CN104031978A - ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof - Google Patents
ARMS fluorescent quantitative PCR-based gene mutation kit and method thereof Download PDFInfo
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Abstract
The invention relates to an ARMS fluorescent quantitative PCR-based gene mutation kit and a method thereof. Compared with present gene polymorphism detection kits and methods thereof, the enhanced ARMS fluorescent quantitative PCR-based gene polymorphism mutation kit and the method thereof have the advantages of strong specificity, high sensitivity, low cost, highly reliable result, low background, easy determination of the result, ascendant operation and use, fast and convenient fluorescent quantitative PCR, closed tube detection in the whole course, pollution reduction, and reduction of the unnecessary cost and workload. The fluorescent quantitative PCR can realize the characterization of the amplification efficiency by using a CT value, so the method can realize the detection of the nuclear gene polymorphism heterozygote genotype conditions, and makes the result accurately and reliably determined.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly a kind of test kit and method based on the transgenation of ARMS fluorescence quantitative PCR detection.
Background technology
Human genome polymorphism is being illustrated human body to the susceptibility of disease, poisonous substance and tolerance, the diversity to disease clinical manifestation, and to all playing an important role in the reactivity of pharmacological agent.By the research of gene pleiomorphism, can disclose the function of biologically active substance between mankind's Different Individual and the essence that effect exists difference from gene level.By contacting between research gene pleiomorphism and disease susceptibility, for example, by the chondriogen mtDNA A1555G deafness that sudden change causes with C1494T, mainly use relevant with aminoglycoside medicaments, the gene pleiomorphism of vitamin K epoxide reductase complex body subunit 1 gene (VKORC1) and cytochrome P450 2C9 gene (CYP2C9) are to affect topmost two inherited genetic factorss of warfarin consumption individual difference etc., can illustrate human body to disease, poisonous substance and stress susceptibility, not only to clinical medicine also for the development of preventive medicine brings new field.
Method for detection of gene pleiomorphism is a lot, comprise the direct Sequencing as DNA, restriction fragment length polymorphism (restriction fragment lengthpolymorphisms, RFLP), amplification refractory mutation system (amplification refractory mutation system, ARMS) etc., the gold standard that wherein DNA direct Sequencing detects for sudden change, but long because of its required time, expense is high, to drawing materials, require strictly, so clinical application is very limited.Restriction fragment length polymorphism is only applicable to exist near mutational site the situation of suitable specific limited restriction endonuclease recognition sequence, and application limitation is larger.
Amplification refractory mutation system is one of conventional classical way of confirmatory detection DNA sequence dna variation.ARMS-PCR genotype tests generally includes two complementary PCR reactions, use identical DNA profiling and identical total primer and a reaction conditions, it is different from the ARMS primer of total primer pairing that difference is only, wild-type ARMS primer 3` end and wild-type template matches, saltant type ARMS primer 3` end and saltant type template matches, make two reaction preferencies specific DNA profiling that increases, be that the extension that 3` end does not mate primer is hindered, conventionally also in 2-4 base of 3` end, a base mismatch is set, to improve the selection amplification property of mispairing primer.But because human gene group DNA's molecular template concentration is generally higher, cause ARMS primer still very high through selective amplification rear backdrop, and for particular sequence, ARMS primer is not enough to form obvious amplification difference, is unfavorable for result judgement.
Summary of the invention
In order to solve the detection of gene pleiomorphism, the object of the present invention is to provide a kind of test kit based on the transgenation of ARMS fluorescence quantitative PCR detection.
Another object of the present invention is to provide a kind of method based on the transgenation of ARMS fluorescence quantitative PCR detection.
In the present invention, described transgenation mainly comprises: point mutation, deletion mutantion and insertion mutation etc.In the present invention, described deletion mutantion can comprise the sudden change that lacks one or more bases, and similarly, described insertion mutation can comprise the sudden change of inserting one or more bases.
The present invention has integrated ARMS round pcr and fluorescent quantitative PCR technique.The invention is characterized in, in same reaction system, (for example, comprise sample DNA, ROX, TaqMan probe, Mg
2+, dNTP, polysaccharase and damping fluid etc.) in, use respectively the enhancement type for wild-type template of the present invention (enhanced) ARMS primer and for the enhanced arm S primer of saltant type template and shared upstream or the downstream primer of answering with described enhanced arm S primer pair, with two reaction tubess, to detecting with a sample to be tested, by the size of CT value and △ CT value, judge sample genotype.
The present invention is according to the principle of traditional ARMS-PCR technology, carried out increasing the base mismatch number of ARMS primer 3` end and region intermediate, enhanced arm S primer pair saltant type sample for wild-type template is not increased or amplification efficiency very low, and for the enhanced arm S primer pair wild-type sample of saltant type template do not increase or amplification efficiency very low, so greatly reduce background signal, make result be easier to judgement.
In one aspect, the invention provides a kind of test kit based on the transgenation of ARMS fluorescence quantitative PCR detection, wherein, described transgenation is point mutation, and described test kit comprises:
For the ARMS primer of wild-type template, the 3` end of described primer terminates in site to be measured and mates with wild-type sequence, wherein, in 3` end 2-4 position and the 7-13 position of described primer, one or more base mismatch is set respectively;
For the ARMS primer of saltant type template, the 3` end of described primer terminate in site to be measured and with saltant type sequences match, wherein, in 3` end 2-4 position and the 7-13 position of described primer, one or more base mismatch are set respectively;
Share upstream or downstream primer, described primer respectively with the described ARMS primer for wild-type template or described for the one section of sequence that comprises testing gene mutational site that increases together with the ARMS primer of saltant type template; With
TaqMan probe, described probe for being designed for the amplification efficiency that detects ARMS primer pair template in ARMS primer amplification fragment.
In one aspect of the method, the invention provides a kind of test kit based on the transgenation of ARMS fluorescence quantitative PCR detection, wherein, described transgenation is deletion mutantion or insertion mutation, and described test kit comprises:
ARMS primer for wild-type template, the 3` end of described primer terminates in disappearance or base place, 2-5, insertion point downstream on bearing of trend, and comprising site to be measured mates with wild-type sequence in interior 3` end base (comprising the several bases in disappearance or insertion point and downstream), wherein, described primer arranges respectively one or more base mismatch in disappearance or 2-4 position, insertion point upstream and 6-8 position on bearing of trend;
ARMS primer for saltant type template, the 3` end of described primer terminates in disappearance or base place, 2-5, insertion point downstream on bearing of trend, and comprise site to be measured in interior 3` end base (comprising the several bases in disappearance or insertion point and downstream) and saltant type sequences match, wherein, described primer arranges respectively one or more base mismatch in disappearance or 2-4 position, insertion point upstream and 6-8 position on bearing of trend;
Share upstream or downstream primer, described primer respectively with the described ARMS primer for wild-type template or described for the one section of sequence that comprises testing gene mutational site that increases together with the ARMS primer of saltant type template; With
TaqMan probe, described probe for being designed for the amplification efficiency that detects ARMS primer pair template in ARMS primer amplification fragment.
In the present invention, preferably, in the described ARMS primer for wild-type template and the ARMS primer for saltant type template, the position of introducing base mismatch is identical with type.
In the present invention, more preferably, except the base at place, mutational site, the described ARMS primer for wild-type template is identical with the described ARMS primer for saltant type template.
In the present invention, preferably, described test kit further comprises: ROX correcting fluid, Mg
2+, dNTP, polysaccharase and damping fluid.
In the present invention, preferably, in described test kit, the end of described TaqMan probe is carried out to fluorescent mark, for example, described probe is carried out to 5 ' end FAM mark.
In the present invention, preferably, described point mutation is for example, deaf-related gene 12SrRNA1494C>T point mutation, warfarin medication related locus VKORC13730G>A, platinum medicine genes involved XRCCIArg194Trp; Described deletion mutantion is for example the GJB235delG deletion mutantion relevant to deafness.
In the present invention, preferably, described test kit is for detection of the GJB235delG deletion mutantion relevant to deafness, wherein, the described downstream ARMS primer for wild-type template as shown in SEQ ID NO:7, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:8; And/or described shared upstream primer is as shown in SEQ ID NO:9; And/or described probe is as shown in SEQ ID NO:10.
In the present invention, preferably, described test kit is for detection of deaf-related gene 12SrRNA1494C>T point mutation, wherein, the described upstream ARMS primer for wild-type template as shown in SEQ ID NO:13, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:14; And/or described shared downstream primer is as shown in SEQ ID NO:15; And/or described probe is as shown in SEQ ID NO:16.
In the present invention, preferably, described test kit suddenlys change for detection of warfarin medication related locus VKORC13730G>A, wherein, the described downstream ARMS primer for wild-type template as shown in SEQ ID NO:19, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:20; And/or described shared upstream primer is as shown in SEQ ID NO:21; And/or described probe is as shown in SEQ ID NO:22.
In the present invention, preferably, described test kit is for detection of platinum medicine genes involved XRCCI Arg194Trp sudden change, wherein, the described downstream ARMS primer for wild-type template as shown in SEQ ID NO:25, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:26; And/or described shared upstream primer is as shown in SEQ ID NO:27; And/or described probe is as shown in SEQ ID NO:28.
According to another object of the present invention, a kind of method based on the transgenation of ARMS fluorescence quantitative PCR detection is provided, described method comprises: in identical reaction system, (comprise sample DNA, ROX, TaqMan probe, Mg
2+, dNTP, polysaccharase, share upstream or downstream primer and damping fluid) in, use the enhanced arm S primer for wild-type template of the present invention and for the enhanced arm S primer of saltant type template respectively to detecting with a sample to be tested, by the size of CT value and △ CT value, judge sample genotype.
The method of the invention is further comprising the steps:
(1) design and screen taqman probe, enhanced arm S primer, pairing primer, the reaction system that contains site to be measured, wherein, described taqman probe, enhanced arm S primer, pairing primer are as mentioned above;
(2) from sample, extract human gene group DNA as template DNA;
(3) carry out real-time fluorescence quantitative PCR amplification: the detection of each pleomorphism site divides 2 pipes to carry out, in every pipe, add identical ROX correcting fluid, Mg
2+, dNTP, polysaccharase, damping fluid, taqman probe, pairing primer and template DNA, in every pipe, add respectively a kind of of wild-type ARMS primer and saltant type ARMS primer, the enhanced arm S-qPCR that carries out polymorphic detection detects;
(4) result is judged
According to Cut-off △ Ct value result of determination.
Calculate the △ Ct value of wild-type and saltant type sudden change detection reaction for sample, △ Ct value=Ct saltant type-Ct wild-type.
If saltant type and wild-type detection system all have amplified signal, according to formula △ Ct value=Ct saltant type-Ct wild-type, calculate △ Ct value; If △ is Ct>8, be judged to be wild homozygous; If-1< △ is Ct<1, be judged to be heterozygous; If △ is Ct<-8, be judged to be sudden change homozygous;
If saltant type has, amplified signal and Ct value are less than 36, wild-type is without amplified signal, are judged to be sudden change homozygous; If saltant type Ct value is greater than 36, illustrate that sample DNA extracting concentration is too low or exceed present method sensing range, need to again extract test sample;
If wild-type has, amplified signal and Ct value are less than 36, saltant type is without amplified signal, are judged to be wild homozygous; If wild-type Ct value is greater than 36, illustrate that sample DNA extracting concentration is too low or exceed present method sensing range, need to again extract test sample;
If saltant type and wild-type detection system Ct value are greater than 36, illustrate that sample DNA extracting concentration is too low or exceed present method sensing range, need to again extract test sample.
Compare with existing gene pleiomorphism detecting method, test kit based on enhanced arm S fluorescence quantitative PCR detection gene pleiomorphism of the present invention and method be high specificity not only, and susceptibility is high, and cost is low, result height is reliable, background is low, and result is easy to judgement, and also has unique advantage in operation and use, fluorescence quantitative PCR detection is efficient and convenient, omnidistance stopped pipe detects, and reduces and pollutes, and has reduced unnecessary expense and workload.Particularly quantitative fluorescent PCR can characterize amplification efficiency by CT value, so this method can detect the heterozygote genotype situation of nuclear gene polymorphism, makes the result judgement can be accurately and reliably.
Accompanying drawing explanation
Fig. 1 is that the detection effect that the conventional ARMS primer of the use of embodiment 1 and enhanced arm S primer of the present invention detect deaf sample GJB2 gene 35delG deletion mutantion compares, this sample turns out to be wild-type through order-checking, wherein, sign 35delWR1,35delWR2,35delWR3,35delWR4,35delMR1,35delMR2,35delMR3,35delMR4 represent to use respectively 8 pairs of primer pairs in embodiment 1 with the detected result of a clinical sample.
Fig. 2 is that the detection effect that the conventional ARMS primer of the use of embodiment 2 and enhanced arm S primer of the present invention detect deaf sample 12SrRNA1494C>T base substitution mutation compares, this sample turns out to be wild-type through order-checking, wherein, sign 1494WR1,1494WR2,1494WR3,1494WR4 represents to use respectively 4 pairs of primer pairs in embodiment 2 with the detected result of a clinical sample.
Fig. 3 is that the conventional ARMS primer of use of embodiment 3 and the detection effect of enhanced arm S primer of the present invention detection chronic atrial fibrillation sample VKORC1 gene 3730G>A base substitution mutation compare, this sample turns out to be wild-type through order-checking, wherein, sign V3WF1, V3WF2, V3MF1, V3MF2 represents to use respectively 4 pairs of primer pairs in embodiment 3 with the detected result of a clinical sample.
Fig. 4 is that the conventional ARMS primer of use of embodiment 4 and the detection effect of enhanced arm S primer of the present invention detection rectum cancer sample XRCCI Gene A rg194Trp base substitution mutation compare, this sample turns out to be wild-type through order-checking, wherein, XRCC6WR1, XRCC6WR2, XRCC6MR1, XRCC6MR2 represent to use respectively 4 pairs of primer pairs in embodiment 4 with the detected result of a clinical sample.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described, but the present invention is not limited to following examples.
In following embodiment, if no special instructions, be this area ordinary method.
In following examples, archaeal dna polymerase is purchased from QIAGEN company (PCR damping fluid, Mg
2+all with enzyme, supply), dNTP, ROX correcting fluid are purchased from Japanese Takara company, and the primer and probe are synthetic by Shanghai biotechnology Services Co., Ltd, and poba gene group DNA extraction test kit is purchased from Tian Gen bio tech ltd, Beijing.
In the following example, all use poba gene group DNA extraction test kit to prepare sample DNA.
Embodiment 1 is used conventional ARMS primer and enhanced arm S primer of the present invention to detect and deaf relevant GJB235delG deletion mutantion
The present invention is on the basis of conventional ARMS primer, make primer base place, 6-8 of upstream at deletion segment on bearing of trend that a base mismatch is set again, in real-time fluorescence quantitative PCR, reduce thus the amplification efficiency of wild-type enhanced arm S primer pair saltant type sample and the amplification efficiency of saltant type enhanced arm S primer pair wild-type sample, make result be easier to judgement.
1) ARMS primer and enhanced arm S design of primers
Experimental design 3 pairs of wild-type ARMS primers, 3 pairs of saltant type ARMS primers, 1 pair of wild-type enhanced arm S primer and 1 pair of saltant type enhanced arm S primer, shared upstream primer is 35delF, correlation parameter is: 55.0 ℃-60.0 ℃ of Tm values, GC value 40.0%-60.0%, primer size 20 ± 3bp.According to the singularity in 35delG site, 3 ' end of ARMS primer and enhanced arm S primer is located on bearing of trend the 5th the base place in deletion segment downstream, 6 bases of end match with wild-type and mutated genes respectively, for improving specificity, on bearing of trend, in deletion segment upstream, the 2nd base place introduces a base mismatch to ARMS primer, and enhanced arm S primer on bearing of trend, in deletion segment upstream, the 2nd base place and base place, the 6th of upstream introduce respectively a base mismatch, designed ARMS primer and enhanced arm S primer sequence are as follows:
Downstream ARMS primer: 35delWR1GTTTGTTCACA
acCCCCA (SEQ ID NO:1)
35delMR1GTTTGTTCACA
ACCCCA(SEQ?ID?NO:2)
35delWR2GTTTGTTCACA
TCCCCCA(SEQ?ID?NO:3)
35delMR2GTTTGTTCACA
TCCCCA(SEQ?ID?NO:4)
35delWR3GTTTGTTCACA
GCCCCCA(SEQ?ID?NO:5)
35delMR3GTTTGTTCACA
GCCCCA(SEQ?ID?NO:6)
Downstream enhanced arm S primer:
35delWR4GTTTGT
GCACA
GCCCCCA(SEQ?ID?NO:7)
35delMR4GTTTGT
GCACA
GCCCCA(SEQ?ID?NO:8)
Share upstream primer 35delF:GTGTGCATTCGTCTTTTCCAG (SEQ ID NO:9)
In primer, underscore partly schematically illustrates position and the base type of the base mismatch of introducing except disappearance base.These 8 primers have been composed of quadruplet primer with upstream primer together, every cover primer comprises 2 downstream primers (wild-type and saltant type) and upstream primer 35delF, be used for setting up the scheme that detects GJB235delG deletion mutantion, select to be more suitable in 4 cover primers for gene type, make two parallel fluorescent quantitative PCR result CT value <35 and the △ CT value >8 of template DNA.
2) probe design
Designing probe in ARMS primer amplification fragment, correlation parameter is: 68.0 ℃-70.0 ℃ of Tm values, GC value 40.0%-70.0%, carries out 5 ' end FAM mark to probe, and its sequence is as follows:
35del probe: CAGCGTGCCCCAATCCATCTTC (SEQ ID NO:10)
3) ARMS fluorescent quantitative PCR reaction
PCR response procedures is: 95 ℃ 5 minutes; 40 circulations: 95 ℃ 15 seconds, 60 ℃ 45 seconds (collection fluorescence).Instrument is ABI stepone.
Test-results as shown in Figure (1) shows, in the quantitative fluorescent PCR reaction of parallel amplification, the △ CT=9.55 of enhanced arm S saltant type primer of the present invention and enhanced arm S wild-type primer, than large 3 the CT values of △ CT of other 3 cover ARMS primers, can more effectively distinguish sample genotype, facilitate the judgement of somatotype result.
Embodiment 2 is used conventional ARMS primer and enhanced arm S primer of the present invention to detect deaf-related gene 12SrRNA1494C>T point mutation
1) ARMS primer and enhanced arm S design of primers
Experimental design 1 pair of wild-type ARMS primer, 1 pair of saltant type ARMS primer, 1 pair of wild-type enhanced arm S primer and 1 pair of saltant type enhanced arm S primer, shared upstream primer is 12SrRNAF, correlation parameter is: 55.0 ℃-60.0 ℃ of Tm values, GC value 40.0%-60.0%, primer size 20 ± 3bp.The 3` terminal bases of ARMS primer and enhanced arm S primer is positioned at site to be measured and matches with wild-type and mutated genes respectively, for improving specificity, at the 3` of ARMS primer end, the 4th base place introduces a base mismatch, and introduce respectively a base mismatch at the 4th base place of 3` end and the 13rd the base place of enhanced arm S primer, designed ARMS primer and enhanced arm S primer sequence are as follows:
Downstream ARMS primer:
1494WR1CTTTGAAGTATACTTGAG
AAGG(SEQ?ID?NO:11)
1494MR1CTTTGAAGTATACTTGAG
AAGA(SEQ?ID?NO:12)
Downstream enhanced arm S primer:
1494WR2CTTTGAAGT
CTACTTGAG
AAGG(SEQ?ID?NO:13)
1494MR2CTTTGAAGT
CTACTTGAG
AAGA(SEQ?ID?NO:14)
Share upstream primer: 12SrRNAF GGTCGAAGGTGGATTTAGCAG (SEQ ID NO:15)
In primer, underscore partly schematically illustrates position and the base type of introduced base mismatch.These 4 primers have been composed of two cover primers with upstream primer together, every cover primer comprises 2 downstream primers (wild-type and saltant type) and upstream primer 12SrRNAF, be used for setting up the scheme that detects 12SrRNA1494C>T, select to be more suitable in 3 cover primers for gene type, make two parallel fluorescent quantitative PCR result CT value <35 and the △ CT value >8 of template DNA.
2) probe design
Designing probe in ARMS primer amplification fragment, correlation parameter is: 68.0 ℃-70.0 ℃ of Tm values, GC value 40.0%-70.0%, carries out 5 ' end FAM mark to probe, and its sequence is as follows:
12SrRNA probe: CCTGAAGCGCGTACACACCGCCT (SEQ ID NO:16)
3) ARMS fluorescent quantitative PCR reaction
PCR response procedures is: 95 ℃ 5 minutes; 40 circulations: 95 ℃ 15 seconds, 60 ℃ 45 seconds (collection fluorescence).Instrument is ABI stepone.
Test-results as shown in Figure 2, in the quantitative fluorescent PCR reaction of parallel amplification, enhanced arm S primer for saltant type template of the present invention and for the △ CT=11.72 of the enhanced arm S primer of wild-type template, be compared to large 1.2 the CT values of △ CT of the ARMS primer of contrast, and saltant type ARMS primer amplification signal is very low, can effectively distinguish sample genotype, facilitate the judgement of somatotype result.
Embodiment 3 is used conventional ARMS primer and enhanced arm S primer of the present invention to detect warfarin medication related locus VKORC13730G>A
1) ARMS primer and enhanced arm S design of primers
Experimental design 1 pair of wild-type ARMS primer, 1 pair of saltant type ARMS primer, 1 pair of wild-type enhanced arm S primer and 1 pair of saltant type enhanced arm S primer, shared downstream primer is V3R, correlation parameter is: 55.0 ℃-60.0 ℃ of Tm values, GC value 40.0%-60.0%, primer size 20 ± 3bp.The 3` terminal bases of ARMS primer and enhanced arm S primer is positioned at site to be measured and matches with wild-type and mutated genes respectively, for improving specificity, at ARMS primer 3` end, the 2nd base place introduces a base mismatch, and introduce respectively a base mismatch at the 2nd base place of enhanced arm S primer 3` end and the 7th base place, designed ARMS primer and enhanced arm S primer sequence are as follows:
Upstream ARMS primer:
V3WF1CCCTCCTCCTGCCATACC
AG(SEQ?ID?NO:17)
V3MF1CCCTCCTCCTGCCATACC
AA(SEQ?ID?NO:18)
Upstream enhanced arm S primer:
V3WF2CCCTCCTCCTGCC
GTACC
AG(SEQ?ID?NO:19)
V3MF2CCCTCCTCCTGCC
GTACC
AA(SEQ?ID?NO:20)
Share downstream primer: V3R CCTTCCCTCCCTGGGCAATG (SEQ ID NO:21)
In primer, underscore partly schematically illustrates position and the base type of introduced base mismatch.These 4 primers and shared downstream primer have been composed of two cover primers, every cover primer comprises 2 downstream primers (wild-type and saltant type) and upstream primer V3R, be used for setting up the scheme that detects VKORC13730G>A, select to be more suitable in 2 cover primers for gene type, make two parallel fluorescent quantitative PCR result CT value <35 and the △ CT value >8 of template DNA.
2) probe design
Designing probe in ARMS primer amplification fragment, correlation parameter is: 68.0 ℃-70.0 ℃ of Tm values, GC value 40.0%-70.0%, carries out 5 ' end FAM mark to probe, and its sequence is as follows:
V3 probe: TGCCACACGCTCGCTCTTTTTTACA (SEQ ID NO:22)
3) ARMS fluorescent quantitative PCR reaction
PCR response procedures is: 95 ℃ 5 minutes; 40 circulations: 95 ℃ 15 seconds, 60 ℃ 45 seconds (collection fluorescence).Instrument is ABI stepone.
As shown in Figure 3, in the reaction of the quantitative fluorescent PCR of parallel amplification, the enhanced arm S primer pair wild-type template for saltant type template of the present invention does not increase test-results, can effectively distinguish sample genotype, is more conducive to the judgement of somatotype result.
Embodiment 4 use present method detect platinum medicine genes involved XRCCI Arg194Trp
1) ARMS primer and enhanced arm S design of primers
Experimental design 1 pair of wild-type ARMS primer, 1 pair of saltant type ARMS primer, 1 pair of wild-type enhanced arm S primer and 1 pair of saltant type enhanced arm S primer, shared upstream primer is XRCC6AF, correlation parameter is: 55.0 ℃-60.0 ℃ of Tm values, GC value 40.0%-60.0%, primer size 20 ± 3bp.The 3` terminal bases of ARMS primer and enhanced arm S primer is positioned at site to be measured and matches with wild-type and mutated genes respectively, for improving specificity, at ARMS primer 3` end, the 3rd base place introduces a base mismatch, and introduce respectively a base mismatch at the 3rd base place of enhanced arm S primer 3` end and the 10th base place, designed ARMS primer and enhanced arm S primer sequence are as follows:
Downstream ARMS primer:
XRCC6WR1GGGGATGTCTTGTTGAT
ACG(SEQ?ID?NO:23)
XRCC6MR1GGGGATGTCTTGTTGAT
ACA(SEQ?ID?NO:24)
Downstream enhanced arm S primer:
XRCC6WR2GGGGATGTCT
GGTTGAT
ACG(SEQ?ID?NO:25)
XRCC6MR2GGGGATGTCT
GGTTGAT
ACA(SEQ?ID?NO:26)
Share upstream primer: XRCC6AF TCACTCCCCATGGCCTTCT (SEQ ID NO:27)
In primer, underscore partly schematically illustrates introduced mispairing position and base type.These 4 primers have been composed of two cover primers with upstream primer together, every cover primer comprises 2 downstream primers (wild-type and saltant type) and upstream primer V3R, be used for setting up the scheme that detects XRCCI Arg194Trp, select to be more suitable in 2 cover primers for gene type, make two parallel fluorescent quantitative PCR result CT value <35 and the △ CT value >8 of template DNA.
2) probe design
Designing probe in ARMS primer amplification fragment, correlation parameter is: 68.0 ℃-70.0 ℃ of Tm values, GC value 40.0%-70.0%, carries out 5 ' end FAM mark to probe, and its sequence is as follows:
XRCC6 probe: CCTCTCCACCCCCACCTGCCA (SEQ ID NO:28)
3) ARMS fluorescent quantitative PCR reaction
PCR response procedures is: 95 ℃ 5 minutes; 40 circulations: 95 ℃ 15 seconds, 60 ℃ 45 seconds (collection fluorescence).Instrument is ABI stepone.
Test-results as shown in Figure 4, in the quantitative fluorescent PCR reaction of parallel amplification, enhanced arm S saltant type primer of the present invention and enhanced arm S wild-type primer △ CT=14.86, be compared to large 2.4 the CT values of △ CT of the ARMS primer of contrast, can effectively distinguish sample genotype, facilitate the judgement of somatotype result.
Claims (10)
1. the test kit based on the transgenation of ARMS fluorescence quantitative PCR detection, wherein, described transgenation is point mutation, described test kit comprises:
For the ARMS primer of wild-type template, the 3` end of described primer terminates in site to be measured and mates with wild-type sequence, wherein, in 3` end 2-4 position and the 7-13 position of described primer, one or more base mismatch is set respectively;
For the ARMS primer of saltant type template, the 3` end of described primer terminate in site to be measured and with saltant type sequences match, wherein, in 3` end 2-4 position and the 7-13 position of described primer, one or more base mismatch are set respectively;
Share upstream or downstream primer, described primer respectively with the described ARMS primer for wild-type template or described for the one section of sequence that comprises testing gene mutational site that increases together with the ARMS primer of saltant type template; With
TaqMan probe, described probe for being designed for the amplification efficiency that detects ARMS primer pair template in ARMS primer amplification fragment.
2. the test kit based on the transgenation of ARMS fluorescence quantitative PCR detection, wherein, described transgenation is deletion mutantion or insertion mutation, described test kit comprises:
ARMS primer for wild-type template, the 3` end of described primer terminates in disappearance or base place, 2-5, insertion point downstream on bearing of trend, and comprising site to be measured mates with wild-type sequence in interior 3` end base, wherein, described primer arranges respectively one or more base mismatch in disappearance or 2-4 position, insertion point upstream and 6-8 position on bearing of trend;
ARMS primer for saltant type template, the 3` end of described primer terminates in disappearance or base place, 2-5, insertion point downstream on bearing of trend, and comprise site to be measured in interior 3` end base and saltant type sequences match, wherein, described primer arranges respectively one or more base mismatch in disappearance or 2-4 position, insertion point upstream and 6-8 position on bearing of trend;
Share upstream or downstream primer, described primer respectively with the described ARMS primer for wild-type template or described for the one section of sequence that comprises testing gene mutational site that increases together with the ARMS primer of saltant type template; With
TaqMan probe, described probe for being designed for the amplification efficiency that detects ARMS primer pair template in ARMS primer amplification fragment.
3. test kit according to claim 1 and 2, wherein, in the described ARMS primer for wild-type template and the ARMS primer for saltant type template, the position of introducing base mismatch is identical with type; Preferably, except the base at place, mutational site, the described ARMS primer for wild-type template is identical with the described ARMS primer for saltant type template.
4. test kit according to claim 1 and 2, wherein, described test kit further comprises: ROX correcting fluid, Mg
2+, dNTP, polysaccharase and damping fluid.
5. test kit according to claim 1 and 2, wherein, described test kit is for detection of the GJB235delG deletion mutantion relevant to deafness, wherein, the described downstream ARMS primer for wild-type template as shown in SEQ ID NO:7, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:8; And/or described shared upstream primer is as shown in SEQ ID NO:9; And/or described probe is as shown in SEQ ID NO:10.
6. test kit according to claim 1 and 2, wherein, described test kit is for detection of deaf-related gene 12SrRNA1494C>T point mutation, wherein, the described upstream ARMS primer for wild-type template as shown in SEQ ID NO:13, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:14; And/or described shared downstream primer is as shown in SEQ ID NO:15; And/or described probe is as shown in SEQ ID NO:16.
7. test kit according to claim 1 and 2, wherein, described test kit suddenlys change for detection of warfarin medication related locus VKORC13730G>A, wherein, the described downstream ARMS primer for wild-type template as shown in SEQ ID NO:19, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:20; And/or described shared upstream primer is as shown in SEQ ID NO:21; And/or described probe is as shown in SEQ ID NO:22.
8. test kit according to claim 1 and 2, wherein, described test kit suddenlys change for detection of platinum medicine genes involved XRCCI Arg194Trp, wherein, the described downstream ARMS primer for wild-type template as shown in SEQ ID NO:25, for the downstream ARMS primer of saltant type template as shown in SEQ ID NO:26; And/or described shared upstream primer is as shown in SEQ ID NO:27; And/or described probe is as shown in SEQ ID NO:28.
9. the method based on the transgenation of ARMS fluorescence quantitative PCR detection, described method comprises: in identical reaction system, use for the enhanced arm S primer of wild-type template and for the enhanced arm S primer of saltant type template respectively to detecting with a sample to be tested, size by CT value and △ CT value is judged sample genotype, wherein, the described enhanced arm S primer for wild-type template is identical with the definition in claim 1 or 2 with the definition of the described enhanced arm S primer for saltant type template.
10. method according to claim 9, further comprising the steps:
(1) design and screen taqman probe, enhanced arm S primer, pairing primer, the reaction system that contains site to be measured, wherein, described taqman probe, enhanced arm S primer, pairing primer are as mentioned above;
(2) from sample, extract human gene group DNA as template DNA;
(3) carry out real-time fluorescence quantitative PCR amplification: the detection of each pleomorphism site divides 2 pipes to carry out, in every pipe, add identical ROX correcting fluid, Mg
2+, dNTP, polysaccharase, damping fluid, taqman probe, pairing primer and template DNA, in every pipe, add respectively a kind of of wild-type ARMS primer and saltant type ARMS primer, the enhanced arm S-qPCR that carries out polymorphic detection detects;
(4) result is judged
According to Cut-off △ Ct value result of determination.
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