CN102618626A - Method and kit for rapidly detecting single nucleotide polymorphisms (SNPs) - Google Patents
Method and kit for rapidly detecting single nucleotide polymorphisms (SNPs) Download PDFInfo
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- CN102618626A CN102618626A CN2011100323837A CN201110032383A CN102618626A CN 102618626 A CN102618626 A CN 102618626A CN 2011100323837 A CN2011100323837 A CN 2011100323837A CN 201110032383 A CN201110032383 A CN 201110032383A CN 102618626 A CN102618626 A CN 102618626A
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Abstract
The invention relates to an SNPs detection method, and concretely relates to a method for detecting specifically amplified products through a one-step PCR reaction and a nucleic acid detection test paper strip in a same reaction system. The method comprises the following steps: nonspecifically amplifying fragments containing SNPs sites through first PCR temperature cycling; specifically amplifying bases containing the SNPs sites through AS-PCR (allele specific polymerase chain reaction); and detecting the specifically amplified products by the nucleic acid detection test paper strip. The method can also be used for detecting mononucleotide mutation. The method which has the characteristics of high sensitivity, strong specificity, simple operation, short time, and rapid and reliable detection result combines respective advantages of proteins and nucleic acids. The invention also relates to applications of a kit in the detection of known drug-resistant gene of pathogens, the mating selection of a donor and an acceptor in organ transplantation, and the discrimination of the identity of a criminal or paternity tests in forensic researches.
Description
Technical field
The present invention relates to a kind of detection method of SNP; Specifically, the technology that relates to a kind of rapid detection SNP of the technological and nucleic acid test strip detection technique of the pcr amplification that utilizes two cycling programs of non-specific amplification circulation and special detection circulation to accomplish.
Background technology
SNPs is meant on genomic level, has two or more different base on the specific nucleotide position, and the frequency of wherein any allelotrope in colony is not less than 1%.SNPs become the restriction enzyme digestion fragment length that continues polymorphic (Restriction Fragment Length Polymorphism, RFLP), STR (Short Tandem Repeat, STR) third generation molecular genetic marker behind the polymorphic mark.Therefore, the detection of SNP is just becoming the focus of extensive concern.At present, the method that is used for the SNPs detection is the basis mostly with the round pcr, and combines with methods such as fluorescence, mass spectroscopy, gene chip or order-checkings.These method complicated operations, cost an arm and a leg, detection time is long, therefore be not suitable for basic hospital and use.The present invention utilizes a kind of simple to operate, the cheap detection technique of PCR nucleic acid amplification technologies and the invention of nucleic acid test strip detection technique.Therefore, can be widely used in and the relevant field of SNP detection.Summarize with regard to several kinds of existing SNP detection techniques below.
1 restriction enzyme digestion fragment length polymorphic (RFLP)
Utilize the specificity of the restriction enzyme site of restriction enzyme, act on same segment, and judge the base type in SNPs site according to the result of agarose gel electrophoresis with different restriction enzymes.
2 oligonucleotide connection analysis (Oligos Ligation Assay, OLA)
Two oligonucleotide of design are after hybridizing with target sequence in advance, and ligase enzyme can make it with covalently bound, detect connecting product through methods such as gel electrophoresises then, can judge the base type in SNPs site.
3 gene chips (Gene chip)
The probe of the known array through dense arrangement on chip is hybridized the back with some target sequences and is detected, thereby reaches the purpose that detects SNPs site base type.
4 real-time fluorescence quantitative PCRs (Real-time Quantitive PCR)
According to the principle of fluorescence resonance, utilize fluorescence detection device to detect change in fluorescence, thereby carry out the detection of SNPs site base type.
5.DNA PCR sequencing PCR
Dna sequencing can accurately, directly reflect the difference of sequence, but this method needs 3-4 days from the processing of sample to providing the result at least, and price is also relatively more expensive.
6. fluorescence polarization detection (Fluorescence polarization)
Utilize the hybridization of specific probe and amplified production, make 3 ' end of probe connect a ddNTP who indicates the specific fluorescent element under the effect of polysaccharase, judge SNPs site base type according to detected resorcinolphthalein kind and intensity of polarization light then.
7. hybridize two fluorescence probe round pcrs
In the PCR reaction system, add fluorescence labeling probe, and judge SNPs site base type through melting curve (melting curve) analysis across the mutational site.
Along with the discovery of SNPs and building up of some SNPs DBs, specific SNPs becomes to attach most importance to the research of some physiology or pathological state relation with frequency analysis and SNPs in the checking of special group.But the whole bag of tricks in the market needs special analytical instrument mostly, not only costs an arm and a leg, and complicated operation, and is strongly professional, therefore on using, received certain restriction.
Summary of the invention
The present invention is on the basis of patented claim 200610003429.1 " nucleic acid membrane chromatographic fast detecting method and test strip thereof and uses thereof " patent; (Polymerase Chain Reaction PCR) invents out a kind of new quick SNPs detection method-PCR-nucleic acid test strip rapid detection SNPs technology in conjunction with the polymerase chain reaction.Purpose is that to people provide a kind of simple to operate cheap and result detects known SNPs site or single base mutation method accurately.
Primary and foremost purpose of the present invention is to provide a kind of detection method of quick SNP.
Second goal of the invention of the present invention is to provide a kind of detection kit of quick SNP.
In order to realize goal of the invention of the present invention, the technical scheme of employing is:
The present invention relates to a kind of detection method of quick SNP, said detection method is employed in the same reaction system method that a step PCR reaction and detection of nucleic acids test strip detect specific amplification products; The fragment that at first contains the SNPs site through first kind of PCR temperature cycle non-specific amplification contains the base in single nucleotide polymorphism site then through the AS-PCR specific amplification, through the detection of nucleic acids test strip specific amplification products is detected at last.
First optimal technical scheme of the present invention is: in same reaction, comprise the primer and the probe of different effects.The different primers and the effect of probe are respectively:
(1) a pair of amplification comprise the SNPs site template sequence not with the primer A of any mark, non-specific amplification target template is to increase the target template number; Primer A can carry out specific amplification to the template sequence that comprises the SNPs site, and is not with any mark;
(2) carry out the Auele Specific Primer B of specific amplification to the Different Alkali base type on the SNPs site, when primer B and template sequence fully during complementation, primer B could extend;
(3) carry out the probe C of specific hybrid with primer B extension products, thereby accomplish detection SNPs site base type.
Second optimal technical scheme of the present invention is: said Auele Specific Primer B marked has antigen or haptin; Said antigen or haptin are selected from vitamin H, digoxin or optical dye; Said optical dye is selected from Cy3, Cy5, Tetramethylrhodamine, AlexaFluor, Rhodamine, Fam or FitC, preferred vitamin H or digoxin.
The 3rd optimal technical scheme of the present invention is: said probe C marked has antigen or haptin; Said antigen or haptin are selected from vitamin H, digoxin or optical dye; Said optical dye is selected from Cy3, Cy5, Tetramethylrhodamine, AlexaFluor, Rhodamine, Fam or FitC, preferred optical dye.
The 4th optimal technical scheme of the present invention is: the condition of PCR is in the said same system: under 90～98 ℃ of conditions, be incubated 1～4 minute earlier; Under 90～96 ℃ of conditions 5～15 seconds then, under 66～75 ℃ of conditions 10～20 seconds, totally 30～50 circulations; Then under 90～96 ℃ of conditions 10～20 seconds, under 40～50 ℃ of conditions 15～25 seconds, totally 5～20 circulations; Under 92～98 ℃ of conditions, be incubated 1～4 minute at last.
The 5th optimal technical scheme of the present invention is: the condition of PCR is in the said same system: under 92～95 ℃ of conditions, be incubated 1.5～2.5 minutes earlier; Under 92～95 ℃ of conditions 8～12 seconds then, under 66～72 ℃ of conditions 12～18 seconds, totally 35～45 circulations; Then under 92～95 ℃ of conditions 12～18 seconds, under 45～50 ℃ of conditions 18～22 seconds, totally 8～15 circulations; Under 92～95 ℃ of conditions, be incubated 1.5～3 minutes at last; Further preferred elder generation is incubated 2 minutes under 95 ℃ of conditions; Under 94 ℃ of conditions 10 seconds then, following 15 seconds of 72 ℃ of conditions, totally 40 circulations; Then under 94 ℃ of conditions 15 seconds, following 20 seconds of 45 ℃ of conditions, totally 10 circulations; Under 95 ℃ of conditions, be incubated 2 minutes at last.
The 6th optimal technical scheme of the present invention is: because the annealing temperature that primer B, probe C and primer A design is different, in first non-specific amplification working cycle, primer B and probe C do not participate in reaction; And when accomplishing non-specific amplification target template and reaching some amount, during second circulation beginning, the primer B of low temperature thermal oxidation participates in reacting with probe C, reaches the result of specific detection.
The 7th optimal technical scheme of the present invention is: the annealing temperature of primer A is 55 ℃～72 ℃, and the annealing temperature of primer B and probe C is 35 ℃～50 ℃, preferred 65 ℃～72 ℃ of the annealing temperature of primer A, preferred 40 ℃～45 ℃ of the annealing temperature of primer B and probe C.
The 8th optimal technical scheme of the present invention is: said detection of nucleic acids test strip is provided with primer B/ probe C hybridizes the antigen of product institute mark or the detection line that hapten specificity bonded antibody forms.
The 9th optimal technical scheme of the present invention is: the pcr amplification system of PCR is in the said same system:
Template: 2～4 μ l;
The non-specific primer A:0.025 of forward～0.05 μ mol;
Reverse non-specific primer A:0.1～0.2 μ mol;
Auele Specific Primer B:0.1～0.2 μ mol;
Specific probe C:0.025～0.05 μ mol;
dNTP： 0.1～0.2mmol；
(NH
4)
2SO
4： 5～10mmol；
KCl： 5～10mmol；
Tris-HCl:5～10mmol of pH 9.0
Triton-100： 0.5％～1.0％；
MgCl： 1.25～2.5mmol；
Taq archaeal dna polymerase: 1～2 unit;
TV is 10～20 microlitres.
Said amplification system is preferably:
Template: 4 μ l;
The non-specific primer A:0.05 of forward μ mol;
Reverse non-specific primer A:0.2 μ mol;
Auele Specific Primer B:0.2 μ mol;
Specific probe C:0.05 μ mol;
dNTP： 0.2mmol；
(NH
4)
2SO
4： 10mmol；
KCl： 10mmol；
The Tris-HCl:10mmol of pH 9.0
Triton-100： 1.0％；
MgCl： 2.5mmol；
Taq archaeal dna polymerase: 2 units;
TV is 20 microlitres.
The tenth optimal technical scheme of the present invention is: according to the Auele Specific Primer B of the design of the Different Alkali base type on SNPs site different IPs nucleotide sequence, only contain a kind of Auele Specific Primer B of nucleotide sequence in each pcr amplification system.
The invention still further relates to the application of this detection method in detecting single nucleotide mutation.
The invention still further relates to a kind of detection kit of quick SNP, said test kit comprises:
(1) the detection of nucleic acids test strip device of room temperature preservation;
(2)-20 the pcr amplification system of the present invention of ℃ preservation:
(3)-20 the positive control solution of ℃ preservation;
(4)-20 the negative controls of ℃ preservation;
(5)-20 the ddH of ℃ preservation
2O.
Wherein, when amplification reaction solution was 20 person-portions, positive control solution was 40ul, and negative controls is 40ul, and ddH20 is 140ul.
Wherein, Positive control solution described in the present invention contains can make test strip produce the template sequence that comprises the SNPs site of positive reaction, and described negative controls is not for containing the liquid that makes test strip produce the template sequence that comprises the SNPs site of positive reaction.
Have only when primer B extension products and probe C is specific when combining, could on the detection line of detection of nucleic acids test strip, form colored band; Simultaneously, if, so just can't carry out specific the combination, also just can't on detection line, form colored band with probe C when not extension of primer B.
The invention still further relates to the discriminating of criminal's identity or the application in the paternity test in the pairing selection between donor and acceptor, the legal medical expert's research in the detection of the known drug resistant gene of pathogenic agent, organ transplantation of this test kit.
The invention still further relates to the discriminating of criminal's identity or the application in the paternity test in the pairing selection between donor and acceptor, the legal medical expert's research in the detection of the known drug resistant gene of pathogenic agent, organ transplantation of the detection method of this quick SNP.
Further specify in the face of technical scheme of the present invention down.
Cardinal principle of the present invention is for containing earlier the fragment in SNPs site through pcr amplification, then to the base type of SNPs carry out allele specific amplification (Allele specific Polymerase Chain Reaction, AS-PCR); The AS-PCR product can be hybridized with specific probe, and the hybridization product can finally be detected by the nucleic acid test strip.Reaction principle and the step of PCR-nucleic acid test strip method detection SNPs are as shown in Figure 1.
The method that the present invention detects SNPs mainly comprises following four steps:
1) through PCR the target template that comprises the SNPs site is increased.The annealing temperature of PCR reaction is between 66 ℃～72 ℃, and cycle number is 35～40.In this step, pcr amplification is not distinguished the base type in SNPs site, only for the enriched target sequence so that participate in the AS-PCR in the step 2.Simultaneously, the amplimer that is applied in this step does not carry out any mark, and its Tm should be about 66 ℃～72 ℃.
2) carry out AS-PCR to the base type in SNPs site.The annealing temperature of AS-PCR reaction is between 45 ℃-50 ℃, and cycle number is 5-10.Through the reaction annealing temperature of control AS-PCR, make and have only when primer of participating in AS-PCR and the complete complementation of target template, could make that primer is able to extend.Therefore, can carry out specific amplification to the Different Alkali base type in SNPs site.During the design primer, can be with the allele-specific primers of vitamin H (Biotin) mark according to the base type design 5 ' end in SNPs site, and with the 3 ' end of SNPs site design at this primer.The Tm of primer participates in the step 1 to avoid it about 45 ℃～50 ℃, thereby improves the specificity of reaction.
3) amplified production of AS-PCR and specific probe are hybridized.(fluorescein isothiocyanate, FITC), Tm is about 50 ℃～60 ℃ for 3 ' terminal band fluorescein isothiocyanate of specific probe.Can form the hybridization complex that has Biotin and FITC mark simultaneously after the amplified production hybridization of probe and AS-PCR.
4) utilize the nucleic acid test strip that the hybridization product is detected.During detection, the hybridization complex that has Biotin and FITC mark simultaneously combines with the red granules of surface band avidin mark earlier, forms the red granules mixture of band FITC mark.This red granules mixture flows along the absorption limit of tunica fibrosa to the nucleic acid test strip through capillarity under the effect of damping fluid.When it ran into the Anti-FITC on the nucleic acid test strip detection line, because the interaction of FITC and Anti-FITC can be fixed on the coloured particle mixture on the detection line, and forms macroscopic red lines, this was positive.If but in AS-PCR; Allele-specific primers does not obtain extending; The hybridization complex that has Biotin and FITC mark simultaneously can't hybridized and form to this primer just with specific probe so; Also just can't on the detection line of nucleic acid test strip, form red lines, this is negative.
The detection method of a kind of quick SNP of the present invention is employed in the same reaction system step PCR reaction and detection of nucleic acids test strip specific amplification products is detected; The fragment that at first contains the SNPs site through first kind of PCR temperature cycle non-specific amplification contains the base in single nucleotide polymorphism site then through the AS-PCR specific amplification, through the detection of nucleic acids test strip specific amplification products is detected at last.In same reaction system, comprise the primer and the probe of different effects.Two or more the different base that go up to exist to same SNPs designs the nucleotide sequence with the corresponding primer B of this site Nucleotide respectively; Parallel pcr amplification system is set; The Nucleotide that a kind of primer B is arranged in each pcr amplification system; In testing process, two of testing sample addings or above parallel PCR system are increased, adopt the nucleic acid test strip to detect after reaction is accomplished, thus the detected result of obtaining.
It is 200610109620.4 the anti-pollution proofing unit of totally-enclosed nucleic acid that the nucleic acid test strip that the present invention adopts can adopt number of patent application, this proofing unit have totally-enclosed detection, high specificity, simple to operate fast, the amplification back detects without any need for plant and instrument.This device detects nucleic acid amplification and analyzes all and accomplish at closed state, reduces the danger of crossed contamination in the conventional TRAP to greatest extent.This test kit is simple to operate, the time short, has also lowered the detection cost simultaneously, makes detected result more fast, reliably.Principle the narrating in another patented claim 200610003429.1 of applicant of the test strip in its device.
Patented claim 200610003429.1 discloses a kind of nucleic acid membrane chromatographic fast detecting method and test strip thereof and uses thereof.This patented claim discloses a kind of detection method of specific nucleic acid sequence, and this method is adsorbed in coloured particle with a strain specific antibodies A, forms antibody sandwich at particle surface; Another kind of specific antibody-colourless anti-B antibody is fixed in forms detection line on the film; When determined nucleic acid increases; Employed probe or primer with A antigen or B antigenic mark, are formed the mixture of amplified material, probe, primer, antigen A, antigen B, this mixture is combined with the antibody A that is adsorbed with coloured particles; When this coloured particle mixture that obtains passes through upwards mobile antibody B detector bar of capillary vessel phenomenon tunica fibrosa oculi in solution; Combine with the antibody B on the lines, thereby be trapped on the detection line, form macroscopic coloured lines.This test strip is included in has sample pad, coloured particle binding substances pad and absorbent filter pad in order on the liner that has non-setting adhesive; Each part mentioned above is overlapped at adjacent; Also be provided with detection line and nature controlling line on the tunica fibrosa, wherein the coloured particle on the coloured particle binding substances pad has anti-A antibody to encapsulate, and has anti-B antibody to encapsulate on the detection line; Anti-A antibody is arranged on the nature controlling line, and coloured particle is selected from colloid gold particle, latex particle.
Wherein, detection of nucleic acids test strip of the present invention is provided with primer B/ probe C and hybridizes the antigen of product institute mark or the detection line that hapten specificity bonded antibody forms.
The present invention is to be detection platform with the detection of nucleic acids test strip, to providing the result, only needs 2～3 hours from sample extraction.Interpretation simultaneously is simple, again without any need for specific apparatus, makes Operating Complexity and detection cost reduce greatly, therefore detects in general molecule laboratory and hospital laboratory completion.Table 1 is present method and gene chip, the comparison of SNPs detection techniques such as gene sequencing:
Table 1
| Item compared | Gene chip | Gene sequencing | Quantitative fluorescent PCR | The inventive method |
| Homozygote/heterozygote detects | Ability | Ability | Ability | Ability |
| The gene of single-time measurement | A plurality of | A plurality of | One | One |
| Operational cycle | 4-5 hour | Three days | 2-3 hour | 2-3 hour |
| Operating Complexity | Higher | High | Higher | Low |
| Requirement to experiment condition | High | Very high | High | Low |
This method is simple to operate, the time is short, has also reduced the detection cost simultaneously, makes detected result more fast, reliably, has protein and diagnostic nucleic acid reagent advantage separately.The present invention can increase the accuracy of clinical diagnosis greatly in the use of Clinical Laboratory, shortens the time that the patient seeks medical advice, and the overall cost of medical health system is reduced.
It is special with it, simply, characteristic can obtain to use very widely efficiently that PCR-nucleic acid test strip method detects SNPs, as:
1. the human genetic disease's directly related molecular diagnosis with SNPs;
2. can confirm examination and the somatotype of the SNPs of human health hereditary basis among the crowd;
3. the human body medicine Low Response opposite sex detects and examination;
4. the examination of certain diseases predisposing gene among the crowd;
5. the detection of the known drug resistant gene of pathogenic agent;
6. the pairing between donor and acceptor is selected in the organ transplantation;
7. the discriminating and the paternity test of criminal's identity in legal medical expert's research.
Description of drawings
Fig. 1 detects SNP reaction principle synoptic diagram for PCR-AS-PCR;
Fig. 2 is the detected result figure of the sick Mitochondrial DNA G11778A of Leber ' the s SNP of sample 1; Experimental result shows that the test strip that detects genotype G shows the positive reaction result, and the test strip that detects genotype A type shows negative experimental result, explains that the detected result in 11778 sites is G, is wild-type; Corresponding sequencing result shows that 11778 sites are G;
Fig. 3 is the detected result figure of the sick Mitochondrial DNA G11778A of Leber ' the s SNP of sample 2; Experimental result shows that the test strip that detects genotype G shows the negative reaction result, and the test strip that detects genotype A type shows positive experimental result, explains that the detected result in 11778 sites is A, is mutant.The right is corresponding sequencing result, shows that 11778 sites are A;
Fig. 4 is the KatG 315 site mutation detected result figure of sample 1 Mycobacterium tuberculosis; Wherein, the positive experimental result of first left test strip, second, third test strip all is negative experimental result, the genotype that sample 1 is described is a wild-type; The right is corresponding sequencing result, and sequencing result is AGC;
Fig. 5 is the KatG 315 site mutation detected result figure of sample 2 Mycobacterium tuberculosiss; Wherein, first left, the 3rd the negative result of test strip, second is positive, and the genotype that this sample is described is that the result that A detects is AAC; The right is corresponding sequencing result, and sequencing result is AAC;
Fig. 6 is the KatG 315 site mutation detected result figure of sample 3 Mycobacterium tuberculosiss; Wherein, the negative result of first, second test strip of the left side, the 3rd is positive, and the genotype that this sample is described is the C type; The right is corresponding sequencing result, and sequencing result is ACC.
Embodiment of the present invention is advanced to be limited to technical scheme of the present invention is done further explanation and explanation, technical scheme of the present invention is not constituted restriction.
Embodiment
The detection and the somatotype of the sick Mitochondrial DNA G11778A of embodiment 1 Leber ' s SNP
Leber ' s hereditary optic neuropathy (Leber ' s hereditary optic neuropathy, LHON) be a kind of eyes optic nerve disease of matrocliny.People such as W allace in 1988 are report line mitochondrial DNA (mtDNA) the 11778th nucleotide site primary sudden change (G → A) can cause LHON at first.Find existing 25 of the mtDNA site mutation relevant so far, so the molecular biology genetic test becomes the sick prefered method of Leber ' s of differentiating the different causes of disease with this disease.In view of for the diagnostic significance of plastosome G11778A site somatotype, use the neoteric SNPS test strip of this patent the G11778A loci polymorphism is detected for Leber ' s hereditary optic neuropathy.Practical implementation is following:
(1) sampling
Examinee's peripheral vein anticoagulation (frozen) 5 microlitres are used the minim DNA rapid extraction test kit that Yousida Biological Technology Co., Ltd., Hangzhou produced and are carried out nucleic acid extraction, are used for the specific fragment amplification.
(2) detection line mitochondrial DNA (mtDNA) the 11778th nucleotide site primary sudden change (reaction system of G → A):
Template: seized genomic dna 20ng/ul 4ul;
Non-specific primer A:LEBSFP 0.05 little rubbing, nucleotides sequence is classified as shown in the SEQ ID NO:1;
LEBSRP 0.2 little rubbing, nucleotides sequence are classified as shown in the SEQ ID NO:2;
The Auele Specific Primer B that detects genotype G is: LEBPF5B 0.2 little rubbing, its nucleotides sequence are classified the substituted SEQ ID of 5 ' vitamin H NO:3 as;
The Auele Specific Primer B that detects genotype A is: LEBPF5B10.2 is little to rub, and its nucleotides sequence is classified the substituted SEQ IDNO:4 of 5 ' vitamin H as;
Specific probe C:LEBPR3F 0.05 little rubbing, its nucleotides sequence are classified the substituted SEQ ID of 3 ' FITC NO:5 as;
SEQ ID NO:1 is 5 '-CACCGGCGCAGTCATTCTCATAATCG-3 ';
SEQ ID NO:2 is 5 '-GGTAAGGCGAGGTTAGCGAGGCTTG-3 ';
The substituted SEQ ID of 5 '-vitamin H NO:3 is 5 '-Biotin-CACTCACAGTCGC-3 ';
The substituted SEQ ID of 5 '-vitamin H NO:4 is 5 ' Biotin-CACTCACAGTCAC-3 ';
The substituted SEQ ID of 3 ' FITC NO:5 is 5 '-GAGAGGATTATGAT-FITC-3 '.
The reaction system that detects genotype G is:
LEBSFP 0.05 little rubbing
LEBSRP 0.2 little rubbing
LEBPR3F 0.05 little rubbing
LEBPF5B 0.2 little rubbing
DNTP:0.2 rubs in the least
(NH
4)
2SO
410 millis rub
KCl 10 millis rub
Tris-HCL (PH 9.0) 10 millis rub
Triton-100 1.0％
MgCL 2.5 millis rub
Taq archaeal dna polymerase: 2 units
Seized genomic dna 20ng/ul 4 microlitres
TV is 20 microlitres
The reaction system that detects genotype A is:
LEBSFP 0.05 little rubbing
LEBSRP 0.2 little rubbing
LEBPR3F 0.05 little rubbing
LEBPF5B1 0.2 little rubbing
DNTP:0.2 rubs in the least
(NH
4)
2SO
410 millis rub
KCl 10 millis rub
Tris-HCL (PH 9.0) 10 millis rub
Triton-100 1.0％
MgCL 2.5 millis rub
Taq archaeal dna polymerase: 2 units
Seized genomic dna 20ng/ul 4 microlitres
TV is 20 microlitres
The PCR response procedures is following:
95 ℃, 2 minutes
Then
Last 95 ℃, 2 minutes
(3) detected result:
Reaction product is all dripped on the nucleic acid test strip, 10 minutes sentence read result.Undergo mutation in examinee's plastosome ND4 gene 11 778 sites, by G → A.The nucleic acid test strip detects SNP result and sees Fig. 2 and Fig. 3; The left side is two test strip, and the first left test strip is for detecting the test strip of genotype G, and the second left test strip is for detecting the test strip of genotype A; The right be corresponding sequencing result, the base of line be the site that will detect; Sequencing result is consistent with SNP test strip detected result.
Fig. 2 is the detected result figure of the sick Mitochondrial DNA G11778A of Leber ' the s SNP of sample 1; Experimental result shows that the test strip that detects genotype G shows the positive reaction result, and the test strip that detects genotype A type shows negative experimental result, explains that the detected result in 11778 sites is G, is wild-type; Corresponding sequencing result shows that 11778 sites are G;
Fig. 3 is the detected result figure of the sick Mitochondrial DNA G11778A of Leber ' the s SNP of sample 2; Experimental result shows that the test strip that detects genotype G shows the negative reaction result, and the test strip that detects genotype A type shows positive experimental result, explains that the detected result in 11778 sites is A, is mutant.The right is corresponding sequencing result, shows that 11778 sites are A.
Embodiment 2. Mycobacterium tuberculosis vazadrine resistance sudden change KatG 315 sudden changes detect
The vazadrine is the important drugs of treatment Mycobacterium tuberculosis, can produce resistance but take for a long time.The sudden change of Mycobacterium tuberculosis specific gene is to produce the drug-fast first cause in vazadrine, and its sudden change is mainly the AGC → AAC and the AGC → ACC in 315 sites of Kat G gene.Therefore, KatG 315 sudden changes of dynamic monitoring Mycobacterium tuberculosis have important directive significance to timely adjustment regimen, rational use of drug and raising curative effect.
(1) sampling
Mycobacterium tuberculosis patient sputum is used the DNA extraction test kit that Yousida Biological Technology Co., Ltd., Hangzhou produced and is carried out nucleic acid extraction, in order to gene amplification.
(2) KatG 315 sudden changes of PCR-AS-PCR nucleic acid test strip method detection branches bacillus:
Non-specific primer A:AGCPF/AGCPR
The non-specific primer A:TBLPF315 of forward, its nucleotides sequence classify as shown in the SEQ ID NO:6;
Reverse non-specific primer A:TBLPR315, its nucleotides sequence classify as shown in the SEQ ID NO:7;
The Auele Specific Primer B that detects the AGC wild-type is respectively: TBLDF5F315-W, its nucleotides sequence classify the substituted SEQ ID of 5 ' end vitamin H NO:8 as;
The Auele Specific Primer B that detects AGC → AAC sudden change is respectively: TBLDF5F315-M1, its nucleotides sequence classify the substituted SEQ ID of 5 ' end vitamin H NO:9 as;
The Auele Specific Primer B that detects AGC → ACC sudden change is respectively: TBLDF5F315-M2, its nucleotides sequence classify the substituted SEQ ID of 5 ' end vitamin H NO:10 as;
Specific probe C TBLRD3F315, its nucleotides sequence classify the substituted SEQ ID of 3 ' end FITC NO:11 as;
SEQ ID NO:6 is 5 '-TGGAAGAGCTCGTATGGCACCGG-3 ';
SEQ ID NO:7 is 5 '-CCCATTTCGTCGGGGTGTTCGTC-3 ';
The substituted SEQ ID of 5 ' vitamin H NO:8 is 5 ' (Biotin) CGCGATCACCAGC 3 ';
The substituted SEQ ID of 5 ' vitamin H NO:9 is 5 ' (Biotin) CGCGATCACCACC 3 ';
The substituted SEQ ID of 5 ' vitamin H NO:10 is 5 ' (Biotin) CGCGATCACCAAC 3 ';
The substituted SEQ ID of 3 ' FITC NO:11 is 5 ' CATACGACCTCGAT-FITC;
The reaction system that detects wild-type is:
TBLPF315 0.05 little rubbing
TBLPR315 0.2 little rubbing
TBLDF5F315-W 0.2 little rubbing
TBLRD3F315 0.05 little rubbing
DNTP:0.2 rubs in the least
(NH
4)
2SO
410 millis rub
KCl 10 millis rub
Tris-HCL (PH 9.0) 10 millis rub
Triton-100 1.0％
MgCL 2.5 millis rub
Taq archaeal dna polymerase: 2 units
DNA 4 microlitres that extract
TV is 20 microlitres
The reaction system that detects AGC → ACC sudden change is:
TBLPF315 0.05 little rubbing
TBLPR315 0.2 little rubbing
TBLDF5F315-M1 0.2 little rubbing
TBLRD3F315 0.05 little rubbing
DNTP:0.2 rubs in the least
(NH
4)
2SO
410 millis rub
KCl 10 millis rub
Tris-HCL (PH 9.0) 10 millis rub
Triton-100 1.0％
MgCL 2.5 millis rub
Taq archaeal dna polymerase: 2 units
DNA 4 microlitres that extract
TV is 20 microlitres
The reaction system that detects AGC → AAC sudden change is:
TBLPF315 0.05 little rubbing
TBLPR315 0.2 little rubbing
TBLDF5F315-M2 0.2 little rubbing
TBLRD3F315 0.05 little rubbing
DNTP:0.2 rubs in the least
(NH
4)
2SO
410 millis rub
KCl 10 millis rub
Tris-HCL (PH 9.0) 10 millis rub
Triton-100 1.0％
MgCL 2.5 millis rub
Taq archaeal dna polymerase: 2 units
DNA 4 microlitres that extract
TV is 20 microlitres
The PCR response procedures is following:
95 ℃, 2 minutes
Then
Last 45 ℃, 2 minutes
(3) detected result: reaction product is all dripped on the nucleic acid test strip sentence read result after 10 minutes.Fig. 4～Fig. 6 is the KatG 315 site AGC → AAC and the AGC → ACC sudden change detected result figure of Mycobacterium tuberculosis; 3 on the left side is the test strip detected result, and the right is that corresponding order-checking detects; Wherein, the first left test strip is that the detection genotype is the test strip of G type-wild-type, and second left is that the detection genotype is the test strip of A type, and third left is that the detection genotype is the test strip of C type; Sequencing result confirms consistent with the test strip detected result.
Fig. 4 is the KatG 315 site mutation detected result figure of sample 1 Mycobacterium tuberculosis; Wherein, the positive experimental result of first left test strip, second, third test strip all is negative experimental result, the genotype that sample 1 is described is a wild-type; The right is corresponding sequencing result, and sequencing result is AGC;
Fig. 5 is the KatG 315 site mutation detected result figure of sample 2 Mycobacterium tuberculosiss; Wherein, first left, the 3rd the negative result of test strip, second is positive, and the genotype that this sample is described is that the result that A detects is AAC; The right is corresponding sequencing result, and sequencing result is AAC;
Fig. 6 is the KatG 315 site mutation detected result figure of sample 3 Mycobacterium tuberculosiss; Wherein, the negative result of first, second test strip of the left side, the 3rd is positive, and the genotype that this sample is described is the C type; The right is corresponding sequencing result, and sequencing result is ACC.
Claims (15)
1. the detection method of a quick SNP (SNPs) is characterized in that, said detection method is employed in the same reaction system method that a step PCR reaction and detection of nucleic acids test strip detect specific amplification products; The fragment that at first contains the SNPs site through first kind of PCR temperature cycle non-specific amplification contains the base in single nucleotide polymorphism site then through the AS-PCR specific amplification, through the detection of nucleic acids test strip specific amplification products is detected at last.
2. quick SNPs detection method according to claim 1 is characterized in that, comprises following primer and probe:
Amplification comprise the SNPs site template sequence not with the primer A of any mark, non-specific amplification target template is to increase the target template number;
Carry out the Auele Specific Primer B of specific amplification to the Different Alkali base type on the SNPs site, when primer B and template sequence were complementary fully, primer B could extend;
Carry out the probe C of specific hybrid with primer B extension products, thereby accomplish detection SNPs site base type.
3. the detection method of quick SNP according to claim 2; It is characterized in that; Said Auele Specific Primer B marked has antigen or haptin; Said antigen or haptin are selected from vitamin H, digoxin or optical dye, and said optical dye is selected from Cy3, Cy5, Tetramethylrhodamine, AlexaFluor, Rhodamine, Fam or FitC, preferred vitamin H or digoxin.
4. the detection method of quick SNP according to claim 2; It is characterized in that; Said probe C marked has antigen or haptin; Said antigen or haptin are selected from vitamin H, digoxin or optical dye, and said optical dye is selected from Cy3, Cy5, Tetramethylrhodamine, AlexaFluor, Rhodamine, Fam or FitC, preferred optical dye.
5. the detection method of quick SNP according to claim 1 is characterized in that, the condition of PCR is in the said same reaction system: under 90～98 ℃ of conditions, be incubated 1～4 minute earlier; Under 90～96 ℃ of conditions 5～15 seconds then, under 66～75 ℃ of conditions 10～20 seconds, totally 30～50 circulations; Then under 90～96 ℃ of conditions 10～20 seconds, under 40～50 ℃ of conditions 15～25 seconds, totally 5～20 circulations; Under 92～98 ℃ of conditions, be incubated 1～4 minute at last.
6. the detection method of quick SNP according to claim 5 is characterized in that, the condition of PCR is in the said same system: under 92～95 ℃ of conditions, be incubated 1.5～2.5 minutes earlier; Under 92～95 ℃ of conditions 8～12 seconds then, under 66～72 ℃ of conditions 12～18 seconds, totally 35～45 circulations; Then under 92～95 ℃ of conditions 12～18 seconds, under 45～50 ℃ of conditions 18～22 seconds, totally 8～15 circulations; Under 92～95 ℃ of conditions, be incubated 1.5～3 minutes at last; Further preferred elder generation is incubated 2 minutes under 95 ℃ of conditions; Under 94 ℃ of conditions 10 seconds then, following 15 seconds of 72 ℃ of conditions, totally 40 circulations; Then under 94 ℃ of conditions 15 seconds, following 20 seconds of 45 ℃ of conditions, totally 10 circulations; Under 95 ℃ of conditions, be incubated 2 minutes at last.
7. according to the detection method of the described quick SNP of the arbitrary claim of claim 1～6, it is characterized in that in first non-specific amplification working cycle, primer B and probe C do not participate in reaction; And when accomplishing non-specific amplification target template and reaching some amount, during second circulation beginning, the primer B of low temperature thermal oxidation participates in reacting with probe C, reaches the result of specific detection.
8. the detection method of quick SNP according to claim 7; It is characterized in that; The annealing temperature of primer A is 55 ℃～72 ℃; The annealing temperature of primer B and probe C is 35 ℃～50 ℃, preferred 65 ℃～72 ℃ of the annealing temperature of primer A, preferred 40 ℃～45 ℃ of the annealing temperature of primer B and probe C.
9. the detection method of quick SNP according to claim 1 is characterized in that, said detection of nucleic acids test strip is provided with and the antigen of primer B or probe C hybridization product institute mark or the detection line that hapten specificity bonded antibody forms.
10. the detection method of quick SNP according to claim 1 is characterized in that, the pcr amplification system of PCR is in the said same system:
Template: 2～4 μ l;
The non-specific primer A:0.025 of forward～0.05 μ mol;
Reverse non-specific primer A:0.1～0.2 μ mol;
Auele Specific Primer B:0.1～0.2 μ mol;
Specific probe C:0.025～0.05 μ mol;
dNTP： 0.1～0.2mmol；
(NH
4)
2SO
4： 5～10mmol；
KCl： 5～10mmol；
Tris-HCl:5～10mmol of pH 9.0
Triton-100： 0.5％～1.0％；
MgCl： 1.25～2.5mmol；
Taq archaeal dna polymerase: 1～2 unit;
TV is 10～20 microlitres.
11. the detection method of quick SNP according to claim 10; It is characterized in that; According to the Auele Specific Primer B of the design of the Different Alkali base type on SNPs site different IPs nucleotide sequence, only contain a kind of Auele Specific Primer B of nucleotide sequence in each pcr amplification system.
12. the application of the described detection method of claim 1 in detecting single nucleotide mutation.
13. the described detection method of claim 1 is the discriminating of criminal's identity or the application in the paternity test in the pairing selection between donor and acceptor, the legal medical expert's research in the detection of the known drug resistant gene of pathogenic agent, organ transplantation.
14. the detection kit of a quick SNP is characterized in that, said test kit comprises:
(1) the detection of nucleic acids test strip device of room temperature preservation;
(2)-20 the described pcr amplification system of the claim 10 of ℃ preservation:
(3)-20 the positive control solution of ℃ preservation;
(4)-20 the negative controls of ℃ preservation;
(5)-20 the ddH of ℃ preservation
2O.
15. the described test kit of claim 14 is the discriminating of criminal's identity or the application in the paternity test in the pairing selection between donor and acceptor, the legal medical expert's research in the detection of the known drug resistant gene of pathogenic agent, organ transplantation.
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