3) intelligence
Isothermal amplification reactions liquid；(4) fluorescent color-developing agent；(5) positive control and negative control.
5. the intelligent constant-temperature amplification detection kit of detection Human epidermal growth factor receptor gene 21L858R point mutation according to claim 4,
It is characterized in that：The archaeal dna polymerase is Bst archaeal dna polymerases；The mispairing associated proteins are Tap Muts mispairing combination eggs
In vain.
6. the intelligent constant-temperature amplification detection kit of detection Human epidermal growth factor receptor gene 21L858R point mutation according to claim 4,
It is characterized in that：The intelligent constant-temperature amplification reaction solution contains：28mM dNTPs, 10%DMSO, 40mM Tris-HCl
(pH8.8)、20mM KCl、20mM(NH4)2SO4、16mM MgSO4, 0.2%20。
7. the intelligent constant-temperature amplification detection kit of detection Human epidermal growth factor receptor gene 21L858R point mutation according to claim 4,
It is characterized in that：Positive control is the carrier T gram containing detection Human epidermal growth factor receptor gene extron 21L858R catastrophe point genetic fragments
Grand, negative control is water or other solvents without Human epidermal growth factor receptor gene extron 21L858R catastrophe point genetic fragments.
8. the intelligent constant-temperature amplification detection kit of detection Human epidermal growth factor receptor gene 21L858R point mutation according to claim 4,
It is characterized in that：The fluorescent color-developing agent is what 1/50000 stoste dilutedGreen I。
9. a kind of intelligent constant-temperature amplification detection method for detecting Human epidermal growth factor receptor gene 21L858R point mutation, comprises the following steps：
(1) measuring samples DNA extraction；
(2) intelligent constant-temperature amplified reaction：The measuring samples DNA extracted in step (1) is added into intelligent constant-temperature amplification reaction system
In, centrifuged after mixing, and 40min is reacted in 63 DEG C；Contain in the intelligent constant-temperature amplification reaction system such as the institute of claim 1 or 2
The intelligent constant-temperature amplimer group stated；
(3) result judges：Amplification is judged by the amplification curve for observing ESE-tubescanner amplification instruments.
10. the intelligent constant-temperature amplification detection method of detection Human epidermal growth factor receptor gene 21L858R point mutation according to claim 9,
Characterized in that, the system of intelligent constant-temperature amplified reaction used is：Contain each 1.6 μ of TP and EP primers in 25 μ L reaction systems
Each 0.5 μm of ol/L, dNTP 0.5mmol/L of 0.8 μm of ol/L, OP1 and OP2 primer of mol/L, BP primer, glycine betaine 0.6mol/L,
(NH4)2SO4mmol/L、MgCl2 2.5mmol/L、KCl 10mmol/L、MgSO4 2mmol/L、Green I 0.5μ
L, pH for 8.8 Tris-HCl 20mmol/L, 0.2%20th, 6U Bst archaeal dna polymerases, 1 μ g Tap Muts, 1 μ
The measuring samples DNA extracted in L DNA steps (1), with deionized water polishing to 25 μ L.
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