CN1303221C - Method for detecting target nucleic acid using affinity amplifying integrated signal group - Google Patents
Method for detecting target nucleic acid using affinity amplifying integrated signal group Download PDFInfo
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- CN1303221C CN1303221C CNB031024971A CN03102497A CN1303221C CN 1303221 C CN1303221 C CN 1303221C CN B031024971 A CNB031024971 A CN B031024971A CN 03102497 A CN03102497 A CN 03102497A CN 1303221 C CN1303221 C CN 1303221C
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Abstract
The present invention relates to a method of detecting target nucleic acid in samples, more especially to a method of detecting target nucleic acid via using affinity amplified integrated signal groups. The method of the present invention improves the signal generation obviously; thus, the detection efficiency and the sensitivity are obviously enhanced without lowering the specific condition.
Description
Invention field
The present invention generally relates to the method for target nucleic acid in the test sample.More particularly, the present invention relates to use the target nucleic acid detection method of the integrated signal group of affine amplification.Method of the present invention has been improved the generation of positive signal significantly, thereby not reducing under the specific condition, has improved detection efficiency and sensitivity significantly.
Background of invention
At present, detect the method for target nucleic acid existence in RNA or the DNA sample basically all based on the complementary nucleic acid hybridizing method.In these methods, generally all be to use and produce chemoluminescence or electrochemiluminescence under given conditions, and the material that exists with the individual molecule form, so as at least a portion complementary oligonucleotide probe of mark and target nucleotide.Under hybridization conditions, when the probe of mark contacts with the sample that contains target nucleotide sequences, target sequence will with the probe hybridization of said mark.Then, normally after separating probe hybridization and not hybridization, the existence that is labeled thing and the amount of light that produces by reporter molecule or photosignal indication and test sample hybridization, thereby quantitatively or determine the existence of target nucleic acid sequence in the sample qualitatively.
In the prior art, generally all be that the individual signals mark is connected on the nucleoside monomers, and then one or more such monomers are incorporated in the probe.For example, at present chemosynthesis contain deoxidation urine purine ribonucleoside triphosphote (dUTP) analogue of biotin moiety and be incorporated in the polynucleotide, can again this biotin labeled Nucleotide be incorporated in the nucleic acid probe then.In addition, the someone proposes to make marker to be connected to the method for the labeled nucleotide probe on the Nucleotide at random, or connects a plurality of marks on the single nucleoside monomers of oligonucleotide probe, in the hope of improving the detectability of label probe.
Yet prove that the Nucleotide of the many this marks of use can reduce the stability of the crossbred of probe sequence and target nucleotide sequences formation on probe.Evidence suggests that the reduction of this crossbred stability equally also sees the nucleic acid probe that is connected with a plurality of biotin molecules.
Described many a plurality of reporter molecules are incorporated into method in the oligonucleotide so far, for example added the phosphide acid amides or the analogue of mark comprising linearity, and the method for introducing amino group.Yet all more complicated and common lab of these methods is difficult to finish.
" branch " DNA, particularly the production of " branch " Nucleotide oligomer provides condition for introducing a plurality of marks, for example can use non-nucleosides phosphatidyl aminated compounds to introduce in newborn oligomer has two 5 ' terminal " branch " structures, thereby can after synthesizing, make the number of labels that is added be increased to n through n time
2Individual.Perhaps can use poly functional reagent with higher coupling efficiency and marker productive rate.But the synthetic polynucleotide synthetic technology that all needs to use complexity of non-nucleosides phosphatidyl aminated compounds and so-called poly functional reagent.
Goal of the invention
An object of the present invention is to provide the method that target nucleic acid exists in a kind of test sample, this method may further comprise the steps:
(1) with chemistry or physics method capture probe is fixed on the solid phase carrier;
(2) add target nucleic acid sample and make it to contact and hybridize, then flush away free capture probe and other nucleotide sequences of not hybridizing with capture probe with fixed capture probe;
(3) under hybridization conditions, add one or more terminal affinity groupsization, and contain the detection probes with target nucleic acid complementary nucleotide sequence;
(4) be suitable under the affine bonded condition, in said mixture, adding by joint arm or the direct signal group that is connected with affinity groups;
(5) detect the intensity of the signal that said signal group launches, so as to the existence and the amount thereof of target nucleic acid in the specific hybrid of judging said target nucleic acid and said report probe and the definite sample.
According to a preferred embodiment of the inventive method, wherein step (2) and (3) can be carried out simultaneously, and promptly said target nucleic acid sample can add simultaneously with said report probe.
According to a preferred embodiment of the inventive method, wherein step (2), (3) and (4) can be carried out simultaneously, and promptly said target nucleic acid sample can add simultaneously with said report probe and said signal group.
According to a preferred embodiment of the inventive method, the target nucleic acid complementary sequence of wherein said one or more report probes and the target nucleic acid complementary sequence of said capture probe are non-overlapping each other.
According to a preferred embodiment of the inventive method, the target nucleic acid complementary sequence of wherein said a plurality of report probes is non-overlapping each other.
According to a preferred embodiment of the inventive method, hybridize with zone one or more complementary region in addition of capture probe hybridization on wherein said detection probes and the target nucleic acid sequence.
According to a preferred embodiment of the invention, wherein said affinity groups is vitamin H and avidin.
According to a preferred embodiment of the inventive method, wherein said reporter group is the group that can produce detectable chemoluminescence or electrochemiluminescence under the condition of said target nucleic acid and said report probe hybridization.
According to a preferred embodiment of the inventive method, wherein said reporter group is can be under the condition of said target nucleic acid and said detection probe, by the affine group that combination between biological example element/avidin is produced detectable signal.
According to a preferred embodiment of the inventive method, wherein said reporter group is a high polymer micro balloons, and this microballoon carries the material of a large amount of generation detectable signals, for example fluorescent substance by embedding or mode such as crosslinked.
According to a preferred embodiment of the inventive method, wherein said joint arm is the straight-chain paraffin or derivatives thereof with 1-20 carbon atom of two ends difference covalently bound affinity groups (for example avidin) and signal group.
According to a preferred embodiment of the inventive method, wherein said solid phase carrier is the aperture of microtiter plate, or high polymer micro balloons or biochip.
According to a preferred embodiment of the inventive method, wherein said capture probe is fixed on the solid phase carrier with the chemical coupling method.
Another object of the present invention provides and is used for the test kit that the test sample target nucleotide exists, said test kit comprise (1) be fixed on the solid phase carrier can with the strand Nucleotide capture probe of target nucleotide hybridization, (2) can with the strand Nucleotide detection probes of one or more affinity groupsization of the different sections hybridization of target nucleotide chain; (3) but by the joint arm or the detection moiety that directly is connected with affinity groups.
Test kit preferred embodiment according to the present invention, wherein said joint arm are the straight-chain paraffin or derivatives thereofs with 1-20 carbon atom of two ends difference covalently bound affinity groups (for example avidin) and signal group.
Test kit preferred embodiment according to the present invention, wherein said signal group is a high polymer micro balloons, this microballoon carries the material that can produce signal in a large number by the embedding mode. fluorescent substance for example.
Brief Description Of Drawings
Accompanying drawing 1 is the synoptic diagram that shows the principle of target nucleic acid detection method of the present invention.
The detailed description of invention
The present invention relates to detect the method with quantitative analysis sample amplifying nucleic acid, particularly relate to the integrated reporter group of a plurality of probes of use and affine amplification as detecting reagent, method and the kit of detection and quantitative analysis specific target nucleic acid in the solid phase detection system. Method of the present invention can be in the situation that need not increase to target nucleic acid, detects target nucleic acid by the integrated signal that has amplified, thereby can being unlikely to reduce under the specific condition, improve significantly detection efficiency and sensitiveness.
That the term that uses in this specification " target nucleotide " or " target " refer to exist in the sample and can be by any nucleic acid substances that contains of qualitative or quantitative detection. For the target nucleotide that exists with double chain form under the nature, can in the presence of acid or alkali, after making strand, heat treated detect according to method of the present invention again.
Term " oligonucleotide probe " or " probe " refer to preferentially to identify any nucleic acid compound that contains of specific nucleotide sequence in the sample. In general, probe mainly comprises widow or the polynucleotide probes for DNA operation (separation, amplification and cut-grafting) or detection (as detecting special pathogen). Term " capture probe " refers to and can and can be fixed, can with the nucleotide sequence of another nucleotide fragments hybridization. Capture probe can directly be attached on the solid phase surface by covalent bond, or by ionic bond, absorption or bispecific combination (such as antigen/antibody, hormone/acceptor, biotin/avidin etc.) is attached on the solid phase indirectly. Term " detector probe " refers to can be with the nucleotide sequence hybridization of part beyond the complementation of capture probe in the target nucleic acid sequence hybridization section, and the oligonucleotide sequence of terminal affinity groups (for example biotinylation).
The term that uses in this specification " joint arm " or " joint " refer to connect two molecules or functional group, and do not disturb molecule or the group of character or the function of its functional groups that connects. Be the object of the invention, applicable joint arm or linkers be comprise 1-20 carbon atom, two terminal all with linear paraffin and its derivative of suitable reactive group.
The term that uses in this specification " signal group " refers to the chemiluminescence of available any physical method detection that can be quantitative or the generating source of electrochemical luminescence signals. Said signal for example can be scattered light, utilizing emitted light, fluorescence and electromagnetic wave. Among the present invention preferably by the be stimulated fluorescence of specific wavelength of rear generation of fluorescent material. Particularly, the preferred signal group microparticle, particularly nano particle of time-resolved fluorescence europium complex that be embedding among the present invention. And wherein said signal group refers to produce under given conditions the aggregate of all kinds of light emitting molecules integrated.
It is affine in conjunction with right that the term that uses in this specification " affinity groups " refers to be selected from the specificity of antigen/antibody, hormone/acceptor, biotin/avidin. It is affine in conjunction with right that term " affinity groups " refers to that then the end of molecule (for example target nucleic acid among the present invention and/or detector probe) combines respectively said specificity. Wherein, the affine combination of the preferred specificity of the present invention is to being biotin and Avidin (avidin).
According to a preferred embodiment of the inventive method, wherein said signal group is can be under the condition of said target nucleic acid and said detection probe, by biotin/avidin in conjunction with between in conjunction with the group that produces detectable fluorescence. And according to the fluorescence intensity that produces after the reaction, the combined standard curve is judged existence and the amount thereof of specific nucleic acid in the test sample.
Some biological molecule is usually utilized in various diagnosis and screening technique the pathoklisis of other molecules. When certain biological molecule has binding specificity to another kind of molecule performance, two kinds of intermolecular ligand/receptor bondings that just form. This ligand/receptor bonding comprises the bonding between antigen and its antibody, acceptor and its part, biotin and the Avidin (avidin). Wherein, fabulous example is the noncovalent interaction between protein Avidin and the little molecular biosciences element. Interaction between biotin and Avidin has been widely used in quantitative analysis and the purifying of nucleic acid and protein. In biological technical field, biotin is the reagent that is usually used in mark, detection and protein purification and nucleic acid. And these marks, detection and purification process all are on the significant affinity basis that is based upon between biotin and the Avidin. Dissociation constant between the two is about 10-15M is known bispecific in conjunction with centering in conjunction with the strongest to one of. Biotin and its are mainly contributed by the Hydrogenbond of uride nitrogen in the molecule in conjunction with combining closely between the object.
An object of the present invention is to provide the method that target nucleic acid exists in a kind of test sample, the method may further comprise the steps:
(1) with chemistry or physics method capture probe is fixed on the solid phase carrier;
(2) add target nucleic acid sample and make it contact with fixing capture probe and hybridize, then the flush away capture probe of dissociating and other nucleotide sequences of not hybridizing with capture probe;
(3) under hybridization conditions, add one or more terminal affinity groups (for example biotinylation) and contain the detector probe of the complementary nucleotide sequence of other the different sections beyond the target nucleic acid section from the hybridization of said capture probe;
(4) be suitable under the condition of affine combination, add in the said mixture by the joint arm or directly with the affinity groups signal group that is connected of Avidin for example;
(5) detect the intensity of the signal that said signal group launches, so as to existence and the amount thereof of target nucleic acid in the specific hybrid of judging said target nucleic acid and said report probe and the definite sample.
According to a preferred embodiment of the inventive method, wherein said detection is carried out in solid system. Solid phase can exist in any form, for example can be reaction vessel such as test tube wall or its part, also can be filter membrane such as nitrocellulose or various high polymer particle or latex particle. High molecular polymerization composition granule preferably wherein. To the not strict restriction of the size of carrier granular, but granular size should be easy to separate with the method such as centrifugal. In general, the mean particle size scope should be between 1nm-100 μ m.
Particularly, detect in order to realize high flux, can finish according to target nucleic acid of the present invention at any commercially available commercial biochip and detect.
The oligonucleotide probe that available various known method will design in advance and prepare is fixed on said solid phase carrier for example on the above-mentioned high polymer particle. The probe that is applicable to the inventive method comprises double-stranded DNA, RNA and protein nucleic acid (PNA), and other nucleic acid analogs that contain uncharged skeleton. According to a preferred embodiment of the inventive method, the length of probe sequence is generally 8-40 base, because in this scope, protokaryon and most eukaryotes and people's minimum peculiar dna sequence dna all can be found. But particularly preferred probe length is about 15-30 base.
Then in the reaction system that contains stationary probe, add the target nucleic acid that derives from biological sample to be checked. In general, can finish the hybridization between the complementary base in about 1-5 minute and form hybridization complex in about 15-25 ℃ of lower reaction.
Because method of the present invention is quite sensitive, so generally do not need target nucleic acid is carried out the PCR amplification. But in some specific situation, very few (for example concentration is lower than 10 if any more pollution or sample size-8M) time, also can suitably add the pcr amplification step.
For the sample target nucleic acid of removing not hybridization and reduce as much as possible background signal, can be after hybridization reaction be finished be connected of the present invention and the affine detector probe that is connected of signal group before, preferably include an abundant washing process. Can use the hybridization buffer with standard salt concentration to finish washing step. Then in system, add the biotinylated detector probe of end of the present invention.
Said detector probe has the complementary nucleotide sequence of other the different sections beyond the target nucleic acid section of hybridizing from said capture probe. For accuracy and the sensitivity that improves detection, can in reaction system, add more than one detector probe. These detector probe have different nucleotide sequences separately, and are complementary to respectively the different sections of target nucleotide. In general, corresponding to a target nucleotide sequences less than 100bp, but the terminal biotinylated detector probe of choice for use 2-3 the about 15bp of length for example. Using in the situation of an above detector probe, the signal group of the different fluorescent material marks that can use emission different colours light and different detector probe affinity combinations is beneficial to improve the specificity of detection.
According to a preferred embodiment of the invention, wherein said signal group is the group that can produce chemoluminescence or electrochemiluminescence under the condition of said target and said probe hybridization.The particularly preferred signal group of the present invention is method and one or more groups fluorescent substance of polymer micropellet bonded with physics or chemistry, and said polymer micropellet also to be connected with dual specific affine in conjunction with right member-avidin.
Said microparticle generally is meant diameter between 1nm-100nm, between the 20nm-20 micron, is preferably in the nano particle between the 20nm-5 micron more fortunately.
The same with the aforementioned bearer particle, be used for nano particle of the present invention and preferably also make by polymer materialss such as polystyrene or polyacrylic acid.The linking agent such as the divinylbenzene that can contain appropriate amount (as 1-3%) in the particle.Particle can contain and help and joint arm or the direct additional surface functional group that is connected with intercalator, for example groups such as carboxylic acid, ester, alcohol, aldehyde, amine.In the practice, can in polymkeric substance, mix the material that contains carboxyl such as acrylate or methacrylate in particle, adding these groups, and utilize these groups of polymer surfaces to connect the compound that contains reactive amine or sulfydryl.
Being used for fluorescent substance of the present invention is the time resolved fluorescence europium complex that wavelength of transmitted light is about 614nm.But under any specified conditions, various materials that can be luminous all can use.Be applicable to that dyestuff of the present invention for example comprises but is not only limited to the blue or green dyestuff of europium complex, phthalein, croconic acid derivative, cyclobutenedione derivatives etc.Can select suitable luminophore according to the light emission of fluorescence and absorbent properties and hydrophobic property.Can be as required, use following three kinds of different currently known methodss to prepare fluorescent grain: (1) is covalently bound to luminophore on the particulate surface; (2) luminophore is mixed in inside during polymer polymerizing forms particle; (3) with diffusion process behind the aggregation of particles and dye particle (respectively referring to United States Patent (USP) 4,774,189,4,717,655 and 5,723, No. 218).For example, can in round-bottomed flask, stir 1% solution of nano particle, then to wherein adding the dyestuff that is dissolved in organic solvent.When particles no longer absorbing dye solution, stop to add dyestuff and removal of solvent under reduced pressure.
Can use chemical process well known by persons skilled in the art the avidin molecule directly to be coupled on the fluorescent grain that as above makes, perhaps be connected on the fluorescent grain that has reactive group (for example carboxyl) equally by a joint arm and by its terminal-reactive group.
According to a preferred embodiment of the inventive method, wherein said joint arm is that two ends can covalently bound embedding molecule and the straight-chain paraffin with 1-8 carbon atom and its derivative of signal group, as alcohol, aldehyde, acid, ester and amine etc.Be purpose of the present invention, particularly preferred linkers is a glutaraldehyde.
Basic skills of the present invention comprises three reactions steps in (2)-(4) as indicated above: promptly at first make the hybridization of immobilised capture probe and nucleic acid target; Be the hybridization of detection probes and nucleic acid target then; Be the vitamin H of detection probes connection and the association reaction between signal group bonded avidin at last.Yet for convenience's sake, also can finish detection in the practice by two step methods based on ultimate principle of the present invention.Promptly after fixing capture probe, in reaction system, add target nucleic acid and the terminal detection probes that is connected with vitamin H simultaneously, and then add the labelling groups such as the fluorescence dye that are connected with avidin and the Nano microsphere that dyes.Perhaps, more easy is, can also finish detection based on ultimate principle of the present invention by a step method.Promptly after fixing capture probe, labelling groups such as the fluorescence dye that adding sample target nucleic acid, the detection probes of vitamin H connection are connected with avidin in reaction the Nano microsphere that dyes simultaneously, the color (wavelength) and the intensity of the fluorescence that sends after being stimulated according to microballoon are then determined the existence and the relative quantity of tested target nucleic acid.
Another object of the present invention provides and is used for the test kit that the test sample target nucleotide exists, said test kit comprise place one or more containers (1) can with the strand Nucleotide capture probe of target nucleotide hybridization, (2) but target nucleotide connects the one or more biotinylated strand Nucleotide detection probes of the different sections hybridization of chain; (3) by joint arm or the direct detection moiety that is connected with avidin.
Test kit preferred embodiment according to the present invention, wherein said joint arm are the straight-chain paraffin or derivatives thereofs with 1-8 carbon atom of covalently bound respectively avidin in two ends and signal group.
Test kit preferred embodiment according to the present invention, wherein said signal group is the fluorescence dye that is carried on the nano particle.
In embodiments of the invention 1, employed signal group is direct and avidin link coupled time resolved fluorescence nano particle.
Generate and present element owing to improved the signal in the nucleic acid detection system, promptly utilized respectively and combined right-biotin/avidin with detection probes and signal group bonded dual specific, thereby the signal of having realized system effectively amplifies, in addition because the use of above-mentioned integrated signal group can make strength of signal improve thousands of times even more completely.In addition, according to method of the present invention, owing to can suitably increase the number of the detection probes of the complementary sequence that comprises the different sections of target nucleic acid, thus improved the susceptibility and the detection efficiency of detection method significantly.Moreover, can three step method of the present invention be merged into two steps or a step method in conjunction with particular case in the practice, thereby can further simplify trace routine, improve detection speed and efficient.
Method of the present invention is particularly suitable for the existence of lower concentration target nucleic acid in the test sample and possible base mispairing thereof, thereby is identified pathogenic micro-organism and various heredopathia wild or variation, and the clinical reliable diagnostic foundation that provides is provided.Because having improved signal in the detection system takes place and presents element, it is the fluorescent radiation after intercalator embeds of being no longer dependent on of hybridization signal, and no longer only rely on a few molecules as the signal generation group in the detection method, but replace the integrated signal group that its intensity exceeds hundreds if not thousands of times.So method of the present invention has not only kept its inherent specificity, and the susceptibility and the detection efficiency that detect have been significantly improved.In addition, because the highly sensitive of the inventive method, thus the pcr amplification that needn't as conventional method, waste time and energy as a rule, promptly can highly sensitive, low cost and high-throughput (for example on chip) finish detection.
The following example is intended to further set forth for example the present invention, but these embodiment and constitute the await the reply restriction of claim to the present invention never in any form.
Embodiment
Embodiment 1:
Present embodiment is an example with the sample to be checked that contains hepatitis B virus (HBV), describes the high detection efficient and the susceptibility of the inventive method for example.
(1) preparation of single strand oligonucleotide probes (capture probe and detection probes) and fixing
Have capture probe P1:5 '-AGA CCA CCA AAT GCC CCT ATC-3 ' and the P2:GTATCT TTT GGA ATG TGG ATT CGC ACT CCT CCC GCT TAC AGACCA CCA AAT GCC CCT ATC TTA TCA ACA CTT CCG GAA ACTACT ATT GTT AGA CGA CGA GGC that shows sequence down based on the known nucleotide sequences Design of hepatitis B virus is also synthetic, and detection probes 5 '-vitamin H-TCCCCT AGA AGA AGA ACT C-3 '.
Nitrocellulose filter is soaked and be clipped in 37 ℃ of oven dry between two glass sheets with 2 * SSC solution.Then, can on the exsiccant filter membrane, drip the solution that contains proper concn (66 μ g/ml) capture probe P1 and P2.Behind the application of sample, with filter membrane clip between two glass sheets 80 ℃ the baking about 2 hours standby.
(2) sample to be checked gives processing
Get the serum sample 0.5ml of clinical patient, to wherein adding equal-volume sample extracting solution (1%TrionX-100,1%NP40,10mM Tris-HCl PH8.0), 96 ℃ of water-baths 10 minutes, 12000rpm centrifuging and taking supernatant is tested.
In the experiment, be experimental group with the hepatitis B human serum sample who has made a definite diagnosis; To pass through pcr amplification and the same probe solution of handling as positive controls; To pass through the same normal human serum sample of handling as negative control group.
(3) prehybridization and hybridization
Then, with 0.5ml and the sample handled and 1ml hybridization solution (by 2ml dimethyl formamide, 1ml 20 * SSC damping fluid, 0.4ml 50 * Denhardts solution, 0.2ml1M PBS, pH 6.4 forms) behind the thorough mixing, get 0.1ml and drip 37 ℃ of prehybridizations on the above-mentioned nitrocellulose filter and under air-proof condition 30 minutes.After prehybridization is finished, with the solution that contains 2 * SSC and 0.1%SDS with the washing 3 times down of 37 ℃ of above-mentioned nitrocellulose filters, and then with the solution washing that contains 0.1 * SSC and 0.1%SDS three times.
After the washing, behind 0.5ml (1 μ g/ml) detection probes and the above-mentioned hybridization solution thorough mixing of 1ml, get 0.1ml and drip 37 ℃ of hybridization on the above-mentioned nitrocellulose filter and under air-proof condition 30 minutes.After hybridization is finished, with the solution that contains 2 * SSC and 0.1%SDS with the washing 3 times down of 37 ℃ of above-mentioned nitrocellulose filters, and then with the solution washing that contains 0.1 * SSC and 0.1%SDS three times.After the washing, use the Tris-HCL that contains 2%BSA, the lock solution of pH 7.5 is sealed filter membrane 30 minutes down for 37 ℃.After the sealing, wash (each 5 minutes) again three times with above-mentioned same washing soln.
(4) compatible reaction
With the filter membrane of washing air-dry after, add avidin connection bonded time resolved fluorescence europium nano particle at suitable application of sample location point.Behind the concussion mixing, lucifuge was reacted about 15 minutes under room temperature.After reaction is finished, with washing soln A and B the filter membrane washing is respectively washed 3 times respectively.
(6) detection of time resolved fluorescence intensity and result thereof
After the washing, differentiate the fluorescence readout instrument duration of service sample is detected (exciting light 340nm, emission light 614nm).The following tabulation 1 of result and shown in Figure 2.
The nucleic acid detection method that table 1. uses signal of the present invention to amplify detects the result of HBV blood sample
| Time resolved fluorescence intensity | Blank | Negative sample | Positive sample 1 | Positive sample 2 |
| Long probe P2 | 420 | 529 | 8649 | 794734 |
| Short probe P1 | 431 | 608 | 9854 | 1003261 |
| PCR result | 0 copy/ml | 0 copy/ml | 105.4 copy/ml | 107.9 copy/ml |
From the result shown in the table 1 as can be seen, when the nucleic acid detection method that uses signal of the present invention to amplify detects pathogenic agent (hepatitis B virus) nucleic acid, needn't carry out under the condition of loaded down with trivial details pcr amplification, can high specific and hypersensitivity ground acquisition expection detected result.Compare with currently known methods of the prior art, method of the present invention improves detection speed and efficient greatly, is particularly useful for high risk population's use on a large scale.
Sequence table
＜110〉Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd.
＜120〉the target nucleic acid detection method of the integrated signal group of the affine amplification of use
<140>
<141>
<160>2
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
AGACCACCAA ATGCCCCTAT C
<210>2
<211>100
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
GTATCTTTTG GAATGTGGAT TCGCACTCCT CCCGCTTACA GACCACCAAA
TGCCCCTATC TTATCAACAC TTCCGGAATA CTATTGTTAG ACGACGAGGC
Claims (10)
1, the method that target nucleic acid exists in a kind of test sample, this method may further comprise the steps:
(1) with chemistry or physics method capture probe is fixed on the solid phase carrier;
(2) add target nucleic acid sample and make it to contact and hybridize, then flush away free capture probe and other nucleotide sequences of not hybridizing with capture probe with fixed capture probe;
(3) under hybridization conditions, add terminal affinity groupsization and contain the detection probes of the complementary nucleic acid sequence of other the different sections beyond the target nucleic acid section with the hybridization of said capture probe;
(4) be suitable under the affine bonded condition, in said mixture, adding by joint arm or the direct integrated signal group that is connected with affinity groups;
(5) detect the intensity of the signal that said signal group launches, so as to the existence and the amount thereof of target nucleic acid in the specific hybrid of judging said target nucleic acid and said detection probes and the definite sample.
2, according to the process of claim 1 wherein that said step (2) and (3) can carry out simultaneously, promptly said target nucleic acid sample can add simultaneously with said detection probes.
3, according to the process of claim 1 wherein that said step (2), (3) and (4) can carry out simultaneously, promptly said target nucleic acid sample can add simultaneously with said detection probes and said signal group.
4, according to the process of claim 1 wherein that the target nucleic acid complementary sequence of said one or more detection probes and the target nucleic acid complementary sequence of said capture probe are non-overlapping copies.
5, according to the process of claim 1 wherein that the target nucleic acid complementary sequence of said a plurality of detection probes is non-overlapping copies each other.
6, according to the process of claim 1 wherein that said signal group is to produce detectable chemoluminescence or the luminous integrated group of electricity under the condition of said target nucleic acid and said detection probe.
7, according to the process of claim 1 wherein that said signal group is can be under the condition of said target nucleic acid and said detection probe, by affinity groups between in conjunction with producing detectable signal group.
8, according to the process of claim 1 wherein that said signal group is to be carried on detectable signal group on the microparticle.
9, according to the process of claim 1 wherein that said joint arm is the straight-chain paraffin with 1-20 carbon atom or its alcohol, aldehyde, acid, ester and the amine of covalently bound respectively affinity groups in two ends and integrated signal group.
10, be used for the test kit that the test sample target nucleotide exists, said test kit comprise place one or more containers (1) can with the single-chain nucleic acid capture probe of target nucleic acid hybridization, (2) can with the one or more biotinylated single-chain nucleic acid detection probes of the different sections hybridization of target nucleic acid chain; (3) but by the joint arm or the detection moiety that directly is connected with affinity groups.
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Citations (1)
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| CA1271705A (en) * | 1985-01-02 | 1990-07-17 | Hans Soderlund | Method for the identification of nucleic acids |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA1271705A (en) * | 1985-01-02 | 1990-07-17 | Hans Soderlund | Method for the identification of nucleic acids |
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