CN102618626B - 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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- CN102618626B CN102618626B CN201110032383.7A CN201110032383A CN102618626B CN 102618626 B CN102618626 B CN 102618626B CN 201110032383 A CN201110032383 A CN 201110032383A CN 102618626 B CN102618626 B CN 102618626B
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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 single nucleotide polymorphism, specifically, relate to the technology of a kind of rapid detection single nucleotide polymorphism of utilize non-specific amplification circulation and specific detection to circulate pcr amplification technology that two cycling programs complete and nucleic acid test strip detection technique.
Background technology
SNPs refers in genomic level, has two or more different base on specific nucleotide position, and wherein the frequency of any allelotrope in colony is not less than 1%.SNPs has become polymorphic (the Restriction Fragment Length Polymorphism of the restriction enzyme digestion fragment length that continues, RFLP), third generation molecular genetic marker after the polymorphic mark of STR (Short Tandem Repeat, STR).Therefore, the detection of SNP is just becoming the focus of extensive concern.At present, the method detecting for SNPs be basic mainly with round pcr greatly, and is combined with the method such as fluorescence, mass spectroscopy, gene chip or order-checking.These method complicated operations, expensive, detection time is long, is therefore not suitable for basic hospital and uses.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 to single nucleotide polymorphism and detect relevant field.Summarize with regard to several 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 with different restriction enzymes, and judge the base type in SNPs site according to the result of agarose gel electrophoresis.
2 oligonucleotide linking parsings (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, then detect connecting product by methods such as gel electrophoresises, can judge the base type in SNPs site.
3 gene chips (Gene chip)
The probe of the known array by dense arrangement on chip, hybridizes rear detection with some target sequences, thereby reaches the object 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 sequencing
DNA sequencing can accurately, directly reflect the difference of sequence, but the method at least needs 3-4 days from the processing of sample to providing result, and price is also more expensive.
6. fluorescence polarization detection (Fluorescence polarization)
Utilize specific probe and amplified production hybridization, make 3 ' end of probe under the effect of polysaccharase, connect a ddNTP who indicates specific fluorescent element, then judge SNPs site base type according to the fluorescein kind detecting and intensity of polarization light.
7. the two fluorescence probe round pcrs of hybridization
In PCR reaction system, add the fluorescence labeling probe across mutational site, and judge SNPs site base type by melting curve (melting curve) analysis.
Along with the discovery of SNPs and building up of some SNPs databases, 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 expensive, and complicated operation, strongly professional, therefore in application, has been subject to certain restriction.
Summary of the invention
The present invention is on the basis of patent application 200610003429.1 " nucleic acid membrane chromatographic fast detecting method and test strip thereof and uses thereof " patent, invent a kind of new quick SNPs detection method-PCR-nucleic acid test strip rapid detection SNPs technology in conjunction with polymerase chain reaction (Polymerase Chain Reaction, PCR).Object is that cheap and result detects known SNPs site or single base mutation method accurately for people provide a kind of simple to operate.
Primary and foremost purpose of the present invention is to provide a kind of detection method of quick single nucleotide polymorphism.
The second goal of the invention of the present invention is to provide a kind of detection kit of quick single nucleotide polymorphism.
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 single nucleotide polymorphism, described detection method adopts the method that a step PCR reacts and detection of nucleic acids test strip detects specific amplification products in same reaction system; First the fragment that contains SNPs site by the first PCR temperature cycle non-specific amplification, the base that then contains single nucleotide polymorphism site by AS-PCR specific amplification, finally detects specific amplification products by detection of nucleic acids test strip.
The first optimal technical scheme of the present invention is: in same reaction, comprise not primer and the probe of same-action.Different primers and the effect of probe are respectively:
(1) the not primer A with any mark of the template sequence that pair for amplification comprises SNPs site, non-specific amplification target template is to increase target template number; Primer A can carry out specific amplification to the template sequence that comprises SNPs site, and is not with any mark;
(2) carry out the Auele Specific Primer B of specific amplification for the Different Alkali base type on SNPs site, in the time of primer B and template sequence complete complementary, primer B could extend;
(3) carry out the probe C of specific hybrid with primer B extension products, thereby complete the detection to SNPs site base type.
The second optimal technical scheme of the present invention is: on described Auele Specific Primer B, be marked with antigen or haptens, described antigen or haptens are selected from vitamin H, digoxin or fluorescence dye, described fluorescence dye is selected from Cy3, Cy5, Tetramethylrhodamine, AlexaFluor, Rhodamine, Fam or FitC, preferably vitamin H or digoxin.
The 3rd optimal technical scheme of the present invention is: on described probe C, be marked with antigen or haptens, described antigen or haptens are selected from vitamin H, digoxin or fluorescence dye, described fluorescence dye is selected from Cy3, Cy5, Tetramethylrhodamine, AlexaFluor, Rhodamine, Fam or FitC, preferably fluorescence dye.
The 4th optimal technical scheme of the present invention is: in described same system, the condition of PCR is: first under 90～98 ℃ of conditions, be incubated 1～4 minute; Then under 90～96 ℃ of conditions 5～15 seconds, 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; Finally under 92～98 ℃ of conditions, be incubated 1～4 minute.
The 5th optimal technical scheme of the present invention is: in described same system, the condition of PCR is: first under 92～95 ℃ of conditions, be incubated 1.5～2.5 minutes; Then under 92～95 ℃ of conditions 8～12 seconds, 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; Finally under 92～95 ℃ of conditions, be incubated 1.5～3 minutes; Further preferably first under 95 ℃ of conditions, be incubated 2 minutes; Then under 94 ℃ of conditions 10 seconds, lower 15 seconds of 72 ℃ of conditions, totally 40 circulations; Then under 94 ℃ of conditions 15 seconds, lower 20 seconds of 45 ℃ of conditions, totally 10 circulations; Finally under 95 ℃ of conditions, be incubated 2 minutes.
The 6th optimal technical scheme of the present invention is: because primer B, probe C are different with the annealing temperature of primer A design, in first non-specific amplification working cycle, primer B and probe C do not participate in reaction; And when completing non-specific amplification target template and reaching some amount, when second circulation starts, the primer B of low temperature thermal oxidation and probe C participate in reaction, reach 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 ℃, preferably 65 ℃～72 ℃ of the annealing temperatures of primer A, preferably 40 ℃～45 ℃ of the annealing temperatures of primer B and probe C.
The 8th optimal technical scheme of the present invention for: in described detection of nucleic acids test strip, be provided with the detection line that the antibody of being combined with antigen or the hapten specificity of primer B/ probe C hybridization product institute mark forms.
The 9th optimal technical scheme of the present invention is: in described same system, the pcr amplification system of PCR is:
Template: 2～4 μ l;
The non-specific primer of forward A:0.025～0.05 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;
Cumulative volume is 10～20 microlitres.
Described 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;
Cumulative volume is 20 microlitres.
The tenth optimal technical scheme of the present invention is: according to the Auele Specific Primer B of the Different Alkali base type design different IPs nucleotide sequence on SNPs site, only contain a kind of Auele Specific Primer B of nucleotide sequence in each pcr amplification system.
The invention still further relates to this detection method in the application detecting in single nucleotide mutation.
The invention still further relates to a kind of detection kit of quick single nucleotide polymorphism, described test kit comprises:
(1) the detection of nucleic acids test strip device of room temperature preservation;
(2) the pcr amplification system of the present invention of-20 ℃ of preservations:
(3) positive control solution of-20 ℃ of preservations;
(4) negative controls of-20 ℃ of preservations;
(5) ddH of-20 ℃ of preservations
2o.
Wherein, in the time that amplification reaction solution is 20 person-portion, positive control solution is 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 SNPs site of positive reaction, and described negative controls is not contain the liquid that makes test strip produce the template sequence that comprises SNPs site of positive reaction.
Only have when primer B extension products and probe C is specific while combining, could on the detection line of detection of nucleic acids test strip, form coloured band; , do not extend if work as primer B meanwhile, so just cannot carry out specific combination with probe C, also just cannot on detection line, form coloured band.
The invention still further relates to the application in discriminating or the paternity test of criminal's identity in the pairing selection between donor and acceptor, 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 application in discriminating or the paternity test of criminal's identity in the pairing selection between donor and acceptor in the detection of the known drug resistant gene of pathogenic agent, organ transplantation of the detection method of this quick single nucleotide polymorphism, legal medical expert's research.
Below technical scheme of the present invention is described in further detail.
Cardinal principle of the present invention is the fragment that first contains SNPs site by pcr amplification, then carries out allele specific amplification (Allele specific Polymerase Chain Reaction, AS-PCR) for the base type of SNPs; AS-PCR product can be hybridized with specific probe, and hybridization product can finally be detected by nucleic acid test strip.The reaction principle of PCR-nucleic acid test strip method detection SNPs and step are as shown in Figure 1.
The method that the present invention detects SNPs mainly comprises following four steps:
1) by PCR, the target template that comprises 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 enriched target sequence to participate in the AS-PCR in step 2.Meanwhile, the amplimer being applied in this step does not carry out any mark, and its Tm should be 66 ℃～72 ℃ left and right.
2) carry out AS-PCR for the base type in SNPs site.The annealing temperature of AS-PCR reaction is between 45 ℃-50 ℃, and cycle number is 5-10.By controlling the reaction annealing temperature of AS-PCR, make to only have in the time of the primer of participation AS-PCR and target template complete complementary, just can make primer be extended.Therefore, can carry out specific amplification for the Different Alkali base type in SNPs site.Design is when primer, can be according to the allele-specific primers of the base type design in SNPs site 5 ' end band vitamin H (Biotin) mark, and by the design of SNPs site the 3 ' end at this primer.The Tm of primer, 45 ℃～50 ℃ left and right, to avoid it to participate in step 1, thereby improves the specificity of reaction.
3) amplified production of AS-PCR and specific probe are hybridized.3 ' end band fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) of specific probe, Tm is 50 ℃～60 ℃ left and right.After the amplified production hybridization of probe and AS-PCR, can form the hybridization complex with Biotin and FITC mark simultaneously.
4) utilize nucleic acid test strip to detect hybridization product.When detection, be first combined with the red granules of surface band avidin mark with the hybridization complex of Biotin and FITC mark simultaneously, form the red granules mixture with FITC mark.This red granules mixture is under the effect of damping fluid, and the absorption limit by capillarity along tunica fibrosa to nucleic acid test strip flows.In the time that it runs into the Anti-FITC on nucleic acid test strip detection line, because the interaction of FITC and Anti-FITC can be fixed on coloured particle mixture on detection line, and form macroscopic red lines, this is positive.If but in AS-PCR, allele-specific primers is extended, the hybridization complex with Biotin and FITC mark simultaneously just cannot be hybridized and form to this primer with specific probe so, also just cannot on the detection line of nucleic acid test strip, form red lines, this is negative.
The detection method of a kind of quick single nucleotide polymorphism of the present invention, employing step PCR in same reaction system reacts and detection of nucleic acids test strip detects specific amplification products; First the fragment that contains SNPs site by the first PCR temperature cycle non-specific amplification, the base that then contains single nucleotide polymorphism site by AS-PCR specific amplification, finally detects specific amplification products by detection of nucleic acids test strip.In same reaction system, comprise not primer and the probe of same-action.Design respectively the nucleotide sequence of the primer B corresponding with this site Nucleotide for upper two or more the different base existing of same SNPs, parallel pcr amplification system is set, in each pcr amplification system, there is the Nucleotide of a kind of primer B, testing sample is added two or more parallel PCR systems to increase in testing process, react rear employing nucleic acid test strip and detected, 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, detect without any need for plant and instrument after amplification.This device all completes nucleic acid amplification determination and analysis at closed state, reduce to greatest extent the danger of crossed contamination in conventional TRAP.This test kit is simple to operate, the time is short, has also lowered testing cost simultaneously, makes detected result more fast, reliably.Principle the narrating in another patent application 200610003429.1 of applicant of the test strip in its device.
Patent application 200610003429.1 discloses a kind of nucleic acid membrane chromatographic fast detecting method and test strip thereof and uses thereof.This Patent Application Publication a kind of detection method of specific nucleic acid sequence, a strain specific antibodies A is adsorbed in coloured particle by the method, forms antibody coated at particle surface; Another kind of specific antibody-colourless anti-B antibody is fixed on and on film, forms detection line; When determined nucleic acid increases, by used probe or Staphylococal Protein A or B antigenic mark for primer, form the mixture of amplified material, probe, primer, antigen A, antigen B, this mixture is combined with the antibody A of absorption coloured particle, when this coloured particle mixture obtaining upwards flows an antibody B detector bar by capillary vessel phenomenon tunica fibrosa oculi in solution, antibody B on lines is combined, thereby is trapped on detection line, forms macroscopic colored line.This test strip is included in having in order sample pad, coloured particle binding substances pad and absorbent filter pad on the liner of non-setting adhesive, each part mentioned above partly overlaps at adjacent, on tunica fibrosa, be also provided with detection line and nature controlling line, wherein the coloured particle on coloured particle binding substances pad has anti-A antibody coated, on detection line, there is anti-B antibody coated, on nature controlling line, have anti-A antibody, coloured particle is selected from colloid gold particle, latex particle.
Wherein, in detection of nucleic acids test strip of the present invention, be provided with the detection line of hybridizing the antigen of product institute mark or the antibody formation that hapten specificity is combined with primer B/ probe C.
The present invention is take detection of nucleic acids test strip as detection platform, from sample extraction to providing result, only needs 2～3 hours.Interpretation is simultaneously simple, again without any need for specific apparatus, Operating Complexity and testing cost is reduced greatly, therefore completes detection at general molecule laboratory and hospital laboratory.Table 1 is present method and gene chip, the comparison of the SNPs detection techniques such as gene sequencing:
Table 1
| Item compared | Gene chip | Gene sequencing | Quantitative fluorescent PCR | The inventive method |
| Homozygote/heterozygote detects | Energy | Energy | Energy | Energy |
| The gene of single-time measurement | Multiple | Multiple | One | One |
| Operational cycle | 4-5 hour | Three days | 2-3 hour | 2-3 hour |
| Operating Complexity | Higher | High | Higher | Low |
| To the requirement of experiment condition | High | Very high | High | Low |
This method is simple to operate, the time is short, has also reduced testing cost simultaneously, makes detected result more fast, reliably, has advantages of protein and diagnostic nucleic acid reagent separately.The present invention, in the use of Clinical Laboratory, can increase the accuracy of clinical diagnosis greatly, shortens the time that patient seeks medical advice, and also can make the overall cost of medical health system.
It is special with it, simply, characteristic can obtain very widely application efficiently that PCR-nucleic acid test strip method detects SNPs, as:
1. the human genetic disease's directly related with SNPs molecular diagnosis;
2. in crowd, can determine examination and the somatotype of the SNPs of human health hereditary basis;
3. the human body medicine Low Response opposite sex detects and examination;
4. the examination of certain diseases predisposing gene in crowd;
5. the detection of the known drug resistant gene of pathogenic agent;
6. in organ transplantation, the pairing between donor and acceptor is selected;
7. discriminating and the paternity test of criminal's identity in legal medical expert's research.
Accompanying drawing explanation
Fig. 1 is that PCR-AS-PCR detects SNP reaction principle schematic diagram;
Fig. 2 is the detected result figure of the sick Mitochondrial DNA G11778A of Leber ' the s single nucleotide polymorphism of sample 1; Experimental result shows, detects the test strip demonstration positive reaction result of genotype G, and the test strip that detects genotype A type shows negative experimental result, illustrates that in the detected result in 11778 sites be 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 single nucleotide polymorphism of sample 2; Experimental result shows, detects the test strip demonstration negative reaction result of genotype G, and the test strip that detects genotype A type shows positive experimental result, illustrates that in the detected result in 11778 sites be A, is saltant type.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 is all negative experimental result, the genotype that sample 1 is described is 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 C type; The right is corresponding sequencing result, and sequencing result is ACC.
The specific embodiment of the present invention is entered to be limited to technical scheme of the present invention is made further explanation, and technical scheme of the present invention is not construed as limiting.
Embodiment
Detection and the somatotype of the sick Mitochondrial DNA G11778A of embodiment 1 Leber ' s single nucleotide polymorphism
Leber ' s hereditary optic neuropathy (Leber ' s hereditary optic neuropathy, LHON) be a kind of eyes optic nerve disease of matrocliny.The people such as W allace in 1988 first report line mitochondrial DNA (mtDNA) the 11778th nucleotide site primary sudden change (G → A) can cause LHON.Find so far existing 25 of the mtDNA site mutation relevant with this disease, therefore molecular biology genetic test becomes the prefered method of Leber ' the s disease of differentiating different pathogeny.In view of the diagnostic significance for Leber ' s hereditary optic neuropathy for plastosome G11778A site somatotype, the neoteric SNPS test strip of application this patent detects G11778A loci polymorphism.Specifically be implemented as follows:
(1) sampling
Examinee's peripheral vein anticoagulation (frozen) 5 microlitres, the minim DNA rapid extraction test kit that application Yousida Biological Technology Co., Ltd., Hangzhou produces carries out nucleic acid extraction, increases for specific fragment.
(2) detection line mitochondrial DNA (mtDNA) the 11778th nucleotide site primary sudden change (G → A) reaction system:
Template: tested genomic dna 20ng/ul 4ul;
Micro-the rubbing of non-specific primer A:LEBSFP 0.05, nucleotides sequence is classified as shown in SEQ ID NO:1;
LEBSRP 0.2 is micro-to rub, and nucleotides sequence is classified as shown in SEQ ID NO:2;
The Auele Specific Primer B that detects genotype G is: LEBPF5B 0.2 is micro-to rub, and its nucleotides sequence is classified the SEQ ID NO:3 that 5 ' vitamin H replaces as;
The Auele Specific Primer B that detects genotype A is: LEBPF5B10.2 is micro-to rub, and its nucleotides sequence is classified the SEQ IDNO:4 that 5 ' vitamin H replaces as;
Specific probe C:LEBPR3F 0.05 is micro-to rub, and its nucleotides sequence is classified the SEQ ID NO:5 that 3 ' FITC replaces as;
SEQ ID NO:1 is 5 '-CACCGGCGCAGTCATTCTCATAATCG-3 ';
SEQ ID NO:2 is 5 '-GGTAAGGCGAGGTTAGCGAGGCTTG-3 ';
The SEQ ID NO:3 that 5 '-vitamin H replaces is 5 '-Biotin-CACTCACAGTCGC-3 ';
The SEQ ID NO:4 that 5 '-vitamin H replaces is 5 ' Biotin-CACTCACAGTCAC-3 ';
The SEQ ID NO:5 that 3 ' FITC replaces is 5 '-GAGAGGATTATGAT-FITC-3 '.
The reaction system that detects genotype G is:
LEBSFP 0.05 is micro-to rub
LEBSRP 0.2 is micro-to rub
LEBPR3F 0.05 is micro-to rub
LEBPF5B 0.2 is micro-to rub
DNTP:0.2 milli rubs
(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
Tested genomic dna 20ng/ul 4 microlitres
Cumulative volume is 20 microlitres
The reaction system that detects genotype A is:
LEBSFP 0.05 is micro-to rub
LEBSRP 0.2 is micro-to rub
LEBPR3F 0.05 is micro-to rub
LEBPF5B1 0.2 is micro-to rub
DNTP:0.2 milli rubs
(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
Tested genomic dna 20ng/ul 4 microlitres
Cumulative volume is 20 microlitres
PCR response procedures is as follows:
95 ℃, 2 minutes
Then
Last 95 ℃, 2 minutes
(3) detected result:
Reaction product is all dripped on nucleic acid test strip, 10 minutes sentence read result.Undergo mutation in examinee's plastosome ND4 gene 11 778 sites, by G → A.Nucleic acid test strip detects SNP and the results are shown in Figure 2 and Fig. 3, the left side is two test strip, and first left test strip is to detect the test strip of genotype G, and second left test strip is to detect the test strip of genotype A, the right is corresponding sequencing result, the site of the base of line for detecting; Sequencing result is consistent with SNP ELISA test strip result.
Fig. 2 is the detected result figure of the sick Mitochondrial DNA G11778A of Leber ' the s single nucleotide polymorphism of sample 1; Experimental result shows, detects the test strip demonstration positive reaction result of genotype G, and the test strip that detects genotype A type shows negative experimental result, illustrates that in the detected result in 11778 sites be 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 single nucleotide polymorphism of sample 2; Experimental result shows, detects the test strip demonstration negative reaction result of genotype G, and the test strip that detects genotype A type shows positive experimental result, illustrates that in the detected result in 11778 sites be A, is saltant type.The right is corresponding sequencing result, shows that 11778 sites are A.
Embodiment 2. Mycobacterium tuberculosis Isoniazid-resistant sudden change KatG 315 sudden changes detect
Vazadrine is the important drugs for the treatment of Mycobacterium tuberculosis, but long-term taking can produce resistance.The sudden change of Mycobacterium tuberculosis specific gene is the first cause producing Isoniazid-resistant, and its sudden change is mainly AGC → AAC and the AGC → ACC in 315 sites of Kat G gene.Therefore, the KatG 315 of dynamic monitoring Mycobacterium tuberculosis suddenlys change to timely adjustment treatment plan, rational use of drug and improves curative effect and has important directive significance.
(1) sampling
The DNA extraction test kit that Mycobacterium tuberculosis patient sputum application Yousida Biological Technology Co., Ltd., Hangzhou produces carries out nucleic acid extraction, in order to gene amplification.
(2) KatG 315 of PCR-AS-PCR nucleic acid test strip method detection branches bacillus suddenlys change:
Non-specific primer A:AGCPF/AGCPR
The non-specific primer A:TBLPF315 of forward, its nucleotides sequence is classified as shown in SEQ ID NO:6;
Reverse non-specific primer A:TBLPR315, its nucleotides sequence is classified as shown in SEQ ID NO:7;
The Auele Specific Primer B that detects AGC wild-type is respectively: TBLDF5F315-W, and its nucleotides sequence is classified the SEQ ID NO:8 that 5 ' end vitamin H replaces as;
The Auele Specific Primer B that detects AGC → AAC sudden change is respectively: TBLDF5F315-M1, and its nucleotides sequence is classified the SEQ ID NO:9 that 5 ' end vitamin H replaces as;
The Auele Specific Primer B that detects AGC → ACC sudden change is respectively: TBLDF5F315-M2, and its nucleotides sequence is classified the SEQ ID NO:10 that 5 ' end vitamin H replaces as;
Specific probe C TBLRD3F315, its nucleotides sequence is classified the SEQ ID NO:11 that 3 ' end FITC replaces as;
SEQ ID NO:6 is 5 '-TGGAAGAGCTCGTATGGCACCGG-3 ';
SEQ ID NO:7 is 5 '-CCCATTTCGTCGGGGTGTTCGTC-3 ';
The SEQ ID NO:8 that 5 ' vitamin H replaces is 5 ' (Biotin) CGCGATCACCAGC 3 ';
The SEQ ID NO:9 that 5 ' vitamin H replaces is 5 ' (Biotin) CGCGATCACCACC 3 ';
The SEQ ID NO:10 that 5 ' vitamin H replaces is 5 ' (Biotin) CGCGATCACCAAC 3 ';
The SEQ ID NO:11 that 3 ' FITC replaces is 5 ' CATACGACCTCGAT-FITC;
The reaction system that detects wild-type is:
TBLPF315 0.05 is micro-to rub
TBLPR315 0.2 is micro-to rub
TBLDF5F315-W 0.2 is micro-to rub
TBLRD3F315 0.05 is micro-to rub
DNTP:0.2 milli rubs
(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
Cumulative volume is 20 microlitres
The reaction system that detects AGC → ACC sudden change is:
TBLPF315 0.05 is micro-to rub
TBLPR315 0.2 is micro-to rub
TBLDF5F315-M1 0.2 is micro-to rub
TBLRD3F315 0.05 is micro-to rub
DNTP:0.2 milli rubs
(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
Cumulative volume is 20 microlitres
The reaction system that detects AGC → AAC sudden change is:
TBLPF315 0.05 is micro-to rub
TBLPR315 0.2 is micro-to rub
TBLDF5F315-M2 0.2 is micro-to rub
TBLRD3F315 0.05 is micro-to rub
DNTP:0.2 milli rubs
(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
Cumulative volume is 20 microlitres
PCR response procedures is as follows:
95 ℃, 2 minutes
Then
Last 45 ℃, 2 minutes
(3) detected result: reaction product is all dripped on nucleic acid test strip to sentence read result after 10 minutes.Fig. 4～Fig. 6 is KatG 315 site AGC → AAC and AGC → ACC sudden change detected result figure of Mycobacterium tuberculosis; 3, the left side is ELISA test strip result, and the right is that corresponding order-checking detects; Wherein, first left test strip is that detection genotype is the test strip of G type-wild-type, and second left is that detection genotype is the test strip of A type, and third left is that detection genotype is the test strip of C type; Sequencing result confirms consistent with ELISA test strip 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 is all negative experimental result, the genotype that sample 1 is described is 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 C type; The right is corresponding sequencing result, and sequencing result is ACC.
Claims (12)
1. a test kit for rapid detection single nucleotide polymorphism, is characterized in that, the method that described test kit adopts in same reaction system two kinds of PCR reactions and detection of nucleic acids test strip to detect specific amplification products; First the fragment that contains SNPs site by the first PCR temperature cycle non-specific amplification, the base that then contains single nucleotide polymorphism site by AS-PCR specific amplification, finally detects specific amplification products by detection of nucleic acids test strip;
Described test kit comprises following primer and probe: the not primer pair A with any mark of the template sequence that amplification comprises SNPs site, and non-specific amplification target template is to increase target template number; Carry out the Auele Specific Primer B of specific amplification for the Different Alkali base type on SNPs site, in the time of primer B and template sequence complete complementary, primer B could extend; Carry out the probe C of specific hybrid with primer B extension products, thereby complete the detection to SNPs site base type;
On described Auele Specific Primer B, be marked with antigen or haptens, described antigen or haptens are selected from vitamin H, digoxin or fluorescence dye, and described fluorescence dye is selected from Cy3, Cy5, tetramethyl-rhodamine (Tetramethylrhodamine), AlexaFluor fluorescence dye, rhodamine (Rhodamine), Fam or FitC;
On described probe C, be marked with antigen or haptens, described antigen or haptens are selected from vitamin H, digoxin or fluorescence dye, and described fluorescence dye is selected from Cy3, Cy5, tetramethyl-rhodamine (Tetramethylrhodamine), AlexaFluor fluorescence dye, rhodamine (Rhodamine), Fam or FitC;
Described test strip is included in having in order sample pad, coloured particle binding substances pad and absorbent filter pad on the liner of non-setting adhesive, each part mentioned above partly overlaps at adjacent, on tunica fibrosa, be also provided with detection line and nature controlling line, wherein the coloured particle on coloured particle binding substances pad has anti-A antibody coated, on detection line, there is anti-B antibody coated, on nature controlling line, have anti-A antibody, coloured particle is selected from colloid gold particle, latex particle; In described detection of nucleic acids test strip, be provided with and the antigen of primer B or probe C hybridization product institute mark or the detection line of the antibody formation that hapten specificity is combined.
2. the test kit of rapid detection single nucleotide polymorphism according to claim 1, is characterized in that, on described Auele Specific Primer B, is marked with vitamin H or digoxin.
3. the test kit of rapid detection single nucleotide polymorphism according to claim 1, is characterized in that, on described probe C, is marked with fluorescence dye.
4. the test kit of rapid detection single nucleotide polymorphism according to claim 1, is characterized in that, in described same reaction system, the condition of PCR is: first under 90～98 ℃ of conditions, be incubated 1～4 minute; Then under 90～96 ℃ of conditions 5～15 seconds, 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; Finally under 92～98 ℃ of conditions, be incubated 1～4 minute.
5. the test kit of rapid detection single nucleotide polymorphism according to claim 4, is characterized in that, in described same system, the condition of PCR is: first under 92～95 ℃ of conditions, be incubated 1.5～2.5 minutes; Then under 92～95 ℃ of conditions 8～12 seconds, 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; Finally under 92～95 ℃ of conditions, be incubated 1.5～3 minutes.
6. the test kit of rapid detection single nucleotide polymorphism according to claim 5, is characterized in that, in described same system, the condition of PCR is: first under 95 ℃ of conditions, be incubated 2 minutes; Then under 94 ℃ of conditions 10 seconds, lower 15 seconds of 72 ℃ of conditions, totally 40 circulations; Then under 94 ℃ of conditions 15 seconds, lower 20 seconds of 45 ℃ of conditions, totally 10 circulations; Finally under 95 ℃ of conditions, be incubated 2 minutes.
7. according to the test kit of the rapid detection single nucleotide polymorphism described in 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 completing non-specific amplification target template and reaching some amount, when second circulation starts, the primer B of low temperature thermal oxidation and probe C participate in reaction, reach the result of specific detection.
8. the test kit of rapid detection single nucleotide polymorphism according to claim 7, is characterized in that, the annealing temperature of primer pair A is 55 ℃～72 ℃, and the annealing temperature of primer B and probe C is 35 ℃～50 ℃.
9. the test kit of rapid detection single nucleotide polymorphism according to claim 8, is characterized in that, the annealing temperature of primer pair A is 65 ℃～72 ℃.
10. the test kit of rapid detection single nucleotide polymorphism according to claim 8, is characterized in that, the annealing temperature of primer B and probe C is 40 ℃～45 ℃.
The test kit of 11. rapid detection single nucleotide polymorphism according to claim 1, is characterized in that, in described same system, the pcr amplification system of PCR is:
Template: 2～4 μ l;
The non-specific primer of forward A:0.025～0.05 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 pH9.0
Triton-100：0.5%～1.0%；
MgCl2:1.25～2.5mmol; Taq archaeal dna polymerase: 1～2 unit; Cumulative volume is 10～20 microlitres.
The test kit of 12. rapid detection single nucleotide polymorphism according to claim 1, is characterized in that, described test kit comprises:
(1) the detection of nucleic acids test strip device of room temperature preservation;
(2) the pcr amplification system described in the claim 11 of-20 ℃ of preservations:
(3) positive control solution of-20 ℃ of preservations;
(4) negative controls of-20 ℃ of preservations;
(5) ddH2O of-20 ℃ of preservations.
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