CN109266723A - Rare mutation detection method, its kit and application - Google Patents
Rare mutation detection method, its kit and application Download PDFInfo
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Abstract
The invention discloses a kind of rare mutation detection method, its kit and application, the method is U-ARMS method.Detection method provided by the present invention combines ARMS and lock nucleic acid (LNA) mutation enrichment and Taqman-MGB detection technique of fluorescence, enhance mutating alkali yl recognition capability using LNA, ARMS primer pair mutated target sequence carries out specific PCR amplification, Taqman-MGB probe detects amplified production, specific mutation is identified on the basis of fluorescent PCR, compared with the mutation detection techniques such as such method and sanger be sequenced, chip detection, the sequencing of two generations, the present invention has that at low cost, high specificity, sensitivity be high, the advantages such as quickly easy to operate for detection in Gene Mutation.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of rare mutation detection method, its kit and
Using.
Background technique
Polymerase chain reaction (PCR) in the Kary B Mullis teaching inventive by heredity portion of PE company of the U.S. in 1985,
Primary great leap is brought for gene amplification technology.Since this technology can pass through 20-50 by becoming in 1.5-3 hours
Property, annealing and extend three-step reaction constitute circulation specifically amplifying target nucleic acid sequence is to millions of times, therefore, short several years
It is interior to be just used widely in fields such as molecular biology, medicine, microbiology, science of heredity.
Nucleic acid is the key substance for determining hereditary information, with the continuous progress of life science, it has been found that pass through
It whether there is certain (a little) nucleic acid and its sequence variations in test sample, so that it may judge whether test object carries certain disease
Pathogenic microorganism and its drug resistance suffer from certain disease or in certain genetic state etc..Rare mutation refers to a large amount of wild type bases
Because of few mutated gene under background, the especially gene of single base mutation.The ratio of general mutated gene and wild type gene
Rate is less than 1/1000.Such as the micro fetus mutated gene contained in pregnant woman's peripheral circulation blood, in tumour patient blood or tumour
The a small amount of tumour somatic mutation contained in tissue.
Rare detection method of gene mutation commonly used at present is DNA direct Sequencing, two generation PCR sequencing PCRs (NGS) and amplification
Hinder abruptly-changing system (Amplification Refractory Mutation System, ARMS) etc..DNA direct Sequencing is prominent
Become the goldstandard of detection.The disadvantages of that there are detection cycles is longer for this method, and non-stopped pipe operation is easy to pollute, and flux is not high, and it is sensitive
Degree only 20%.Two generation PCR sequencing PCRs are emerging detection methods, have many advantages, such as high throughput, highly sensitive, sensitivity is reachable
1%, but detection cycle is longer, and is readily incorporated mutation in detection process.ARMS detection cycle is short, stopped pipe operation, high sensitivity,
Detectability is 1%, but 3 ' terminal bases of ARMS primer are different to different mispairing separating capacities, therefore to the area of some mutation
The ability of dividing is limited.
Summary of the invention
Therefore, the purpose of the present invention is to overcome the defects in the prior art, provide a kind of rare mutation detection method, its
Kit and application.Detection method of the invention is directed to rare mutation, the especially detection method of non-disease diagnostic properties.
Before illustrating the content of present invention, it is as follows to define term used herein:
Term " U-ARMS " refers to: using the amplification refractory mutation system of universal primer and general probe, i.e., universal expansion
Increase and hinders abruptly-changing system (Universal-Amplification Refractory Mutation System).
To achieve the above object, the first aspect of the present invention provides a kind of rare mutation detection method, and the method is
U-ARMS method, comprising the following steps:
(1) ARMS primer, the upstream primer or downstream primer of the ARMS primer are designed according to target gene mutational site
3 ' ends be mutational site, carry out LNA modification, and mutational site identification primer 5 ' ends extend one section of universal sequence；
(2) Taqman-MGB probe, the Taqman-MGB probe specific recognition amplification are designed according to the universal sequence
The antisense strand of product, 5 ' end mark fluorophors of the probe, 3 ' ends of the probe are combined with minor groove binding molecule；
(3) universal primer is designed according to the universal sequence；
(4) template DNA is extracted, Taqman-MGB probe and step obtained by ARMS primer, step (2) obtained by step (1) are utilized
Suddenly universal primer obtained by (3) carries out fluorescent PCR detection to template DNA.
According to method of the first aspect of the present invention, wherein in the step (1), the mutational site is EGFR gene
2369 missense mutation, the ARMS primer are nucleotide sequence shown in SEQ ID No:2 and SEQ ID No:6.
According to method of the first aspect of the present invention, wherein in the step (2), the Taqman-MGB probe is SEQ
Nucleotide sequence shown in ID No:7.
According to method of the first aspect of the present invention, wherein in the step (3), the universal primer is SEQ ID No:8
Shown in nucleotide sequence.
According to method of the first aspect of the present invention, wherein in the step (4), in the universal primer and ARMS primer
Mutation identification primer additional proportion be 10~100:1, preferably 30~50:1, most preferably 40:1.
The second aspect of the present invention provides a kind of rare mutation detection kit, and the kit includes testing goal base
ARMS primer, universal primer and the Taqman-MGB probe in the mutational site of cause；
Wherein, 3 ' ends of the upstream primer or downstream primer of the ARMS primer are mutational site, carry out LNA modification,
And extend one section of universal sequence in 5 ' ends of mutational site identification primer；
The antisense strand of the Taqman-MGB probe specific recognition amplified production, 5 ' end mark fluorescent bases of the probe
Group, 3 ' ends of the probe are combined with minor groove binding molecule；
5 ' end universal sequences of the mutational site the universal primer specific recognition ARMS identification primer.
Kit according to a second aspect of the present invention, wherein the ARMS primer is SEQ ID No:2 and SEQ ID No:
Nucleotide sequence shown in 6；
Preferably, the Taqman-MGB probe is nucleotide sequence shown in SEQ ID No:7；
It is highly preferred that the universal primer is nucleotide sequence shown in SEQ ID No:8.
Kit according to a second aspect of the present invention, wherein the kit by PCR buffer mix, primer pair,
Taqman-MGB probe and template DNA composition, wherein the primer pair includes the special upstream and downstream primer of ARMS and universal primer；
Preferably, final concentration of when each component reacts:
Final concentration of the 1 of PCR buffer mix ×；
The final concentration of 250-900nM of the special upstream and downstream primer of ARMS；
The final concentration of 250-900nM of universal primer；
The final concentration of 50-250nM of Taqman-MGB probe；And/or
The final concentration of 0.05-1.5ng/ μ l of template DNA.
Kit according to a second aspect of the present invention, wherein the mutational site the ARMS identification primer is final concentration of
12.5nM, the final concentration of 500nM of the non-mutated sites identification primer；
The final concentration of 500nM of the universal primer；And/or
The final concentration of 250nM of the Taqman-MGB probe.
The third aspect of the present invention provides method described in first aspect or exists according to kit described in second aspect
Preparation is for the application in the product of detection in Gene Mutation；Preferably, the gene mutation is rare mutation.
The present invention provides one kind based on the method under high wild type DNA background, detecting low-content gene mutation DNA.This
Detection of the new U-ARMS method for rare mutation is invented, solving detection in the prior art, rare mutation program is cumbersome, expense
With costly, time consuming length, easy to pollute and resolution capability is limited the problems such as.
For achieving the above object, technical solution are as follows:
U-ARMS method for rare mutation detection, comprising the following steps:
(1) ARMS primer, the upstream primer or downstream primer of the ARMS primer are designed according to target gene mutational site
3 ' ends be mutational site, carry out LNA modification, and mutational site identification primer 5 ' ends extend one section of universal sequence；
(2) Taqman-MGB probe, the Taqman-MGB probe specific recognition amplification are designed according to the universal sequence
The antisense strand of product, 5 ' end mark fluorophors of the probe, 3 ' ends of the probe are combined with minor groove binding molecule；
(3) universal primer is designed according to the universal sequence；
(4) template DNA is extracted, Taqman-MGB probe and step obtained by ARMS primer, step (2) obtained by step (1) are utilized
Suddenly universal primer obtained by (3) carries out fluorescent PCR detection to template DNA.
Preferably, in the step (1), the mutational site is 2369 missense mutation of EGFR gene, and the ARMS draws
Object is nucleotide sequence shown in SEQ ID No:2 and SEQ ID No:6.
Preferably, in the step (2), the Taqman-MGB probe is nucleotide sequence shown in SEQ ID No:7.
Preferably, in the step (3), the universal primer is nucleotide sequence shown in SEQ ID No:8.
Preferably, the mutation identification primer additional proportion in the universal primer and ARMS primer is 40:1.
Preferably, the kit is made of PCR buffer mix, primer pair, Taqman-MGB probe and template DNA, respectively
It is final concentration of when component reaction:
Preferably, the PCR buffer mix, primer pair, Taqman-MGB probe and template DNA final concentration composition are as follows:
The U-ARMS technology is based on Real-time PCR platform, in conjunction with LNA and ARMS mutation enrichment and Taqman-MGB
Detection technique of fluorescence.Enhance mutating alkali yl recognition capability using LNA, ARMS primer pair mutated target sequence carries out specific PCR expansion
Increase, Taqman-MGB probe carries out specific detection to amplified production, identifies specific mutation on the basis of Real-time PCR.
Method of the invention can have but be not limited to it is following the utility model has the advantages that
The method of U-ARMS technology detection gene mutation typing disclosed by the invention has high specificity, LNA enhancing mutation
Base recognition capability, ARMS primer is just for gene mutation sequence, specific amplification mutagenesis template DNA；This method also has spirit
The mutation of the high feature of sensitivity, 3-5 copy can detect, and the sensitivity for detecting gene mutation can achieve 1%；Detection process
A possibility that reacting for stopped pipe, and introduce UDG/dUTP system, reducing pollution, easy to operate quick, entire PCR reaction process is only
It needs 90 minutes.It is high to detect flux, the multiple genes of multiple samples can be carried out while be detected on 96 orifice plates, as a result accurately
It is errorless.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows U-ARMS design schematic diagram.Wherein, UF is general upstream primer, and UP is general probe, F be with
The special upstream ARMS primer of universal sequence, R are special downstream primer.
Base mismatch is introduced Fig. 2 shows different location and mutational site LNA modifies result figure.W1, W2, W3, WL difference
Wild type matter is expanded for special primer, the introducing base mismatch of mutational site upstream the 2nd and 3 and mutational site LNA Mdification primer
The curve of grain；M2, M3, ML are respectively the introducing base mismatch of mutational site upstream the 2nd and 3 and mutational site LNA Mdification primer
Expand the curve of mutant plasmids.
Fig. 3 shows different primers comparative example result figure.NTC is negative control；Other are respectively universal primer and mutation
Site LNA Mdification primer ratio is the curve that 10:1,20:1,30:1,40:1,50:1 expand mutant plasmids.
Fig. 4 shows the sensitivity results figure of U-ARMS technology.NTC is negative control；Other are respectively kit detection
The amplification curve that mutant proportion is 0%, 1%, 5%, 10%, 20%, 50%, 100%.
Specific embodiment
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for specifically describing in more detail.
This part carries out general description to the material and test method that arrive used in present invention test.Although being
It realizes many materials used in the object of the invention and operating method is it is known in the art that still the present invention still uses up herein
It may detailed description.It will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and behaviour
It is well known in the art as method.
Reagent and instrument used in the following embodiment are as follows:
Reagent:
Mutant plasmids, wild plasmid, Taqman-MGB probe interrogate the limited public affairs of biotechnology share purchased from Suzhou letter
Department；PCR Buffer Mix is purchased from vazyme.
Instrument:
Fluorescent PCR instrument is purchased from Hangzhou BIOER Technology Co., Ltd, model FQD-96A.
Embodiment 1
According to 2369 missense mutation of EGFR gene, i.e., 2369 become T (abbreviation T790M) from C, by genetic engineering point
Not Gou Jian wild plasmid and mutant plasmids as template, wild plasmid sequence is as shown in SEQ ID No:1, plasmid measurement
After concentration, it is diluted to 1ng/ul with TE buffer, the plasmid of 1ng/ul is then subjected to 10 times of gradient dilutions with TE buffer, it is dilute
It releases concentration and is followed successively by 10-1、10-2、10-3、10-4、10-5、10-6Ng/ul is spare, and 10-6Gene copy number contained by ng plasmid and 1ng
Target gene copy number contained by genomic DNA is suitable.
Mutant plasmids after taking dilution mix in varing proportions with wild plasmid, obtain wild type sample and (do not contain
Mutant plasmids), 1% sudden change sample (ratio of mutant plasmids and wild plasmid be 1:99), (mutation of 5% sudden change sample
The ratio of type plasmid and wild plasmid is 5:95), (ratio of mutant plasmids and wild plasmid is 10% sudden change sample
10:90), 20% sudden change sample (ratio of mutant plasmids and wild plasmid is 20:80), 50% sudden change sample (saltant type
The ratio of plasmid and wild plasmid is 50:50), 100% sudden change sample (be free of wild plasmid), it is spare.
According to T790M mutation type, ARMS primer pair is designed, missense mutation site is made to be located at the last of the end of upstream primer 3 '
One, and mutational site upstream the 1st and 2 positions will be located at and introduce mispairing mutating alkali yl, or missense mutation site progress LNA is repaired
Decorations, particular sequence information is as shown in table 1, downstream primer are as follows: 5 '-GAGCAGGTACTGGGAGCC-3 ' (SEQ ID No:2)
1 ARMS upstream primer sequence of table
Respectively using mutant plasmid and wild plasmid as template, the ARMS primer pair of T790M mutation type, SEQ ID are utilized
Nucleotide shown in No:7 is that nucleotide shown in universal primer and SEQ ID No:8 is that Taqman-MGB probe progress fluorescent PCR is anti-
Answer that (final concentration of each component is respectively 1 × PCR Buffer Mix, 500nM ARMS primer pair, 500nM logical in 20 μ l systems
With primer, 250nM Taqman-MGB probe, 10-6Mutant plasmid/10 ng-6Ng wild plasmid and surplus are water), fluorescent PCR
Reaction condition are as follows: 37 DEG C sludge digestion 2 minutes, one circulation；95 DEG C initial denaturation 5 minutes, one circulation；40 amplification cycles,
95 DEG C are denaturalized 10 seconds, and 60 DEG C are annealed and extended 1 minute, are annealing and are extending stage collection fluorescence signal, as shown in Figure 2.By Fig. 2
It is found that the introducing base mismatch of mutational site upstream the 2nd and 3 and mutational site LNA modification detection mutant plasmid amplification efficiency are equal
It is fabulous, but only have LNA Mdification primer to rise without curve when detection wild plasmid.Therefore, nucleosides shown in SEQ ID No:6
Acid sequence is that ARMS upstream primer mutation resolving effect is best, and specificity is best.
Embodiment 2
Using mutant plasmids as template, the nucleotides sequence of SEQ ID No:6 and SEQ ID No:2 is classified as ARMS primer, SEQ
Nucleotide shown in ID No:7 is that nucleotide shown in universal primer and SEQ ID No:8 is that Taqman-MGB probe carries out fluorescence
PCR, in Fluorescence PCR universal primer and the additional proportion of AMRS upstream primer be followed successively by 10:1,20:1,30:1,40:1,
50:1, remaining constituent concentration identical (in 20 μ l the systems final concentration of 1 × PCR Buffer Mix of each component, 500nM ARMS
Downstream primer, 500nM universal primer, 250nM Taqman-MGB probe, 10-6Ng mutant plasmid and surplus are water).Fluorescent PCR
Reaction condition is same as Example 1, as a result as shown in Figure 3.From the figure 3, it may be seen that when universal primer and ARMS upstream primer ratio are
When 40:1, amplification efficiency is best.
Embodiment 3
It is tested using the site T790M, 100% sudden change sample, 50% sudden change sample, 20% prepared with embodiment 1
(additional amount is 10 for sudden change sample, 10% sudden change sample, 5% sudden change sample, 1% sudden change sample and wild type sample-6Ng) it is
Template, using in embodiment 2 primer and primer ratio and Taqman-MGB probe carry out fluorescent PCR verify its sensitivity, tie
Fruit is as shown in Figure 4.As shown in Figure 4, the detection sensitivity of detection method disclosed by the invention can achieve 1%.
Embodiment 4
60 patients with lung cancer FFPE samples are collected, DNA is extracted as template and is drawn with the detection T790M mutation of U-ARMS technology
Object is the nucleotide sequence of SEQ ID No:6, SEQ ID No:2 and SEQ ID No:7, the nucleotide sequence of probe such as SEQ ID
Shown in No:8, each component final concentration is respectively 1 × PCR Buffer Mix, 12.5nM ARMS in 20 μ l Fluorescence PCR systems
Upstream primer, 500nM ARMS downstream primer, 500nM universal primer, 250nM Taqman-MGB probe, 10ng DNA profiling and
Surplus is water.Fluorescence PCR condition are as follows: 37 DEG C sludge digestion 2 minutes, one circulation；95 DEG C initial denaturation 5 minutes, one is followed
Ring；40 amplification cycles, 95 DEG C are denaturalized 10 seconds, and 60 DEG C are annealed and extended 1 minute, in annealing and extend stage collection fluorescence letter
Number.Simultaneously by this detection method compared with sanger sequencing.Testing result of the present invention and sanger sequencing result are completely the same: 60
There are 4 to detect that T790M is mutated in example sample.
Detection time of the present invention only needs 90 minutes, and it is 24-48 hours that detection time, which is sequenced, in sanger, therefore the present invention is time saving
Laborsaving, accuracy is high, can meet the quick diagnosis of mutation.Moreover, the coincidence rate of fluorescence PCR method and traditional sequencing methods result
Be 100%, but fluorescent PCR sensitivity and selective enumeration method ability are higher than traditional sequencing methods, in 1ng sample DNA containing 1% it is prominent
Becoming DNA can detect, and traditional sanger sequencing can only mutation of the detection ratio higher than 20%.
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right
It is required that range comprising the equivalent replacement of each factor.
Sequence table
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Claims (10)
1. a kind of rare mutation detection method, which is characterized in that the method is U-ARMS method, comprising the following steps:
(1) ARMS primer is designed according to target gene mutational site, the 3 ' of the upstream primer of the ARMS primer or downstream primer
End is mutational site, carries out LNA modification, and extend one section of universal sequence in 5 ' ends of mutational site identification primer；
(2) Taqman-MGB probe, the Taqman-MGB probe specific recognition amplified production are designed according to the universal sequence
Antisense strand, 5 ' end mark fluorophors of the probe, 3 ' ends of the probe are combined with minor groove binding molecule；
(3) universal primer is designed according to the universal sequence；
(4) template DNA is extracted, Taqman-MGB probe and step (3) obtained by ARMS primer, step (2) obtained by step (1) are utilized
Gained universal primer carries out fluorescent PCR detection to template DNA.
2. the method according to claim 1, wherein the mutational site is EGFR gene in the step (1)
2369 missense mutation, the ARMS primer are nucleotide sequence shown in SEQ ID No:2 and SEQ ID No:6.
3. method according to claim 1 or 2, which is characterized in that in the step (2), the Taqman-MGB probe
For nucleotide sequence shown in SEQ ID No:7.
4. method according to any one of claim 1-3, which is characterized in that in the step (3), the universal primer
For nucleotide sequence shown in SEQ ID No:8.
5. method according to any of claims 1-4, which is characterized in that in the step (4), the universal primer
It is 10~100:1, preferably 30~50:1, most preferably 40:1 with the mutation identification primer additional proportion in ARMS primer.
6. a kind of rare mutation detection kit, which is characterized in that the kit includes the mutational site of testing goal gene
ARMS primer, universal primer and Taqman-MGB probe；
Wherein, 3 ' ends of the upstream primer or downstream primer of the ARMS primer be mutational site, carry out LNA modification, and
Mutational site identifies that 5 ' ends of primer extend one section of universal sequence；
The antisense strand of the Taqman-MGB probe specific recognition amplified production, 5 ' end mark fluorophors of the probe,
3 ' ends of the probe are combined with minor groove binding molecule；
5 ' end universal sequences of the mutational site the universal primer specific recognition ARMS identification primer.
7. kit according to claim 6, which is characterized in that the ARMS primer is SEQ ID No:2 and SEQ ID
Nucleotide sequence shown in No:6；
Preferably, the Taqman-MGB probe is nucleotide sequence shown in SEQ ID No:7；
It is highly preferred that the universal primer is nucleotide sequence shown in SEQ ID No:8.
8. kit according to claim 6 or 7, which is characterized in that the kit is by PCR buffer mix, primer
To, Taqman-MGB probe and template DNA composition, wherein the primer pair includes the special upstream and downstream primer of ARMS and general draws
Object；
Preferably, final concentration of when each component reacts:
Final concentration of the 1 of PCR buffer mix ×；
The final concentration of 250-900nM of the special upstream and downstream primer of ARMS；
The final concentration of 250-900nM of universal primer；
The final concentration of 50-250nM of Taqman-MGB probe；And/or
The final concentration of 0.05-1.5ng/ μ l of template DNA.
9. kit according to claim 8, it is characterised in that: the mutational site the ARMS identification primer is final concentration of
12.5nM, the final concentration of 500nM of the non-mutated sites identification primer；
The final concentration of 500nM of the universal primer；And/or
The final concentration of 250nM of the Taqman-MGB probe.
10. any one of claim 1-3 the method or the kit according to any one of claim 4-9 are used in preparation
Application in the product of detection in Gene Mutation；Preferably, the gene mutation is rare mutation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628883A (en) * | 2019-10-31 | 2019-12-31 | 北京安智因生物技术有限公司 | Kit and method for detecting gene polymorphism based on shared primer probe and application |
| CN113308519A (en) * | 2021-06-30 | 2021-08-27 | 上海伯杰医疗科技有限公司北京分公司 | Primer and probe for detecting single base mutation site and detection method |
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