Fluorescence detection method for DNA and kit thereof
Technical Field The invention relates to the technical field of biology, in particular to a method for fluorescence detection of DNA kit thereof. Background Art Gene (DNA or RNA) genetic information carrier is, in in individual growth, growth, reproduction, such as genetic and the variation in the course of life plays an extremely important role. Gene detection in clinical diagnostic, single nucleotide polymorphism analysis, gene sequencing, environmental analysis, such as counter has important significance in the field. With the growing concern for their own health, to tumor, cancer and other major disease early diagnosis technology there are increasingly high requirements, there is therefore an urgent need for development of high-sensitivity gene detection technique. Gene mutation is one of the forms of single nucleotide polymorphism, with various diseases of occurrence of relationship is closely, the latest research shows that, with four hundred diseases related to the gene mutation. In order to detect the early onset of pathogenic gene can be the existence of specific, often need to conduct PCR amplification of the gene to be tested or by other forms of signal amplification method, such as gold amplification, conjugated high molecular amplification, enzyme-linked amplification, (L.Pang, J.Li, J.Jiang, G.Shen, R.Yu, Biochem. 2006,358 Anal., 99 ; D.Q.Tang, D.J. Zhang, D.Y.Tang, H.Ai, J.Immunol.Methods 2006,316,144; X.Yao, X.Li, F. Toledo, C. Zurita-Lopez, M.Gutova, J.Momand, F.Zhou, Anal.Biochem. 2006, 354,220). CN1808101A of the applicant discloses number of Patent applications discloses a method for fluorescence detection of DNA and its kit, the detection method comprises: using is connected to the capture probe on the magnetic particles to be detected in DNA from a biological sample magnetic enrichment and separation; then adding fluorescent group labeled signal probe (FAM), after the magnetic separation and concentration of the DNA to be detected with the signal probe matching, and the low-salt buffer solution used for washing; then adding water-soluble electrically conductive high molecular material, in other words conjugated high molecular (such as polyfluorenes derivatives PF) realizing signal multiplication, the conductive high molecular material is made of the fluorescence spectrum of the excitation wavelength, fluorescence resonance energy transfer (FRET) the method for detecting the detection and analysis of DNA to be detected. The method has high specificity and sensitivity. However, on the one hand low-salt washing process will increase the step of the operation, the reaction and detection time; on the other hand, low-salt solution will also make the fully complementary part of the double-stranded DNA melting, so that the complementary with mutated but discrimination reduced, that is, will reduce the selectivity of the mutation. Content of the invention The invention aims to solve the technical problem of providing a better selectivity of the DNA fluorescence detection method, and the method has high sensitivity, without amplification, quick, simple and convenient operation, high in overall performance. Other to be solved by the invention a technical problem of providing a method for adopting the above-mentioned DNA reagent kit for the detection of. The present inventors in the above-mentioned CN1808101A on the basis of the technical scheme disclosed, as a result of further research, found that adopt the competition mechanism, magnetic particles the enrichment of the target DNA, separation, and water-soluble conjugated polymer with a fluorescent group combination of technical FRET between, can greatly improve specificity and sensitivity of DNA detection, shortens the reaction time, comprehensive better performance, can especially be used for the gene and detection of mutant gene, used in some of the more promising gene mutation-related disorders, such as tumor diagnosis. Wherein biological detection in competition mechanisms are frequently used for improving the selectivity of a detection method, such as ELISA, such as competition and competitive PCR. In detection of the gene (DNA), often adopt this kind of method. Compared with the linear DNA molecules, stem-loop structure specific DNA probe is because of the stem-loop structure can be formed, and its complementary target DNA with the more narrow melt temperature range and more high selectivity (G.Bonnet, S.Tyagi, A.Libchaber, F.Kramer, Proc.Natl.Acad.Sci.U.S.A. 1999, 96, 6171), commonly used in the detection of the single base mismatch. Therefore, the introduction of competition probe stem-loop structure will be further enhanced the comprehensive performance of the DNA biological sensor. Therefore, the present invention solve the above-mentioned 1st technical problems, the invention adopts the technical scheme is:a method for fluorescence detection of DNA, comprising the following steps: 1) the capture probe is connected to the surface of the magnetic particles; 2) the target DNA to be measured; and fluorescein modified signal probe, the capture probe can be a section of the target DNA nucleotide sequence hybridization, the signal probe with the target DNA can be another section of the nucleotide sequence of the hybridization, the hybridization of the DNA, forming the magnetic particle-targeting DNA-fluorescein modification of the sandwich structure of the signal probe; 3) finally, remove the free fluorescein modified after the signal probe, with signal multiplication function added to the water soluble conjugated polymer, on the probe to the signal of the fluorescence signal of the amplified detection, so as to detect the target DNA; Wherein step 2) is also added in the hybridization of the capture probe or the signal probe to the competitive probe, competition binding with the target DNA. Preferably, the capture probes can be stem-loop structure of the probe DNA, at the same time, the signal probe is linear DNA probe, the competitive probe without any modification, its sequence with the capture probe only by one base. Preferably, the capture probe can be a linear DNA probe, the signal at the same time, the stem-loop structure of the probe is probe DNA, the competitive probe without any modification, and the sequence of the signal probe only by one base. Preferably, step 2) in the temperature of the hybridization reaction is the 29-39 the [...][...] , preferably 37 the left and right [...]. By controlling reaction temperature condition, the magnetic particle-targeting DNA-fluorescein-modified signal in the sandwich structure of the probe containing the single-point mutation of the double-chain under conditions such that the melting, become a free state, and fully complementary double-chain is not affected. Preferred, the competitive probe may be added an equimolar amount of competition probe 1-7 times, preferably 2 times. According to the present invention, step 1) said magnetic particle with the existing technology, such as the above-mentioned CN1808101A in, it has already been widely used for the enrichment of biologically active substances, separation, drug magnetic targeting and and the diagnosis of disease treatment. Functionalization of the surface of the magnetic particles not only can the ligand of the receptor (or receptors) (or ligand) the peculiar interaction between, such as avidin-biotin (biotin) to realize the rapid separation of a target biological target, but also can be used as a solid-phase carrier is used for connecting the biological molecule. Compared with the traditional separation method, the use of magnetic particles the separation of the speed is obviously shortened, in 30 seconds fast separation can be realized. Furthermore, using magnetic particles of the target substance can be realized when analyzing the detection of the reaction, the reaction system to avoid the interference of the other component. The magnetic particles of the present invention usually the city of streptavidin coated magnetic beads (MMPs) micron-sized goods, can be according to the diameter of the existing method for synthesizing 50-100nm magnetic nano particle. When adopting the micron level of the MMPs, in order to avoid magnetic bead light scattering, fluorescence detection is usually required before the is fixed on the magnetic bead on the DNA denatured melting, and the selected transgendered melting method can be of conventional method, such as by strong alkali such as NaOH (specific can refer to CN1808101A). According to the present invention, step 3) of the conjugated polymer is preferably water-soluble polyfluorenes (PF). This invention adopts the micron-sized magnetic bead MMPs as an example to illustrate, containing modified separation of the signal probe in of the supernatant, adding water soluble conjugated high molecular PF then fluorescence detection, PF and the fluorescein by fluorescence resonance energy transfer between the signal amplification. This invention solve the above-mentioned 2nd technical problems, the invention adopts the technical scheme is:a a kit for the detection of the fluorescent DNA, including: 1) magnetic particles to the capture probe, fluorescein modified signal probe; 2) the above-mentioned capturing probe or the signal probe competitive probe; 3) water-soluble polyfluorenes. The invention introduces the competition mechanism, in particular the stem-loop structure with high selectivity of the competitive probe, the selectivity of the detecting DNA; functionalized magnetic particles at the same time as a magnetic particle, to realize the quick separation of the free components, to further improve the selectivity. At the same time, the invention chooses fluorescein modified signal probe, detection is directly added in the final conjugated high molecular, through its and the fluorescein fluorescence resonance energy transfer between, realizing signal multiplication. Not only increases the sensitivity of the detecting DNA, and do not need to carry out amplification of the signal probe, the operation is simple. The present invention can remarkably improve the DNA mutation detection DNA and, in particular, in gene, such as breast cancer susceptibility gene BRCA1 and its mutant gene detection sensitivity and selectivity, in biological detection, early diagnosis and treatment of disease is of important significance in. The Figure illustrates: Figure 1 is the working principle diagram of this invention introduces the competitive capture probe method for detecting DNA; wherein A complementary target DNA for detecting the working principle of the Figure, for detecting the mutation targeting DNA B the working principle of the Figure. Figure 2 is the working principle diagram of this invention introduces the competitive signal probe method for detecting DNA; wherein A complementary target DNA for detecting the working principle of the Figure, for detecting the mutation targeting DNA B the working principle of the Figure. Figure 3 for introducing competitive capture probe competition reaction temperature is 37 the targeting DNA when [...] (a) and mutation targeting DNA (b) fluorescence detection signal, for target density 1 nm. Figure 4 for separately introducing two kinds of competition mechanism the reaction temperature is 37 the targeting DNA when [...] and mutation targeting DNA fluorescence detection signal. A, respectively b competitive capture probe when the introduction of the targeting DNA and mutation targeting DNA fluorescence detection signal; c, d respectively of the signal probe is competitive to introduce the targeting DNA and mutation targeting DNA fluorescence detection signal. Mode of execution BRCA1 breast cancer susceptibility gene for the lower portion thereof for the implementation of detection of mutant gene ' to further illustrate the invention, but the invention is not limited to detecting breast cancer susceptibility gene mutant gene thereof, the embodiment is not affected by other conditions in the restriction. The following is some the embodiment of the invention, the apparatus and equipment used, other not specifically specified in accordance with the experiment conditions of the conventional or drugs or to the conditions recommended by the manufacturers. Fluorescence detection instrument used for the fluorescence spectrophotometer (Hitachi F-4500, Japan). Fluorescence spectrum measuring conditions: xenon lamp excitation, the excitation and emission slender peak width is 2.5 nm, voltage PMT 950V, response time 2S, excitation wavelength is 380 nm, emission wavelength scanning range 390-700nm; with 3 ml quartz cuvette is measured, sample volume 1 ml; room temperature. Streptavidin modified super-paramagnetic particles (magnetic micro-particles, MMPs) in Promega Corporation, particle diameter is 1.0 the left and right m, solid content is 1 mg/mL, the binding capacity of 1.25 nmol probe/mg MMPs. Water soluble conjugated polymer is cationic the polyfluorenes derivatives (PF), according to the literature (F.Huang, H. Wu, D.Wang, W.Yang, Y.Cao, Chem.Mater. 2004, 16,708) synthesis, its structural formula is as follows: wherein R= (CH2)3 N+ (CH3)2 CH2 CH3 Br. Various DNA sequence as shown in table 1 illustrated, are purchased from Shanghai biological engineering technology limited. Table 1 oligonucleotide base composition table The embodiment with various solution components as shown in table 2 is shown. Table 2 various solution components used by the table Embodiment 1 As shown in Figure 1, according to the product specification requirements, MMPs in the first using 0.5 ×SSC buffer washing 3 times, then the stem-loop structure of the biotin-labeled capture probe DNA 1 TTL buffer solution comprising in MMPs, gently mixed contract 10 minutes. The capture probe is about surface density 4-6 × 1011 chain/cm2. Then the complex of MMPs and capture probe (MMPs-capture probe, recorded as MMPs-cp 1) for liquid cleaning two buffer TTA, in suspended in hybridization, 4 the refrigerated [...] spare. Then the stem-loop structures competitive capture probe DNA 2, targeting DNA 4 (or a mutant target DNA 5) and a linear signal probe DNA 3 of the mixture into the pre-prepare a good MMPs-cp 1 in the suspension, wherein the targeting DNA or a mutant target DNA 5 is a concentration of 1 nm, competitive capture probe DNA2 the DNA capture probe [...] 1 the targeting complementary [...] DNA4 or mutant target DNA 5 the signal probe [...] DNA3 molar concentration ratio is about 2 the [...] the 1 [...] the 1 [...] 2. In the 37 the reaction [...] 30 minutes. After the reaction, magnetic separation to remove free component. Then washing with a washing solution 3-5 time, until the washing the supernatant fluid in the separation of the non-fluorescent signal. The complex is then added to the magnetic particles in the 100 L concentration is 50 mm of NaOH, denatured at room temperature 5 minutes, magnetic separation, collection transgendered supernatant, adding equivalent HCL to neutralize, to the 200 L 10 mm Tris-HCl (pH 8.0) buffer solution and 400 L H2 O fluorescence detection (Ex: 480 nm, Em: 490-700nm), finally added the solution makes the PF to the final concentration of 200 nm, high molecules conjugated to the detection of signal amplification (Ex: 380 nm, Em: 390-700nm). As a result in Figure 3. 37 the reaction [...] , containing single-point mutation of the DNA double-strand with, and fully complementary double-chain is not affected. The signal ratio of 3.8, has a significant difference, the complementary and mutation can be distinguished from the signal. With the prior art in (CN1808101A Patent application) complementary targeting DNA with mutation of the signal ratio is 2.2 compared with, we can see that the invention greatly improves the selective detection of DNA. Embodiment 2 With reference to embodiment 1 in the reaction conditions, the difference lies in the temperature of the hybridization reaction for the 29 [...]. The result is that complementary targeting DNA with mutation to signal ratio of 2.25. Embodiment 3 With reference to embodiment 1 in the reaction conditions, the difference lies in the temperature of the hybridization reaction for the 39 [...]. The result is that complementary targeting DNA with mutation of the signal ratio is 2.31. Embodiment 4 With reference to embodiment 1 in the reaction conditions, is characterized in that the different: DNA2 the competitive capture probe DNA capture probe [...] 1 the targeting complementary [...] DNA4 or mutant target DNA 5 the signal probe [...] DNA3 molar concentration ratio is about 7 the [...] the 1 [...] the 1 [...] 2. The result is that complementary targeting DNA with mutation to signal ratio of 2.36. Embodiment 5 With reference to embodiment 1 in the reaction conditions, is characterized in that the different: DNA2 the competitive capture probe DNA capture probe [...] 1 the targeting DNA complementary [...] 4 or mutant target DNA 5 the signal probe [...] DNA3 molar concentration ratio of about to 1 the [...] the 1 [...] the 1 [...] 2. The result is that complementary targeting DNA with mutation of the signal ratio is 2.97. Embodiment 6 With reference to embodiment 1 in the reaction conditions, is characterized in that the different: DNA using a linear capture probe 6 preparation of the complex of MMPs and capture probe (MMPs-capture probe, recorded as MMPs-cp 6), the stem-loop structure of the signal probe DNA 7, competition probe DNA for the stem-loop structure 8, the DNA targeting DNA be detected 4 or mutation targeting DNA 9, the two 37 the left and right [...] reaction. The concentration of DNA is also the example treats examines 1 nm. Other reagents and step and the embodiment 1 the same. Its reaction principle as shown in Figure 2. As a result in Figure 4. Result display, for introduction into a competitive strategy of the signal probe, the signal probe is a stem-loop structure, the formation of the stem-loop structures stem the enhanced quenching of FAM base pair part, therefore FAM fluorescence emission efficiency is low, and the occurring between PF FRET, FRET efficiency is low. Furthermore, the presence of a stem-loop structure of the sterically hindered FRET efficiency is also one of the important factors. The result shows that introduce competitive capture probe to the single-point mutation of better selectivity. This is because the introduction of the strategy of competitive capture probe, the signal probe is a linear structure, the secondary structure does not exist, so its fluorescence emission efficiency is high, and the occurring between PF FRET, FRET efficiency is high. The invention discloses a DNA fluorescent detection process and its agent box. In the method, target direction DNA is captured through cross-fertilizing between DNA after the capturing detecting probe is coupled to the magnetic particle surface, another nucleotide sequence of target direction DNA is cross-fertilized with semaphore detecting probe modified by luciferin to form sandwich filler structure of magnetic particle-target direction DNA-fluorescence labeling semaphore detecting probe marked, meanwhile competitiveness detecting probe of above capture detecting probe or semaphore detecting probe is added to combine with target direction DNA, finally, water-solubility conjugate high molecule with semaphore multiply function is added to amplify and detect the signal in order to detect the target direction DNA. The invention can improve greatly the selectivity and sensitivity of gene and gene mutation detection without amplifying and increasing nucleic acid and with quick and easy operation, has important meaning in biological detection, morbid early diagnosis and treatment. 1, a method for fluorescence detection of DNA, comprising the following steps: 1) the capture probe is connected to the surface of the magnetic particles; 2) the target DNA to be measured; and fluorescein modified signal probe, the capture probe can be a section of the target DNA nucleotide sequence hybridization, the signal probe with the target DNA can be another section of the nucleotide sequence of the hybridization, the hybridization of the DNA, forming the magnetic particle-targeting DNA-fluorescein modification of the sandwich structure of the signal probe; 3) finally, remove the free fluorescein modified after the signal probe, with signal multiplication function added to the water soluble conjugated polymer, on the probe to the signal of the fluorescence signal of the amplified detection, so as to detect the target DNA; The utility model is characterized in that step 2) is also added in the hybridization of the capture probe or the signal probe to the competitive probe, competition binding with the target DNA. 2, fluorescent detection method according to Claim 1, which is characterized in that the capturing probe is stem-loop structure of the probe DNA, the signal probe is linear DNA probe, the competitive probe without any modification, its sequence with the capture probe only by one base. 3, fluorescence detection method according to Claim 1, which is characterized in that the capture probe is a linear DNA probe, the signal probe is stem-loop structure of the probe DNA, the competitive probe without any modification, and the sequence of the signal probe only by one base. 4, fluorescence detection method according to Claim 1, which is characterized in that step 2) the temperature of the hybridization reaction is in the 29-39 the [...][...]. 5, according to Claim 4 fluorescence detection method, which is characterized in that the hybrid reaction temperature is the 37 [...]. 6, any one according to Claim 1-5 of the fluorescence detection method, which is characterized in that the the molar amount of the added competitive probe of the probe is competition 1-7 times. 7, fluorescent detection method according to Claim 6, which is characterized in that the the molar amount of the added competitive probe competition probe is 2 times. 8, fluorescent detection method according to Claim 1, which is characterized in that the conjugate polymer is a water-soluble polyfluorenes. 9, a fluorescent a kit for the detection of the DNA, which is characterized in that it includes: 1) magnetic particles to the capture probe, fluorescein modified signal probe; 2) the above-mentioned capturing probe or the signal probe competitive probe; 3) water-soluble polyfluorenes. DNA1 DNA2 DNA3 DNA4 DNA5 DNA6 DNA7 DNA8 DNA9 The 5 [...] -CGC GCT CCT ATG AAC TCA CGA TAT GCG5-BIOTIN -3 the [...] the 5 [...] -CGC A TCA AAC GCT CCT CGA A GCG-3 the TAT G[...] 5 the ATA ATT [...] -FAM-GTA AAA-3 the ATC TGA [...] 5 the TAG [...] -GAG ACA GGT TCT TTC CAT TTT CTT TAA TGG TTC TGA C-3 the ATA TTA [...] 5 the AC [...] -GAG TAG CAT GGT TTC T TTT TGG TCT TTC CTT TAA TGA C-3 the ATA TTA [...] the 5 [...] -GAA CTC ACC TGT ATG CTA TTT TTT TTT T5-BIOTIN -3 the [...] 5 the TGA GTA GCG [...] -FAM-TAC ATA CGC ATT GT-3 the AAA ATC [...] the 5 [...] -TAC TGA GCG ATC GTA AAA A CGC AT GT-3 the ATA [...] 5 the TAG [...] -GAG CAT GGT TCT TTC TGG ACA CTT TGA TTT TTC T T ATA TTA C-3 the A[...] TTL TTA 0.5 ×SSC hybridization washing liquid 75 mm NaCl, 7.5 mm sodium citrate, pH 7.4 100 mm Tris-HCl, 0.1% Tween 20, 1M LiCl, pH 8.0 250 mm Tris-HCl, 0.1% Tween 20, 5% BSA, pH 7.4 750 mm NaCl, 75 mm sodium citrate, pH 7.4 10 mm Tris-HCl, 50 mm NaCl, pH 7.4