CN105886501A - Nucleic acid signal amplification sequence and method - Google Patents
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- CN105886501A CN105886501A CN201610267807.0A CN201610267807A CN105886501A CN 105886501 A CN105886501 A CN 105886501A CN 201610267807 A CN201610267807 A CN 201610267807A CN 105886501 A CN105886501 A CN 105886501A
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention relates to the field of molecular biology, in particular to a nucleic acid signal amplification sequence and a nucleic acid signal amplification method. The nucleic acid signal amplification sequence comprises a leader sequence and a repetitive sequence in a plurality of times; the leader sequence and an objective target drone sequence complement each other; and the repetitive sequence and a reporter probe complement each other. An objective target drone can be captured and hybridized by the leader sequence of the nucleic acid signal amplification sequence, and then by the repetitive sequence in a plurality of times and corresponding reporter probes, signals can be amplified, so that nucleic acid signals of the objective target drone are detected. Compared with a PCR technology which needs enzyme to maintain a hybrid system, the nucleic acid signal amplification sequence can detect nucleic acid substances on the premise of not increasing concentration of a detected object, and is good in stability, high in repeatability, low in cost and short in detection technological process.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of nucleic acid signal amplification sequence and nucleic acid signal amplification side
Method.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR) is a kind of specific for amplifying amplification
The Protocols in Molecular Biology of DNA fragmentation, it is considered as the special DNA replication dna of in vitro, and the DNA of trace can significantly be increased by it
Add.Therefore, the either remains of the extinct plants and animal in fossil, historical personage, in murder case, assailant is left over the most decades ago
Hair, skin or blood, as long as the DNA of a wee bit can be isolated, just can be amplified with PCR, be compared.
The ultimate principle of round pcr is similar to the natural reproduction process of DNA, and its specificity depends on mutual with target sequence two ends
The oligonucleotide primers mended.PCR is by degeneration--annealing--, and three fundamental reaction steps of extension are constituted: the 1. degeneration of template DNA: mould
After plate DNA is heated to about 93 DEG C certain times, make template DNA double-strand or through PCR amplification formed double-stranded DNA dissociate, make
Become strand, in order to it is combined with primer, for lower whorl reaction prepare；2. template DNA and the annealing (renaturation) of primer: template
After the heated degeneration of DNA becomes strand, temperature is down to about 55 DEG C, and primer combines with the complementary series pairing of template DNA strand；③
The extension of primer: DNA profiling--primer conjugate 72 DEG C, under the effect of archaeal dna polymerase (such as Taq DNA polymerase), with dNTP
For reaction raw materials, target sequence is template, by base pair complementarity and semiconservative replication principle, synthesizes a new and template DNA
The semiconservative replication chain that chain is complementary, repetitive cycling degeneration--annealing--extends three processes and is achieved with more " semiconservative replication
Chain ", and this new chain can become the template of circulation next time.Often completing a circulation to need 2～4 minutes, 2～3 hours with regard to energy
Genes of interest to be expanded amplification is amplified millions of times.
But, stability and the repeatability of round pcr are poor, there is serious Aerosol Pollution defect, although follow-up
Stopped pipe fluorescent PCR solves the problem of cross-contamination, but still could not solve the congenital defect problem of round pcr.Secondly PCR becomes
This is higher, and PCR reaction needs to build the Special experimental room of higher air cleaning rank, experiment is wanted partition management, manages into
Originally it is greatly increased；And the oversize time cost increase causing every time detecting of response time flow process.Additionally, Taq enzyme in PCR reaction
Or polymerase is also easy to be affected by the material in sample, false positive or false-negative result is caused to occur.
Summary of the invention
In view of this, present invention aims to the defect of prior art provide a kind of nucleic acid signal amplification sequence and
Its preparation method, and utilize the method that this sequence carries out nucleic acid signal amplification.
In order to realize the purpose of the present invention, the present invention adopts the following technical scheme that.
A kind of nucleic acid signal amplification sequence, the repetitive sequence including a targeting sequencing and some multiples is constituted, institute
State targeting sequencing complementary with purpose target sequence, described repetitive sequence and reporter probe complementary.
In some embodiments, in nucleic acid signal amplification sequence of the present invention, the Tm value of described targeting sequencing is 55
℃-62℃；The Tm value of described repetitive sequence is less than 55 DEG C.
In some embodiments, the end of targeting sequencing 5 ' described in nucleic acid signal amplification sequence of the present invention also includes sulfur
For the fragment after the restricted enzyme I restriction enzyme site enzyme action of adenosine triphosphate protection, the enzyme action position of described restricted enzyme I
It is sticky end after some enzyme action.
Further, in some embodiments, described in nucleic acid signal amplification sequence of the present invention, nucleic acid signal is put
Big sequence 3 ' end also includes the fragment after the restriction enzyme site enzyme action of restricted enzyme II；The enzyme action of described restricted enzyme II
It it is blunt end after the enzyme action of site.
In some embodiments, in nucleic acid signal amplification sequence of the present invention, described restricted enzyme I is EcoR
I、BamH I、HindII、HindIII；Described restricted enzyme II is EcoR V, Alu I, BsuR I, Bal I, HalIII,
HPa I、Sma I。
Further, in some embodiments, described in described nucleic acid signal amplification sequence, the multiple of repetitive sequence is 5
-20 times again.
Present invention also offers the preparation method of described nucleic acid signal amplification sequence, specifically include following steps:
A, according to purpose drone design nucleic acid signal targeting sequencing and the repetitive sequence of some multiples, and respectively in leading sequence
Row 5 ' end is held plus restricted enzyme II plus the restriction enzyme site of restricted enzyme I, the repetitive sequence 3 ' at some multiples
Restriction enzyme site；The restriction enzyme site of described restricted enzyme I is connected, after enzyme action with the targeting sequencing of nucleic acid signal amplification sequence
For sticky end；The restriction enzyme site of described restricted enzyme II is connected with the repetitive sequence of nucleic acid signal amplification sequence, enzyme action
It is blunt end afterwards；
Carry out clone after B, composition sequence insertion vector to replicate；
After C, restricted enzyme I and restricted enzyme II enzyme action, thio triphosphates adenosine is to Restriction Enzyme simple stage property end
Protect；
D, exonuclease III digestion obtain nucleic acid signal amplification sequence.
Wherein, in the preparation method of described nucleic acid signal amplification sequence, described carrier be pGEM-3ZF (+) carrier.
Present invention also offers a kind of method that nucleic acid signal amplifies, microwell plate will be coated on containing the sample of purpose target
On, it is subsequently adding the hybridization of above-mentioned nucleic acid signal amplification sequence, adds chemical luminous substrate after adding reporter probe hybridization afterwards and read
Take result.
Further, in some embodiments, the method that nucleic acid signal of the present invention amplifies also includes believing with nucleic acid
Repetitive sequence in number amplification sequence is the step that targeting sequencing carries out secondary signal amplification.
As shown from the above technical solution, the invention provides a kind of nucleic acid signal amplification sequence and preparation method thereof, and
Utilize the method that this sequence carries out nucleic acid signal amplification.Nucleic acid signal amplification sequence of the present invention include a targeting sequencing and
The repetitive sequence of one some multiple is constituted, and described targeting sequencing is complementary with purpose target sequence, described repetitive sequence and report
Probe sequence is complementary.The capture of purpose target can be hybridized by nucleic acid signal amplification sequence of the present invention by targeting sequencing, then
Repetitive sequence through some multiples combines corresponding reporter probe can realize signal amplification, thus purpose target detected
Nucleic acid signal.Relative to round pcr need enzyme to maintain hybridization system, before the present invention can realize not increasing detectable substance concentration
Putting the detection realizing nucleic acid substances, and good stability, repeatability is high, and low cost, testing process is short.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 shows nucleic acid signal amplification sequence structural representation；
Fig. 2 shows that exonuclease III digestion obtains the schematic diagram of nucleic acid signal amplification sequence；
Fig. 3 show pGEM-3ZF (+) Vector map；
Fig. 4 shows that primary signal is amplified and adds secondary signal amplification conformational map.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all
Belong to the scope of protection of the invention.
In order to realize the purpose of the present invention, the present invention adopts the following technical scheme that.
A kind of nucleic acid signal amplification sequence, the repetitive sequence including a targeting sequencing and some multiples is constituted, institute
State targeting sequencing complementary with purpose target sequence, described repetitive sequence and reporter probe complementary.
Nucleic acid signal amplification sequence of the present invention is for purpose drone design a kind of non-natural existence that synthesizes
Gene order, this gene order is to be made up of the repetitive sequence of a targeting sequencing and some multiples.Its structure such as Fig. 1 institute
Show.
Tm value is DNA melting temperature, refers to the double-spiral structure of DNA ultraviolet absorption value during thermal denaturation to reach maximum
Value 1/2 time temperature.
Targeting sequencing described in nucleic acid signal amplification sequence of the present invention can be complementarily shaped to double with purpose target sequence
Helical structure.Described some repetitive sequences can form double-spiral structure with reporter probe complementary.
In order to ensure stablizing of structure, the Tm value of targeting sequencing described in nucleic acid signal amplification sequence of the present invention ensures
At about 60 DEG C.In some embodiments, the Tm value of described targeting sequencing is 55 DEG C-62 DEG C.
Same, repetitive sequence described in nucleic acid signal amplification sequence described in nucleic acid signal amplification sequence of the present invention
Tm value less than 55 DEG C, to guarantee the specific hybrid of repetitive sequence.
As, targeting sequencing described in the most described nucleic acid signal amplification sequence is specially
ACGTTTTAGAGAGAGATATGAT, described repetitive sequence is TCCCGGGAGGCGAGCA.
As, targeting sequencing described in the most described nucleic acid signal amplification sequence is specially
TCCCTGAATGCCTAACGAT, described repetitive sequence is AGGTCTTTACCACCAATATTCTTTT.
In some embodiments, the end of targeting sequencing 5 ' described in described nucleic acid signal amplification sequence also includes sulfur generation three phosphorus
Fragment after the restricted enzyme I restriction enzyme site enzyme action of adenosine monophosphate protection, the restriction enzyme site enzyme action of described restricted enzyme I
It is sticky end afterwards.The most described targeting sequencing 5 ' end connects the restriction enzyme site of restrictive restriction endonuclease I, and this restriction enzyme site is through enzyme
Forming sticky end after cutting, sticky end polishing after thio triphosphates adenosine is protected is flat end subsequently, it is thus achieved that include
Fragment after the restricted enzyme I restriction enzyme site enzyme action of thio triphosphates adenosine protection.
In some embodiments, nucleic acid signal amplification sequence 3 ' of the present invention end also includes restricted enzyme II's
Fragment after restriction enzyme site enzyme action；It is blunt end after the restriction enzyme site enzyme action of described restricted enzyme II.
It will be understood by those skilled in the art that in described nucleic acid signal amplification sequence, described restricted enzyme I can be
Any enzyme of sticky end is formed, as EcoR I, BamHI, HindII, HindIII etc. after any one enzyme action.One
In a little embodiments, described restricted enzyme I is HindII.
Described restricted enzyme II can be any enzyme forming blunt end after any one enzyme action, as
EcoR V, Alu I, BsuR I, Bal I, HalIII, HPa I, Sma I etc..In some embodiments, described restricted interior
Cutting enzyme II is EcoR V.
Described in nucleic acid signal amplification sequence of the present invention, the multiple of repetitive sequence is 5 times-20 times.In some embodiments
In, described repetitive sequence is 20 times.In further embodiments, described repetitive sequence is 10 times.
Present invention also offers the preparation method of above-mentioned nucleic acid signal amplification sequence, specifically include following steps:
A, according to purpose drone design nucleic acid signal targeting sequencing and the repetitive sequence of some multiples, and respectively in leading sequence
Row 5 ' end is held plus restricted enzyme II plus the restriction enzyme site of restricted enzyme I, the repetitive sequence 3 ' at some multiples
Restriction enzyme site；The restriction enzyme site of described restricted enzyme I is connected, after enzyme action with the targeting sequencing of nucleic acid signal amplification sequence
For sticky end；The restriction enzyme site of described restricted enzyme II is connected with the repetitive sequence of nucleic acid signal amplification sequence, enzyme action
It is blunt end afterwards；
Carry out clone after B, composition sequence insertion vector to replicate；
After C, restricted enzyme I and restricted enzyme II enzyme action, thio triphosphates adenosine is to Restriction Enzyme simple stage property end
Protect；
D, exonuclease III digestion obtain nucleic acid signal amplification sequence.
Preparation method of the present invention is to be protected the fragment after enzyme action by thio triphosphates adenosine, utilizes nucleic acid
ExonucleaseⅢ can not cut the feature containing thio triphosphates adenosine sequence, the fragment that will do not protected by thio triphosphates adenosine
Cut into single-stranded probe.
Exonuclease III has 3 ' → 5 ' 5 prime excision enzyme activities, and this enzyme acts on double-stranded DNA, from 3 ' OH end directions by
Step cuts mononucleotide.Enzyme-to-substrate combines catalysis every time, and the most several nucleotide are removed, thus produces in DNA molecular group
Raw progressive disappearance.Although can act on double-stranded DNA nicking produce single stranded gaps, but the suitableeest substrate of this enzyme be generally flush with end or
3 ' recessed ends DNA.Due to inactive to single stranded DNA, therefore this enzyme cannot cut 3 ' protruding terminuses.And exonuclease III
3 ' → 5 ' 5 prime excision enzyme activities the cutting degree of substrate is changed with the length of 3 ' protruding terminuses, four bases or longer prominent
End can not be cut completely.
Owing to exonuclease III can not act on sticky end, preparation method of the present invention need to be protected that
On bar chain first-stage polymerization after several thio triphosphates adenosines, exonuclease III can only cut that chain unprotected, and protects
Stay that chain that thio triphosphates adenosine is protected, thus prepare strand specific probe, i.e. nucleic acid signal amplification sequence.Specifically
As shown in Figure 2.
Wherein, described thio triphosphates adenosine has α-dAtp, α-dTtp, α-dGtp and α-dCtp four kinds, and structural formula is respectively
As follows:
In the preparation method of nucleic acid signal amplification sequence of the present invention, described carrier be pGEM-3ZF (-) carrier.Sequence
After insertion vector, named pGEM-3ZF (+)-Amplifier.Collection of illustrative plates is as shown in Figure 3.
In the preparation method of nucleic acid signal amplification sequence of the present invention, step C is specially and uses restricted enzyme I mono-
Enzyme action carrier sequence produces 5 ' sticky ends, uses Kelnow Large fragment polymerase and thio triphosphates adenosine to 5 ' sticky ends
Carry out filling-in operation；With restricted enzyme II, purpose band is excised from carrier again.
In the preparation method of nucleic acid signal amplification sequence of the present invention, step D is specially and uses exonuclease III pair
The double-strand of sulfur generation protection carries out strand digestion, it is ensured that the sequence of 5 ' sulfur generation protections can be converted into single-stranded probe structure.
Present invention also offers a kind of method that nucleic acid signal amplifies, microwell plate will be coated on containing the sample of purpose target
On, it is subsequently adding the hybridization of above-mentioned nucleic acid signal amplification sequence, adds chemical luminous substrate after adding reporter probe hybridization afterwards and read
Take result.
Wherein, described nucleic acid signal amplification sequence hybridization temperature be 50 DEG C～55 DEG C, described hybridization time be 40～
50min。
And in the method that described nucleic acid signal amplifies, with the condition of reporter probe hybridization be 51 DEG C～53 DEG C reactions 30～
40min。
Described in the method that nucleic acid signal of the present invention amplifies, chemical luminous substrate can be that any one can identify
The chemical luminous substrate of reporter probe.In some embodiments, described chemical luminous substrate is lumi-Phos Plus.
In the method that nucleic acid signal of the present invention amplifies, experimental result uses chemiluminescent analyzer to read data.
Further, the method that nucleic acid signal of the present invention amplifies also includes with the repetition in nucleic acid signal amplification sequence
Sequence is the step that targeting sequencing carries out secondary signal amplification.Concrete conformation is as shown in Figure 4.
On the basis of primary signal is amplified, carry out secondary signal amplify the accumulation amplification that can form multiple, thus enter one
Step provides the sensitivity of detection.It is 20 times as primary nucleic acid signal amplifies repetitive sequence, and secondary signal amplifies 20 times put again
Greatly, primary adds secondary signal amplification accumulation can reach 400 times of amplifications, substantially increases detection sensitivity.
As shown from the above technical solution, the invention provides a kind of nucleic acid signal amplification sequence and preparation method thereof, and
Utilize the method that this sequence carries out nucleic acid signal amplification.Nucleic acid signal amplification sequence of the present invention include a targeting sequencing and
The repetitive sequence of one some multiple is constituted, and described targeting sequencing is complementary with purpose target sequence, described repetitive sequence and report
Probe sequence is complementary.The capture of purpose target can be hybridized by nucleic acid signal amplification sequence of the present invention by targeting sequencing, then
Repetitive sequence through some multiples combines corresponding reporter probe can realize signal amplification, thus purpose target detected
Nucleic acid signal.Relative to round pcr need enzyme to maintain hybridization system, before the present invention can realize not increasing detectable substance concentration
Putting the detection realizing nucleic acid substances, and good stability, repeatability is high, and low cost, testing process is short.
In order to be further appreciated by the present invention, below in conjunction with specific embodiment, the present invention will be described in detail.As without special
Illustrating, reagent used in the present invention and instrument are reagent commonly used in the art and instrument.Method involved in the present invention is
The universal method of this area.Wherein chemical luminous substrate is lumi-Phos Plus, purchased from U.S. Beckman Coulter
Lumigen。
Embodiment 1, the preparation of nucleic acid signal amplification sequence
Design targeting sequencing according to primer sequence 1, design the repetitive sequence of different multiples simultaneously, and respectively at targeting sequencing
5 ' ends, plus the restriction enzyme site of HindII, at repetitive sequence 3 ' end plus the restriction enzyme site of EcoR V, synthesize different conformational sequence
Rear insertion pGEM-3ZF (-) carry out clone after carrier and replicate；HindII single endonuclease digestion carrier sequence produces 5 ' sticky ends, uses
Kelnow Large fragment polymerase and thio triphosphates adenosine carry out filling-in to 5 ' sticky ends, then with EcoR V by purpose band from
Excising on carrier, exonuclease III digestion becomes ssDNA probe.Each sequence is as shown in table 1.
The each sequence of table 1
| Sequence name | Sequence |
| Purpose target | ATCGTTAGGCATTCAGGGA |
| Targeting sequencing | TCCCTGAATGCCTAACGAT |
| Composition sequence 1 | TCCCTGAATGCCTAACGAT-20X-AGGTCTTTACCACCAATATTCTTTT-- |
| Composition sequence 2 | TCCCTGAATGCCTAACGAT-10X-AGGTCTTTACCACCAATATTCTTTT-- |
| Composition sequence 3 | TCCCTGAATGCCTAACGAT-5X-AGGTCTTTACCACCAATATTCTTTT-- |
Embodiment 2, primary signal are amplified
1, the purpose target in the table 1 of 100pmol concentration is coated on microwell plate；
2, synthesize each sequence according to the method for embodiment 1 and be prepared as single-stranded probe, by composition sequence 1, composition sequence 2 and
The probe of composition sequence 3 preparation is respectively designated as pre-amplification 1, pre-amplification 2, pre-amplification 3；
3, controlling hybridization temperature and control between 50 DEG C～55 DEG C, every hole adds the concentration of pre-amplification 1,2,3 and is
100pmol/ μ L, hybridizes 40～50 minutes；
4, be separately added into reporter probe (AAAAGAATATTGGTGGTAAAGACCT-AP), 51 DEG C～53 DEG C reactions 30～
40min；
5, add chemical luminous substrate lumi-Phos Plus (Beckman Inc.), read experimental result.
Wherein fabric swatch designs such as table 2, corresponding data result such as table 3.
Table 2 fabric swatch designs
Table 3 corresponding data result
| 575 | 13575 | 6575 | 3275 |
| 620 | 13655 | 6625 | 3000 |
| 585 | 14110 | 6195 | 3175 |
| 600 | 12995 | 6360 | 3065 |
| 630 | 13050 | 6030 | 2985 |
| 580 | 13530 | 5990 | 3215 |
| 575 | 12855 | 6100 | 3065 |
| 590 | 14050 | 6395 | 2985 |
From table 3 result, pre-amplification 1,2,3 be about exaggerated on the basis of background luminescence value respectively 20 times, 10 times,
The chemiluminescence signal value of 5 times, is consistent with desired design.
Embodiment 3, secondary signal amplify
A kind of nucleic acid signal amplification sequence-composition sequence 4 of redesign, its targeting sequencing and enforcement on the basis of embodiment 2
The repetitive sequence of example 2-in-1 one-tenth sequence is complementary, and the sequence of composition sequence 4 is particularly as follows: AAAAGAATATTGGTGGTAAAGACCT-
20X-GATATCATTTCTTGATGGC)。
Method:
1, by 100pmol concentration table 1 in purpose target be coated on microwell plate；
2, synthesize each sequence be prepared as single-stranded probe according to the method for embodiment 1, by composition sequence 1, composition sequence 2,
The probe of composition sequence 3 and composition sequence 4 preparation is respectively designated as pre-amplification 1, pre-amplification 2, pre-amplification 3 and amplifies 1；
3, controlling hybridization temperature and control between 50 DEG C～55 DEG C, every hole adds the concentration of pre-amplification 1,2,3 and is
100pmol/ μ L, hybridizes 40～50 minutes；
4, control hybridization temperature control between 50 DEG C～55 DEG C, every hole add 50pmol/ μ L amplification 1, hybridization 40～
50 minutes；
5, reporter probe (GCCATCAAGAAATGATATC-AP) is added, 51 DEG C～53 DEG C reactions 30～40min.
6, add chemical luminous substrate lumi-Phos Plus (Beckman Inc.), read experimental result.
Wherein fabric swatch designs such as table 4, corresponding data result such as table 5.
Table 4 fabric swatch designs
Table 5 corresponding data result
| 865 | 237565 | 105210 | 26205 |
| 930 | 238965 | 106055 | 24050 |
| 880 | 246925 | 109120 | 25400 |
| 900 | 227415 | 101760 | 24520 |
| 945 | 228375 | 96485 | 23880 |
| 870 | 236775 | 95840 | 25725 |
| 865 | 224965 | 97605 | 24520 |
| 885 | 245875 | 102325 | 23880 |
From table 5 result, pre-amplification 1,2,3 is about exaggerated 330 respectively with amplifying 1 on the basis of background luminescence value
Times, 160 times, the chemiluminescence signal value of 40 times, be consistent with desired design.
Claims (10)
1. a nucleic acid signal amplification sequence, the repetitive sequence including a targeting sequencing and some multiples is constituted, described
Targeting sequencing is complementary with purpose target sequence, described repetitive sequence and reporter probe complementary.
Nucleic acid signal amplification sequence the most according to claim 1, the Tm value of described targeting sequencing is 55 DEG C-62 DEG C；Described
The Tm value of repetitive sequence is less than 55 DEG C.
Nucleic acid signal amplification sequence the most according to claim 1 and 2, described targeting sequencing 5 ' end also includes thio triphosphates
Fragment after the restricted enzyme I restriction enzyme site enzyme action of adenosine protection, after the restriction enzyme site enzyme action of described restricted enzyme I
For sticky end.
4., according to the nucleic acid signal amplification sequence described in claim 1-3 any one, described nucleic acid signal amplification sequence 3 ' is held
Also include the fragment after the restriction enzyme site enzyme action of restricted enzyme II；After the restriction enzyme site enzyme action of described restricted enzyme II
For blunt end.
5., according to the nucleic acid signal amplification sequence described in claim 1-4 any one, described restricted enzyme I is EcoR
I、BamH I、Hind II、HindIII；Described restricted enzyme II is EcoR V, Alu I, BsuR I, Bal I,
HalIII、HPa I、Sma I。
6. according to the nucleic acid signal amplification sequence described in claim 1-5 any one, the multiple of described repetitive sequence is 5 times-
20 times.
7. the preparation method of nucleic acid signal amplification sequence described in claim 1, comprises the following steps:
A, according to purpose drone design nucleic acid signal targeting sequencing and the repetitive sequence of some multiples, and respectively at targeting sequencing 5 '
End holds the enzyme plus restricted enzyme II plus the restriction enzyme site of restricted enzyme I, the repetitive sequence 3 ' at some multiples
Cut site；The restriction enzyme site of described restricted enzyme I is connected with the targeting sequencing of nucleic acid signal amplification sequence, is viscous after enzyme action
Property end；The restriction enzyme site of described restricted enzyme II is connected with the repetitive sequence of nucleic acid signal amplification sequence, after enzyme action is
Blunt end；
Carry out clone after B, composition sequence insertion vector to replicate；
After C, restricted enzyme I and restricted enzyme II enzyme action, Restriction Enzyme simple stage property end is carried out by thio triphosphates adenosine
Protection；
D, exonuclease III digestion obtain nucleic acid signal amplification sequence.
Preparation method the most according to claim 7, described carrier be pGEM-3ZF (+) carrier.
9. the method that nucleic acid signal amplifies, is coated on the sample containing purpose target on microwell plate, is subsequently adding right
Require nucleic acid signal amplification sequence hybridization described in 1-6 any one, add at the bottom of chemiluminescence after adding reporter probe hybridization afterwards
Thing reads result.
Method the most according to claim 9, also includes with the repetitive sequence in nucleic acid signal amplification sequence as targeting sequencing
Carry out the step of secondary signal amplification.
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| CN108504738A (en) * | 2018-04-27 | 2018-09-07 | 郑州科蒂亚生物技术有限公司 | A kind of prostate cancer detection probe sequence and its application |
| CN108796134A (en) * | 2018-07-12 | 2018-11-13 | 郑州科蒂亚生物技术有限公司 | A kind of cervical carcinoma detection probe sequence and its application |
| CN116256519A (en) * | 2023-02-17 | 2023-06-13 | 成都诺森医学检验有限公司 | Antigen hypersensitive detection method |
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| CN108504738A (en) * | 2018-04-27 | 2018-09-07 | 郑州科蒂亚生物技术有限公司 | A kind of prostate cancer detection probe sequence and its application |
| CN108504738B (en) * | 2018-04-27 | 2021-09-10 | 郑州科蒂亚生物技术有限公司 | Prostate cancer detection probe sequence and application thereof |
| CN108796134A (en) * | 2018-07-12 | 2018-11-13 | 郑州科蒂亚生物技术有限公司 | A kind of cervical carcinoma detection probe sequence and its application |
| CN108796134B (en) * | 2018-07-12 | 2021-09-10 | 郑州科蒂亚生物技术有限公司 | Cervical cancer detection probe sequence and application thereof |
| CN116256519A (en) * | 2023-02-17 | 2023-06-13 | 成都诺森医学检验有限公司 | Antigen hypersensitive detection method |
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