CN101993956A - Method for detecting HBV DNA and single base mutation by resonance energy transfer based on quantum dots - Google Patents

Method for detecting HBV DNA and single base mutation by resonance energy transfer based on quantum dots Download PDF

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CN101993956A
CN101993956A CN2010100030475A CN201010003047A CN101993956A CN 101993956 A CN101993956 A CN 101993956A CN 2010100030475 A CN2010100030475 A CN 2010100030475A CN 201010003047 A CN201010003047 A CN 201010003047A CN 101993956 A CN101993956 A CN 101993956A
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dna
sample
hbv dna
single base
hbv
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毛红菊
王祥
钟新华
赵建龙
金庆辉
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East China University of Science and Technology
Shanghai Institute of Microsystem and Information Technology of CAS
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East China University of Science and Technology
Shanghai Institute of Microsystem and Information Technology of CAS
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Abstract

The invention relates to a method for detecting HBV DNA and single base mutation by resonance energy transfer based on quantum dots, which comprises the following steps: preparing CdSe/Zns core/shell structured quantum dots coated with mercaptopropionic acid MPA; designing and synthesizing an HBV DNA detection probe; preparing a QDs-DNA probe cross-linked matter; preparing an HBV DNA clinical sample; and using a microplate reader to carry out high-throughput detection on complementary and single base mutation HBV target DNA. The method is simple for operation and does not need to carry out separation and purification on the DNA which is not linked in a hybrid system; as Cy5 can not directly produce a fluorescence signal under the selected exciting light (485nm), the background interference is small and the signal is strong; and the method further has the characteristics of being specific and rapid and having high resolution, high sensitivity and high throughput, and can be used for HBV DNA and single base mutation high-throughput and multi-target detection in the clinical medicine.

Description

Shift the method that detects HBV DNA and single base mutation based on quantum dot resonance energy
Technical field
The invention belongs to the detection range of target DNA or single base mutation, particularly relate to a kind of method that detects HBV DNA and single base mutation that shifts based on quantum dot resonance energy.
Background technology
Hepatitis B is that (infection of hepatitis B virus is a global health problem for hepatitis B virus, the disease that HBV) causes by hepatitis B virus.The whole world has the chronic carrier of 3.5 hundred million HBV nearly, and there are 1.2 hundred million HBV virus carriers in China, and in rising trend.The infection of HBV can cause multiple disease, such as: chronic hepatitis, liver cirrhosis and primary hepatocarcinoma.Aspect the treatment of HBV, suppressing virus replication by anti-HBV medicine is to treat the most important and effective means of chronic hepatitis B (CHB) at present.Be widely used in the treatment of anti-HBV such as medicines such as lamivudine and Adefovirs.But after the medicine prolonged application, virus produces transgenation, and then the variation that causes and resistance problem have become the thorny problem in clinical.Therefore, detection by quantitative HBV DNA and base mutation are significant in HBV fundamental research and clinical practice.Present modal detection HBV infects and the method for single base mutation comprises: gel electrophoresis polymerase chain reaction (polymerase chain reaction, PCR), branched DNA signal amplification method (branched DNA signal ampli-fication assay), the DNA hybridizing method (allele-specific DNA hybridization) that equipotential is special, connect or primer extension (ligation or primer extension), based on the FRET (fluorescence resonance energy transfer) (molecular-beacon-based fluorescence resonance energy transfer) of molecular beacon, Electrochemical Detection (electroche mical detect-ing) etc.But these some defectives of method ubiquity, perhaps complicated operating process, perhaps sensitivity is not high, and is perhaps more time-consuming, lacks the ability of many targets high throughput testing.Therefore the method for setting up the detection of simple, special, quick and high-throughout target DNA and single base mutation is the key issue that solves clinical application.
(quantum dots QDs), is a kind of novel fluorescent probe to quantum dot.Since CdSe/ZnS core/shell structure QDs that first report use to be modified demarcated biological sample as fluorescent marker, QDs had caused in numerous bioanalysiss and physiological process research in recent years and pays close attention to widely and use.QDs has the photophysical property of many uniquenesses, as the absorption spectrum of broadness and narrow emmission spectrum, this makes them can be used as the excellent energy donor and is applied to that (Fluorescence resonance energy transfer is in transmitter FRET) based on FRET (fluorescence resonance energy transfer).This is because these fluorescent characteristics allow us to select certain excitation wavelength to excite in the absorption spectrum of QDs broadness, thereby avoids directly exciting dye acceptor (as Cy5); And can select suitable spectral filter that donor is effectively separated with the acceptor emitted fluorescence.Up to now, investigators have made up multiple QDs nano-sensor based on FRET.These transmitters successfully have been used for the detection of multiple analytes, comprise the variation of nutritive substance, explosive substance, protein, enzyme and pH value etc.Meanwhile, also have research to concentrate on the identification that is applied to oligonucleotide (DNA) based on the QDs-DNA nano-sensor of FRET, thereby the variation by the FRET fluorescent emission signals that causes behind the oligonucleotide hybridization reach identification and detection to oligonucleotide (DNA).But the preparation process complexity of many QDs-DNA transmitters, the cost height, and also the probe for preparing can not prolonged preservation.In most cases, the QDs-DNA FRET transmitter of structure is what to realize the detection of target DNA on the luminoscope of routine, but the method for utilizing common luminoscope to detect is all more loaded down with trivial details and time-consuming usually.Some investigators are also arranged by QDs being implanted on the nanotube or in conjunction with the single molecular fluorescence detection technique, can obtain the very high DNA transmitter of detection sensitivity, yet the complicacy of transmitter preparation process in these methods, and the instrument that needs smart your complexity, all make the application of these methods be restricted.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method based on quantum dot fluorescence resonance energy transfer detection HBV DNA and single base mutation, to solve defective of the prior art.
A kind of method based on quantum dot resonance energy transfer detection HBV DNA and single base mutation of the present invention comprises:
(1) preparation of the preparation of detection probes and HBV DNA clinical sample
(1) preparation of the CdSe/ZnS core/shell structure quantum dot of thiohydracrylic acid parcel
Get the toluene solution of the CdSe/ZnS core/shell structure QDs of the trioctylphosphine oxide that the 0.5mL fluorescence emission peak is 575～615nm (TOPO) parcel, add 2～4mL chloroform, dropwise add 2～4mL thiohydracrylic acid (MPA) solution again, stirring at room 30～60 minutes adds 5～8mL water, concussion repeatedly, divide and remove organic layer, in water layer, add acetone, after the centrifugation, the QDs that obtains is dissolved in the Milli-Q ultrapure water, prepares the water-soluble CdSe/ZnSQDs of MPA parcel;
(2) design of HBV DNA detection probe and synthetic
The variation of HBV polymerase site rtM204I/V (YMDD) is relevant with bounce-back and the transaminase level of serum HBV DNA.When M becomes V or I, can cause the flexibility of amino acid side chain to lower, and between HBV polysaccharase and lamivudine triphosphate, form sterically hinderedly, its bonding force with the lamivudine triphosphoric acid is descended, cause viral resistance lamivudine.The present invention is according to the dna sequence dna design detection probes of YMDD (rtM204M) and anomaly YVDD (rtM204V), wherein two probes that are connected are designed in each site, the length of two probes all is 15 bases, single base polymorphic site design is at 3 ' end of first probe, and 5 ' the terminal modified Cy5 (being signal probe), another probe will be as the crosslinked probe of QDs-DNA, 3 ' not end process amino functional modification of probe, so that combine with the quantum dot of having modified carboxyl, all dna sequence dnas of using see Table 1, and the synthetic of probe finished by precious biological (TAKARA) company limited in Dalian;
Table 1.HBVDNA target sequence and probe
Figure G2010100030475D00031
(3) preparation of QDs-DNA probe cross-linking agent
With the QDs of the MPA parcel of the N-hydroxy-succinamide (NHS) of 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) of 0.5g/L, 0.5g/L and 0.05 μ M is 1～2: 1～2 by volume: 2～6 mixed, activate 0.5～2 hour, add above-mentioned-NH then 2The ssDNA probe that functional modification is crossed in 37 ℃ of hatchings 0.5～2 hour, prepares QDs-DNA probe cross-linking agent;
(4) preparation of HBV DNA clinical sample
Adopt HBV genomic dna in the cracking process extracting positive serum sample, then HBV DNA is carried out pcr amplification, make the clinical sample to be tested of HBV DNA;
(2) use the detection of microplate reader to complementary target HBV DNA and single base mutation
(1) detection of complementary target HBV DNA and single base mutation
The Taq that the PCR sample to be tested of the HBV DNA of 10 μ L is joined 115 μ L (20mM) connects in the damping fluid, through 95 ℃ of sex change 5 minutes, it is separated be strand on the PCR instrument; Subsequently this mixed solution is put in the refrigerator, ice bath is 5 minutes in frozen water; In this mixed solution, add the signal probe-1 (10 μ M) of the Cy5 mark of QDs-DNA probe among the 70 μ L above-mentioned ()/(3), 10 μ L, the Taq DNA ligase of 5 μ L (1 unit) then;
With this mixed solution totally 210 μ L in 42 ℃ of down reactions 15～30 minutes, after coupled reaction is finished, with this reaction mixture on Eppendorf PCR instrument 85 ℃ of following heat denatured 5 minutes, two strands is dissociated becomes strand; After the sex change, reaction mixture was placed the frozen water ice bath 5 minutes immediately;
Behind the ice bath reaction mixture is transferred to rapidly in the 96 orifice plate microplate reader, on Wallac 1420 type microplate reader, adopt the 485nm optical excitation, detect the fluorescent emission intensity at hybridization reaction solution 670nm place, thereby realize detection the HBV DNA clinical sample of complete complementary or single base mutation;
(2) result of complementary target HBV DNA and single base mutation detection judges
1. based on the detection by quantitative of the HBV DNA wild-type of QDs-Cy5 FRET (fluorescence resonance energy transfer)
The detected result of 670nm place fluorescent emission intensity in above-mentioned (1) is compared with the detected result that does not add the check sample of PCR sample to be tested, if the fluorescent emission intensity at 670nm place has obvious enhancing than check sample in above-mentioned (1), judge that then the HBV DNA in this sample does not undergo mutation, be wild-type, at this moment fluorescent emission intensity and the typical curve of this sample in the 670nm place can be contrasted, can determine the concentration of HBV DNA in the sample, thereby realize detection by quantitative wild-type HBV DNA;
2. based on the detection of the HBV dna single base mutation of QDs-Cy5 FRET (fluorescence resonance energy transfer)
The detected result of 670nm place fluorescent emission intensity in above-mentioned (two)/(1) is compared with the detected result that does not add the check sample of PCR sample to be tested, if the fluorescence intensity of the fluorescence intensity of sample and blank noise is suitable, judge that then single base mutation has taken place the HBV DNA in this sample;
3. the judgement of the mutation type of HBV single base mutation sample
Sample to be tested in replacement step (two)/(1) is the single base mutation sample of above-mentioned HBV DNA in 2., and the signal probe-1 of the Cy5 mark in this step is replaced with the signal probe-2 of Cy5 mark, and additive method is identical to be detected;
The detected result of sudden change sample in 670nm place fluorescent emission intensity compared with the detected result that does not add the check sample of PCR sample to be tested, if the fluorescence intensity ratio check sample of sample has obvious enhancing, judge that then the YVDD sudden change has taken place the HBVDNA in this sample, otherwise then be the sudden change of other types.
Thiohydracrylic acid solution in described step ()/(1) is the methanol solution of thiohydracrylic acid, and concentration 30mmol/L is 9～11 with the Tetramethylammonium hydroxide adjust pH;
QDs in described step ()/(1) is connected by amido linkage with the dna probe that directly links to each other, and promptly the QDs employing carboxyl of TOPO parcel (COOH) replace, displacement back and amino (NH by part (such as MPA) 2) dna probe modified is connected; Vice versa, i.e. the QDs employing-NH of TOPO parcel 2Part is replaced, and displacement back is connected with the dna probe of-COOH modification;
In described step ()/(3)-NH 2SsDNA probe that functional modification is crossed and the mol ratio of MPA-QDs are 10～500: 1;
Pcr amplification system in described step ()/(4) is: 10 * damping fluid, 1.5 μ l, Taq enzyme (1 unit), 25mmol/L MgCl 20.8 μ l, 25mmol/L dNTP 1 μ l, each 0.5 μ l of upstream and downstream primer, the primer final concentration is 0.5pmol/L; Amplification condition: 94 ℃ of sex change 4 minutes; 94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 4 minutes;
The mol ratio of the QDs-DNA probe in described step (two)/(1) and the signal probe of Cy5 mark is 1: 1;
Use multi-functional microplate reader in described step (two)/(1), to the HBV DNA enforcement detection of complementary target HBV DNA and single base mutation.
The present invention is according to the spectral signature of fluorescence quantum, with the CdSe/ZnS core/shell structure quantum dot of thiohydracrylic acid parcel as energy donor, its fluorescence emission peak is 575～615nm, quantum yield is greater than 20%, finishing has a plurality of carboxylic groups, can connect by the amido linkage covalency with amido modified dna probe; The QDs-DNA probe that obtains after the connection forms the hybridization combination of " sandwich " structure with the signal dna of Cy5 mark and HBV target DNA three to be measured again, thereby realizes the FRET (fluorescence resonance energy transfer) between QDs and the Cy5.
Detection method of the present invention is the crossbred by optical excitation " sandwich " structure of using 485nm, detects it in fluorescent emission realization at 670nm place.Finishing the fluorescence QDs and the amidized DNA of carboxyl be combined into the crosslinked probe of QDs-DNA, under the condition that HBV DNA target sequence exists, be combined into the crossbred of " sandwich " structure with the signal dna of having modified Cy5.When the HBV target DNA was wild-type, the QDs-DNA probe will be in the hybridization system directly be connected with the Cy5 signal dna under the effect of Taq dna ligase, thereby becomes a single stranded DNA.After " sandwich " structure crossbred unwind through thermally denature, QDs and Cy5 just can link together by a DNA chain, under the exciting light of 485nm resonance energy transfer will take place, thereby detect the fluorescent emission of the Cy5 at 670nm place like this.In view of the above, the power of the fluorescent emission by detecting the 670nm place can also reach the purpose of wild-type HBV DNA being carried out detection by quantitative.Yet when the HBV target DNA was the single base mutation type, because base complementary pairing fully, even there is the Taq dna ligase in the hybridization system, the QDs-DNA probe can not be connected to become a single stranded DNA with the Cy5 signal dna yet.After " sandwich " structure crossbred unwind through thermally denature, QDs and Cy5 just can't link together like this, thereby just detected the fluorescent emission less than the Cy5 of 670nm place under the exciting light of 485nm.In view of the above, can effectively detect by the HBV target DNA of this method single base mutation.
The present invention connects chemistry by carbodiimide, sets up a kind of QDs-DNA transmitter, and has developed a kind of novel method that can be used for HBV DNA and single base mutation detection based on FRET.Than existing method, the preparation process by amido linkage banded QDs-DNA transmitter in present method is simple, convenient and stability is high; The testing process of target DNA and single base mutation does not need detection architecture is carried out purifies and separates, thereby simple, quick; And present method can become a kind of universal method that detects target DNA and single base mutation; In addition, owing to used microplate reader, thereby present method fast, efficiently, it is ageing to make that this method has concurrently, and can be used for high-throughput and the detection of many targets, become the novel method of a kind of clinically HBV of being used for target DNA and the chemical sproof single base mutation DNA detection of generation thereby make present method be expected to develop.
Beneficial effect
This method is simple to operate and have versatility, and the preparation process of QDs-DNA transmitter is simple, convenient and stability is high; Testing process does not need detection architecture is carried out purifies and separates, thereby simple, quick; Because Cy5 can directly not produce fluorescent signal under the 485nm optical excitation, so background interference is little, signal is strong; That this method also has is special, high resolving power, highly sensitive characteristics; In addition, owing to used microplate reader, thereby present method fast, efficiently, it is ageing to make that this method has concurrently, and can be used for the detection of high-throughput and many targets, thereby make present method be expected to develop to become a kind of clinically HBV of being used for target DNA and produce the novel method of chemical sproof single base mutation DNA detection.
Description of drawings
Fig. 1 is the testing process of the HBV target DNA (circuit 2) of complementary HBV target DNA (circuit 1) and single nucleotide variation; (wherein, figure A be the energy transfer process principle schematic of " sandwich " structure crossbred, when QDs excites with the light of 485nm, the energy transfer takes place, and energy is from passing to acceptor Cy5 to body QDs, thereby causes the fluorescent emission of Cy5);
Fig. 2 is the different detected result synoptic diagram of the target HBV DNA of complementary and single base mutation;
Than the fluorescence intensity of blank, the fluorescence intensity of single base mutation type HBV target DNA sample is suitable with it, and the fluorescence intensity of wild-type sample is better than it;
Fig. 3 shifts the typical curve of the hybridization Cy5 fluorescent emission intensity that combination produced with wild-type HBV DNA change in concentration for the QDs-DNA energy.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
Shift the structure that detects HBV DNA and single base mutation method based on quantum dot resonance energy
(1) the CdSe/ZnS core/shell structure quantum dot of preparation thiohydracrylic acid parcel
At first according to people's such as Yang method (Yang Y.A.et al.Angew.Chem.Int.Ed.2005,44,6712) fluorescence emission peak of synthetic trioctylphosphine oxide (TOPO) parcel is the CdSe/ZnS nuclear/shell quantum dot of 605nm, get the toluene solution of the above-mentioned synthetic TOPO-QDs well of 0.5mL then, to wherein adding the 3mL chloroform, thiohydracrylic acid (MPA) solution (being dissolved in the methyl alcohol, is 10.0 with the Tetramethylammonium hydroxide adjust pH in advance) that dropwise adds the 30mmol/L of 3mL again; This mixture was at room temperature stirred 30 minutes, then to wherein adding 5mL water, repeatedly after the concussion, after water layer separated from organic layer, to wherein adding acetone, the QDs in the water layer is precipitated, with this mixing solutions centrifugal after, again sedimentary QDs is dissolved in the Milli-Q ultrapure water, prepares the water-soluble CdSe/ZnS QDs of MPA parcel; With the QDs aqueous solution that obtains ultrasonic after, through syringe filtering, finally obtain uniformly MPA-QDs of particle diameter;
(2) design of HBV DNA detection probe and synthetic
The variation of HBV polymerase site rtM204I/V (YMDD) is relevant with bounce-back and the transaminase level of serum HBV DNA.When M becomes V or I, can cause the flexibility of amino acid side chain to lower, and between HBV polysaccharase and lamivudine triphosphate, form sterically hinderedly, its bonding force with the lamivudine triphosphoric acid is descended, cause viral resistance lamivudine.The present invention is according to the dna sequence dna design detection probes of YMDD rtM204 and anomaly rtV204 YVDD, wherein two probes that are connected are designed in each site, the length of two probes all is 15 bases, single base polymorphic site design is at 3 ' end of first probe, and 5 ' the terminal modified Cy5 (being signal probe), another probe will be as the crosslinked probe of QDs-DNA, 3 ' of probe is not held through amino functional modification, so that combine with the quantum dot of having modified carboxyl, sequence sees Table 2, and the synthetic of probe finished by precious biological (TAKARA) company limited in Dalian.
Table 2.HBV DNA target sequence and probe
Figure G2010100030475D00071
(3) preparation of QDs-DNA probe cross-linking agent
With 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC, 0.5g/L), N-hydroxy-succinamide (NHS, 0.5g/L) and MPA-QDs (0.05 μ M) be 1: 1: 2 mixed by volume, activate 0.5 hour, subsequently adding-NH in the quantum dot aqueous solution after this activation 2The ssDNA probe that functional modification is crossed (mol ratio of dna probe: QDs is 50: 1); Above-mentioned solution is 37 ℃ of down hatchings 0.5 hour, by on the QDs-COOH and dna probe on-NH 2Between the generation of acid amides covalent linkage, thereby make dna probe be connected to the QDs surface, prepare QDs-DNA probe cross-linking agent;
(4) based on the HBV DNA wild-type of quantum dot resonance energy transfer and the detection of single base mutation
2 target sequences (the HBV DNA (YVDD) of complementary target HBV DNA (YMDD) and single base mutation) of synthetic HBV DNA in each 10 μ L above-mentioned (2) are joined respectively in 2 Taq connection damping fluids of 115 μ L (20mM), in these 2 mixed solutions, all add the QDs-DNA probe among the 70 μ L above-mentioned (3), the signal dna probe (10 μ M) of 10 μ L, the Taq DNA ligase of 5 μ L (1 unit) subsequently separately;
With the mixed solution of these 2 each 210 μ L all in 42 ℃ of reactions 30 minutes down, after coupled reaction is finished, with reaction mixture on the EppendorfPCR instrument 85 ℃ of following heat denatured 5 minutes, two strands is dissociated becomes strand; After the sex change, reaction mixture was placed the frozen water ice bath 5 minutes immediately;
Adopt with top identical mode and do a blank (being that the HBVDNA target sequence substitutes with isopyknic water) that does not add any target sequence;
Behind the ice bath 3 reaction mixtures are transferred to rapidly in 96 orifice plates, on Wallac 1420 type microplate reader, adopt the 485nm optical excitation, detect the fluorescent emission intensity separately at each hybridization reaction solution 670nm place.
1. HBV single base mutation test result of samples is judged:
The mixed solution of the HBV DNA target sequence (YVDD) of above-mentioned single base mutation is suitable in the fluorescence intensity of the detected result of 670nm place fluorescent emission intensity and blank, this has just verified our experimental program, the HBV target DNA crossbred that is single base mutation is after thermally denature is unwind, QDs and Cy5 can't link together, thereby just detect the fluorescent emission less than the Cy5 of 670nm place under the exciting light of 485nm;
2. the detection of HBV wild-type sample:
The mixed solution of above-mentioned complementary target HBV DNA (YMDD) is more eager to excel in whatever one does than check sample in 670nm place fluorescent emission intensity, illustrate that then the HBV DNA in these samples does not undergo mutation, be wild-type (complementary type fully), this is that wild-type is consistent with our designed sequence really, and this has confirmed our experimental program too.The fluorescent emission intensity reference standard curve of mixed solution at the 670nm place with complementary target HBVDNA contrasts subsequently, the concentration of final definite this complementary HBV dna sequence dna is 10.5 μ M, the known add-on of the 10 μ M that added with us matches, and has shown that this method is used for the feasibility and the accuracy of HBV DNA detection by quantitative.
Embodiment 2
The detection of HBV DNA clinical sample
(1) the CdSe/ZnS core/shell structure quantum dot of preparation thiohydracrylic acid parcel
Method is as described in the example 1;
(2) design of HBV DNA detection probe and synthetic
The variation of HBV polymerase site rtM204I/V (YMDD) is relevant with bounce-back and the transaminase level of serum HBV DNA.When M becomes V or I, can cause the flexibility of amino acid side chain to lower, and between HBV polysaccharase and lamivudine triphosphate, form sterically hinderedly, its bonding force with the lamivudine triphosphoric acid is descended, cause viral resistance lamivudine.The present invention is according to the dna sequence dna design detection probes of YMDD rtM204 and anomaly rtV204YVDD, wherein two probes that are connected are designed in each site, the length of two probes all is 15 bases, single base polymorphic site design is at 3 ' end of first probe, and 5 ' the terminal modified Cy5 (being signal probe), another probe will be as the crosslinked probe of QDs-DNA, 3 ' of probe is not held through amino functional modification, so that combine with the quantum dot of having modified carboxyl, sequence sees Table 3, and the synthetic of probe finished by precious biological (TAKARA) company limited in Dalian;
Table 3.HBVDNA target sequence and probe
Figure G2010100030475D00091
(3) preparation of QDs-DNA probe cross-linking agent
With 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC, 0.5g/L), N-hydroxy-succinamide (NHS, 0.5g/L) and MPA-QDs (0.05 μ M) be 1: 1: 2 mixed by volume, activate 0.5 hour, subsequently adding-NH in the quantum dot aqueous solution after this activation 2The ssDNA probe that functional modification is crossed (mol ratio of dna probe: QDs is 50: 1); Above-mentioned solution is 37 ℃ of down hatchings 0.5 hour, by on the QDs-COOH and dna probe on-NH 2Between the generation of acid amides covalent linkage, thereby make dna probe be connected to the QDs surface, prepare QDs-DNA probe cross-linking agent;
(4) preparation of HBV DNA sample to be tested
HBV genomic dna in 5 positive serum samples of cracking process extracting, increase to HBV DNA with PCR method then:
With the positive serum cracking centrifugal after, get 1.5 μ l, add distilled water to 15 μ l; PCR system: 10 * damping fluid, 1.5 μ l, Taq enzyme (1 unit), 25mmol/LMgCl 20.8 μ l, 25mmol/L dNTP 1 μ l, each 0.5 μ l of upstream and downstream primer, the primer final concentration is 0.5pmol/L; Amplification condition: 94 ℃ of sex change 4 minutes; 94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 4 minutes.Finally obtain 5 PCR product samples (being designated as sample 1～sample 5); (5) based on the HBV DNA wild-type of quantum dot resonance energy transfer and the detection of single base mutation
The Taq that the PCR sample to be tested 1 of the HBV DNA of 10 μ L is joined 115 μ L (20mM) connects in the damping fluid, through 95 ℃ of sex change 5 minutes, it is separated be strand on the PCR instrument; Subsequently this mixed solution is put in the refrigerator, ice bath is 5 minutes in frozen water; In this mixed solution, add the QDs-DNA probe among the 70 μ L above-mentioned (3), the signal dna (10 μ M) of 10 μ L, the Taq DNA ligase of 5 μ L (1 unit) then;
With this mixed solution totally 210 μ L in 42 ℃ of down reactions 30 minutes, after coupled reaction is finished, with this reaction mixture on Eppendorf PCR instrument 85 ℃ of following heat denatured 5 minutes, two strands is dissociated becomes strand; After the sex change, reaction mixture was placed the frozen water ice bath 5 minutes immediately;
Adopt with top identical mode to PCR sample 2～sample 5 operation that experimentizes; Do a blank (being that the PCR sample substitutes with isopyknic water) that does not add any PCR sample simultaneously;
Behind the ice bath 6 reaction mixtures are transferred to rapidly in 96 orifice plates, on Wallac 1420 type microplate reader, adopt the 485nm optical excitation, detect the fluorescent emission intensity separately at each hybridization reaction solution 670nm place.
1. the detection of HBV wild-type sample:
Above-mentioned sample 1, sample 3 and sample 4 are more eager to excel in whatever one does than check sample in 670nm place fluorescent emission intensity, illustrate that then the HBV DNA in these samples does not undergo mutation, and are wild-type (YMDD type).Subsequently sample 1, sample 3 and the sample 4 fluorescent emission intensity reference standard curve at the 670nm place is contrasted, determine that finally the concentration of HBVDNA in these three samples is respectively 35.4 μ M, 7.2 μ M and 46.8 μ M;
2. the detected result of HBV single base mutation sample is judged:
Above-mentioned sample 2, sample 5 are suitable in the fluorescence intensity of the detected result of 670nm place fluorescent emission intensity and check sample, illustrate that then single base mutation has taken place the HBV DNA in these two samples, and promptly sample 2 and sample 5 are the sudden change sample;
3. the judgement of the mutation type of HBV single base mutation sample:
Below in order to detect the sudden change which kind of type has taken place for sample 2 and sample 5, we designed one section with the YVDD type list nucleotide variation sequence signal dna probe-2 of another section of complementary-Cy5 mark (seeing Table 3) mutually, utilize the thinking identical that it is detected then with detection wild-type sample.Method is as follows:
The Taq that the PCR sample to be tested 2 of the HBV DNA of 10 μ L is joined 115 μ L (20mM) connects in the damping fluid, through 95 ℃ of sex change 5 minutes, it is separated be strand on the PCR instrument; Subsequently this mixed solution is put in the refrigerator, ice bath is 5 minutes in frozen water; In this mixed solution, add the QDs-DNA probe among the 70 μ L above-mentioned (3), the signal probe-2 (10 μ M) of 10 μ L Cy5 marks, the TaqDNA ligase of 5 μ L (1 unit) then; With this mixed solution totally 210 μ L in 42 ℃ of down reactions 30 minutes, after coupled reaction is finished, with this reaction mixture on the EppendorfPCR instrument 85 ℃ of following heat denatured 5 minutes, two strands is dissociated becomes strand; After the sex change, reaction mixture was placed the frozen water ice bath 5 minutes immediately;
Adopt with top identical mode to PCR sample 5 operation that experimentizes; Do a blank (being that the PCR sample substitutes with isopyknic water) that does not add any PCR sample simultaneously;
Behind the ice bath 3 reaction mixtures are transferred to rapidly in 96 orifice plates, on Wallac 1420 type microplate reader, adopt the 485nm optical excitation, detect the fluorescent emission intensity separately at each hybridization reaction solution 670nm place.Detected result shows: above-mentioned sample 2, sample 5 are more eager to excel in whatever one does than check sample in 670nm place fluorescent emission intensity, the base sequence that HBV DNA in these samples then is described is complementary fully with the signal probe-2 of-Cy5 mark, in view of the above can judgement sample 2 and sample 5 sudden change of YVDD type has taken place.

Claims (5)

1. one kind shift to be detected the method for HBV DNA and single base mutation based on quantum dot resonance energy, comprising:
(1) preparation of the preparation of detection probes and HBV DNA clinical sample
(1) preparation of the CdSe/ZnS core/shell structure quantum dot of thiohydracrylic acid parcel
Get the 0.5mL fluorescence emission peak and be the toluene solution of CdSe/ZnS core/shell structure QDs of the trioctylphosphine oxide TOPO parcel of 575～615nm, add 2～4mL chloroform, dropwise add 2～4mL thiohydracrylic acid MPA solution again, stirring at room 30～60 minutes adds 5～8mL water, concussion repeatedly, divide and remove organic layer, in water layer, add acetone, after the centrifugation, the QDs that obtains is dissolved in the Milli-Q ultrapure water, prepares the water-soluble CdSe/ZnS QDs of MPA parcel;
(2) design of HBV DNA cloning primer and detection probes is synthetic
The pcr amplification primer:
PCR upstream primer: 5 '-ACGACCGACCTTGAGGCATACTTC-3 ';
PCR downstream primer: 5 '-CAGAGCAGAGGCGGTGTCG-3 ';
Detection probes:
-NH 2The ssDNA probe of modifying: 5 '-TGG ATG ATG TGG TAT (T) 10(CH 2) 3-(NH 2)-3 ';
Signal dna probe-the 1:5 ' of-Cy5 mark-Cy5-TGG CTT TCA GTT ATA-3 ';
Signal dna probe-the 2:5 ' of-Cy5 mark-Cy5-TGG CTT TCA GTT ATG-3 ';
(3) preparation of QDs-DNA probe cross-linking agent
With the QDs of the MPA parcel of the N-hydroxy-succinamide NHS of 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide EDC, 0.5g/L of 0.5g/L and 0.05 μ M is 1～2: 1～2 by volume: 2～6 mixed, activate 0.5～2 hour, add above-mentioned-NH then 2The ssDNA probe that functional modification is crossed in 37 ℃ of hatchings 0.5～2 hour, prepares QDs-DNA probe cross-linking agent;
(4) preparation of HBV DNA clinical sample
Adopt HBV genomic dna in the cracking process extracting positive serum sample, then HBV DNA is carried out pcr amplification, make the clinical sample to be tested of HBV DNA;
(2) use the detection of microplate reader to complementary target HBV DNA and single base mutation
(1) detection of complementary target HBV DNA and single base mutation
The Taq that the PCR sample to be tested of the HBV DNA of 10 μ L is joined 115 μ L (20mM) connects in the damping fluid, through 95 ℃ of sex change 5 minutes, it is separated be strand on the PCR instrument; Subsequently this mixed solution is put in the refrigerator, ice bath is 5 minutes in frozen water; In this mixed solution, add the signal probe-1 (10 μ M) of the Cy5 mark of QDs-DNA probe among the 70 μ L above-mentioned ()/(3), 10 μ L, the Taq DNA ligase of 5 μ L (1 unit) then;
With this mixed solution totally 210 μ L in 42 ℃ of down reactions 15～30 minutes, after coupled reaction is finished, with this reaction mixture on Eppendorf PCR instrument 85 ℃ of following heat denatured 5 minutes, two strands is dissociated becomes strand; After the sex change, reaction mixture was placed the frozen water ice bath 5 minutes immediately;
Behind the ice bath reaction mixture is transferred to rapidly in the 96 orifice plate microplate reader, on Wallac 1420 type microplate reader, adopt the 485nm optical excitation, detect the fluorescent emission intensity at hybridization reaction solution 670nm place, thereby realize detection the HBV DNA clinical sample of complete complementary or single base mutation;
(2) result of complementary target HBV DNA and single base mutation detection judges
1. based on the detection by quantitative of the HBV DNA wild-type of QDs-Cy5 FRET (fluorescence resonance energy transfer)
The detected result of 670nm place fluorescent emission intensity in above-mentioned (two)/(1) is compared with the detected result that does not add the check sample of PCR sample to be tested, if the fluorescent emission intensity at 670nm place has obvious enhancing than check sample in above-mentioned (1), judge that then the HBV DNA in this sample does not undergo mutation, be wild-type, at this moment fluorescent emission intensity and the typical curve of this sample in the 670nm place can be compared, can determine the concentration of HBV DNA in the sample, thereby realize detection by quantitative wild-type HBV DNA;
2. based on the detection of the HBV dna single base mutation of QDs-Cy5 FRET (fluorescence resonance energy transfer)
The detected result of 670nm place fluorescent emission intensity in above-mentioned (1) is compared with the detected result that does not add the check sample of PCR sample to be tested, if the fluorescence intensity of the fluorescence intensity of sample and blank noise is suitable, judge that then single base mutation has taken place the HBV DNA in this sample;
3. the judgement of the mutation type of HBV single base mutation sample
Sample to be tested in replacement step (two)/(1) is the single base mutation sample of above-mentioned HBV DNA in 2., and the signal probe-1 of the Cy5 mark in this step is replaced with the signal probe-2 of Cy5 mark, and additive method is identical to be detected;
The detected result of sudden change sample in 670nm place fluorescent emission intensity compared with the detected result that does not add the check sample of PCR sample to be tested,, then judge the HBV in this sample if the fluorescence intensity ratio check sample of sample has obvious enhancing
The YVDD sudden change has taken place in DNA, otherwise then is other sudden changes.
2. a kind of method that detects HBV DNA and single base mutation that shifts based on quantum dot resonance energy according to claim 1, it is characterized in that: the thiohydracrylic acid solution in described step ()/(1) is the methanol solution of thiohydracrylic acid, concentration 30mmol/L is 9～11 with the Tetramethylammonium hydroxide adjust pH.
3. a kind of method based on quantum dot resonance energy transfer detection HBV DNA and single base mutation according to claim 1 is characterized in that: in described step ()/(3)-NH 2SsDNA probe that functional modification is crossed and the mol ratio of MPA-QDs are 10～500: 1.
4. a kind of method that detects HBV DNA and single base mutation that shifts based on quantum dot resonance energy according to claim 1, it is characterized in that: the pcr amplification system in described step ()/(4) is: 10 * damping fluid, 1.5 μ l, Taq enzyme (1 unit), 25mmol/L MgCl 20.8 μ l, 25mmol/L dNTP 1 μ l, each 0.5 μ l of upstream and downstream primer, the primer final concentration is 0.5pmol/L; Amplification condition: 94 ℃ of sex change 4 minutes; 94 ℃ 30 seconds, 54 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations, 72 ℃ were extended 4 minutes.
5. a kind of method based on quantum dot resonance energy transfer detection HBV DNA and single base mutation according to claim 1, it is characterized in that: the mol ratio of the QDs-DNA probe in described step (two)/(1) and the signal probe of Cy5 mark is 1: 1.
CN2010100030475A 2010-01-03 2010-01-03 Method for detecting HBV DNA and single base mutation by resonance energy transfer based on quantum dots Pending CN101993956A (en)

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WO2013107041A1 (en) * 2012-01-20 2013-07-25 East China University Of Science And Technology Reversibly water-soluble nanocrystals capped by nucleotides and/or nucleosides
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WO2013107041A1 (en) * 2012-01-20 2013-07-25 East China University Of Science And Technology Reversibly water-soluble nanocrystals capped by nucleotides and/or nucleosides
CN103207166A (en) * 2012-12-25 2013-07-17 西安交通大学 Preparation method of fluorescence resonance system for rapid detection of ATP
CN103207166B (en) * 2012-12-25 2015-06-03 西安交通大学 Preparation method of fluorescence resonance system for rapid detection of ATP
CN107505310A (en) * 2017-08-15 2017-12-22 深圳市易瑞生物技术股份有限公司 Bio-energy shifts luminescence analyzer
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