CN109852670A - A kind of high specific nucleic acid detection reagent and its application method - Google Patents
A kind of high specific nucleic acid detection reagent and its application method Download PDFInfo
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Abstract
The present invention relates to the high specific detection of nucleic acids in basic research (gene expression regulation, biological evolution, Species estimation, Genotyping) and application study (drug development, forensic identification, the supervision of food drug safety, customs inspection quarantine etc.) field, and in particular to arrives a kind of high specific nucleic acid detection reagent and its application method.The main component of the detection reagent includes detecting the single-chain nucleic acid enzyme of the specific probe of target nucleic acid chain and the single nucleic acid strands that can degrade.High specific nucleic acid detection reagent of the invention is easy to operate, rapid and convenient, cost performance are high, can satisfy the needs of automation, time saving and energy saving, is easy to implement scale experiment and basic research.
Description
Technical field
The present invention relates to basic research (gene expression regulation, biological evolution, Species estimation, Genotyping) and application studies
High specific detection of nucleic acids in (drug development, forensic identification, the supervision of food drug safety, customs inspection quarantine etc.) field, tool
Body is related to a kind of high specific nucleic acid detection reagent and its application method.
Background technique
The weight of modern biology (botany, zoology, microbiology, marine biology, paleontology, paleoanthropology etc.)
Want research contents first is that Nucleic Acid Identification and detection of nucleic acids.Complete genomic information can make the mankind at the genetic level over the ground
The origin of various species become extinct on ball and existing, evolve, migrate and be destroyed carry out profound level, multi-angle of view,
Comprehensive analysis and interpretation because the nucleotide sequence and nucleotide diversity of genome determine Species Characteristics and species into
Change.
The basic principle of detection of nucleic acids is to utilize base pair complementarity principle.Nucleic acid probe based on genomic information design
Stable heteroduplex is formed with target nucleic acid fragment to be detected, it then will be in conjunction with nucleic acid probe using Amplification Technologies
Micro target nucleic acid fragment amplification to be easy to detection level.The success of detection of nucleic acids depends on the stability of heteroduplex,
And the stability of heteroduplex is strong by the ion in the length of nucleic acid chains, the nucleotide component of nucleic acid chains and sequence, solution
The influence of the factors such as the base complementrity degree of degree, hybridization temperature and heteroduplex.Under conditions of other conditions are fixed, hybridization is double
The base complementrity degree of chain is higher, and the stability of heteroduplex is also higher, i.e. detection specificity is higher.But work as target nucleic acid
When differing only by one or several nucleotide between the nucleotide sequence of segment and the nucleotide sequence of non-targeted nucleic acid fragment, core
Acid probe is difficult to distinguish target nucleic acid chain and non-targeted nucleic acid fragment, thereby produces false positive detection signal.Such vacation
Positive findings to detection there is subtle nucleotide diversity to propose huge challenge.
Subtle nucleotide variation includes simple point mutation, single nucleotide polymorphism (the single nucleotide of gene
Polymorphism, abbreviation SNP), methylation, microRNA (microRNA) family member etc..These subtle nucleotide diversities are not
The variation of gene level can be only influenced, but also species characteristics can be influenced in epigenetic level.
The method of these subtle mononucleotide variations of currently used detection has nucleic acid sequencing (sequencing), high score
Distinguish solubility curve (high resolution melting, abbreviation HRM), Restrictive fragment length polymorphism
(restriction fragment length polymorphism, abbreviation RFLP), single-strand conformation polymorphism (single
Strand conformation polymorphism, abbreviation SSCP), the oligonucleotide hybridization of allele specific
(allele specific oligonucleotide hybridization, abbreviation ASO), ApoE gene
(allele specific PCR) etc..Although these technologies can detect SNP, simple point mutation etc. to a certain extent,
But they there are certain technological deficiencies.For example, nucleic acid sequencing is considered as the goldstandard of detection of nucleic acids, but nucleic acid sequencing
It is with high content of technology, need professional technician to operate, be sequenced it is costly, be not easy to popularize.SSCP and RFLP must be by solidifying
Gel electrophoresis is detected, it is difficult to meet the needs of automation, time and effort consuming is not easy to realize scale experiment.Allele is special
Different PCR is then faced with the rational limitation of design of primers.In recent years, people also utilize nucleic acid and protein to interact and invent
The detection technique of SNP and simple point mutation, such as detection method based on single-chain nucleic acid enzyme.But these methods require it is right first
Determined nucleic acid carries out PCR amplification, then recycles nuclease that pcr amplification product hydrolysis is become different segments, to influence
The high specific of detection of nucleic acids.Therefore, either basic research or practical application, there is an urgent need to the cores of high specific by people
Sour detection method and technology.
Summary of the invention
In view of the existing technical defect, the present invention provides a kind of high specific nucleic acid detection reagent and its users
Method.
In order to achieve the object of the present invention, following technical scheme is provided.
A kind of high specific nucleic acid detection reagent, the main component of the detection reagent include the special of detection target nucleic acid chain
Property the probe and single-chain nucleic acid enzyme of single nucleic acid strands of can degrading.
A kind of high specific nucleic acid detection reagent application method, specifically includes the following steps.
Step 1: 1. 2. specific probe is added in reaction buffer with containing nucleic acid chains to be detected, is become
Property annealing.
Step 2: by single-chain nucleic acid enzymeIt is added in reaction buffer, specific time is incubated at a temperature of optimization, it is excellent
Change temperature and depends on the probe length of specific probe, the nucleotide component of composition specific probe and type and single-stranded core
The most suitable operating temperature of sour enzyme；Its reaction time depends on the digesting efficiency of single-chain nucleic acid enzyme.
Step 3: after reaction, reaction system is incubated for 20 minutes at 95 DEG C.
Step 4: by reaction system Temperature fall to room temperature.
Step 5: the reaction system that single-chain nucleic acid enzymatic treatment is crossed carries out detection of nucleic acids.
Further, the specific probe of the detection target nucleic acid chain is that length is no less than ten, not more than 30
The single nucleic acid strands of nucleotide；Specific probe can form hybrid nucleic acid double-strand with the segment in nucleic acid chains to be detected；It constitutes
The nucleotide of specific probe can be nucleotide, deoxynucleotide and nucleotide derivative, including but not limited to lock nucleic acid,
Peptide nucleic acid, sulphur nucleic acid, dideoxy nucleotide, methoxyl group nucleotide etc..
Further, the single-chain nucleic acid enzyme of the single nucleic acid strands that can degrade has non-in degradation hybrid nucleic acid double-strand
The nuclease of duplex structure.
Further, the nuclease of the non-duplex structure, non-duplex structure include do not meet standard base pairing (A:T,
A:U and G:C) principle base-pair, or the balloon-shaped structure or cyclic structure or nucleic acid that are formed due to nucleotides inserted or missing
The single stranded gaps of double-strand.
Further, the single-chain nucleic acid enzyme of the single nucleic acid strands that can degrade, including but not limited to S1 nuclease (S1
Nuclease), Mung bean nuclease (Mung Bean Nuclease), P1 nuclease (P1 Nuclease), BAL bal 31 nuclease bal
(31 Nuclease of BAL), ribonuclease A (Ribonuclease A), celery nuclease (CEL I Nuclease) etc..
Further, the nucleic acid chains to be detected, can be nucleic acid double chain or single nucleic acid strands, can be longer nucleic acid chain or
Oligonucleotide segment can be linear nucleic acid chain or annular nucleic acid chains.The nucleic acid chains to be detected can be DNA chain, RNA chain
Or the nucleic acid chains containing nucleotide derivative.
Further, the reaction system that single-chain nucleic acid enzymatic treatment is crossed carries out detection of nucleic acids, and detection technique includes appointing
What a kind of detection technique dependent on nucleic acid amplification method or detection technique independent of nucleic acid amplification method.
Further, the detection technique dependent on nucleic acid amplification method, detection technique can be based on thermal cycle core
Sour amplification technique is based on constant temperature nucleic acid amplification technology.
Compared with prior art, the present invention has the advantages that.
1) excellent detection specificity: the nucleic acid detection reagent will directly be reacted with determined nucleic acid sample first, be protected
The primordial condition for having held sample to be tested nucleic acid sequence, avoid the prior art first expands the strategy recognized afterwards, therefore can be real
The specific detection of existing hi-fi.
2) huge sample treatment ability: the nucleic acid detection reagent can examine a variety of different types of nucleic acid samples
It surveys, nucleic acid samples to be measured can be nucleic acid double chain or single nucleic acid strands, can be DNA chain or RNA chain, can be longer
Nucleic acid chains or very short oligonucleotide segment (such as microRNA).This is that current any nucleic acid detection technique institute is unreachable
's.
3) testing cost is cheap: the nucleic acid detection reagent is low in cost, and detection method is easily operated, needs not move through profession
Technical staff's operation.It can be combined with naked eye detection method, the low cost detection of instrument and equipment is implemented without, thus real
Now detection of nucleic acids real-time and on the spot.
Detailed description of the invention
Fig. 1 is the operation principle schematic diagram of high specific nucleic acid detection reagent.Wherein 1.: specific probe；2. to be detected
Nucleic acid chains；The heteroduplex of complete complementary；: non-fully complementary heteroduplex；: single-chain nucleic acid enzyme.
Fig. 2 is the microRNA let- using rolling circle amplification (RCA) detection high specific nucleic acid detection reagent processing
The flow chart of 7 family members.Wherein 1.: specific probe probe 7E；2. nucleic acid chains let-7a ~ let-7f to be detected；The heteroduplex probe 7E/let-7e of complete complementary；: non-fully complementary heteroduplex probe 7E/let-7(is not
Including let-7e)；: single-chain nucleic acid enzyme CEL I；: annular template；: the archaeal dna polymerase with strand displacement function
phi29.Step 1: 1. specific probe probe 7E is mixed with the sample containing nucleic acid chains to be detected.Denaturation annealing is special
1. 2. specific probes become heteroduplex with let-7 family member to be detected.Wherein specific probe probe 7E and sample
In let-7e form the heteroduplex of complete complementary, and other let-7 in specific probe probe 7E and sample
Family member forms non-fully complementary heteroduplex.Step 2: by single-chain nucleic acid enzymeIt is added to sample mixture
In, specific time is incubated at a temperature of optimization.Single-chain nucleic acid enzymeThe heteroduplex probe 7E/ of complete complementary cannot be hydrolyzed
let-7e, and can be by non-fully complementary heteroduplex: probe 7E/let-7a, probe 7E/let-7b,
The hydrolysis of the single stranded portion of probe 7E/let-7c, probe 7E/let-7d and probe 7E/let-7f.Small pieces after hydrolysis
Section it is unstable, be dissociated into it is single-stranded after finally all hydrolyzed.Step 3: by the sample mixture of single-chain nucleic acid enzymatic treatment at 95 DEG C
It is incubated for 20 minutes, single-chain nucleic acid enzyme is inactivated.Step 4: by sample mixture Temperature fall into room temperature.Step 5: will be single-stranded
The sample mixture of nucleic acid enzymatic treatment is added to RCA reaction system (annular template, polymerase) in detected.Not by
The let-7e of hydrolysis can be used as primer starting RCA reaction.
Fig. 3 is two samples (sample 1: only let-7e；Sample 2: other let-7 family members' without let-7e
Mixture) without the testing result of high specific nucleic acid detection reagent treated let-7 family member.
Fig. 4 is two samples (sample 1: only let-7e；Sample 2: other let-7 family members' without let-7e
Mixture) by the testing result of high specific nucleic acid detection reagent treated let-7 family member.
Fig. 5 be the mixing sample that is formed in varing proportions of sample 1 and sample 2 without high specific nucleic acid detection reagent at
The testing result of let-7e after reason.
Fig. 6 is that the mixing sample that sample 1 and sample 2 are formed in varing proportions is handled by high specific nucleic acid detection reagent
The testing result of let-7e afterwards.
Fig. 7 is the RT-qPCR result that sample 1 and sample 2 are handled without CEL I.
Fig. 8 is sample 1 and sample 2 by CEL I treated RT-qPCR result.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, the explanation of following embodiment is merely used to help understand the present invention
Core concept.It is noted that those of ordinary skill in the art, in the premise for not departing from the invention patent principle
Under, can to the invention patent, some improvement and modification can also be carried out, these improvement and modification are also in the invention patent claim
Within protection scope.
Embodiment high specific nucleic acid detection reagent combination RCA technology detects microRNA let-7 family member let-
7e。
MicroRNA let-7 is that a 22-25 nucleotide is long, has 9 members (let-7a ~ let-7i)
MicroRNA family, they take part in each stage in life process, are important one of gene expression regulation molecule.But
(1 ~ 3 nucleotide) of slight difference between family member, accurate detection go out family member with certain challenge.This example
After high specific nucleic acid detection reagent detection processing sample, rolling circle amplification (rolling circle is recycled
Amplification, abbreviation RCA) detect the microRNA family member let-7e.
One, it designs specific probe and prepares sample.
With microRNA let-7 family (let-7a ~ let-7f) for test object, according to the core of let-7 family member
Nucleotide sequence design dna probe probe 7E, is shown in Table 1.
The nucleotide of red-label is the mismatch site formed after the microRNA hybridizes with specific probe probe 7E.
Sample 1:let-7e；Concentration 100nM.
Sample 2: the mixture 100nM of other let-7 family members without let-7e.
Sample 3: a series of samples 1 and sample 2 are shown in Table 2 according to the mixture of different proportion.
Two, the annular template of RCA is prepared.
Annular template DNA(Circular-Template DNA, CT-DNA for RCA reaction) and connection DNA few nucleosides
The nucleotide sequence of sour chain (Ligation DNA, abbreviation L-DNA) is as shown in table 3.
Underscore is the annular template DNA end sequence complementary with connection DNA.
Overstriking italic is the sequence complementary with miRNA let-7e sequence.
The cyclisation of linear oligonucleotide template is become into circular template.20 μ l of cyclization system total volume is anti-containing 1 × connection
Answer buffer (30 mM pH8.0 Tris-HCl, 4 mM MgCl2, 10 mM (NH4)2SO4, 1.2 mM EDTA, 100 μM of NAD
+, 0.005%BSA), 10 μM of CT-DNA, 10 μM of L-DNA, EDligase 120U.16 DEG C of cyclization temperature, it is cyclized the time
16 hours.After reaction, system is warming up to 95 DEG C, be incubated for 20 minutes, inactivate EDligase.
After cyclization, exonuclease III(exonuclease III, exo-III are utilized) the no cyclization of removal
CT-DNA and L-DNA, obtain the cyclisation product (pure CT-DNA, pCT-DNA) of purifying.20 μ of endonuclease reaction system volume
L contains 1 × exo-III endonuclease reaction buffer (50 mM pH8.0 Tris-HCl, 5 mM MgCl2, 1 mM DTT), wherein
Exo-III 200U, the CT-DNA(circularized CT-DNA, cCT-DNA of cyclisation) 1 μM (37 DEG C of digestion temperature, digestion
The 4 hours time).
After endonuclease reaction, cyclization result and endonuclease reaction result are detected using PAGE silver staining.Digestion is purified
Cyclisation product pCT-DNA to be diluted to 100 nM spare.
Three, let-7 member is detected using the examination of high specific detection of nucleic acids and RCA method.
1. handling sample using high specific nucleic acid detection reagent.
Form heteroduplex: by the specific probe probe 7E of 200 nM, the 5 μ l sample 1 with 100 nM, 5 μ l respectively
It is mixed with 100 nM, 5 μ l sample 2, is warming up to 95 DEG C, be incubated for 5 minutes.Temperature fall is to room temperature.
Endonuclease reaction is gone unless purpose nucleic acid: 10 μ l endonuclease reaction liquid A are added separately to the mixture and sample of sample 1
The mixture of product 2.Endonuclease reaction liquid A include 2 × CEL I buffer (37 DEG C of 66 mM Tris-acetate pH, 7.9 at,
20 mM magnesium acetates, 132 mM potassium acetates, 0.2% (v/v) Tween, 20,2 mM DTT) and 2 μ l of CEL I nuclease.Instead
Answer 55 DEG C of temperature, the reaction time 60 minutes.
Inactivation nuclease: digestion products are warming up to 95 DEG C, incubation time 20 minutes.
2.RCA amplified reaction.
Prepare 100 μ l RCA reaction systems: 1 × phi29 reaction buffer (33 mM Tris-acetate pH 7.9
Under the conditions of 37 DEG C of temperature, 10 mM magnesium acetates, 66 mM potassium acetates, 0.1% (v/v) Tween, 20,1 mM DTT), 1:
10000 SYBR Green II, 1 nM pCT-DNA, every kind of dNTP concentration of dNTPs(be 125 μM), phi29 archaeal dna polymerase
2.5U。
Sample-adding: 100 μ l RCA reaction systems are added in each hole in 96- hole elisa Plates, are kept for 40 DEG C of temperature.
RCA reaction: by the digestion products of the digestion products of 1 μ l sample 1 and 1 μ l sample 2 respectively with to be added to RCA anti-
It answers in system, starting RCA reaction.
Utilize the change in fluorescence of microplate reader detection RCA reaction.Excitation wavelength is 480 nm, and launch wavelength is 530 nm.
3. handling sample 3(Mix1-Mix11 using same step 1 and step 2).
4. testing result.
Using the testing result of high specific nucleic acid detection reagent as shown in Fig. 3-Fig. 6.
Fig. 3 is sample without high specific nucleic acid detection reagent treated testing result.Fig. 3 shows fluorescence intensity
As RCA reaction is gradually risen, but the fluorescence intensity of sample 1 and the result of sample 2 are about the same, show to start RCA
The primer of reaction has target nucleic acid let-7e and non-targeted nucleic acid (other let-7 members) in sample.
Fig. 4 is sample by high specific nucleic acid detection reagent treated testing result.Fluorescence curve shows only sample
Product 1 have obtained RCA amplification, and the fluorescence curve of sample 2 and negative control are essentially identical, react without RCA.The experimental results showed that
Handled by high specific nucleic acid detection reagent, remaining let-7 family member (let-7a, let-7b, let-7c, let-7d and
Let-7f it) is hydrolyzed, RCA reaction cannot be started, realize the effect of high specific detection reagent.
Fig. 5 is sample 3 without high specific nucleic acid detection reagent treated testing result.Sample 3 is sample 1 and sample
Product 2 according to different proportion a series of mixtures.RCA reaction does not distinguish the let-7 family member in mixture.
Fig. 6 is sample 3 by high specific nucleic acid detection reagent treated testing result.As let-7e is in aggregate sample
Being stepped up of ratio (each step is 10%) in product, the fluorescence curve of RCA reaction and constantly increase, have with expected result
One-to-one concentration-dependent relation.
The sharp high specific nucleic acid detection reagent combination round pcr of embodiment 2 detects microRNA let-7 family member
let-7e。
This example recycles inverse first with the sample 1 and sample 2 in high specific nucleic acid detection reagent Processing Example 1
Transcribe realtime quantitative inspection (RT-quantitative polymerase chain reaction, abbreviation RT-
QPCR let-7e) is detected.Without CEL I handle sample as a control group.
Taqman probe RT-qPCR kit in experiment using Thermo-Fisher Scientific company is to above-mentioned
Sample is detected.
1. handling sample using high specific nucleic acid detection reagent.
Form heteroduplex: by the specific probe probe 7E of 200 nM, the 5 μ l sample with 100 nM, 5 μ l respectively
The mixing of 1 and 100 nM, 5 μ l sample 2, is warming up to 95 DEG C, is incubated for 5 minutes.Temperature fall is to room temperature.
Endonuclease reaction is gone unless purpose nucleic acid: 10 μ l endonuclease reaction liquid A are added separately to the mixture and sample of sample 1
2 mixture.Endonuclease reaction liquid A include 2 × CEL I buffer (37 DEG C of 66 mM Tris-acetate pH, 7.9 at, 20
MM magnesium acetate, 132 mM potassium acetates, 0.2% (v/v) Tween, 20,2 mM DTT) and 2 μ l of CEL I nuclease.Reaction
55 DEG C of temperature, the reaction time 60 minutes.
Inactivation nuclease: digestion products are warming up to 95 DEG C, incubation time 20 minutes.
2. reverse transcription reaction (Taqman probe RT- of the following procedure according to Thermo-Fisher Scientific company
QPCR kit operation instructions) 3 μ 5 × RT of l primer and 5 μ l digestion products are mixed, 85 DEG C are warming up to, is incubated for 5 points
Clock.Alternating temperature is incubated for 5 minutes to 60 DEG C.Mixing sample is placed in spare on ice.Configuration reverse transcription reaction liquid is shown in Table 4 on ice.
Reverse transcription reaction liquid is mixed with prefabricated sample, soft concussion mixes, and stands 5 minutes after centrifugation on ice.
Reaction solution after 15 μ l are mixed is put into PCR instrument, and set temperature cycling condition is shown in Table 5.
3.PCR amplified reaction (Taqman probe RT- of the following procedure according to Thermo-Fisher Scientific company
QPCR kit operation instructions) by 10 times of reverse transcription reaction product dilution, it is placed in spare on ice.Preparation qPCR reaction solution is shown in Table
6。
PCR reaction solution is softly shaken to mixing, is put into the detection of Roche LightCycler480 real-time fluorescence quantitative PCR
Instrument, setting thermal circulation parameters are shown in Table 7, starting PCR reaction.
4. testing result.
Fig. 7 is the RT-qPCR result that sample is handled without high specific nucleic acid detection reagent.The CT of sample 1 and sample 2
Value is 24 or so almost without difference, and the CT value of negative control group is greater than 40.The experimental results showed that RT-qPCR method is not
The sample 1(let-7e without the processing of high specific detection reagent can be distinguished) and sample 2(other let-7 members).
Fig. 8 is the RT-qPCR testing result that sample passes through the processing of high specific nucleic acid detection reagent.The CT value of sample 1 does not have
There is difference, is still 24, and the CT value of sample 2 obviously moves back.The experimental results showed that by high specific nucleic acid detection reagent
The specificity of detection microRNA can be improved in treated sample, RT-qPCR method.
SEQUENCE LISTING
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Claims (9)
1. a kind of high specific nucleic acid detection reagent, which is characterized in that the main component of the detection reagent includes detection mesh
Mark the single-chain nucleic acid enzyme of the specific probe of nucleic acid chains and the single nucleic acid strands that can degrade.
2. a kind of application method of high specific nucleic acid detection reagent, which is characterized in that specifically include the following steps:
Step 1: 1. 2. specific probe is added in reaction buffer with containing nucleic acid chains to be detected, carries out denaturation and moves back
Fire；
Step 2: by single-chain nucleic acid enzymeIt is added in reaction buffer, specific time, optimization temperature is incubated at a temperature of optimization
Degree depends on probe length, the nucleotide component for constituting specific probe and the type and single-chain nucleic acid enzyme of specific probe
Most suitable operating temperature；Its reaction time depends on the digesting efficiency of single-chain nucleic acid enzyme；
Step 3: after reaction, reaction system is incubated for 20 minutes at 95 DEG C；
Step 4: by reaction system Temperature fall to room temperature；
Step 5: the reaction system that single-chain nucleic acid enzymatic treatment is crossed carries out detection of nucleic acids.
3. high specific nucleic acid detection reagent as described in claim 1, which is characterized in that the detection target nucleic acid chain
Specific probe is the single nucleic acid strands that length is no less than ten, not more than 30 nucleotide；Specific probe can with it is to be checked
Segment in the nucleic acid chains of survey forms hybrid nucleic acid double-strand；The nucleotide for constituting specific probe can be nucleotide, deoxidation core
Thuja acid and nucleotide derivative, including but not limited to lock nucleic acid, peptide nucleic acid, sulphur nucleic acid, dideoxy nucleotide, methoxy yl nucleosides
Acid.
4. high specific nucleic acid detection reagent as described in claim 1, which is characterized in that can degrade the single-stranded of single nucleic acid strands
Nuclease has the nuclease of the non-duplex structure in degradation hybrid nucleic acid double-strand.
5. high specific nucleic acid detection reagent as described in claim 1, which is characterized in that the core of the non-duplex structure
The non-duplex structure of sour enzyme includes the base-pair for not meeting standard base pairing (A:T, A:U and G:C) principle, or due to nucleosides
The single-stranded of the single-stranded nick or nucleic acid double chain of balloon-shaped structure or cyclic structure or nucleic acid double chain that acid insertion or missing are formed lacks
Mouthful.
6. high specific nucleic acid detection reagent as described in claim 1, which is characterized in that the single nucleic acid strands that can degrade
Single-chain nucleic acid enzyme include but is not limited to S1 nuclease (S1 Nuclease), Mung bean nuclease (Mung Bean
Nuclease), P1 nuclease (P1 Nuclease), BAL bal 31 nuclease bal (31 Nuclease of BAL), ribonuclease A
(Ribonuclease A), celery nuclease (CEL I Nuclease).
7. the high specific nucleic acid detection reagent as described in claim 1, which is characterized in that the core to be detected
Sour chain can be nucleic acid double chain or single nucleic acid strands, can be longer nucleic acid chain or oligonucleotide segment, can be linear nucleic acid chain or ring
Shape nucleic acid chains；The nucleic acid chains to be detected can be DNA chain, RNA chain or the nucleic acid chains containing nucleotide derivative.
8. the application method of high specific nucleic acid detection reagent as claimed in claim 2, which is characterized in that by single-chain nucleic acid enzyme
Processed reaction system carries out detection of nucleic acids and includes any detection technique dependent on nucleic acid amplification method or do not depend on
In the detection technique of nucleic acid amplification method.
9. the application method of high specific nucleic acid detection reagent as claimed in claim 2, which is characterized in that described depends on
The detection technique of nucleic acid amplification method is based on thermal cycle nucleic acid amplification technologies or based on constant temperature nucleic acid amplification technology.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110779970A (en) * | 2019-09-18 | 2020-02-11 | 南京农业大学 | Electrochemical detection method for chicken infectious bronchitis virus H120 strain |
| CN111254206A (en) * | 2019-11-21 | 2020-06-09 | 辽宁中晨优智医疗技术有限公司 | Detection method of mycobacterium tuberculosis drug-resistant strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667252A (en) * | 2012-09-10 | 2014-03-26 | 思洛生物技术股份有限公司 | Nucleic acid amplification method |
| CN103866025A (en) * | 2014-03-18 | 2014-06-18 | 中国海洋大学 | Pre-amplification method for nucleic acid and application thereof |
| CN106119371A (en) * | 2016-06-12 | 2016-11-16 | 深圳市人民医院 | A kind of method utilizing strand specific nucleic acid enzyme detection microRNA content |
-
2019
- 2019-03-04 CN CN201910160705.2A patent/CN109852670A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667252A (en) * | 2012-09-10 | 2014-03-26 | 思洛生物技术股份有限公司 | Nucleic acid amplification method |
| CN103866025A (en) * | 2014-03-18 | 2014-06-18 | 中国海洋大学 | Pre-amplification method for nucleic acid and application thereof |
| CN106119371A (en) * | 2016-06-12 | 2016-11-16 | 深圳市人民医院 | A kind of method utilizing strand specific nucleic acid enzyme detection microRNA content |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110779970A (en) * | 2019-09-18 | 2020-02-11 | 南京农业大学 | Electrochemical detection method for chicken infectious bronchitis virus H120 strain |
| CN110779970B (en) * | 2019-09-18 | 2022-04-12 | 南京农业大学 | Electrochemical detection method for chicken infectious bronchitis virus H120 strain |
| CN111254206A (en) * | 2019-11-21 | 2020-06-09 | 辽宁中晨优智医疗技术有限公司 | Detection method of mycobacterium tuberculosis drug-resistant strain |
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