CN102796817A - Method for rapidly detecting mutation of KRAS gene - Google Patents
Method for rapidly detecting mutation of KRAS gene Download PDFInfo
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- CN102796817A CN102796817A CN2012102337504A CN201210233750A CN102796817A CN 102796817 A CN102796817 A CN 102796817A CN 2012102337504 A CN2012102337504 A CN 2012102337504A CN 201210233750 A CN201210233750 A CN 201210233750A CN 102796817 A CN102796817 A CN 102796817A
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Abstract
The invention provides a method for rapidly detecting the mutation of KRAS gene, to overcome the disadvantages of low sensitivity and inaccuracy in conventional probe melting curve assay. The method for rapidly detecting the mutation of KRAS gene comprises the following steps: (1) designing and synthesizing molecular beacon probe and template amplification primers; (2) extracting the KRAS genome DNA of a detected sample; (3) preparing a reaction system; (4) performing PCR reaction; performing melting curve analysis after the PCR reaction is finished, and determining if a KRAS mutation occurs in the detected sample through the Tm value of a melting peak. The method combines the molecular beacon probe and extension retardance primers, and uses the DNA polymerase without 5'->3' exonuclease activity to detect KRAS mutation rapidly in vitro, thereby saving consumables simultaneously having the characteristics of high sensitivity and specificity which ensure a more reliable detection result.
Description
Technical field
The present invention relates to a kind of method of the KRAS of detection transgenation.
Background technology
Transgenation is meant the change that the genomic dna molecule based composition takes place or put in order on structure function, mainly comprise substituting and segmental insertion disappearance of base, is one of major reason that causes the genotype disease.Gene mutation analysis especially has important effect in genotype medical diagnosis on disease and pathological study in biomedical research.The albumen of KRAS genes encoding is participated in the intracellular signal transduction pathway by EGF-R ELISA (EGFR) mediation, influences propagation, growth and the transfer of cell; The albumen of normal KRAS genes encoding is activated after the stream signal activation accepting, and will after signal passes to the downstream cytokine, recover unactivated state; And the albumen of KRAS coded by said gene of sudden change need not the stream signal activation and just is in state of activation all the time, causes the improper propagation and the growth of cell, causes malignant tumour.Anti-EGFR class tumour medicine is blocked the cell transduction path through specificity retardance EGFR and receptors bind, thereby reaches the purpose of anticancer propagation; Multinomial discovering accepted in the colorectal cancer patients of anti-EGFR pharmacological agent, and the patient of KRAS gene wild-type more can be benefited from treatment than KRAS genic mutation type patient: have objective reactivity of higher medicine and long survival time.Therefore, can accurately detect the KRAS mutation status to instructing the colorectal cancer patients clinical application significant.
The method of traditional detection KRAS transgenation is varied; Wherein classics are dideoxy sequencing technology the most, comprise allele specific oligonucleotide hybridization (ASO), ligase enzyme detection reaction (LDR), the restricted length polymorphism analysis of polymerize chain reaction (PCR-RFLP) and TaqMan technology in addition.Whether exist though these methods can detect sudden change, most methods is not sure of the type of sudden change and can only be detected the part of sudden change; Most methods needs PCR post-processing detection ability analytical resultss such as agarose gel electrophoresis simultaneously, and accuracy and sensitivity are restricted.In recent years; Allele-specific PCR (Allele-specific PCR; ARMS), high resolution solubility curve (HRM), dhplc analysis (dHPLC) and tetra-sodium sequencing technologies methods such as (Pyrosequencing) improved the sensitivity and the specificity that detect, but still has shortcoming such as the serious and complex operation of false positive phenomenon.
Molecular beacon probe (Molecular Beacon) is a kind of double-tagging oligonucleotide probe that can spontaneous formation loop-stem structure, and its 5 ' end mark has fluorophor, and 3 ' end mark has quenching group; After the neck ring structure forms, fluorophor and quenching group space length near, FRET (FRET) takes place, the photon that fluorophor sends after being excited by external light source is absorbed by quenching group, this moment, probe can not be inspired fluorescence; If there is the template that is complementary with probe sequence in the system; After molecular beacon probe and template hybridization, its hairpin structure is opened, and makes fluorophor and quenching group space length increase; The fluorescence that this moment, fluorophor was inspired can detect fluorescent signal not by cancellation; Simultaneously owing to the amount of template in fluorescence intensity and the system is proportional, so molecular beacon probe can be used for the real-time quantitative PCR reaction.After molecular beacon and template strand hybridization, can cause probe to separate once more if continue elevated temperature with template, reduce thereby make fluorescence intensity raise with temperature.Fluorescence intensity differentiated to the temperature variation time just obtains melting curve, and the peak value of melting curve is fluorescent signal changing down corresponding temperature when maximum, is template and probe bonding strength (Tm value).Because the difference of sequence, the bonding strength of probe and wild-type template and mutant template has difference, shows the difference at melting curve (fusion peak).Traditional melting curve method sensitivity is lower, makes detected result inaccurate.
Summary of the invention
Low in order to overcome the sensitivity of conventional probe melting curve method, detect coarse shortcoming, the present invention proposes a kind of method of rapid detection KRAS transgenation, has not only saved consumptive material, has highly sensitive simultaneously, the characteristics of high specific, detected result is more reliable.
For realizing above goal of the invention, the present invention provides following basic technical scheme.
A kind of method of rapid detection KRAS transgenation may further comprise the steps:
(1) molecular beacon probe and template amplification primer design and synthetic
Upstream primer (25nt):
5’-GATAGTGTATTAACCTTATGTGTGA-3’
Extend retardance primer (38nt):
5 '-molecular beacon probe (27nt):
(2) the KRAS genomic dna of extraction tested sample;
(3) preparation reaction system
The KRAS genomic dna, upstream primer, extension retardance primer, UNG enzyme (uridylic-N-glycosylase), 5 ' → 3 ' the 5 prime excision enzyme activity disappearance polysaccharase (TIANNIUM that in reaction tubes, add tested sample
TMTaq DNA Polymerase), dNTPs (deoxyribonucleoside triphosphate), dUTP (deoxyuridine triphosphate), molecular beacon probe, 10 * PCR Buffer (TIANNIUM
TMTaq PCR Buffer);
(4) PCR reaction
The reaction system for preparing is increased on the fluorescence real-time quantitative PCR appearance and fuses, carry out the melting curve analysis after the PCR reaction is accomplished, judge through the Tm value at fusion peak whether the KRAS sudden change has taken place in the tested sample.
Based on above general planning, for obtaining better technique effect, the present invention also can make following optimization and limit.
In single tube reaction system 25 μ l, add upstream primer 500nM in the reaction tubes, extend retardance primer 50nM, UNG enzyme (uridylic-N-glycosylase) 0.2U, 5 ' → 3 ' 5 prime excision enzyme activity disappearance polysaccharase (TIANNIUM
TMTaq DNA Polymerase) 0.5U, dNTPs (deoxyribonucleoside triphosphate) 200 μ M, dUTP (deoxyuridine triphosphate) 400 μ M, molecular beacon probe 250nM, 10 * PCRBuffer (TIANNIUM
TMTaq PCR Buffer) 2.5 μ l, ddH2O supply volume 25 μ l.
Under the situation that the total amount of reaction system is confirmed (such as 25 μ l), the dimension of above admixture is those skilled in the art's the routine amount of getting mode.
Above-mentioned PCR reaction parameter is provided with as follows: 50 ℃, and 2 ~ 5min; 95 ℃, 12 ~ 15min; 95 ℃, 2 ~ 5sec; 56 ℃, 0 ~ 1sec; 65 ℃, 0 ~ 1sec; 55 ~ 65 circulations; Fusion: 95 ℃, 1 ~ 5min; 50 ℃ → 72 ℃ per seconds are collected fluorescence 15 ~ 20 times, 0.02 ~ 0.05 ℃ of gradient increased temperature.
The present invention compared with prior art has tangible advantage and beneficial effect:
1. high specificity.In the present invention, have very high specificity, guaranteed the specific amplification of template to KRAS mutational site designed molecules beacon probe and combination of primers.
2. highly sensitive.In the present invention, the polysaccharase of use lacks 5 ' → 3 ' 5 prime excision enzyme activity, and molecular beacon probe has all played obstruction wild-type template amplification, the effect of enrichment sudden change with extension retardance primer outside its basic function; Simultaneously polysaccharase has very high enzyme activity, can be in the extremely short time (0 ~ 2 second) mutant that efficiently increases template; Two kinds of effect common guarantee this invention have very high sensitivity.
3. save consumptive material and time.Design based on molecular beacon probe in this experiment; Make this method can save experimental period and consumptive material to a great extent; Combine the response procedures of design again, also optimized expanding effect greatly, PCR reaction and fusion processes only need 60-70min altogether; Each sample only needs a reacting hole simultaneously, need not the extra wild-type template that is provided with as contrast.
4. interpretation of result is simple.The present invention is with respect to traditional high resolution melting curve method, and interpretation of result more easily experimentizes; Only need observe its fusion peak Tm to each sample and just can analyze its sudden change and whether take place, need not reference standard.
Description of drawings
Fig. 1 is a KRAS mutational site sequence information, molecular beacon probe and primer information synoptic diagram.
Fig. 2 is this invention experiment flow and principle of design synoptic diagram.
Fig. 3,4 is applied to KRAS sudden change test experience figure as a result for the present invention.
Embodiment
The present invention combines molecular beacon probe and extension retardance primer; And utilize nothing 5 ' → 3 ' 5 prime excision enzyme activity polysaccharase to carry out external rapid detection KRAS sudden change, and not only saved consumptive material, have highly sensitive simultaneously; The characteristics of high specific make detected result more reliable.
Extending retardance primer (Extension-Refractory primes) is a kind of primer that can form " neck ring " structure with self extension products; Except general primer sequence part, extend the extra one section base sequence that comprises of retardance primer 5 ' end, this section sequence can be extended in the product strand that obtains with self and comprised the mutational site in one section interior base sequence complementation, forms " neck ring " structure; Because sequence is different between wild-type and mutant template, so " neck ring " structural strength (Tm value) of formation has significant difference.
The present invention is directed to high specific molecular beacon probe of No. second exon focus sudden change zone design of KRAS gene, this probe sequence and wild-type KRAS gene mate fully; Upstream and downstream at probe designs a pair of high specific primer, and wherein upstream primer is a general primer, and downstream primer is for extending the retardance primer.In the PCR reaction system, upstream primer concentration is for extending 10 times that block primer concentration; Extending the basic primer part that blocks primer does not have selectivity to template, but its 5 ' end additional sequences part only obtains the complete complementary pairing of product with the extension of wild-type template.In reaction system, add primer to and molecular beacon probe; In the PCR reaction process, select suitable annealing and elongating temperature, control annealing and the time of extending; Probe and wild-type template bonding strength high (68 ℃ of Tm ≈); Can strong mode combine, and probe and mutant template complementary pairing (mispairing of position, mutational site) fully, so the two bonding strength lower (59 ℃ of Tm ≈); Extend retardance primer and the resulting extension products of wild-type template simultaneously and can form " neck ring " structure (70 ℃ of Tm ≈), the extension products of sudden change template then can not; Because the used polysaccharase of PCR reaction lacks 5 ' → 3 ' 5 prime excision enzyme activity; Molecular beacon probe is hindered with the extension that combines and extend the formation of blocking the primer secondary structure to cause upstream primer and extension to block primer respectively of template; This inhibition mainly works to the wild-type template; And the mutant template amplification is not almost had influence, and therefore reach the enrichment sudden change, improve the effect of sensitivity.After the PCR reaction is accomplished, carry out the melting curve analysis, just accurately in the judgement sample whether the KRAS sudden change has taken place through the Tm value of fusing the peak.
Fig. 1 is molecular beacon probe of using among KRAS Gene Partial sequence (containing the mutational site at interior 120nt) information and the present invention and the synoptic diagram that extends the retardance primer;
See also shown in Fig. 1-1, be involved KRAS sequence information and primer and probe location synoptic diagram among the present invention.Marking blue base is the corresponding sequence of primer, and wherein black half arrow in upper left side refers to upstream primer, and length is 25nt; Middle folded beneath formula half arrow refers to and extends the retardance primer, and its basic primer partly is 26nt, and 5 ' end additional sequences (neck ring forms required) is 12nt; The site takes place for the KRAS sudden change in arrow indication base (3 G), and its upper illustration is the corresponding sequence scope of molecular beacon probe.Used molecular beacon is the double-tagging oligonucleotide probe among the present invention, its 5 ' end mark fluorophor (oval sign), 3 ' end mark quenching group (oval sign).Probe 5 ' end has 2nt complementary with template, and 3 ' end has 5nt complementary with template, with template complementary portion sequence be 20nt.
See also shown in Fig. 1-2, be molecular beacon probe among the present invention " neck ring " structural representation.Wherein " neck " is that 6 base complementrity pairings form 56 ℃ of Tm ≈; After the neck ring structure forms, the fluorophor of probe and quenching group group space length near, can not be inspired fluorescence;
See also shown in Fig. 1-3, for extending the formed neck ring structural representation of extension products of retardance primer among the present invention.Wherein " neck " comprises the mutational site by its 5 ' end additional sequences and himself extension products (wild-type template) and forms 70 ℃ of Tm ≈ at interior sequence complementary pairing.Extension retardance primer and mutant template are extended the product that obtains can not form similar secondary structure when PCR extends.
Fig. 2 is this invention involved experiment flow and principle of design synoptic diagram.
See also shown in Fig. 2-1, for detecting the experimental principle figure of KRAS transgenation among the present invention.Because PCR reaction system middle and upper reaches primer concentration for extending 10 times that block primer (downstream primer), in the PCR reaction process, except that normal double-stranded PCR product forms, also has a large amount of upstream primers to extend the strand PCR product that produces simultaneously; These strands can with the molecular beacon probe complementary pairing in the system, after the two combined, probe " neck ring " structure was opened, and is inspired fluorescent signal.Because in the PCR reaction process, strand produces quantity and is the multiple increase in proper order with PCR, strengthens phenomenon so the fluorescence that is excited also presents multiple.After the PCR reaction was accomplished, strand that exists in the system and probe combined, and this moment, fluorescence intensity was the highest.Further afterwards elevated temperature, the bonded probe can break away from single stranded product, and fluorescence intensity descends.When temperature reached the annealing temperature (Tm) of probe, the speed that itself and single stranded product break away from was the fastest.If whole temperature-rise period is asked for the derivative (change in fluorescence speed) of fluorescence intensity and time, just can obtain melting curve figure; The Tm of probe fusion has been indicated at fusion peak wherein, has reacted the bonding strength of probe and template.
See also shown in Fig. 2-2, for extending the synoptic diagram that the retardance primer carries out enrichment to the sudden change template among the present invention.Left part is divided into the extension products that extends retardance primer and the generation of mutant template among the figure, because the complementation (mutational site mispairing) fully of its additional sequences and product sequence can not form " neck ring " structure, so upstream primer can extend normally when extending.Right half is depicted as the extension products that extends retardance primer and the generation of mutant template; Its additional sequences can be complementary fully with the product sequence; When extending, formed " neck ring " structure; Lack 5 ' → 3 ' 5 prime excision enzyme activity because of polysaccharase simultaneously, the extension of upstream primer is hindered, and PCR can not normally carry out.Therefore, the mutant template in the sample can obtain enrichment, and the amplification of wild-type template has received inhibition.
See also shown in Fig. 2-3, for molecular beacon probe among the present invention to the sudden change template synoptic diagram that carries out enrichment.Shown in figure, molecular beacon probe and wild-type template are complementary fully, when PCR extends, can well combine with template, because polysaccharase lacks 5 ' → 3 ' 5 prime excision enzyme activity, the extension of downstream primer has received obstruction, and PCR can not normally carry out.And molecular beacon probe and mutant template can not be fully complementary (mutational site mispairing), when PCR extends can not with the template good combination, the extension of primer can normally be carried out.So the mutant template in the sample can obtain enrichment, and the amplification of wild-type template has received inhibition.
Comprehensive above-mentioned two kinds of effects, the mutant template can obtain good enrichment in the PCR reaction process.
To tested sample, the method for rapid detection KRAS transgenation of the present invention may further comprise the steps: (1) molecular beacon probe and template amplification primer design and synthetic
Upstream primer (25nt):
5’-GATAGTGTATTAACCTTATGTGTGA-3’
Extend retardance primer (38nt):
5 '-molecular beacon probe (27nt):
(2) the KRAS genomic dna of extraction tested sample;
(3) preparation reaction system
The KRAS genomic dna, upstream primer, extension retardance primer, UNG enzyme (uridylic-N-glycosylase), 5 ' → 3 ' the 5 prime excision enzyme activity disappearance polysaccharase (TIANNIUM that in reaction tubes, add tested sample
TMTaq DNA Polymerase), dNTPs (deoxyribonucleoside triphosphate), dUTP (deoxyuridine triphosphate), molecular beacon probe, 10 * PCR Buffer (TIANNIUM
TMTaq PCR Buffer);
In single tube reaction system 25 μ l, upstream primer 500nM extends retardance primer 50nM, UNG enzyme 0.2U; 5 ' → 3 ' 5 prime excision enzyme activity disappearance polysaccharase 0.5U, dNTPs200 μ M, dUTP400 μ M; Molecular beacon probe 250nM, 10 * PCR Buffer2.5 μ l, ddH2O supply volume 25 μ l;
(4) PCR reaction
The reaction system for preparing is increased on the fluorescence real-time quantitative PCR appearance and fuses, and reaction parameter is provided with as follows: 50 ℃, and 2 ~ 5min; 95 ℃, 12 ~ 15min; 95 ℃, 2 ~ 5sec; 56 ℃, 0 ~ 1sec; 65 ℃, 0 ~ 1sec; 55 ~ 65 circulations; Fusion: 95 ℃, 1 ~ 5min; 50 ℃ → 72 ℃ per seconds are collected fluorescence 15 ~ 20 times, 0.02 ~ 0.05 ℃ of gradient increased temperature.Carry out the melting curve analysis after the PCR reaction is accomplished, judge through the Tm value at fusion peak whether the KRAS sudden change has taken place in the tested sample.
Below be to adopt the method proposed by the invention example that sudden change detects to KRAS G12V (35G>T), with further embodiment technique effect of the present invention.
SW480 cell (KRAS G12V) genomic dna of cultivating is extracted, use Nanodrop nucleic acid quantification appearance quantitative; Get part G12V genomic dna and wild type gene group DNA by the 1:19 mixed.With the G12V genomic dna, wild type gene group DNA and mixed type DNA are as three kinds of templates simultaneously.
The amplification system of real-time fluorescence PCR is 25 μ l, has two groups, every group of three reaction tubess; Every Guan Zhongjun adds upstream primer 500nM; Common downstream primer or extension retardance primer 50nM, UNG enzyme (uridylic-N-glycosylase) 0.2U, 5 ' → 3 ' 5 prime excision enzyme activity disappearance polysaccharase (TIANNIUM
TMTaq DNA Polymerase) 0.5U, dNTPs (deoxyribonucleoside triphosphate) 200 μ M, dUTP (deoxyuridine triphosphate) 400 μ M, molecular beacon probe 250nM, 10 * PCR Buffer (TIANNIUM
TMTaq PCR Buffer) 2.5 μ l, ddH2O supply volume 25 μ l.Three pipes add wild-type template, G12V mutant template and mixed type template (G12V5%) respectively in every group.The system for preparing is increased on the fluorescence real-time quantitative PCR appearance and fuses.Reaction parameter is provided with as follows: 50 ℃, and 2 ~ 5min; 95 ℃, 12 ~ 15min; 95 ℃, 2 ~ 5sec, 56 ℃, 0 ~ 1sec, 65 ℃ of 0 ~ 1sec, 55 ~ 65 circulations; Fusion: 95 ℃, 1 ~ 5min, 50 ℃ → 72 ℃ per seconds are collected fluorescence 15 ~ 20 times, 0.02 ~ 0.05 ℃ of gradient increased temperature.The melting curve of observation sample.The result is as shown in Figure 3.
See also shown in Fig. 3-1; When using general primer, the corresponding fusion of G12V sudden change template peak Tm is about 58 ℃, and the corresponding fusion of wild-type template peak Tm is about 68 ℃; And 5%G12V mutant template fusion peak is only obvious at corresponding wild-type fusion place, peak, locates not obvious at 58 ℃; See also shown in Fig. 3-2; When using extension retardance primer; The fusion peak Tm of G12V mutant template and wild-type template does not change, and 5%G12V mutant template then demonstrates " two fusions peak " form, and its corresponding mutant fusion peak (Tm58 ℃) is very obvious; The proof sudden change has obtained enrichment, meets fully with notional result.
For the pattern detection of typical other mutation types (like G12S, G12R, G12C, G12A, G12D, G13D), same etection theory according to the invention; Through experiment, also all can produce a desired effect.
See also shown in Figure 4ly, use the present invention that 5 routine colorectal cancer patients tumor sample DNA are detected, validation sample 5 is the KRAS wild-type, and sample 1,2,3,4 is a mutant.
Above embodiment should not be regarded as the restriction to protection domain of the present invention; The present invention has confirmed best embodiment and parameter in the preparation of template and the link of real-time fluorescence PCR; But those skilled in the art are based on technological thought of the present invention and designed primer system; Operating method (like reagent proportioning, amplification procedure parameter etc.) according to routine is enough to rapid detection KRAS sudden change, and comparing to prior art has marked improvement.
Claims (3)
1. the method for rapid detection KRAS transgenation may further comprise the steps:
(1) molecular beacon probe and template amplification primer design and synthetic
Upstream primer (25nt):
5’-GATAGTGTATTAACCTTATGTGTGA-3’
Extend retardance primer (38nt):
5 '-molecular beacon probe (27nt):
(2) the KRAS genomic dna of extraction tested sample;
(3) preparation reaction system
The KRAS genomic dna, upstream primer, extension retardance primer, UNG enzyme (uridylic-N-glycosylase), 5 ' → 3 ' the 5 prime excision enzyme activity disappearance polysaccharase (TIANNIUMW that in reaction tubes, add tested sample
TMTaq DNA Polymerase), dNTPs (deoxyribonucleoside triphosphate), dUTP (deoxyuridine triphosphate), molecular beacon probe, 10 * PCR Buffer (TIANNIUM
TMTaq PCR Buffer);
(4) PCR reaction
The reaction system for preparing is increased on the fluorescence real-time quantitative PCR appearance and fuses, carry out the melting curve analysis after the PCR reaction is accomplished, judge through the Tm value at fusion peak whether the KRAS sudden change has taken place in the tested sample.
2. the method for rapid detection KRAS transgenation according to claim 1 is characterized in that:
In single tube reaction system 25 μ l, add upstream primer 500nM in the reaction tubes, extend retardance primer 50nM, UNG enzyme (uridylic-N-glycosylase) 0.2U, 5 ' → 3 ' 5 prime excision enzyme activity disappearance polysaccharase (TIANNIUM
TMTaq DNA Polymerase) 0.5U, dNTPs (deoxyribonucleoside triphosphate) 200 μ M, dUTP (deoxyuridine triphosphate) 400 μ M, molecular beacon probe 250nM, 10 * PCRBuffer (TIANNIUM
TMTaq PCR Buffer) 2.5 μ l, ddH2O supply volume 25 μ l.
3. the method for rapid detection KRAS transgenation according to claim 2 is characterized in that:
The PCR reaction parameter is provided with as follows: 50 ℃, and 2 ~ 5min; 95 ℃, 12 ~ 15min; 95 ℃, 2 ~ 5sec; 56 ℃, 0 ~ 1sec; 65 ℃, 0 ~ 1sec; 55 ~ 65 circulations; Fusion: 95 ℃, 1 ~ 5min; 50 ℃ → 72 ℃ per seconds are collected fluorescence 15 ~ 20 times, 0.02 ~ 0.05 ℃ of gradient increased temperature.
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| CN103088137A (en) * | 2013-01-18 | 2013-05-08 | 陕西北美基因股份有限公司 | Method for accurately detecting KRAS gene mutation by use of probe melting technology |
| CN103103269A (en) * | 2013-01-18 | 2013-05-15 | 陕西北美基因股份有限公司 | Method for detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology |
| CN104651529A (en) * | 2015-03-24 | 2015-05-27 | 厦门大学 | Method for detecting deletion mutation of nucleic acid molecule |
| CN106834520A (en) * | 2017-03-27 | 2017-06-13 | 杭州迪安生物技术有限公司 | A kind of kit of utilization molecular beacon melting curve technical appraisement bacterium and its application |
| CN107034277A (en) * | 2017-04-27 | 2017-08-11 | 厦门大学 | A kind of method for detecting low abundance gene mutation |
| CN107385016A (en) * | 2017-06-14 | 2017-11-24 | 苏州承美生物科技有限公司 | A kind of molecular beacon probe and kit for detecting KRAS gene mutation |
| CN112063701A (en) * | 2020-10-13 | 2020-12-11 | 苏州中科先进技术研究院有限公司 | Nucleic acid composition for detecting KRAS gene mutation, kit and detection method thereof |
| CN116426617A (en) * | 2023-04-25 | 2023-07-14 | 无锡市人民医院 | High-sensitivity mutation detection system based on hairpin structure and enzyme digestion mechanism and application |
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| CN103088137A (en) * | 2013-01-18 | 2013-05-08 | 陕西北美基因股份有限公司 | Method for accurately detecting KRAS gene mutation by use of probe melting technology |
| CN103103269A (en) * | 2013-01-18 | 2013-05-15 | 陕西北美基因股份有限公司 | Method for detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology |
| CN103088137B (en) * | 2013-01-18 | 2014-11-05 | 陕西北美基因股份有限公司 | Method for accurately detecting KRAS gene mutation by use of probe melting technology |
| CN103103269B (en) * | 2013-01-18 | 2015-07-15 | 陕西佰美基因股份有限公司 | Method for detecting BRAF (block repeat active flag) gene mutation accurately based on probe fusion technology |
| CN104651529A (en) * | 2015-03-24 | 2015-05-27 | 厦门大学 | Method for detecting deletion mutation of nucleic acid molecule |
| CN106834520A (en) * | 2017-03-27 | 2017-06-13 | 杭州迪安生物技术有限公司 | A kind of kit of utilization molecular beacon melting curve technical appraisement bacterium and its application |
| CN106834520B (en) * | 2017-03-27 | 2020-12-18 | 杭州迪安生物技术有限公司 | Kit for identifying bacteria by using molecular beacon-melting curve technology and application thereof |
| CN107034277A (en) * | 2017-04-27 | 2017-08-11 | 厦门大学 | A kind of method for detecting low abundance gene mutation |
| CN107385016A (en) * | 2017-06-14 | 2017-11-24 | 苏州承美生物科技有限公司 | A kind of molecular beacon probe and kit for detecting KRAS gene mutation |
| CN112063701A (en) * | 2020-10-13 | 2020-12-11 | 苏州中科先进技术研究院有限公司 | Nucleic acid composition for detecting KRAS gene mutation, kit and detection method thereof |
| CN116426617A (en) * | 2023-04-25 | 2023-07-14 | 无锡市人民医院 | High-sensitivity mutation detection system based on hairpin structure and enzyme digestion mechanism and application |
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