CN1460722A - Nucleic acid amplification detection method - Google Patents
Nucleic acid amplification detection method Download PDFInfo
- Publication number
- CN1460722A CN1460722A CN 03123596 CN03123596A CN1460722A CN 1460722 A CN1460722 A CN 1460722A CN 03123596 CN03123596 CN 03123596 CN 03123596 A CN03123596 A CN 03123596A CN 1460722 A CN1460722 A CN 1460722A
- Authority
- CN
- China
- Prior art keywords
- probe
- nucleic acid
- sequence
- nucleotide
- target nucleic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The nucleic acid amplification detection method includes: adding at least one line probe and at least one ring probe into the detection sample, the mole concentration ratio of both them is 1:100 to 100:1 in which the line probe at least contains (1) 5' end and target nucleic acid matched sequence; (2) 3' end and ring probe matched sequence, the ring probe at least contains (1), sequence matched with target nucleic acid and (2), sequence matched with 3' end of line probe; and (3) filling sequence for regulating total length of ring probe. The invention can implement several operations of nucleic acid hybridization, amplification and detection in same reaction tube, by only using one kind of DNA polymerase and by means of one-step constant temp. reaction. Said invention is applicable to detection of RNA and DNA.
Description
Technical field
The present invention relates to the detection of nucleic acids field, more particularly, relate to and a kind ofly come target nucleic acid in the specific detection sample whether to exist and determine the method for its concentration by nucleic acid hybridization and constant-temperature amplification external.
Background of invention
A problem that often runs in medical science and field of biology is to confirm whether exist with the corresponding target nucleic acid of certain disease pathogen and measure the concentration of this target nucleic acid at certain sample (for example be used for the blood sample of transfusing blood or from the blood of patient's withdraw or humoral sample etc.).But, can't directly use Instrument measuring when the concentration of this target nucleic acid is lower than when delimiting or mixing coexistence with the nucleic acid of other multiple different sources.A common method that solves this class problem at present is the specific nucleic acid amplification, and the amplification object can be the sequence of target nucleic acid itself or the sequence of discerning the specific probe of this target nucleic acid.By detecting amplification back nucleic acid product and concentration, can the indirect judgement sample in this target nucleic acid whether exist and original concentration.
The method of specific nucleic acid amplification has some kinds, can whether the needs temperature cycle is divided into two classes according to it.One class needs temperature cycle, as polymerase chain reaction,PCR (Polymerase chain reaction, PCR), ligase chain reaction (Ligase chain reaction, LCR); Another kind ofly do not need temperature cycle and under constant temperature, increase, dubbing system (the transcription-based amplification system that transcribes as dependence, TAS), nucleotide sequence self-replicating system (self-sustained sequence replication, 3SR), Q β replicative enzyme system etc.Though these methods can both be carried out nucleic acid the high power amplification, but on detecting, actual clinical all has separately shortcoming and limitation, poor such as quantitative property, multiplicity (multiplicity) is low, the product crossed contamination easily takes place, only be applicable to and detect a kind of among DNA or the RNA, or needs plurality of enzymes, multistep operation etc.; And the temperature cycle method is compared a distinct disadvantage and is need to use special, expensive temperature cycle instrument with constant temperature method.Above-mentioned a variety of causes has influenced application and the popularization clinically of these methods.So, how to find a kind of method that can overcome external nucleic acid amplification above-mentioned defective, even more ideal and detection just to become the problem that presses for solution.
Have at present a kind of new, rising nucleic acid amplification method be called the rolling circle amplification method (Rolling-circle amplification, RCA).Rolling circle amplification be a kind of under constant temperature, utilize archaeal dna polymerase and strand ring-type dna profiling, the DNA cloning mode that will successively extend with linear mode with the primer of circular template hybridization.This amplification linear rolling circle amplification (LRCA) that is otherwise known as, its amplified production are extremely long single stranded DNA (Fire and Xu, 1995, PNAS Proc. Natl. Acad. Sci.USA 92:4641-45 who is connected into by circular template complementary sequence copy; The periodical 118:1587-94 of Liu et al.1996 J.Am.Chem.Soc. american chemical society).Exist as two kinds of primers, wherein a kind of primer and the complementation of circular template the preceding paragraph sequence, when another fragment sequence of second kind of primer and circular template is identical, can cause the substitution reaction of the two-way multiple branch-like chain of spontaneous, successive, be called using hyper-branched rolling circle amplification (Hyperbranched Rolling-circle amplifoication, HRCA).HRCA is the continuous DNA amplification of index (progression) mode, can increase 10 in more than one hour
9-12Doubly, amplification rate surpasses polymerase chain reaction,PCR (PCR).Final amplified production is that length is that unit is the double-stranded DNA (Lizardi et al.1998 Nature Genet. natural genetics magazine 19:225-32) that integral multiple the increases with the circular template.
Because that RCA reaction has is simple, sensitive, quantitatively, advantage such as high, the no product crossed contamination of multiplicity, thereby extremely rising in the Molecular Detection field.Yet at this stage, the application of RCA in external detection of nucleic acids is also more limited, be mainly used in that signals in situ is amplified and use the open loop probe survey the mononucleotide diversity (Single-nucleotide polymorphism, SNP).When being used for the signals in situ amplification, general formation with an end of a linear nucleus acid probe and attached to the target nucleic acid on the solid phase carrier earlier stablized hybridization, and the probe flush away of will not hybridize then adds the cyclic DNA template, form stable hybridization with the other end of wire probe, cause linear rolling circle amplification; Single stranded DNA product after the amplification again with the probe hybridization that has mark, detect amplified signal directly or indirectly.The weak point of this method is: 1) only use the probe of an identification target nucleic acid, thereby specificity is not high; 2) need the multistep operation, more loaded down with trivial details; 3) washing thoroughly can not cause higher background.
This shows, need defective novel more, that perfect design overcomes existing method, give full play to the advantage of rolling circle amplification principle, enlarge its range of application in detection of nucleic acids.
Summary of the invention
The objective of the invention is problem at above-mentioned existence, provide a kind of use a kind of enzyme, in same reaction tubes, need only be by isothermal reaction can make the target nucleic acid (comprising DNA and RNA) in the sample solution promptly increase once the step, thereby can in very short time, utilize instrument more accurately to measure the method for the concentration of this target nucleic acid.
Another object of the present invention provides a dedicated kit that is used to realize aforesaid method.
The inventor furthers investigate to achieve these goals, found that, on the basis of above-mentioned existing rolling circle amplification technology, hybridize with target nucleic acid fragment and the concentration ratio of two probes is limited in certain scope only using the step that increases again after the hybridization of a kind of probe and target nucleic acid, the flushing to change into to use simultaneously a kind of wire probe and a kind of cycling probe, limit the matched sequence that the two has certain-length each other simultaneously, can achieve the above object, so far just finish the present invention.That is to say that technical scheme of the present invention is as follows:
1. nucleic acid amplification detection method, this method comprises: add nucleic acid probe and other known necessary ancillary components in detected sample, under design temperature, carry out amplified reaction, use instrument detecting amplified signal and quantitative analysis target nucleic acid in reaction is carried out or after finishing, whether there is target nucleic acid and determines its concentration in the judgement sample as a result according to one's analysis, it is characterized in that
Above-mentioned nucleic acid probe is the probe groups that comprises a kind of wire probe and a kind of cycling probe at least, and wherein, the molar concentration rate of wire probe and cycling probe is in 1: 100 to 100: 1 scope; Wherein,
The wire probe comprises following two parts at least: (1) 5 ' end and target nucleic acid paired sequence, and its length is in the scope of 15-35 Nucleotide; (2) 3 ' ends and the short sequence of cycling probe paired, its length is in the scope of 2-10 Nucleotide; And the interval between above-mentioned (1) and (2) two portions is in the scope of 0-5 Nucleotide.
The total length of cycling probe wherein comprises following 3 parts at least in the scope of 35-200 Nucleotide: (1) and target nucleic acid paired sequence, and its length is in the scope of 15-35 Nucleotide; (2) with wire probe 3 ' end paired sequence, its length is in the scope of 2-10 Nucleotide; (3) be used to regulate the padding sequence of cycling probe total length; And the interval between above-mentioned (1) and (2) two portions is in the scope of 0-5 Nucleotide.
2. as technical scheme 1 described method, it is characterized in that the molar concentration rate of wherein said wire probe and cycling probe is 1: 10-10: in 1 the scope.
3. as technical scheme 1 described method, it is characterized in that the length of wherein said wire probe 5 ' end and target nucleic acid paired sequence is in the scope of 18-32 Nucleotide.
4. as technical scheme 1 described method, it is characterized in that, in the wherein said cycling probe with the length of target nucleic acid paired sequence in the scope of 18-32 Nucleotide.
5. as technical scheme 1 described method, it is characterized in that wherein said wire probe can be any in the following three state: (a) free state; (b) be attached on the carrier; (c) part is free state, and part is attached on the carrier; Nucleotide or other composition that also can comprise any modification on the composition.
6. as technical scheme 1 described method, it is characterized in that also comprise at least a wire amplimer in the wherein said probe groups, its length is in the scope of 15-35 Nucleotide; A fragment is identical in all or part of and cycling probe padding sequence of its sequence.
7. as technical scheme 1 described method, it is characterized in that, comprise DNA product detection reagent in the wherein said amplified reaction composition.
8. as technical scheme 1 described method, it is characterized in that wherein said method with instrument detecting nucleic acid amplification signal can be to detect any method of strand or double-stranded DNA use.
9. as each the described method among the technical scheme 1-8, it is characterized in that whole nucleic acid amplification testing process is carried out in same reaction vessel.
10. dedicated kit is used for each described method of technical application scheme 1-9, it is characterized in that, wherein is equipped with to be used for target nucleic acid kind and the required reagent of relative populations that test sample may exist.
Describe the present invention below.
The mechanism of action of method proposed by the invention is roughly as follows: after wire probe and cycling probe and target nucleic acid hybridization, it is template with the cycling probe that 3 ' end of wire probe is made primer, carries out linear rolling circle amplification under the catalysis of DNA polymerase; It is template with the single stranded DNA product hybridization of rolling circle amplification generation and with it constantly that chain in the reaction soln replaces amplimer, carries out the secondary amplification in the mode of chain substitution reaction, produces the single stranded DNA of different lengths continuously; Wire probe hybridization and synthetic final double-stranded DNA product freely in 3 ' end of these secondary single stranded DNA products and the solution; In this process, the wire probe and the cycling probe of the target nucleic acid one group of hybridization in front constantly break away from, and with new one group of wire probe and cycling probe hybridization, cause new round amplification again, and constantly circulation like this is till raw material comes to the greatest extent (seeing Fig. 1-3).
The present invention has designed a kind of novel probe combination of being made up of a wire probe and a cycling probe (Fig. 1).In the combination each part of wire probe and cycling probe can be respectively with target nucleic acid on two adjacent (continuously every) or the pairing of close (one to several Nucleotide at interval) fragment.Each pairing segmentally is generally 15-35 Nucleotide than suitable length, and length preferably is 18-32 Nucleotide.Concrete length is decided according to the composition of amplified reaction temperature and oligonucleotide, generally matches segmental solvent temperature (Tm) than the high 3-6 of amplified reaction temperature degree centigrade.3 ' end short-movie Duan Buyu target nucleic acid pairing of wire probe; Just when wire probe and cycling probe simultaneously with target nucleic acid on the hybridization of correct position after, this fragment just may be matched (see figure 1) with contiguous and corresponding short-movie section on the cycling probe.The segmental suitable length of matching is a 2-10 Nucleotide, concrete length is decided because of this segmental Nucleotide composition, selection principle is: when no target nucleic acid existed, this fragment was not enough to cause rolling cycle replication with cycling probe hybridization under same reaction conditions and temperature.Between two portions of wire probe generally continuously every, but also can be at interval one to several nucleic acids or fill with other composition, do the specificity that perhaps helps to improve reaction in some cases like this.5 ' end of wire probe does not have particular restriction, can add other sequence and modification group, can be connected with surface group with various carriers yet.
Cycling probe in the probe groups roughly is made up of three parts: a fragment pairing on first part and the target nucleic acid.As mentioned above, this fragment is generally 15-35 Nucleotide than suitable length, length preferably is 18-32 Nucleotide, and concrete length is decided according to the composition of amplified reaction temperature and oligonucleotide, generally matches segmental solvent temperature (Tm) than the high 3-6 of amplified reaction temperature degree centigrade; Second section contains and can hold paired short-movie section mutually with 3 ' of wire probe, and pairing short-movie section is a 2-10 Nucleotide than suitable length, and this part is generally in the upstream of first part (5 ' end) and near first part; Remaining third part can have multiple motor-driven function, and for example: 1) this part can comprise and one or more identical sequences of wire primer that are used for the secondary amplification; 2) this part can include one section sequence, its corresponding part in duplicating product can with the probe hybridization that has mark, as a kind of method that detects amplified production; 3) this part can comprise the sequence that is used as other any purposes, as can be used as filling, regulates the total length of cycling probe etc., and usually, the suitable total length of cycling probe is in the scope of 35-200 Nucleotide.Too shortly be unfavorable for that the rolling circle amplification reaction carries out; The oversize difficulty and the cost that can increase the cycling probe making.
Above-mentioned probe groups can also comprise the wire primer of one or more synthetic.The effect of wire primer is to be template with the hybridization of the matched sequence on the one-level amplified production and with it, carries out the secondary amplification in the mode of chain substitution reaction.The wire primer be generally 15-35 Nucleotide than suitable length, length preferably is 18-32 Nucleotide, concrete length is decided according to the composition of amplified reaction temperature and oligonucleotide, generally matches segmental solvent temperature (Tm) than the high 3-6 of amplified reaction temperature degree centigrade.
The main difference that primer that uses among probe combinations provided by the present invention and the common RCA and circular template are right is: 1) wire probe among the present invention and cycling probe all will be hybridized with target nucleic acid; And among the common RCA, only duplicating one of primer and circular template with target nucleic acid hybridization, primer and circular template are simultaneously with target nucleic acid hybridization; 2) 3 ' of the wire probe among the present invention end is shorter with cycling probe paired sequence length, generally is no more than 10 Nucleotide, does not form stable hybridization when no target nucleic acid exists mutually; Usually the segmental length of using among the RCA of pairing of duplicating on primer and the circular template generally is longer than 16 Nucleotide, is not shorter than 10 Nucleotide at least.Just can form stable hybridization in advance between the two, need not the help of target nucleic acid.
The stability of the nucleic acid hybridization that relates among the present invention can be calculated by the method for delivering in the document, as: Lesnick and Freier, 1995, Biochemistry biological chemistry 34:10807-10815; McGraw et al., 1990, Biotechniques biotechnology 8:674-678; Rychliket al., 1990 Nucleic Acids Res. nucleic acids research 18:6409-6412.
Owing to used the novel probe combination, the reaction mechanism of the inventive method reacts different (seeing Fig. 2,3) with reaction kinetics with HRCA.Difference on the reaction mechanism has: 1) in HRCA reaction, and the copy hybridization of each circular template and cause the substitution reaction of another level chain on the wire probe single stranded DNA product that can produce with the chain substitution reaction freely; In the present invention reaction, 3 ' end of the complete single stranded DNA product that the wire probe only produces with the chain substitution reaction, just self copy of wire probe forms the final product of stable hybridization and synthetic dsdna form; 2) in the HRCA reaction, each target nucleic acid molecule generally only causes rolling circle amplification one time; In the present invention's reaction, each target nucleic acid molecule circulation is constantly reacted, is broken away from then hybridization, initiation rolling circle amplification with probe, again with new a pair of probe hybridization (Fig. 3).Can infer that on reaction mechanism this method amplified reaction kinetics will relax than HRCA, can avoid too sensitive defective of HRCA.In addition, target nucleic acid concentration and fluorescence reach the used time of certain intensity and are and can estimate relation, thereby this method is very potential develops into the method that a kind of quantitative nucleic acid detects.
Wire probe among the present invention and wire primer can be the mixed state of one of following two states or two states: 1) free state refers to that wire probe and wire primer are dissolved in the solution, and can have any known mark or modification; 2) be fixed on the carrier, an end (majority refers to 5 ' end parts) that refers to wire probe or wire primer is before reaction or the reaction back is directly or indirectly fixing or attached on the carrier.Suitable set form is that an end (majority refers to 5 ' end parts) of probe or wire primer is fixing, and the other end (referring generally to 3 ' end) freely stretches.Carrier can be made by any material, through any surface treatment, is Any shape, as microballon, microparticle, micropore, chain polymer, plane (chip surface) etc.
Wire probe used among the present invention has different characteristics and purposes when being different states.When locating free state, the hit probability height of nucleic acid hybridization of probe and solution, thereby the speed of surveying is fast, highly sensitive.Probe stationary on carrier, can be caught the target nucleic acid with its hybridization and be separated, and then increase from solution.Can avoid in the sample some composition to the inhibition of amplified reaction like this.Simultaneously, the reaction product of rolling circle amplification can be by being fixed on the carrier probe or primer extension and is produced, by anchored in place on carrier.Utilize this principle multiple different wire probe or primer can be fixed on the carrier a plurality of target nucleic acids of parallel detection.On the DNA chip, use the mixed state of free probe and stationary probe to help accelerating amplified reaction speed and increase the sensitivity that detects.
The target nucleic acid that uses the inventive method to detect comprises RNA and DNA, chain or ring-type, strand or two strands, freely or be fixed on the carrier.When target nucleic acid and probe groups hybridization portion are two strands, generally to heat or add other agent of unwinding earlier and two strands be separated be strand, annealing allows probe follow target nucleic acid hybridization then.
The DNA polymerase that uses in the inventive method can have multiple choices, and DNA polymerase that in principle can the catalytic dna rolling-circle replication all can.These enzymes generally have following characteristic: the activity of 1) unwinding; 2) be difficult for coming off from template DNA; 3) there is not 5 ' to 3 ' DNA 5 prime excision enzyme activity.Some are applicable to that enzyme of the present invention, that meet above-mentioned condition has: the sudden change of the Klenow fragment of DNA polymerase I, phi-29 DNA polymerase, phage M2 DNA polymerase, phage PRD1 DNA polymerase, BST large fragment DNA polymerase, Vent DNA polymerase, T5 DNA polymerase, T4 DNA polymerase and some enzyme or transformation body etc.More suitable enzyme comprises: phi-29 DNA polymerase, Vent DNA polymerase and BST DNA polymerase etc.Wherein Vent DNA polymerase and BST DNA polymerase are thermophilic type DNA polymerase, can allow be reflected under the comparatively high temps and carry out, and help improving the specificity of detection.
In addition, in reaction, add the DNA factor of untwisting and help to unwind, as dna helicase and single stranded DNA attachment protein etc.
The method that detects amplified signal has multiple, and the method that is used for detecting strand or double-stranded DNA can be used substantially.For example, but be not limited to:
1) in amplified reaction, uses the Nucleotide or the nucleotide analog of tape label, marker is integrated directly in the amplified production goes.Mark can be fluorescent mark, haptens or radio-labeling etc.Wherein more suitable fluorescent mark has: Cyanine Dyes (Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Fluorescein (FITC), Rhodamine, Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin and dansyl chloride etc.; More suitable haptens has: vitamin H (biotin), digoxin (digoxigenin) etc.; More suitable radio-labeling has:
32P,
33P,
35S,
125I etc.; The more suitable nucleotide analog that has snoop tag has BrdUrd and BrUTP etc.
2) use has specific fluorescence dye to strand or double-stranded DNA, and a denominator of these fluorescence dyes is obviously to strengthen with the chimeric back of nucleic acid luminous intensity.The fluorescence dye of more suitable detection single stranded DNA product has SYBR Green II etc.; The fluorescence dye of more suitable detection double-stranded DNA product has SYBR Green I etc.
3) use nucleic acid probe and the hybridization of single stranded DNA product that is associated with mark or signal generation material, the signal that certification mark produces.Mark can be but be not limited to fluorophor, pigment group, chemiluminescent groups, haptens, antibody or enzyme labelling etc.Enzyme labelling commonly used has alkaline phosphatase and horseradish peroxidase etc.
4) use molecular beacon (Molecular Beacon), glimmering mark (Amplifluor) and other various fluorescent quenching technology or energy transfer technique designed probe utilized increase, as FRET (fluorescence resonance energy transfer) (fluorescence resonance energy transfer, FRET), the back stagnate fluorescence energy transfer (Delayed Fluorescence Energy Transfer, DEFRET) etc.
Be applicable to that the detection reagent that signal detection and amplified reaction carry out synchronously has, but be not limited to: molecular beacon, the glimmering mark of amplification and chimeric fluorescence dye (as SYBR Green) etc.Other detection method can be selected as required and used by the personnel with general expertise and technical ability, as electrophoretic method etc.
The scope of the invention has also comprised form various biological detection reagent kits and the detection chip of designing according to above-mentioned principle different with purposes.These test kits have all used the foregoing probe combinations of this specification sheets at least directly or indirectly, generally also comprise isocyatic 4 kinds of thymus nucleic acids (dATP, dTTP, dCTP, dGTP), pH buffer composition, the various ions of proper concn, detection of nucleic acids reagent in the reaction mixture, can also comprise suitable method for extracting nucleic acid, concrete collocation can be selected as required by the user.
Compare with existing external nucleic acid detection method, method provided by the invention has shown following superiority and beneficial effect:
The present invention has realized under constant temperature, in same reaction tubes, only uses a kind of archaeal dna polymerase, finishes multi-mode operations such as nucleic acid hybridization, amplification and signal detection by single step reaction.All processes can be finished in about one hour, did not have the product crossed contamination, was equally applicable to detect RNA and DNA.
The inventive method can be with DNA amount amplification 10 in about one hour
10Doubly, detection sensitivity reaches single copy level.On reaction kinetics, relax, more be applicable to detection by quantitative than HRCA.
Provided by the invention pair of probe combinations all has higher specificity than methods such as PCR and RCA on principle and actual effect.Reason is that the probe combinations that the present invention uses not only uses two probes, and the relative position that two probes are hybridized on target nucleic acid had strict control, promptly two probes must be hybridized and just can caused amplification on two adjacent or very close positions on the target nucleic acid.When two probes or one of them during, can can't increase because distance is improper once in a while with target nucleic acid generation non-specific hybridization.By contrast, RCA has only a probe and target nucleic acid hybridization, so the probability of non-specific hybridization is big.Washing thoroughly can not cause false positive yet in addition.Some other uses the RCA form of dna ligase can occur false positive because the non-spontaneity that depends on target nucleic acid connects.Though PCR uses two primers, when any one and target nucleic acid non-specific hybridization in two primers, amplified reaction just might take place.The accuracy rate of the inventive method actual detected can near and reach 100%.
Whether the present invention can be used to detect has specified nucleotide sequence existence and measures its concentration quantitatively in the various biological samples, can be used for polytype rapid gene and detect.Whether for example: detecting has various viruses to exist and concentration in serum or other biological sample; That determines fast bacterium, fungi or other infected by microbes or pollution dyes source etc.Of many uses in fields such as health care, quarantine, food, environmental protection and scientific researches.
Description of drawings
The synoptic diagram of two adjacent fragments hybridization on Fig. 1 probe combinations that to be an example be made up of a wire probe and a cycling probe and the target nucleic acid.Wherein the wire probe is made of 2 parts at least: 3 ' end is 2-10 Nucleotide with cycling probe mating section (1) than suitable length; 5 ' end is 15-35 Nucleotide with target nucleic acid matched sequence (2) than suitable length, and length preferably is 18-32 Nucleotide.Cycling probe is made up of three parts: with target nucleic acid mating section (3), be 15-35 Nucleotide than suitable length, length preferably is 18-32 Nucleotide; With 3 ' end matched sequence (4) of wire probe, be 2-10 Nucleotide than suitable length; Remaining multi-functional part (5) for example can comprise with one or more secondary chains that are used for and replace the identical sequence of amplimer.
Fig. 2 shown when a pair of probe respectively with target nucleic acid on after the hybridization of two adjacent regions, 3 ' end of wire probe utilizes cycling probe to carry out rolling cycle replication for template DNA is polymerization catalyzed under, generation single stranded DNA product.Cycling probe breaks away from target nucleic acid under the archaeal dna polymerase effect; Single stranded DNA product that rolling-circle replication produces and secondary chain replace amplimer hybridization.
Fig. 3 is a routine reaction process synoptic diagram, shown that it is template with the single stranded DNA product hybridization of rolling circle amplification generation and with it constantly that the chain in the reaction soln replaces amplimer, carry out the secondary amplification in the mode of chain substitution reaction, produce the single stranded DNA of different lengths continuously; Wire probe hybridization and synthetic final double-stranded DNA product (process C, D) freely in 3 ' end of these secondary single stranded DNA products and the solution; Simultaneously, the wire probe and the cycling probe of the target nucleic acid one group of hybridization in front constantly break away from, and with new one group of wire probe and cycling probe hybridization, cause new round amplification (process A, B) again; Said process continuously repeats, and stops until reaction terminating or because of raw material exhausts.
Fig. 4 has shown that the single stranded DNA that rolling cycle replication produces can hybridize with label probe, thereby quantitatively indicates target nucleic acid concentration.
Fig. 5 is embodiment 1 result.1) for the negative control of no target nucleic acid; 2) be the relative intensity of fluorescence of laboratory sample.
Fig. 6 has shown the 1%DNA electrophoretogram of reaction result among the embodiment 2.1) negative control of no target nucleic acid; 2) laboratory sample; M) dna molecular amount standard.
Fig. 7 is embodiment 3 experimental results.
Fig. 8 is embodiment 4 experimental results.1) the negative control mean value of no target nucleic acid; 2) hepatitis B virus sequence set; 3) hepatitis C virus sequence set; 4) human macrophage virus sequence group.
Embodiment
Below in conjunction with example and diagram the present invention and uses thereof is further elaborated.The concrete grammar that uses in the example, operation steps, reagent, equipment etc. all can carry out appropriate change and replacement as required, give an actual example going up in all senses and accompanying drawing do not limit the scope of the invention.Target RNA in embodiment 1. test sample (HCV RNA sequence)
This example has been described a kind of described principle of this patent and probe combinations specificity utilized and has been surveyed the hit method of RNA sequence of solution.Select to use BST archaeal dna polymerase amplification of nucleic acid under constant temperature in this example, and use SYBR Green I detection of fluorescent dyes double-stranded DNA amplified production concentration.Whole process comprises that nucleic acid constant-temperature amplification reacts and detection is carried out in same pipe, finished in about 1 hour.
The reaction cumulative volume is 20 microlitres, and reaction solution consists of: 10
6Copy target RNA, 0.5 μ M cycling probe, 0.5 μ M wire probe (ratio 1: 1), 0.5 μ M wire primer, 20mM Tris-Cl (pH8.8,25 ℃), 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,0.5mM dNTP, 0.5U/ μ l BST archaeal dna polymerase, SYBR green I (1X).Do not add target nucleic acid in zero control group.
Operating process is as follows: probe groups with after target nucleic acid solution mixes in microtubule, is put into fluorescence analyser with microtubule and read initial fluorescent intensity, and fluorescence exciting wavelength is 487nm, and emission wavelength is 525nm.95 ℃ were heated 3 minutes then, were cooled to 65 ℃, added an amount of BST archaeal dna polymerase, and 65 ℃ are continued insulation 60 minutes.After finishing, reaction can read reaction back fluorescence intensity immediately, the variation of calculating fluorescence intensity before and after reacting.Fig. 5 has compared and has contained 10
6The relative variation of the experimental group (2) of copy target RNA and zero control group (1) reaction front and back fluorescence intensity.The relative variation (0.97) of the fluorescence intensity of experimental group is higher 193 times than zero control group (0.005) in this example, shown that this method can be at short notice with the amplification of target nucleic acid signal high power, and can detect with common fluorescence detector.
Used nucleotide sequence (5 '-〉 3 ') is as follows in this example; Specific features is seen sequence table: target nucleic acid RNA sequence: GCCACCAUAGAUCACUCCCCUGUGAGGAACUACUGUCUUCACGCAGAAAGCGUCUA GCCAUGGCGUUAGU (SEQ ID NO:1) RNA uses the T7 RNA polymerase synthetic by vitro reactions, and the sequence of dna profiling is: CGAAATTAATACGACTCACTATAGGGCCACCATAGATCACTCCCCTGTGAGGAACT ACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGT (SEQ ID NO:2) cycling probe sequence: target DNA (human macrophage viral DNA sequence) in GATATACAACCACATCAGCTAGACAGTAGTTCCTCACAGGGGAGTGATATCAGCAT AGCAGTAGACTTGCGTACCTAAG (SEQ ID NO:3) wire probe sequence: TGGCTAGACGCTTTCTGCGTGAATAGCTGAT (SEQ ID NO:4) amplimer sequence: TCAGCATAGCAGTAGACTTGCG (SEQ ID NO:5) embodiment 2. test sample
This example has been described a kind of described principle of this patent and probe combinations specificity utilized and has been surveyed the hit method of dna sequence dna of solution.Select to use BST archaeal dna polymerase amplification of nucleic acid under constant temperature in this example, the reaction back shows amplified production with agarose gel electrophoresis.
Operating process is as follows: adding cumulative volume in microtubule is the reaction mixture of 18 microlitres, comprising: 0.5 μ M cycling probe, 0.5 μ M wire probe (ratio 1: 1), 0.5 μ M wire primer, 20mMTris-Cl (pH8.8,25 ℃), 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,0.5mM dNTP contains 10 in the experimental group
4The copy target DNA; Do not add target nucleic acid in zero control group.95 ℃ were heated 3 minutes, were cooled to 65 ℃, added 2 microlitres (16U) BST archaeal dna polymerase then, and 65 ℃ are continued insulation 60 minutes.Reaction is got 10 microlitre reaction solutions after finishing with 1% agarose gel electrophoresis analysis.
Can find out obviously that from the gel electrophoresis spectrum that Fig. 6 shows produced a large amount of double-stranded DNA products in the experimental group behind the amplified reaction (2), its length is incremented with the length of cycling probe, forms a DNA " ladder ", conform to reaction mechanism institute prediction result; Then survey less than any amplified production in zero control group (1).M is a dna molecular amount mark.
Used nucleotide sequence (5 '-〉 3 ') is as follows in this example, and specific features is seen sequence table: target DNA sequence (taking from the human macrophage virus sequence): CCTAATCGCATCCTGCATCAAAGCGTCAATCAGACTTTCGACGTGCGCCAG (SEQ ID NO:6) cycling probe sequence: GATATACAACCACATCAGCTAACGCTTTGATGCAGGATGCGATTTATCAGCATAGC AGTAGACTTGCGTACCTAAG (SEQ ID NO:7) wire probe sequence: GCGCACGTCGAAAGTCTGATTGATAGCTGAT (SEQ ID NO:8) amplimer sequence: TCAGCATAGCAGTAGACTTGCG (SEQ ID NO:5) embodiment 3. usefulness real-time fluorescence PCR instrument record amplified reaction curve
The real-time fluorescence PCR instrument is used to the quantitative assay sample dna content that hits at present.Method provided by the present invention need not temperature cycle, only utilizes the fluorescence intensity of the rapid fluorescence sweep test real time record reaction release of instrument, surveys thereby target nucleic acid in the sample is carried out relative quantification.
Working method and example 1 are basic identical in this example, use BST large fragment DNA polysaccharase and SYBRGREEN I fluorescence dye, and nucleic acid constant-temperature amplification reaction and detection are carried out in same pipe.The reaction soln cumulative volume is 20 microlitres.A plurality of reactions can be carried out in 8,12 pipe bars or 96 orifice plates.
With after target nucleic acid solution mixes in microtubule, 95 ℃ of heating 3 minutes are cooled to 65 ℃ with probe groups, the mixed solution that adds a small amount of BST archaeal dna polymerase etc., at this moment the reaction soln cumulative volume is 20 microlitres, identical in the final composition of reaction soln and the example 1, and the target DNA total amount is about 10
5With 10
2Copy, other adds the 500ng human gene group DNA and makes background.Has only the 500ng human gene group DNA in the negative control, no target nucleic acid.0.2ml PCR pipe is put into 96 hole reactive tanks of ABI 7700 (Perkin Elmer) sequence detection instrument, 65 ℃ of insulations 80 minutes (2 minutes * 40 circulation).Fluorescence exciting wavelength is 487nm, and emission wavelength is 525nm.
Fig. 7 has write down the curve that fluorescence intensity changes in the amplified reaction process.As can be seen, the target nucleic acid amount is many more in the reaction, and fluorescence occurs and to reach the needed time of certain intensity short more, thereby can be used as the basis that relative quantification detects nucleic acid.Identical in used nucleotide sequence and the example 2 in this example.Embodiment 4. detects multiple target nucleic acid simultaneously in a reaction
This example has been described a kind of method of utilizing described principle of this patent and multiple different probe combinations to detect the multiple target nucleic acid sequence that may exist in the solution.In vitro detect multiple target nucleic acid simultaneously and can reduce the nucleic acid samples consumption same, reduce testing cost, increase and detect flux with a reaction.
The target nucleic acid sequence that will detect in this example is taken from hepatitis B virus, hepatitis C virus and human macrophage viral genome respectively.Correspondingly in the reaction soln mixed three kinds of different probe groups, the linear probe in each probe groups has the glimmering mark of amplification of different wave length, and fluorescence exciting wavelength (EX)/emission wavelength (EM) is respectively: hepatitis B virus, FAM 495/519nm; Hepatitis C virus, HEX 530/553nm; Human macrophage virus, TAMRA 560/583nm.Finish the back in reaction and can judge have which kind of target nucleic acid to exist and relative concentration according to fluorescence color and intensity.BST archaeal dna polymerase amplification of nucleic acid under constant temperature is used in reaction.Whole process comprises that nucleic acid constant-temperature amplification reacts and detection is carried out in same pipe, finished in about 1 hour.
Operating process is as follows: probe groups is mixed in 96 orifice plate microtubules with target nucleic acid solution, microtubule is put into the initial fluorescence value that fluorescence analyser reads each wavelength, and 95 ℃ were heated 3 minutes then, were cooled to 65 ℃, add an amount of BST archaeal dna polymerase, 65 ℃ are continued insulation 75 minutes.After finishing, reaction can read reaction back fluorescent value in each respective wavelength immediately.Calculate reaction front and back change in fluorescence value.
The reaction cumulative volume is 40 microlitres, and reaction solution consists of: 0 or 10
6The copy target nucleic acid, 0.5 μ g human gene group DNA, every kind of cycling probe of 0.5 μ M (totally 3 kinds), every kind of amplification of 0.5 μ M glimmering graticule shape probe (totally 3 kinds), the shared wire primer of 1 μ M, 20mM Tris-Cl (pH8.8,25 ℃), 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,0.5mMdNTP, 0.5U/ μ l BST archaeal dna polymerase.
As can be seen from Figure 8, the fluorescence intensity of amplification back experimental group is all than high nearly 200 times of zero control group.Proof is by using different probe groups and fluorescent mark, and the inventive method is used in and detects multiple different target nucleic acid in the same reaction simultaneously.
Used nucleotide sequence (5 '-〉 3 ') is as follows in this example; Specific features is seen sequence table: the hepatitis type B virus target sequence: ACCACATCATCCATATAACTGAAAGCCAGACAGTGGGGGAAAGCCCTACGAACCAC TGAACAAATGGCACTAGTAAACTGAGCCA (SEQ ID NO:9) cycling probe sequence: GATATACAACCACATCAGCTAGCTTTCCCCCACTGTCTGGCTTTTATCAGCATAGC AGTAGACTTGCGTACCTAAG (SEQ ID NO:10) the glimmering graticule shape probe sequence that increases: 5 ' FAM-TCGATGACTGACGGTCATCG (DABCYL-dT) ACTAGTGCCATTTGTTCAGTGGTTCGTAGGTAGCTGAT (SEQ ID NO:11) HCV target nucleic acid RNA sequence is with (SEQ ID NO:1); The cycling probe sequence is with (SEQ IDNO:3); Glimmering graticule shape probe sequence increases: 5 ' HEX-TCGATGACTGACGGTCATCG (DABCYL-dT) ACTTGGCTAGACGCTTTCTGCGTGAATAGCTGAT (SEQ ID NO:12) human macrophage virus target sequence is with (SEQ ID NO:6), and the cycling probe sequence is the glimmering graticule shape probe sequence of (SEQ ID NO:7) amplification together: 5 ' TAMRA-TCGATGACTGACGGTCATCG (DABCYL-dT) ACTGCGCACGTCGAAAGTCTGATTGATAGCTGAT (SEQ ID NO:13) universal amplification primer sequence is with (SEQ ID NO:5)
Sequence table＜110〉Tian Jingdong
Gong Qihong＜120〉nucleic acid amplification detection method＜130〉PFO30007; Deng Dingji,Zhongzi Law Office＜140〉＜141〉＜160〉14＜170〉Patent In Version 3.1＜210〉1＜211〉70＜212〉RNA＜213〉＜223〉RNA＜400〉SEQUENCE：1gccaccauag aucacucccc ugugaggaac uacugucuuc acgcagaaag 50cgucuagcca uggcguuagu 70＜210〉2＜211〉95＜212〉DNA＜213〉＜220〉＜223〉DNA＜400〉SEQUENCE：2cgaaattaat acgactcact atagggccac catagatcac tcccctgtga 50ggaactactg tcttcacgca gaaagcgtct agccatggcg ttagt 95＜210〉3＜211〉79＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 14 ) .. ( 21 )＜223〉＜221〉＜222〉 ( 22 ) .. ( 47 )＜223〉＜221〉＜222〉 ( 49 ) .. ( 71 )＜223〉＜400〉SEQUENCE：3gatatacaac cacatcagct agacagtagt tcctcacagg ggagtgatat 50cagcatagca gtagacttgc gtacctaag 79＜210〉4＜211〉31＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 1 ) .. ( 23 )＜223〉＜221〉＜222〉 ( 24 ) .. ( 31 )＜223〉＜400〉SEQUENCE：4tggctagacg ctttctgcgt gaatagctga t 31＜210〉5＜211〉22＜212〉DNA＜213〉＜220〉＜223〉＜400〉SEQUENCE：5tcagcatagc agtagacttg cg 22＜210〉6＜211〉51＜212〉DNA＜213〉＜220〉＜223〉DNA＜400〉SEQUENCE：6cctaatcgca tcctgcatca aagcgtcaat cagactttcg acgtgcgcca g 51＜210〉7＜211〉76＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 14 ) .. ( 21 )＜223〉＜221〉＜222〉 ( 22 ) .. ( 44 )＜223〉＜221〉＜222〉 ( 47 ) .. ( 68 )＜223〉＜400〉SEQUENCE：7gatatacaac cacatcagct aacgctttga tgcaggatgc gatttatcag 50catagcagta gacttgcgta cctaag 76＜210〉8＜211〉31＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 1 ) .. ( 23 )＜223〉＜221〉＜222〉 ( 24 ) .. ( 31 )＜223〉＜400〉SEQUENCE：8gcgcacgtcg aaagtctgat tgatagctga t 31＜210〉9＜211〉85＜212〉DNA＜213〉＜220〉＜223〉DNA＜400〉SEQUENCE：9accacatcat ccatataact gaaagccagac agtggggga aagccctacg 50aaccactgaa caaatggcac tagtaaactg agcca 85＜210〉10＜211〉76＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 14 ) .. ( 21 )＜223〉＜221〉＜222〉 ( 22 ) .. ( 44 )＜223〉＜221〉＜222〉 ( 47 ) .. ( 68 )＜223〉＜400〉SEQUENCE：10gatatacaac cacatcagct agctttcccc cactgtctgg cttttatcag 50catagcagta gacttgcgta cctaag 76＜210〉11＜211〉59＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 1 )＜223〉FAM＜221〉＜222〉 ( 21 )＜223〉DABCYL＜221〉＜222〉 ( 25 ) .. ( 52 )＜223〉＜221〉＜222〉 ( 53 ) .. ( 59 )＜223〉＜400〉SEQUENCE：11tcgatgactg acggtcatcg tactagtgcc atttgttcag tggttcgtag 50gtagctgat 59＜210〉12＜211〉55＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 1 )＜223〉HEX＜221〉＜222〉 ( 21 )＜223〉DABCYL＜221〉＜222〉 ( 25 ) .. ( 48 )＜223〉＜221〉＜222〉 ( 49 ) .. ( 55 )＜223〉＜400〉SEQUENCE：12tcgatgactg acggtcatcg tacttggcta gacgctttct gcgtgaatag 50ctgat 55＜210〉13＜211〉55＜212〉DNA＜213〉＜220〉＜223〉＜221〉＜222〉 ( 1 )＜223〉TAMRA＜221〉＜222〉 ( 21 )＜223〉DABCYL＜221〉＜222〉 ( 25 ) .. ( 48 )＜223〉＜221〉＜222〉 ( 49 ) .. ( 55 )＜223〉＜400〉SEQUENCE：13tcgatgactg acggtcatcg tactgcgcac gtcgaaagtc tgattgatag 50ctgat 55
Claims (10)
1. nucleic acid amplification detection method, this method comprises: add nucleic acid probe and other known necessary ancillary components in detected sample, under design temperature, carry out amplified reaction, use instrument detecting amplified signal and quantitative analysis target nucleic acid in reaction is carried out or after finishing, whether there is target nucleic acid and determines its concentration in the judgement sample as a result according to one's analysis, it is characterized in that
Above-mentioned nucleic acid probe is the probe groups that comprises a kind of wire probe and a kind of cycling probe at least, and wherein, the molar concentration rate of wire probe and cycling probe is in 1: 100 to 100: 1 scope; Wherein,
The wire probe comprises following two parts at least: (1) 5 ' end and target nucleic acid paired sequence, and its length is in the scope of 15-35 Nucleotide; (2) 3 ' ends and the short sequence of cycling probe paired, its length is in the scope of 2-10 Nucleotide; And the interval between above-mentioned (1) and (2) two portions is in the scope of 0-5 Nucleotide;
The total length of cycling probe wherein comprises following 3 parts at least in the scope of 35-200 Nucleotide: (1) and target nucleic acid paired sequence, and its length is in the scope of 15-35 Nucleotide; (2) with wire probe 3 ' end paired sequence, its length is in the scope of 2-10 Nucleotide; (3) be used to regulate the padding sequence of cycling probe total length; And the interval between above-mentioned (1) and (2) two portions is in the scope of 0-5 Nucleotide.
2. method according to claim 1 is characterized in that, the molar concentration rate of wherein said wire probe and cycling probe is 1: 10-10: in 1 the scope.
3. method according to claim 1 is characterized in that, the length of wherein said wire probe 5 ' end and target nucleic acid paired sequence is in the scope of 18-32 Nucleotide.
4. method according to claim 1 is characterized in that, in the wherein said cycling probe with the length of target nucleic acid paired sequence in the scope of 18-32 Nucleotide.
5. method according to claim 1 is characterized in that, wherein said wire probe can be any in the following three state: (a) free state; (b) be attached on the carrier; (c) part is free state, and part is attached on the carrier; Nucleotide or other composition that also can comprise any modification on the composition.
6. method according to claim 1 is characterized in that, also comprises at least a wire amplimer in the wherein said probe groups, and its length is in the scope of 15-35 Nucleotide; A fragment is identical in all or part of and cycling probe padding sequence of its sequence.
7. method according to claim 1 is characterized in that, comprises DNA product detection reagent in the wherein said amplified reaction composition.
8. method according to claim 1 is characterized in that, wherein said method with instrument detecting nucleic acid amplification signal can be to detect any method of strand or double-stranded DNA use.
9. according to each the described method among the claim 1-8, it is characterized in that whole nucleic acid amplification testing process is carried out in same reaction vessel.
10. dedicated kit is used for implementing each described method of claim 1-9, it is characterized in that, wherein is equipped with to be used for target nucleic acid kind and the required reagent of relative populations that test sample may exist.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03123596 CN1233847C (en) | 2003-05-19 | 2003-05-19 | Nucleic acid amplification detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03123596 CN1233847C (en) | 2003-05-19 | 2003-05-19 | Nucleic acid amplification detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1460722A true CN1460722A (en) | 2003-12-10 |
| CN1233847C CN1233847C (en) | 2005-12-28 |
Family
ID=29591063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03123596 Expired - Fee Related CN1233847C (en) | 2003-05-19 | 2003-05-19 | Nucleic acid amplification detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1233847C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111684077A (en) * | 2018-02-06 | 2020-09-18 | 简·探针公司 | Far-red dye probe formulations |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101595231A (en) * | 2006-12-29 | 2009-12-02 | 霍尼韦尔国际公司 | The reaction buffer that is used for microarray |
-
2003
- 2003-05-19 CN CN 03123596 patent/CN1233847C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111684077A (en) * | 2018-02-06 | 2020-09-18 | 简·探针公司 | Far-red dye probe formulations |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1233847C (en) | 2005-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11884965B2 (en) | Chimeric primers with hairpin conformations and methods of using same | |
| CN1203186C (en) | Method for detection of target nucleic acids using PCR | |
| US20120107820A1 (en) | Multiplexed Quantitative PCR End Point Analysis of Nucleic Acid Targets | |
| US20090191618A1 (en) | Reaction chamber for real time pcr comprising capture probes and permitting detection of the pcr product by hybridisation without opening the pcr vessel | |
| JP2003334097A5 (en) | ||
| KR20160100415A (en) | Hydrolysis probes | |
| Xu et al. | Exponential rolling circle amplification and its sensing application for highly sensitive DNA detection of tumor suppressor gene | |
| WO2020069045A1 (en) | Digital amplification for protein detection | |
| Lan et al. | Polymerization and isomerization cyclic amplification for nucleic acid detection with attomolar sensitivity | |
| CN106319079B (en) | Method for detecting 22q11.2 copy number loss | |
| US20040209291A1 (en) | Method for expressed gene analysis and probe kit for expressed gene analysis | |
| CN1180091C (en) | Composite gene probe structure and use | |
| CN112359143A (en) | Isothermal index amplification method based on Y-type probe set and application thereof | |
| JP7004570B2 (en) | Substrate molecule | |
| WO2011068518A1 (en) | Multiplexed quantitative pcr end point analysis of nucleic acid targets | |
| WO2007055255A1 (en) | Method of amplifying multiple nucleic acid sequences for differentiation | |
| EP1390529B1 (en) | Method of analysing dna methylation using fluorescence polarisation | |
| Oberc et al. | Nucleic acid amplification test (NAAT) conducted in a microfluidic chip to differentiate between various ginseng species | |
| CN1460722A (en) | Nucleic acid amplification detection method | |
| KR102343373B1 (en) | Method for multiplex detection of miRNA and miRNA detection kit using the same | |
| US20040161763A1 (en) | Fluroscence polarisation | |
| CN106916882B (en) | Method for dual allele-specific polymerase chain reaction of genotype identification chip for identifying polymorphism of nucleotide gene | |
| Dong et al. | Polymerization-driven successive collapse of DNA dominoes enabling highly sensitive cancer gene diagnosis | |
| JP2004329209A (en) | Method for expressed gene analysis and probe kit for expressed gene analysis | |
| CN115058493B (en) | DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |