CN105624296A - Universal fluorescent hairpin primers for detecting gene polymorphism on basis of ARMS (amplification refractory mutation system) fluorescent PCR (polymerase chain reaction) process - Google Patents
Universal fluorescent hairpin primers for detecting gene polymorphism on basis of ARMS (amplification refractory mutation system) fluorescent PCR (polymerase chain reaction) process Download PDFInfo
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Abstract
The invention provides universal fluorescent hairpin primers for detecting gene polymorphism on the basis of an ARMS (amplification refractory mutation system) fluorescent PCR (polymerase chain reaction) process. The nucleotide sequences of the primers are disclosed as Seq ID No:1 and 2. The detection primer and fluorescent primer provided by the invention are low in price, and the other required experimental consumables are cheap and accessible. The experiment process does not need sequencing, thereby greatly shortening the detection period and enhancing the detection efficiency on the premise of saving the detection cost. Compared with the TaqMan probe process, a pair of universal fluorescent primers are used in the invention, the synthesis of specific fluorescent primers (probes) for each SNP (single-nucleotide polymorphism) site is not need, and thus, the universal fluorescent primers have higher site adaptability. According to different genes and genotypes, only the synthesis of the common PCR specific amplification primers is needed, thereby greatly lowering the research and development cost.
Description
Technical field
The present invention relates to biology field, specifically, relate to a kind of Universal fluorescence clamp primers detecting gene pleiomorphism based on ARMS fluorescent PCR method.
Background technology
Gene mutation refers to that base pair composition or the change put in order structurally occur gene. Point mutation includes single nucleotide mutation, nucleotide deletion, nucleotide insertion etc. Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by the variation of single core thuja acid in genomic level. Point mutation detection main method has PCR-PFLP detection technique, DNA direct sequencing, ApoE gene (AS-PCR) method, Taqman sonde method, PCR-Manganic pyrophosphate complex initiation method and PCR-chip method etc. PCR-PFLP technology relies on the reason such as digestion with restriction enzyme, electrophoretic analysis, often the response time is long, need open pipe repeatedly, cross-contamination and result misjudgment phenomenon easily occur, affect its Detection accuracy, flux is relatively low simultaneously, is not suitable for rapid screening and the clinical diagnosis of a large amount of crowd and visiting. Although DNA direct sequencing is gene test goldstandard, however it is necessary that the instrument and equipment of costliness and complicated operating process and the feature of detection cycle length, most of laboratorys are difficult to, and are also not suitable for Clinical Laboratory. PCR-chip hybridization methods has multiple advantages, the multiple PCR-chip hybridization methods detection polymorphism test kit of CFDA approved, but chip detection complicated operation, professional standards are required height, chip hybridization length consuming time, and flexibility ratio is low, cost is high, it is necessary to special instrument and equipment. PCR-Manganic pyrophosphate complex initiation method is suitable for various flux, highly sensitive, but detection apparatus expensive, it is relatively more suitable for the determination of larger samples, the mutant proportion all kinds SNP detection higher than 5%. ApoE gene (AS-PCR) electrophoresis method is highly sensitive, but flux is low, is especially suitable for small-size laboratory detection, but since it is desired that electrophoresis, the detection time is long, it is easy to erroneous judgement occur. Taqman sonde method uses fluorescent probe, highly sensitive, simple to operate, and instrument and equipment is easily universal and is suitable for various flux, but fluorescent probe is expensive, and each SNP site need to design two allelic specific probes, and development cost is very high. Therefore, the practical situation according to the detection of China basic hospital, design is a kind of highly sensitive, and detection method is easy, and instrument cost is few and the method for the new detection gene pleiomorphism of applicable various flux is particularly urgent.
ARMS-PCR technology and amplification refractory mutation system��ARMS (AmplificationRefractoryMutationSystemARMS-PCR), it is also called allele specific amplification method (AlleleSpecificAmplification, AS-PCR). Its ultimate principle is two specificity ARMS primers of design, and primer 3 ' holds last base identical with wild type or mutant bases respectively, and other one is universal reverse primer. When carrying out PCR reaction, owing to Taq DNA polymerase lacks 3 ' �� 5 ' 5 prime excision enzyme activities, if primer 3 ' end forms mispairing, chain extension reaction is obstructed, and in certain amplification cycles, not will detect that special amplified production; Otherwise, 3 ' end pairings are then capable of detecting when amplified production.
Fluorescence clamp primers (FluorescenceHairpinPrimer, FHP), also known as LUX primer (Light-UponeXtension primer), it is that a kind of 5 ' and 3 ' ends have complementary palindrome, and is carrying out fluorophor list target primer in the dT base of 3 ' ends. This primer can form hairpin structure (loop-stem structure) under free state, and this DNA conformation itself has the characteristic of self-quenching fluorophor, it is not necessary at other end labelling quenching group. When clamp primers and template are matched and after PCR primer acquisition extension under Taq DNA polymerase effect, namely this hairpin structure is opened, and discharges fluorescence, causes that fluorescence signal dramatically increases, it is possible to carry out quantitatively with quantitative real time PCR Instrument. Due to 3 ' end can the multiple fluorophor of labelling, therefore apply this hair clip type fluorescent primer and can realize multiple fluorescence PCR.
Summary of the invention
It is an object of the invention to provide a kind of Universal fluorescence clamp primers detecting gene pleiomorphism based on ARMS fluorescent PCR method.
It is another object of the present invention to detect expensive, the deficiency such as cycle length, poor efficiency, data analysis are loaded down with trivial details of mthfr gene polymorphism for prior art, provide a kind of based on fluorescent quantitative PCR technique platform, in conjunction with the method for detecting mutation of gene point of ARMS-PCR and fluorescence clamp primers.
In order to realize the object of the invention, the present invention a kind of based on ARMS fluorescent PCR method detect gene pleiomorphism Universal fluorescence clamp primers (described primer includes (SeqIDNo:1-2) for FHP primer, Fig. 1 a):
Wild type fluorescence clamp primers FHP-W:5 '-GATCGAGTACCTACCTTGTTCGATC-3 '
Saltant type fluorescence clamp primers FHP-M:5 '-GACTGAGTAGTTCTGCGTCATCAGTC-3 '
The sequence length of described Universal fluorescence clamp primers is 26-27bp, and sequence and human genome not homology or can not homology completely. This primer is the primer that a kind of 5 ' and 3 ' ends have palindrome, has the complementary series of 4-7 base, can form hairpin structure, and carry out fluorophor list mark in 3 ' end dT bases. The conformation of the hairpin structure that primer is formed under free state itself has the characteristic of self-quenching fluorescence. Due to hair clip fluorescent primer in PCR process easily upon opening, therefore when primer and template are matched time, this hairpin structure discharges fluorescence after opening, cause that fluorescence signal dramatically increases, and extension can be obtained under Taq enzyme effect as PCR primer, produce with fluorescently-labeled PCR primer, it is possible to carry out quantitatively with quantitative real time PCR Instrument. When the �� G optimum range of hairpin structure is-1.6��-5.8kcal/mol, structure is easily upon opening, can with template complementary pairing, it is thus achieved that best fluorescent quenching effect. The melting temperature of clamp primers should between 60 DEG C-68 DEG C. Fluorophor conventional at present includes FAM, TET, JOE, HEX, AlexaFluor546, Alexa594 etc. Fluorophor is preferably used and includes FAM and HEX. Base sequence generally adopts DNA synthesizer synthetic, through polyacrylamide gel electrophoresis or other proper method purification after synthesis.
In the present invention, described ARMS specificity amplification primer refers to the oligonucleotide sequence being made up of a number of dNTPs, generally by DNA synthesizer synthetic, through polyacrylamide gel electrophoresis or other proper method purification after synthesis. In polymerase chain reaction, primer can be combined in the region of complementation with it in purpose nucleic acid chains to be amplified, and its function is the starting point as nucleotide polymerization effect, on 3 '-OH of primer, nucleotide synthesizes with diester linkage form, and therefore 3 '-OH of primer must be free. Archaeal dna polymerase can be proceeded by extension by its 3 ' end, synthesizes new nucleic acid chains. It is, in general, that for identifying that the ARMS specificity amplification primer of point mutation position includes two. Last base is held respectively to identify the base of wild type or saltant type at primer 3 '.
The present invention also provides for the test kit detecting gene pleiomorphism for ARMS fluorescent PCR method containing described Universal fluorescence clamp primers.
Preferably, described test kit also includes dNTPs, Taq DNA polymerase, Mg2+��K+, at least one in Tris-HCl, PCR reaction buffer etc.
The present invention also provides for described Universal fluorescence clamp primers or described test kit and detects the application in gene pleiomorphism in ARMS fluorescent PCR method.
The present invention also provides for detecting the specific primer of gene M THFR polymorphism based on ARMS fluorescent PCR method, including forward primer MTHFR-C677T-W, forward primer MTHFR-C677T-M and a reverse general primer, described forward primer is by sequence label, specific primer sequence and 3 ' end base composition (Fig. 1 b). The base composition of sequence label respectively with two Universal fluorescence clamp primers sequences fully and partially identical (at least above 18bp). Specific primer sequence is identical with institute's amplification gene point mutation position (MTHFRC677T) upstream sequence, for specific recognition with in conjunction with genomic DNA. Article two, 3 ' end bases of ARMS specific primer are identical with wild-type base and mutant bases respectively, are mainly used in ARMS-PCR reaction and hinder Taq DNA polymerase to extend. Article one, reverse general primer, expands for ARMS-PCR, and its sequence 3 ' end can not form complementary pairing with Universal fluorescence clamp primers and ARMS specific primer.
Described primer includes (SeqIDNo:3-5):
Forward primer MTHFR-C677T-W:5 '-GATCGAGTACCTACCTTGTTCGATCGAGAAGGTGTCTGCGGGTGC-3 '
Forward primer MTHFR-C677T-M:5 '-GACTGAGTAGTTCTGCGTCATCAGTCGAGAAGGTGTCTGCGGGCGT-3 '
Reverse general primer R:5 '-CAAAGCGGAAGAATGTGTCAG-3 '
The present invention also provides for the test kit detecting gene M THFR polymorphism for ARMS fluorescent PCR method containing described specific primer.
Preferably, described test kit also includes described Universal fluorescence clamp primers (FHP-W and FHP-M), dNTPs, Taq DNA polymerase, Mg2+��K+, at least one in Tris-HCl, PCR reaction buffer.
Specifically, test kit includes fluorescent PCR-ARMS reactant liquor (MasterMix) and specificity amplification primer mixed liquor (PrimerMix). Fluorescent PCR-ARMS reactant liquor (MasterMix) contains PCR and reacts outside the materials such as necessary Taq DNA polymerase, buffer, magnesium ion, dNTPs, also comprises a pair universal hair clip fluorescent primer. ARMS primer mixed liquor (PrimerMix) then comprises the wild type of the forward of the corresponding point mutation of detection and the reverse general primer R of the specificity amplification primer (MTHFR-C677T-W and MTHFR-C677T-M) of saltant type, corresponding point mutation.
The reaction system of described test kit application is as follows: 2 �� MasterMix5 �� l, PrimerMix2 �� l, template DNA 1-10ng, ddH2O complements to 10 �� l.
Wherein, 2 �� MasterMix is composed as follows: Tris-HCl20mM, KCl50mM, the MgCl of Taq DNA polymerase 0.25U, pH8.324mM, dNTPs0.4mM, wild type fluorescence clamp primers FHP-W0.1-1.0 ��M, saltant type fluorescence clamp primers FHP-M0.1-1.0 ��M.
PrimerMix is composed as follows: forward primer MTHFR-C677T-W0.05-1.0 ��M, forward primer MTHFR-C677T-M0.05-1.0 ��M, reverse general primer R0.1-1.0 ��M.
The response procedures of described test kit application is as follows:
PCR reaction was made up of 3 stages:
First stage, denaturation, 95 DEG C, 10 minutes;
Second stage is made up of 10-15 amplification cycles: 95 DEG C of degeneration 20 seconds; Annealing: initial temperature is 61 DEG C, and target temperature is 55 DEG C, and decrease speed is 0.6 DEG C/sec, and the time is 1 minute;
Phase III is made up of 28-35 amplification cycles: 95 DEG C of degeneration 20 seconds; Anneal 1 minute for 55 DEG C; Now gather fluorescence signal.
Finally, draw melting curve, fluorescence signal acquisition is set.
Article two, the melting curve of Universal fluorescence clamp primers is as shown in Figure 2. In the touchdownPCR process of first stage, the ARMS amplimer of specific amplification wild type and saltant type is competitive to be combined with degeneration template and anneals, owing to Taq DNA polymerase lacks 3 ' �� 5 ' 5 prime excision enzyme activities, when carrying out PCR reaction, if primer 3 ' end formed mispairing, chain extension reaction will because of 3 ', 5 '-phosphodiester bond formed obstacle and be obstructed, causing that PCR primer amount sharply reduces, the primer of correct corresponding gene type of matching then obtains amplification. In second stage PCR process subsequently, corresponding fluorescence clamp primers can with the sequence label specific recognition of initial stage product and pairing, under Taq DNA polymerase effect, product obtains and extends, hairpin structure becomes a part for new product after opening, its hairpin structure opens the release of rear fluorescence, owing to a large amount of hair clip fluorescent primers obtain exponential amplification thus fluorescence signal dramatically increases and amplifies. Another hair clip fluorescent primer unpaired with templet gene type then continues to keep hairpin structure, and fluorescence is by self-quenching. Quantitative real time PCR Instrument carries out after detection by quantitative the kind according to corresponding fluorescence and fluoresced power, it is judged which kind of genotype obtains expands. This method need not go to synthesize special fluorescent primer for each SNP site, universal fluorescent primer ARMS-PCR principle based on oneself uniqueness, the detection of all of site is made finally all to use Universal fluorescence clamp primers to expand, greatly reduce reagent cost, existing ARMS goldstandard accurate, reduce again use cost, than Taqman sonde method, there is better site adaptability.
The present invention further provides the method detecting gene M THFR polymorphism based on ARMS fluorescent PCR method, in order to detect the SNP site C677T (rs1801133) of gene M THFR, the method comprises the following steps:
(1) testing sample processes and DNA profiling extraction
A. extract person's blood sample to be measured, from blood sample, extract DNA. Recommend business-like test kit and extract peripheral blood genomic DNA.
B. DNA concentration and purity are measured, it is desirable to concentration 1ng/ �� l-10ng/ �� l, OD260nm/OD280nm=1.6-2.0.
(2) fluorescent PCR amplification
Template DNA to be detected (1-10ng) is added containing ARMS-Fluorescence PCR liquid (2 �� MasterMix) of the present invention and ARMS specific primer mixed liquor (PrimerMix). After mix homogeneously is centrifuged in fluorescent PCR pipe, puts in quantitative real time PCR Instrument and carry out PCR reaction according to specific temperature cycles and signals collecting program, in order to monitor response availability.
Described fluorescent PCR amplification scheme preferably employs temperature below circulation and signals collecting program carries out PCR reaction:
PCR reaction was made up of 3 stages:
First stage, denaturation, 95 DEG C, 10 minutes;
Second stage is made up of 15 amplification cycles, and its condition is (touchdown PCR, touchdownPCR) 95 DEG C of degeneration 20 seconds; Annealing: initial temperature is 61 DEG C, and target temperature is 55 DEG C, and decrease speed is 0.4 DEG C/sec, and the time is 1 minute;
Phase III is made up of 30-35 amplification cycles, and its condition is: 95 DEG C of degeneration 20 seconds; Anneal 1 minute for 55 DEG C; Now gather fluorescence signal.
Finally, draw melting curve, fluorescence signal acquisition is set.
(3) interpretation of result
Require to carry out the analysis of Ct value according to different Real-timeqPCR programs.
The present invention is shown in Fig. 3 based on the ARMS-Fluorescence PCR principle schematic of Universal fluorescence clamp primers.
The invention have the advantages that
(1) a pair Universal fluorescence clamp primers provided by the invention designs and synthesizes simple. Have only to, holding one fluorophor of labelling in dT base near primer 3 ', utilize the hairpin structure quenching fluorescence that primer self is formed, without picture Taqman probe simultaneously need to labelling quenching probes, saved reagent cost.
(2) a pair Universal fluorescence clamp primers provided by the invention will not form dimer. Before PCR denaturation temperature is increased to denaturation temperature, due to self ready-made special hairpin structure trend be better than between primer formed double-strand, it is difficult to another primer formed double-strand, will not be formed and amplify primer dimer.
(3) specific amplification of fluorescence clamp primers provided by the invention and ARMS-fluorescent PCR specific primer is high. First, the 3 ' of two ARMS-fluorescent PCR specific primers hold last base respectively to identify the base of wild type or saltant type, can the corresponding genotype of specific amplification. Secondly, only when the hairpin structure that target sequence and primer sequence adhesion are formed more than fluorescence clamp primers self, primer could occur hybridization to open with target sequence, and primer just obtains DNA and extends ability, and release fluorescence obtains detection.
(4) detection primer provided by the invention is cheap with fluorescent primer, and other required experiment consumptive material prices are also cheap and easy to get. Experimentation does not need order-checking, while saving testing cost, substantially reduces the detection cycle, improve the efficiency of detection.
(5) detection primer provided by the invention is wide with the fluorescent primer suitability. Compared with Taqman sonde method, the present invention uses universal fluorescent clamp primers, it is not necessary to goes to synthesize special fluorescent primer (probe) for each SNP site, has better site adaptability. According to different genes and genotype, it is only necessary to synthesize common PCR specificity amplification primer, be substantially reduced R&D costs.
(6) amplimer provided by the invention and universal fluorescent clamp primers, adopt Fluorescence PCR assay and other fluorescence analyser platforms, it may be achieved basic, normal, high various flux detect. Practical situation according to current China clinical gene polymorphic detection, as long as having quantitative real time PCR Instrument and other fluorescence analysers, can realize detecting fast and accurately at small-size laboratory completely, greatly reducing inspection technical threshold.
(7) amplimer detecting people's mthfr gene C677T pleomorphism site provided by the invention and corresponding fluorescent primer, its PCR detects specificity and susceptiveness is very high, and adopt Fluorescence PCR assay, reaction is quickly, without repeatedly uncapping, avoiding cross-contamination, all detection reaction can complete in 3 hours, and the easy interpretation of testing result.
Accompanying drawing explanation
Fig. 1 is the present invention universal hair clip fluorescent primer (Fig. 1 a), detects the schematic diagram of the specific primer (Fig. 1 b) of gene M THFR polymorphism C677T based on ARMS fluorescent PCR method.
Fig. 2 is the melting curve of two Universal fluorescence clamp primers of the present invention.
Fig. 3 is the present invention ARMS-Fluorescence PCR principle schematic based on universal hair clip fluorescent primer.
Fig. 4 is to tri-kinds of genetic polymorphism detection results of MTHFR-C677TSNP in the embodiment of the present invention 2.
Fig. 5 is the melting curve of the PCR primer of ARMS-fluorescent PCR detection MTHFR-C677T gene pleiomorphism CT type in the embodiment of the present invention 2.
Fig. 6 is the result that MTHFR-C677TSNP wild type and saltant type detect in the embodiment of the present invention 2 limit.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention. If not specializing, embodiment is experiment condition all conventionally, such as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ&RussellDW, Molecularcloning:alaboratorymanual, 2001) condition, or according to manufacturer's description advised. The quantitative real time PCR Instrument model used in following example is ABI7500.
The design of embodiment 1ARMS specific primer and Universal fluorescence clamp primers and synthesis
Reference sequences (the NG_013351.1 of mthfr gene disclosed in GenBank GenBank according to US National Biotechnology Information center NCBI, NM_005957.4) and the information of C677T pleomorphism site (SNPID:rs180113) disclosed in dbSNP data base, PrimerPremier5 software design primer MTHFR-C677T-W wild primers, MTHFR-C677T-M mutant primers and reverse general primer R (Fig. 1) are adopted. Sequence according to designed primer, is synthesized by Shanghai Sheng Gong company and adopts PAGE mode purification primer. Universal fluorescence clamp primers sequence be one section with human genome without complementary or entirely without complementary oligonucleotide sequence, fluorophor is connected to the primer dT base near 3 ' ends, without quenching group, Shanghai Sheng Gong company synthesize, through HPLC method purification after synthesis. The mark fluorescent group used in the present embodiment is FAM and HEX. Article two, the melting curve of Universal fluorescence clamp primers is as in figure 2 it is shown, be 48-60 DEG C in melting temperature, and fluorescent primer hairpin structure is gradually opened, and fluorescence signal obtains release, can obtain amplification as primer.
Universal fluorescence clamp primers sequence following (SeqIDNo:1-2):
Wild type fluorescence clamp primers FHP-W:5 '-GATCGAGTACCTACCTTGTTCGATC-3 '
Saltant type fluorescence clamp primers FHP-M:5 '-GACTGAGTAGTTCTGCGTCATCAGTC-3 '
ARMS-fluorescent PCR specific primer sequence following (SeqIDNo:3-5):
Forward primer MTHFR-C677T-W:5 '-GATCGAGTACCTACCTTGTTCGATCGAGAAGGTGTCTGCGGGTGC-3 '
Forward primer MTHFR-C677T-M:5 '-GACTGAGTAGTTCTGCGTCATCAGTCGAGAAGGTGTCTGCGGGCGT-3 '
Reverse general primer R:5 '-CAAAGCGGAAGAATGTGTCAG-3 '
Embodiment 2 applies the method for the ARMS-fluorescent PCR method detection gene M THFR polymorphism of Universal fluorescence clamp primers
(1) sample to be tested DNA extraction
The extraction of peripheral blood in patients sample DNA preferably employs commercial kit, or extracts according to " molecular cloning " method.
Measure DNA concentration and purity, it is desirable to genome ultimate density is adjusted to 1ng/ �� l-10ng/ �� l, OD260nm/OD280nm=1.6-2.0.
(2) PCR reactant liquor preparation
By ARMS-Fluorescence PCR liquid (MasterMix) prepared, ARMS specificity amplification primer mixed liquor (PrimerMix) and DNA profiling, ddH2O is separately added in 0.2mlPCR pipe and mixes, centrifugal. Reaction arranges 4, it is known that tri-kinds of genotypic samples of MTHFR-C677T and a negative controls are (with ddH2O is template).
Reaction system is as follows: 2 �� MasterMix5 �� l, PrimerMix2 �� l, template DNA 1-10ng, ddH2O complements to 10 �� l.
Wherein, 2 �� MasterMix is composed as follows: Tris-HCl20mM, KCl50mM, the MgCl of Taq DNA polymerase 0.25U, pH8.324mM, dNTPs0.4mM, wild type fluorescence clamp primers FHP-W0.1-1.0 ��M, saltant type fluorescence clamp primers FHP-M0.1-1.0 ��M.
PrimerMix is composed as follows: forward primer MTHFR-C677T-W0.05-1.0 ��M, forward primer MTHFR-C677T-M0.05-1.0 ��M, reverse general primer R0.1-1.0 ��M.
(3) upper machine testing
For ABI7500 instrument, following temperature cycles and signals collecting program are set:
PCR reaction was made up of 3 stages:
First stage, denaturation, 95 DEG C, 10 minutes;
Second stage is made up of 15 amplification cycles, and its condition is (touchdown PCR, touchdownPCR) 95 DEG C of degeneration 20 seconds; Annealing: initial temperature is 61 DEG C, and target temperature is 55 DEG C, and decrease speed is 0.4 DEG C/sec, and the time is 1 minute;
Phase III is made up of 30-35 amplification cycles, and its condition is: 95 DEG C of degeneration 20 seconds; Anneal 1 minute for 55 DEG C; Now gather fluorescence signal.
Finally, draw melting curve, fluorescence signal acquisition is set, FAM and HEX fluorescence signal is set.
(4) interpretation of result
Enter ABI7500qPCR instrument software, select the fluorescence representated by corresponding allele, be chosen as VIC/HEX as target1 is chosen as FAM, target2. Click " Analysis ", so that whether the amplification curve of FAM and/or HEX fluorescence signal rises (i.e. Ct value) as genotype criterion.
Tri-kinds of genetic polymorphism detection results of MTHFR-C677TSNP are shown in Fig. 4. The melting curve utilizing the PCR primer of ARMS-fluorescent PCR detection MTHFR-C677T gene pleiomorphism CT type is shown in Fig. 5.
(5) detection limit is analyzed
It is that template carries out ARMS-PCR reaction with the human gene group DNA of known MTHFR-C677T wild type CC and saltant type TT. Respectively take the wild type of 33ng, 10ng, 3.3ng, 1.0ng and the DNA of saltant type, detect according to above-mentioned ARMS-PCR reaction kit and method. Testing result is as shown in Figure 6. Analyzing according to detection limit, amplified fluorescence curve should have the S type curve of smoothness, and Ct value regards as the positive between 10 and 20. It is too little that Ct value regards as applied sample amount more than 20. It is excessive that Ct value regards as DNA applied sample amount less than 10, and after all should adjusting concentration, row detects again.
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. detect the Universal fluorescence clamp primers of gene pleiomorphism based on ARMS fluorescent PCR method, it is characterised in that described primer includes:
Wild type fluorescence clamp primers FHP-W:5 '-GATCGAGTACCTACCTTGTTCGATC-3 '
Saltant type fluorescence clamp primers FHP-M:5 '-GACTGAGTAGTTCTGCGTCATCAGTC-3 '.
2. Universal fluorescence clamp primers according to claim 1, it is characterised in that using different fluorophor labellings in 3 ' the end dT bases of primers F HP-W and FHP-M respectively; Described fluorophor includes FAM, TET, JOE, HEX, AlexaFluor546, Alexa594, it is preferable that FAM, HEX.
3. contain the test kit for ARMS fluorescent PCR method detection gene pleiomorphism of Universal fluorescence clamp primers described in claim 1 or 2.
4. test kit according to claim 3, it is characterised in that described test kit also includes dNTPs, Taq DNA polymerase, Mg2+��K+, at least one in Tris-HCl, PCR reaction buffer.
5. Universal fluorescence clamp primers described in claim 1 or 2 or test kit described in claim 3 or 4 detect the application in gene pleiomorphism in ARMS fluorescent PCR method.
6. detect the specific primer of gene M THFR polymorphism C677T based on ARMS fluorescent PCR method, it is characterised in that described primer includes:
Forward primer MTHFR-C677T-W:5 '-GATCGAGTACCTACCTTGTTCGATCGAGAAGGTGTCTGCGGGTGC-3 '
Forward primer MTHFR-C677T-M:5 '-GACTGAGTAGTTCTGCGTCATCAGTCGAGAAGGTGTCTGCGGGCGT-3 '
Reverse general primer R:5 '-CAAAGCGGAAGAATGTGTCAG-3 '.
7. contain the test kit for ARMS fluorescent PCR method detection gene M THFR polymorphism of specific primer described in claim 6.
8. test kit according to claim 7, it is characterised in that described test kit also includes Universal fluorescence clamp primers, dNTPs, Taq DNA polymerase, Mg described in claim 1 or 22+��K+, at least one in Tris-HCl, PCR reaction buffer.
9. test kit according to claim 8, it is characterised in that the reaction system of described test kit application is as follows: 2 �� MasterMix5 �� l, PrimerMix2 �� l, template DNA 1-10ng, ddH2O complements to 10 �� l;
Wherein, 2 �� MasterMix is composed as follows: Tris-HCl20mM, KCl50mM, the MgCl of Taq DNA polymerase 0.25U, pH8.324mM, dNTPs0.4mM, wild type fluorescence clamp primers FHP-W0.1-1.0 ��M, saltant type fluorescence clamp primers FHP-M0.1-1.0 ��M;
PrimerMix is composed as follows: forward primer MTHFR-C677T-W0.05-1.0 ��M, forward primer MTHFR-C677T-M0.05-1.0 ��M, reverse general primer R0.1-1.0 ��M.
10. test kit according to claim 8 or claim 9, it is characterised in that the response procedures of described test kit application is as follows:
PCR reaction was made up of 3 stages:
First stage, denaturation, 95 DEG C, 10 minutes;
Second stage is made up of 10-15 amplification cycles: 95 DEG C of degeneration 20 seconds; Annealing: initial temperature is 61 DEG C, and target temperature is 55 DEG C, and decrease speed is 0.6 DEG C/sec, and the time is 1 minute;
Phase III is made up of 28-35 amplification cycles: 95 DEG C of degeneration 20 seconds; Anneal 1 minute for 55 DEG C; Now gather fluorescence signal.
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