CN104498602B - A kind of nucleic acid detection method based on magnetic bead Yu ultrasonic Separation luminous marker - Google Patents
A kind of nucleic acid detection method based on magnetic bead Yu ultrasonic Separation luminous marker Download PDFInfo
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Abstract
The present invention relates to a kind of nucleic acid detection method based on magnetic bead Yu ultrasonic Separation luminous marker: the method is at magnetic bead surfaces rhetorical function arm molecule；It is designed for detecting the functionalization probe of target nucleic acid fragment, by functionalization probe modification in the magnetic bead surfaces being combined with functionalization arm molecule；By hybridization, target nucleic acid fragment and luminous marker thereof are captured to magnetic bead surfaces；Magneto separate, cleans, has been obtained the magnetic bead of luminous marker；By physical methods such as ultrasonic wave process, luminous marker is separated from magnetic bead surfaces, the luminous marker solution without magnetic bead is carried out luminous detection, obtains target nucleic acid information.The present invention can not only overcome the luminous signal loss that the capture-effect of luminous signal is caused by magnetic bead, and use the physics modes such as ultrasonic wave process, the reagent that chemistry or biological method can be avoided to cause inactivates or other interference, being capable of detecting when minimum 50amol nucleic acid fragment in 20 μ L volumes, detection sensitivity is high.
Description
Technical field
The present invention relates to biochemistry detection and molecular diagnosis field, particularly relate to one based on magnetic bead with ultrasonic
Separate the nucleic acid detection method of luminous marker.Present invention can apply to relate to the neck of hypersensitive nucleic acid molecules detection
Territory.
Background technology
In recent years, owing to pernicious public health event takes place frequently, such as the Escherichia coli (E. of Japan's generation in 1996
Coli) outbreak of epidemic of O157:H7 food poisoning, causes several people dead, infects more than ten thousand people, cause huge
Society is panic；SARS virus wreaking havoc in China in 2003；The harm of Streptococcus suis in 2005；Recently fowl stream
H1N1virus etc. popular in Influenza Virus and worldwide in 2009, all to the health of people and
Life causes serious threat.It addition, it is also to affect China's food hygiene and safety that pathogenic microorganisms pollutes
Main factor, the Poisoning Number that microbes food poisoning causes is most, complete at 2003 and 2004
In the Grave food-poisoning accident of state's report, microbes Grave food-poisoning plays number and number all has increase, point
Do not account for total rising then to count and the 26% of total number of persons, 43.8% and 34%, 58.1%.Food processing, transport,
It is highly prone to pathogenic microorganisms during storage, sale etc. pollute.Harmful pathogenic microorganisms not only affects people's
Health, jeopardizes the life of people, and causes the fear of the whole society, affect normal Socialized Reading.In order to enable
Identifying pathogen rapidly and accurately, make every effort to the harm of people is preferably minimized pathogen, scientists employs very
Multiple authentication method.Traditional microorganism characteristic information detection method has the cultivation of pathogen, biochemical analysis, poison
Element mensuration, serology (antigen or antibody) detection etc., but these detection methods are not only not suitable for quick detection,
And be difficult to detect and identify much information contained in pathogen, such as Disease-causing gene, virulence gene, resistance base
Because of etc..Although have also been developed round pcr and nucleic acid probe hybridization technology in recent years, but can not be to microorganism feature
Information is used for quickly detecting, with less the ability of high flux examination.
Malignant tumour is one of disease of serious harm human health.The World Health Organization and International Contre
The related data of alliance shows, if the incidence of disease of malignant tumour can not effectively be controlled, it is contemplated that pathogenesis of cancer example exists
The year two thousand twenty is up to 16,000,000, and nearly 10,000,000 people are dead.China increases cancer patient about 1,600,000 people every year newly, its
Middle because of number of cancer deaths about 1,300,000 people.Cause cancer death to be primarily due to find early thus
Affect therapic opportunity adversely and tumor cure rate is low, and early detection and effectively treatment are prevention and the pass for the treatment of cancer
Key means.In decades, researchers find, biomarker can be used to carry out early diagnosis of tumor, makes
Obtain the cure rate of cancer, survival rate is greatly improved.In recent years, along with Microrna (micro RNA, miRNA)
Discovery and further research so that it is possible as new biomarker.It has proven convenient that miRNA is outside
Expression in all blood has cancer-related, tissue specificity and expression stability, and peripheral blood miRNA is very
Being probably a kind of preferably tumor markers, the early diagnosis for cancer opens new way.At present, peripheral blood
The detection means of miRNA mainly has miRNA chip technology and Real-Time Fluorescent Quantitative PCR Technique.But two dimension
Chip technology makes nucleic acid hybridization inefficiency so that its detection sensitivity is the most on the low side.And quantitative fluorescent PCR
Detection miRNA needs the stem ring probe that design is complicated, and carries out amplified reaction so that it detects into higher,
Also automation cannot be realized.
Along with developing rapidly of nanometer technology, nano material is gradually applied to life science, nano magnetic
Pearl (magneticbeads, MBs) because having that separating rate is fast, efficiency is high, reusable, simple to operate, easy
Functionalization, easily realize automation and do not affect special physicochemical properties and the biofacies such as activity of separate substance
Capacitive, has been applied to the aspects such as the fixing of cell separation, immunoassays, protein and enzyme and detection of nucleic acids the most.
In recent years, luminescent marking class detection method is as the most extensive in fluorescence labeling, enzyme mark chemiluminescence etc.
It is applied to biomolecule detection and carrys out amplification detection signal to improve detection sensitivity.Operation safety, method simplicity,
Quickly, there are huge potentiality, just becoming the most important field of bioanalysis research and development.Therefore, syncaryon
Acid probe hybridization technique, can produce the molecule of luminous signal by mark and obtain mesh target nucleic acid information, can be real
The quickly detection of existing target nucleic acid and high flux examination.
At present, various nucleic acid detection method based on solid-phase hybridization Yu luminescent marking has been reported.It is basic
Thinking is: at surface of solid phase carriers modification of nucleic acids probe, be marked with the spy to be measured of luminous signal molecule by capture
Heteronuclear acid fragment, then detect luminous signal, after analyzing, can quickly confirm the characteristic of target nucleic acid.
But along with research deeply finds, affect based on magnetic bead and luminescent marking nucleic acid detection method sensitivity
A key factor be: luminous signal can weaken sensitivity decrease significantly due to the bridging effect of magnetic bead
Reach as high as 90%.But prior art only fluorescence detection uses the method that high temperature makes double-strandednucleic acid sex change unwind
Separate fluorescent marker, because fluorescent marker such as fluorescein (Cy3, Cy5) etc. can tolerate high temperature.But it is chemical
The luminous marker of luminous detection method etc. cannot reach this purpose by the way of high temperature, because chemiluminescent labeling
Things etc. cannot tolerate high temperature, can inactivate when high temperature and cannot proceed luminous detection.Therefore, one is developed
Kind general based on magnetic bead and the nucleic acid detection method separating luminous marker, it is possible to be greatly enhanced fluorescence, outstanding
It is chemiluminescent detection sensitivity, and this has great for disease molecules diagnosis, food microorganisms monitoring etc.
Meaning.
Summary of the invention
Technical problem underlying to be solved by this invention be overcome existing based on magnetic bead and luminous marker
The detection sensitivity limitation problem that exists of nucleic acid detection method, its reason is magnetic bead to optical signal to be produced and covers effect
Should, greatly reduce luminous signal intensity.It is therefore proposed that one is based on magnetic bead and ultrasonic Separation luminescent marking
The nucleic acid detection method of thing.
A kind of nucleic acid detection method (Fig. 1) based on magnetic bead Yu ultrasonic Separation luminous marker, including such as
Lower step: (1) modifies the functionalization arm molecule being suitable for connecting nucleic acid probe in magnetic bead surfaces；(2) design is used
In the functionalization probe of detection target nucleic acid fragment, by functionalization probe modification in being combined with functionalization arm molecule
Magnetic bead surfaces；(3) by hybridization, target nucleic acid fragment and luminous marker thereof are captured to magnetic bead surfaces；(4)
Magneto separate, cleans, has the most been obtained the magnetic bead of luminous marker；(5) by physics sides such as ultrasonic wave process
Luminous marker is separated by method from magnetic bead surfaces, i.e. obtains the luminous marker solution without magnetic bead；(6)
This solution is carried out luminous detection, target nucleic acid information can be obtained.The method passes through ultrasonic wave separation luminescent marking
Thing, removes magnetic bead, individually detects luminous marker, overcome magnetic bead completely and lead the capture-effect of luminous signal
The luminous signal loss caused, and use the physics modes such as ultrasonic wave process, chemistry or biological method can be avoided
The reagent inactivation that may cause or other interference etc..It addition, the modification of arm molecule can improve the suspension of magnetic bead
Property, reduce sterically hindered, improve the hybridization free degree, increase probe binding site, thus improve magnetic bead surfaces
Nucleic acid hybridization efficiency.Therefore, the present invention is remarkably improved based on magnetic bead sensitive with the detection of nucleic acids of luminous marker
Degree.
Described surface is modified, and includes but not limited in magnetic bead surfaces with covalent bond, non-covalent bond or physics
Suction type rhetorical function arm molecule.
Described functionalization arm molecule, it is possible to simultaneously with magnetic bead and functionalization probe with covalent bond, non-co-
The polymer that valence link or physical adsorption way combine, includes but not limited to glucan, shitosan, cellulose, gathers
Ethylene glycol and derivative thereof.
Described target nucleic acid fragment is: non-amplification or the nucleic acid fragment after amplification.
Described functionalization probe is: can be with arm molecule covalency, Non-covalent binding or physical absorption formula
In conjunction with, and with the oligonucleotides of target nucleic acid fragment complementary.
Described luminous marker, (1) this label is that the luminescence that can produce being applicable to Molecular Detection is believed
Number molecule, include but not limited to fluorescent marker, chemiluminescent labels etc.；(2) luminescent label
The mode of target nucleic acid, includes but not limited to be marked by target nucleic acid amplification, by reporter probe and target nucleic acid
Fragment hybridization is marked.
Described physical method, the physical method such as including ultrasonic wave process.
Described luminescence is detected as: luminous detection architecture based on molecular labeling, include but not limited to fluorescence,
Chemiluminescence or other wavelength light etc..
A kind of nucleic acid detection method based on magnetic bead Yu ultrasonic Separation luminous marker, its step is as follows:
1. at magnetic bead surfaces rhetorical function arm molecule: (1) magnetic bead amination is modified: by 30mg
Fe3O4@SiO2Magnetic bead Magneto separate, adds 6mL ethanol/water mixed liquor ultrasonic disperse, then takes 12 μ L
Aminopropyl triethoxysilane (aminopropyltriethoxysilane, APTES) adds in above-mentioned mixed liquor,
Shaken at room temperature stirring 7h.Utilize externally-applied magnetic field to be separated by the magnetic-particle that APTES modifies, with ethanol and
PBS (0.1M, pH 7.4, PB) is respectively washed for several times, is dispersed in PB solution, standby；
(2) carboxylated polymer modify magnetic bead (carboxylated polymer-modified magnetic beads,
CP-MBs) preparation: by amination magnetic bead (10mg/mL) Magneto separate, by equal-volume 2-N-morpholino second
Azochlorosulfonate acid hydrate solution (2-(N-morpholino) ethanesulfonic acid hydrate, MES, 25mM, pH
6) cleaning for several times, Magneto separate, the carboxylated polymer (CP) adding the dissolving of 5mg/mL MES solution is molten
Liquid is in the most cleaned magnetic bead, and after mixing, room temperature rotates and hatches 30min, is added immediately and cold uses MES solution
10mg/mL 1-(3-the dimethylaminopropyl)-3-ethyl carbodiimide dissolved
(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, EDC) mixes to magnetic bead mixed liquor,
Rotate at 4 DEG C and hatch more than 2h.Magneto separate abandons supernatant, cleans for several times with PB solution, is dispersed in PB solution
In, i.e. obtain CP-MBs.
2. being designed for detecting specific primer and the functionalization probe of target nucleic acid fragment, probe is with 3 ' ends
Amido modified.Probe modification CP-MBs: CP-MBs (10mg/mL) is clear with equal-volume MES solution
Washing for several times, Magneto separate abandons supernatant, in the probe (2 μMs) of addition MES dilution to washed CP-MBs,
After mixing, room temperature rotates and hatches 30min, is added immediately the cold EDC (10mg/mL) dissolved with MES solution,
Mix to magnetic bead mixed liquor, rotate at 4 DEG C and hatch more than 2h.Magneto separate abandons supernatant, adds Tris solution
(50mM, containing 0.1%Tween-20, pH 7.4) solution is hatched in 15min and unreacted carboxyl groups, then is used
Tris solution cleans for several times, is finally dispersed in PB solution, i.e. obtains being connected with the functionalization magnetic bead of specific probe.
3. pair target nucleic acid fragment carries out PCR amplification: reaction system is 10 × Buffer 2 μ L, Mg2+1.2
The each 1 μ L of μ L, dNTP Mix 1 μ L, upstream and downstream primer, DNA profiling 1 μ L, Taq enzyme 0.5 μ L, addition
Deionized water supplies volume to 20 μ L；Reaction condition is 95 DEG C of 5min, 95 DEG C of 30s, 60 DEG C of 30s 72 DEG C
30s, 35 circulations.
4. nucleic acid hybridization: take 30 μ L probe modification magnetic bead (10mg/mL) in PCR pipe, Magneto separate
Abandon supernatant, add hybridization solution 10 μ L, target nucleic acid fragment PCR primer 1 μ L, add deionized water and supply volume
To 20 μ L；Hybridization reaction condition is 95 DEG C of 5min, 60 DEG C of 10min；Then Magneto separate, cleans magnetic bead
For several times, the magnetic bead of target nucleic acid fragment has the most been obtained.
5. luminescent label target nucleic acid fragment: (1) is marked by PCR amplification: in step
Adding biotin-11-dUTP in dNTP Mix described in 3, other are with step 3, then carry out step 4；(2)
Marked by nucleic acid hybridization: design and the reporter probe of target nucleic acid fragment complementary, this report probe end is repaiied
It is decorated with luminous marker；After step 4 completes, carry out second time nucleic acid hybridization: by the hybrid product magnetic of step 4
Separate, abandon supernatant, add hybridization solution 10 μ L, reporter probe solution 1 μ L, add deionized water and supply volume
To 20 μ L；Hybridization reaction condition is 95 DEG C of 5min, 60 DEG C of 10min；Then Magneto separate, cleans magnetic bead
For several times, the magnetic bead of the target nucleic acid fragment of band luminous marker has the most been obtained.
6. pair obtain the magnetic bead of the target nucleic acid fragment of band luminous marker, frequency modulation ultrasonic wave to 500kHz
(100W), process 20min, Magneto separate, obtain supernatant with pipettor, i.e. obtain sending out without magnetic bead
Signal thing solution.Wherein, 500kHz (100W) is a preferably Ultrasonic Conditions, it is intended that explain this
Invention, the form not being changed Ultrasonic Conditions is interpreted as limiting the scope of the present invention.
7. pair luminous marker solution carries out luminous detection, obtains target nucleic acid information.
Compared with prior art, the invention have the characteristics that:
Functionalization probe is connected to magnetic bead surfaces as molecule arm by big molecule polymer, improves particle and suspends
Property, reduce sterically hindered, add probe binding site and add the hybridization free degree, thus improve
Hybridization efficiency.The luminous marker of corresponding target nucleic acid fragment is separated from magnetic bead surfaces and detects, it is to avoid
Magnetic bead capture-effect, improves luminous signal intensity.Meanwhile, use ultrasonic physics mode of Denging, can avoid
Reagent inactivation that chemistry or biological method may cause or other interference etc..Therefore, present invention greatly enhances
Detection sensitivity.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described:
Fig. 1 is a kind of nucleic acid detection method flow process signal based on magnetic bead with ultrasonic Separation luminous marker
Figure.
Fig. 2 is HBV nucleic acid PCR amplified production electrophoretogram.
Fig. 3 is the chemiluminescence detection dynamic curve of HBV nucleic acid.
Detailed description of the invention
Below in conjunction with example, the invention will be further described:
The invention discloses that a kind of characteristic utilizing magnetic bead to be prone to Magneto separate combines hybridization and ultrasonic Separation is luminous
The strategy of label, carries out luminous detection, obtains target nucleic acid information the luminous marker solution without magnetic bead
Method.In one of the present invention preferably example, with Fe3O4@SiO2Magnetic bead is as carrier, on its surface
Rhetorical function arm molecule, functionalization probe in connection, with biotin labeling target nucleic acid fragment, passes through nucleic acid
Hybridization will carry biotin labeled target nucleic acid fragment to capture to magnetic bead surfaces, make by hatching with Avidin marker enzyme
Magnetic bead obtains the enzyme molecule of corresponding target nucleic acid fragment, then utilizes ultrasonic wave by the enzyme molecule of corresponding target nucleic acid fragment
Separate from magnetic bead surfaces, finally carry out chemiluminescence detection, obtain target nucleic acid information.
Embodiment 1: modify for functionalization arm molecule in magnetic bead surfaces with Sensor Chip CM 5
1. the amination of magnetic bead is modified: by 1mLFe3O4@SiO2Suspension containing magnetic beads (30mg/mL) magnetic
Separate, add in 6mL ethanol/water (99/1) mixed liquor and ultrasonic disperse, then take 12 μ L aminopropyls three
Ethoxysilane (aminopropyltriethoxysilane, APTES) adds in above-mentioned mixed liquor, shaken at room temperature
Stirring 7h.Utilize externally-applied magnetic field to be separated by the magnetic-particle that APTES modifies, delay with ethanol and phosphate
Dissolved liquid (PB, 0.1M, pH 7.4) is respectively washed for several times, is dispersed in 3mL PB solution, it is thus achieved that amino
Change magnetic bead (aminated MBs, AMBs, 10mg/mL).
2. Sensor Chip CM 5 modifies magnetic bead (carboxymethylated dextran-modified
Magnetic beads, CMD-MBs) preparation: by amination magnetic bead (10mg/mL) Magneto separate, with etc.
Volume 2-N-morpholino ethyl sulfonic acid hydrate soln (2-(N-morpholino) ethanesulfonic acid hydrate,
MES, 25mM, pH 6) clean for several times, Magneto separate, add the carboxymethyl that 2mg/mLMES solution dissolves
In the magnetic bead that glucan (CMD) solution is the most cleaned, after mixing, room temperature rotates and hatches 30min, adds at once
Enter cold 1-(3-the dimethylaminopropyl)-3-ethyl carbodiimide dissolved with MES solution
(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, EDC, 10mg/mL) is to magnetic bead mixed liquor
Middle mixing, rotates at 4 DEG C and hatches more than 2h.Magneto separate abandons supernatant, cleans for several times with PB solution, dispersion
In PB solution, i.e. obtain CMD-MBs.
3. water and radius by dynamic light scattering mensuration magnetic bead can obtain: CMD-MBs's and MBs is flat
All water and radiuses are respectively 657.9nm and 738.5nm, show the modification of CMD add MBs water and
Radius；Meanwhile, can be obtained by the zeta current potential of dynamic light scattering mensuration magnetic bead: AMBs and CMD-MBs
Zeta current potential be respectively+13.7mV and 12.6mV, show AMBs through CMD modify after, on CMD
Carboxyl make magnetic bead surfaces be changed into negative electrical charge by positive charge.Above-mentioned experimental result explanation CMD successfully repaiies
Decorations are in magnetic bead surfaces.
Embodiment 2: modify for functionalization arm molecule in magnetic bead surfaces with carboxymethylcellulose calcium
1. the amination of magnetic bead is modified: the method modified with the amination of magnetic bead described in embodiment 1.
2. carboxymethylcellulose calcium modifies magnetic bead (carboxymethylcellulose-modified magnetic
Beads, CMC-MBs) preparation: by amination magnetic bead (10mg/mL) Magneto separate, use equal-volume 2-N-
Morpholino ethyl sulfonic acid hydrate soln (2-(N-morpholino) ethanesulfonic acid hydrate, MES, 25
MM, pH 6) clean for several times, Magneto separate, add the carboxymethylcellulose calcium that 1mg/mL MES solution dissolves
(CMC), in the magnetic bead that solution is the most cleaned, after mixing, room temperature rotates and hatches 40min, is added immediately cold
1-(3-the dimethylaminopropyl)-3-ethyl carbodiimide dissolved with MES solution
(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, EDC, 10mg/mL) is to magnetic bead mixed liquor
Middle mixing, rotates at 4 DEG C and hatches more than 2h.Magneto separate abandons supernatant, cleans for several times with PB solution, dispersion
In PB solution, i.e. obtain CMC-MBs.
3. water and radius by dynamic light scattering mensuration magnetic bead can obtain: CMC-MBs's and MBs is flat
All water and radiuses are respectively 678.2nm and 765.7nm, show the modification of CMC add MBs water and
Radius；Meanwhile, can be obtained by the zeta current potential of dynamic light scattering mensuration magnetic bead: AMBs and CMC-MBs
Zeta current potential be respectively+14.1mV and 15.7mV, show AMBs through CMC modify after, on CMC
Carboxyl make magnetic bead surfaces be changed into negative electrical charge by positive charge.Above-mentioned experimental result explanation CMC successfully repaiies
Decorations are in magnetic bead surfaces.
Embodiment 3: be that object detects with hepatitis B (Hepatitis B virus, HBV) DNA
1. design a pair can the primer of specific amplified HBV X gene fragment and joining with this complementary
To functionalization probe, probe 3 ' hold with amido modified.Primer sequence is as follows:
5’-CCTCTACCGTCCCCTTCTTCA-3’(SEQ ID NO.1)、
5’-ACGTGCAGAGGTGAAGCGAAG-3’(SEQ ID NO.2)；Probe sequence is as follows:
5‘-CACTTCGCTTCACCTCTGCACGTA-(T)15-NH2-3’(SEQ ID NO.3)。
2. probe modification Sensor Chip CM 5 modifies magnetic bead (CMD-MBs): by CMD-MBs (10
Mg/mL) cleaning for several times with equal-volume MES solution, Magneto separate abandons supernatant, adds the probe (2 of MES dilution
μM) in washed CMD-MBs, after mixing, room temperature rotates and hatches 30min, is added immediately cold use
The EDC (10mg/mL) that MES solution dissolves, mixes to magnetic bead mixed liquor, rotates and hatch 2h at 4 DEG C
Above.Magneto separate, abandons supernatant, adds Tris solution (50mM, containing 0.1%Tween-20, pH 7.4) and incubates
Educate in 15min and unreacted carboxyl groups, then clean for several times with Tris solution, be finally dispersed in PB solution,
I.e. obtain being connected with the functionalization magnetic bead of capture probe.
3. pair target nucleic acid fragment carries out PCR amplification: reaction system is 10 × Buffer 2 μ L, Mg2+1.2
μ L, dNTP Mix (dATP:dCTP:dGTP:dTTP:biotin-11-dUTP=25:25:25:23:2) 1 μ L, on
The each 1 μ L of downstream primer, DNA profiling 1 μ L, Taq enzyme 0.5 μ L, add deionized water and supply volume to 20 μ L；
Reaction condition is 95 DEG C of 5min, 95 DEG C of 30s, 72 DEG C of 30s of 60 DEG C of 30s, 35 circulations.Fig. 3 is
HBV nucleic acid PCR amplified production electrophoretogram, target nucleic acid fragment band is single, it was demonstrated that expand successfully.
4. nucleic acid hybridization: take 30 μ L probe modification magnetic bead (10mg/mL) in PCR pipe, Magneto separate,
Abandon supernatant, add hybridization solution 10 μ L, PCR primer 1 μ L, add deionized water and supply volume to 20 μ L；Miscellaneous
Friendship reaction condition is 95 DEG C of 5min, 60 DEG C of 20min；Then Magneto separate, cleans for several times magnetic bead, i.e. obtains
Obtain the magnetic bead of the target nucleic acid fragment of band luminescent marking.
5. chemiluminescence detection: suck supernatant after magnetic bead liquid Magneto separate, adds room temperature after confining liquid mixing
Placing 30min, period constantly mixes.Suck supernatant after Magneto separate, add confining liquid dilution alkaline phosphatase mark
Note Streptavidin (alkaline phosphatase labeled streptavidin, ALP-SA), after mixing, room temperature is incubated
Educate 30min.Cleaning for several times after Magneto separate, then frequency modulation ultrasonic wave is 500kHz (100W), processes 20min,
Shift supernatant with pipettor, supernatant is separated with magnetic bead, be then separately added in supernatant with magnetic bead
0.25mM chemical luminous substrate 3-(2 '-spiral adamantane)-4-methoxyl group-4-(3 "-hydroxyl) benzene-1,2-dioxane
Butane phosphoric acid (3-(2 '-spiroadamantane)-4-methoxy-4-(3 "-phosphoryloxy) phenyl-1,
2-dioxetane,AMPPD).Fully measure chemiluminescence intensity value after mixing stable to it, parallel determination 3
Secondary.The luminous testing result processed using unused ultrasonic wave is as comparison.
Chemiluminescence detection result is as shown in Figure 3.Magnetic bead capture-effect is removed in the luminous value explanation recorded
After, chemiluminescence signal is obviously improved, and brings up to about 7 times relative to the most ultrasonic control group.Micro-at 20 μ L
Volume is capable of detecting when minimum 50amol nucleic acid fragment.Testing in triplicate, result is stable.I.e. by super
The HBV information nucleic acid sensitivity that the chemiluminescence detection of sound separation enzyme molecule obtains is significantly higher than the most ultrasonic comparison
The testing result of group.
Embodiment 4: detect for object with serum miR-21
1. designing the functionalization capture probe with the pairing of miR-21 complementary and reporter probe, capture is visited
Pin 3 ' is held with amido modified, and reporter probe 5 ' is held with biotin modification.Probe sequence is as follows:
5’-CTGATAAGCTA-(T)15-NH2-3’(SEQ ID NO.4)、
5’-Biotin-(T)15-TCAACATCAG-3’(SEQ ID NO.5)。
2. capture probe is modified carboxymethylcellulose calcium and is modified magnetic bead (CMC-MBs): by CMC-MBs
(5mg/mL) cleaning for several times with equal-volume MES solution, Magneto separate abandons supernatant, adds the spy of MES dilution
Pin (2 μMs) is in washed CMC-MBs, and after mixing, room temperature rotates and hatches 30min, is added immediately cold
With MES solution dissolve EDC (10mg/mL), to magnetic bead mixed liquor mix, at 4 DEG C rotate incubate
Educate more than 2h.Magneto separate, abandons supernatant, adds Tris solution (50mM, containing 0.1%Tween-20, pH 7.4)
Hatch in 15min and unreacted carboxyl groups, then clean for several times with Tris solution, be finally dispersed in PB solution,
I.e. obtain being connected with the functionalization magnetic bead of capture probe.
3. for the first time nucleic acid hybridization: (1) take 30 μ L capture probes modify magnetic bead (10mg/mL) in
PCR pipe, Magneto separate, abandon supernatant, add hybridization solution 10 μ L, PCR primer 1 μ L, add deionized water and supply
Volume is to 20 μ L；Hybridization reaction condition is 95 DEG C of 5min, 60 DEG C of 20min；Then Magneto separate, to magnetic bead
Clean for several times, the most obtained the magnetic bead of miR-21 fragment.
4. second time nucleic acid hybridization: Magneto separate, abandons supernatant, and addition hybridization solution 10 μ L, reporter probe are molten
Liquid 1 μ L, adds deionized water and supplies volume to 20 μ L；Hybridization reaction condition be 95 DEG C of 5min, 60 DEG C 10
min；Then Magneto separate, cleans for several times magnetic bead, has the most been obtained the miR-21 sheet of band luminous marker
The magnetic bead of section.
5. chemiluminescence detection: with the method for chemiluminescence detection described in embodiment 3.
Chemiluminescence detection result shows, the beneficial effect of miR-21 detection is with embodiment 3, therefore does not goes to live in the household of one's in-laws on getting married
State.
Claims (4)
1. a nucleic acid detection method based on magnetic bead Yu ultrasonic Separation luminous marker, the method is used for non-diagnostic purpose, and its feature exists
In comprising the following steps: (1) is at Fe3O4@SiO2Magnetic bead surfaces first carries out amination modification, then carries out through carboxylated polymer
Covalent surface is modified, and obtains the magnetic bead that carboxylated polymer is modified；Described carboxylated polymer is Sensor Chip CM 5 or carboxymethyl
Cellulose；(2) being designed for detecting the functionalization probe of target nucleic acid fragment, this probe is modified with 3 ' Amino End Group；Functionalization is visited
The magnetic bead that pin is modified with carboxylated polymer is covalently bound, obtains being connected with the functionalization magnetic bead of specific probe；(3) by hybridization,
Target nucleic acid fragment and luminous marker thereof are captured to magnetic bead surfaces；(4) Magneto separate, cleans, has the most been obtained luminescent marking
The magnetic bead of thing；(5) by ultrasonic processing method, luminous marker is separated from magnetic bead surfaces, i.e. obtain without magnetic bead
Luminous marker solution；(6) this solution is carried out luminous detection, target nucleic acid information can be obtained.
Method the most according to claim 1, it is characterised in that wherein (2) described target nucleic acid fragment, be non-amplification or
Nucleic acid fragment after amplification.
Method the most according to claim 1, it is characterised in that wherein (2) described functionalization probe, is can be with institute
State carboxylated polymer to combine with covalent manner, and with the oligonucleotides of target nucleic acid fragment complementary.
Method the most according to claim 1, it is characterised in that wherein (3) described luminous marker is for being applicable to molecule
The molecule that can produce luminous signal of detection, including fluorescent marker, chemiluminescent labels；Luminescent marking in step (3)
The mode of substance markers target nucleic acid is hybridized with target nucleic acid fragment for being marked by target nucleic acid amplification, by reporter probe and is marked.
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| EP0288618A1 (en) * | 1986-03-20 | 1988-11-02 | Gen-Probe Incorporated | Method for releasing RNA and DNA from cells |
| CN1580283A (en) * | 2003-08-13 | 2005-02-16 | 清华大学 | Method for detecting nucleic acid molecule |
| CN102358910A (en) * | 2011-11-03 | 2012-02-22 | 东南大学 | Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension |
| CN103743722A (en) * | 2014-01-02 | 2014-04-23 | 东南大学 | Aptamer sensor based on nano particles and chemiluminiscence, as well as preparation method and application of sensor |
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| EP0288618A1 (en) * | 1986-03-20 | 1988-11-02 | Gen-Probe Incorporated | Method for releasing RNA and DNA from cells |
| CN1580283A (en) * | 2003-08-13 | 2005-02-16 | 清华大学 | Method for detecting nucleic acid molecule |
| CN102358910A (en) * | 2011-11-03 | 2012-02-22 | 东南大学 | Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension |
| CN103743722A (en) * | 2014-01-02 | 2014-04-23 | 东南大学 | Aptamer sensor based on nano particles and chemiluminiscence, as well as preparation method and application of sensor |
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