CN107663530A - A kind of single-chain nucleic acid detection kit, method and its application - Google Patents
A kind of single-chain nucleic acid detection kit, method and its application Download PDFInfo
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- CN107663530A CN107663530A CN201711107037.4A CN201711107037A CN107663530A CN 107663530 A CN107663530 A CN 107663530A CN 201711107037 A CN201711107037 A CN 201711107037A CN 107663530 A CN107663530 A CN 107663530A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
Abstract
The invention belongs to field of molecular detection, and in particular to a kind of single-chain nucleic acid detection kit, method and its application, shown kit include ring-type DNAzyme and molecular beacon, and the ring-type DNAzyme has DNAzyme sequence.The kit can be under RNase H auxiliary, operated by the simplicity of one-step method, Two-way Cycle amplification is carried out to the fluorescence signal of molecular beacon, so as to realize the high sensitivity specific detection to single-chain nucleic acid, is finally applied in the detections of single-chain nucleic acid such as virus or miRNA.
Description
Technical field
The present invention relates to field of molecular detection, in particular to a kind of single-chain nucleic acid detection kit, method and
It is applied.
Background technology
The nucleic acid detection method advantage such as have high sensitivity, high specificity and stability good, therefore in each research field
In be obtained for and be widely applied.Wherein, nucleic acid signal amplifying technique is that one kind is realized using nucleic acid elements by amplification cycles
The detection technique of the signal enhancing such as optics or electrochemistry in system.Nucleic acid signal amplifying technique can be realized to nucleic acid molecules, egg
The high-sensitivity detection of white matter, small-molecule substance or ion, therefore, it is widely used in Food Science, molecular biosciences in recent years
The numerous areas such as and medical science.
At present, researchers have been developed such as based on nucleic acid polymerase, hydrolase nucleic acid, DNAzyme and nano material
Multiple nucleic acids signal amplification technique.For example most at present is the signal amplification skill based on PCR (PCR)
Art, i.e., molecular beacon is added in PCR system, fluorescence is discharged by the combination of molecular beacon and PCR primer, this method is not
Only need the alternating temperature controller unit of precision and the detection to single stranded oligonucleotide has certain limitation.For another example, researchers
A kind of single cycle nucleic acid signal amplifying technique for being based on ribonuclease H (RNase H) is also developed, i.e., is carried by ring portion
The molecular beacon of RNA sequence hybridizes with target dna, by the RNA partial hydrolysis of molecular beacon in the presence of RNase H, so that
Discharge fluorophor.Although the target sequence of this technology can be single stranded oligonucleotide, because it is that single cycle is linearly believed
Number amplifying technique, signal amplification efficiency is relatively low so as to caused signal intensity deficiency in certain time, cause detection sensitivity compared with
It is low.In addition, for some Two-way Cycles or multi-cycle signal amplification technique, because the element in different circulations interferes with each other, need to lead to
Cross many more manipulations and realize that signal amplifies, add the fussy degree of operation, while also increase the possibility of sample pollution.
Therefore, in view of many weak points in nucleic acid signal amplifying technique, one kind are applied to single-chain nucleic acid and detected at present
Efficient constant-temperature and nucleic acid signal amplifying technique easy to operate urgently develop.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of single-chain nucleic acid detection kit, and the Two-way Cycle constant temperature based on the kit
Nucleic acid signal amplification method, the kit can be operated under RNase H auxiliary, molecule believed by the simplicity of one-step method
Target fluorescence signal carries out Two-way Cycle amplification, so as to realize the high sensitivity specific detection to single-chain nucleic acid, is finally applied to
In the detection of single-chain nucleic acid such as virus or Microrna (miRNA).
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
The present invention relates to a kind of single-chain nucleic acid detection kit, and it includes ring-type DNAzyme and molecular beacon；
The ring-type DNAzyme is connected what is obtained for the sequence of RNA cutting-type DNAzymes with complementary nucleic acid fragment head and the tail
Single stranded circle sequence；
The complementary nucleic acid fragment can be complementary with Single-stranded DNA fragments to be checked, or obtained with RNA reverse transcriptions to be checked
Single-stranded cDNA is complementary；The interlude of the complementary nucleic acid fragment is ribonucleotide site A, and remainder is dezyribonucleoside
Acid, the ribonucleotide site A are RNase H restriction enzyme site；It is described after the ribonucleotide site A is digested
The section of complementary nucleic acid fragment splits the two-arm that part forms the DNAzyme；
The molecular beacon contains complementary region, and the interlude of the complementary region is ribonucleotide site B, the molecule letter
It is deoxyribonucleotide to put in addition to the ribonucleotide site B；It is reserved with the ribonucleotide site B de-
Oxygen ribozyme restriction enzyme site, the complementary region sequence at the DNAzyme restriction enzyme site both ends are mutual with the two-arm of the DNAzyme respectively
Mend；
The molecular beacon also carries fluorophor and quenching group, and the fluorophor and quenching group are located at respectively
The both sides of the DNAzyme restriction enzyme site.
According to an aspect of the present invention, the invention further relates to a kind of single-chain nucleic acid detection method, including：
Measuring samples and ring-type DNAzyme as described above, molecular beacon, and RNase H, reaction buffer are mixed
After conjunction, appropriate time is reacted at a constant temperature and detects reaction system fluorescent value；By the fluorescent value and it is not added with measuring samples
The background fluorescence activity of control group be compared to judge that single-chain nucleic acid to be checked is present.
The concrete principle of the kit and method is shown in Fig. 1, first the complementary series on ring-type DNAzyme (cDz) with it is to be checked
Target dna (Target DNA) combine and carry out digestion using RNase H, obtain the linearly sequence containing DNAzyme；Line
Property DNAzyme and molecular beacon (rMBs) combine, and be two free oligonucleotides by molecular beacon digestion, so as to discharge
Fluorescence.
According to an aspect of the present invention, the invention further relates to kit as described above and method as described above to examine
The application surveyed in single-chain nucleic acid genomic viral or miRNA.
Compared with prior art, beneficial effects of the present invention are：
The present invention is Two-way Cycle signal amplification technique, is amplified compared to single cycle linear signal, signal amplification efficiency is significantly
Improve.Also, because the reaction condition of two circulations is similar and does not interfere with each other, therefore two can be circulated in same system
Carry out simultaneously, this, which allows for reaction, to be detected by single stepping, enormously simplify operating procedure and reduce pollution
Possibility.In addition, the present invention is the nucleic acid detection technique under constant temperature, it is not necessary to accurate variable temperature unit, is had higher
Actual application value.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The required accompanying drawing used is briefly described in embodiment or description of the prior art, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the principle schematic of method provided by the present invention；
Fig. 2 is the preparation principle schematic diagram of ring-type DNAzyme；
Fig. 3 is the signal amplification efficiency result figure of the embodiment of the present invention 1；
Fig. 4 is the signal amplification efficiency result figure of the embodiment of the present invention 2；
Fig. 5 is the signal amplification efficiency result figure of the embodiment of the present invention 3.
Embodiment
The present invention relates to a kind of single-chain nucleic acid detection kit, and it includes ring-type DNAzyme and molecular beacon；
The ring-type DNAzyme is connected what is obtained for the sequence of RNA cutting-type DNAzymes with complementary nucleic acid fragment head and the tail
Single stranded circle sequence；
The complementary nucleic acid fragment can be complementary with Single-stranded DNA fragments to be checked, or obtained with RNA reverse transcriptions to be checked
Single-stranded cDNA is complementary；The interlude of the complementary nucleic acid fragment is ribonucleotide site A, and remainder is dezyribonucleoside
Acid, the ribonucleotide site A are RNase H restriction enzyme site；It is described after the ribonucleotide site A is digested
The section of complementary nucleic acid fragment splits the two-arm that part forms the DNAzyme；
The molecular beacon contains complementary region, and the interlude of the complementary region is ribonucleotide site B, the molecule letter
It is deoxyribonucleotide to put in addition to the ribonucleotide site B；It is reserved with the ribonucleotide site B de-
Oxygen ribozyme restriction enzyme site, the complementary region sequence at the DNAzyme restriction enzyme site both ends are mutual with the two-arm of the DNAzyme respectively
Mend；
The molecular beacon also carries fluorophor and quenching group, and the fluorophor and quenching group are located at respectively
The both sides of the DNAzyme restriction enzyme site.
Preferably, Single-stranded DNA fragments to be checked include genomic DNA, mitochondrial DNA, chloroplast DNA, DNA.
Preferably, RNA to be checked includes miRNA, mRNA, siRNA, snRNA, snoRNA, rRNA, tRNA.
Preferably, the Single-stranded DNA fragments to be checked, or the obtained single-stranded cDNA of RNA reverse transcriptions to be checked length choosing
From 14~40 bases, more preferably 16~30 bases, 18~20 bases can also be selected.
Preferably, single-chain nucleic acid detection kit as described above, the ribonucleotide site A are continuous by 4,5 or 6
Ribonucleotide composition；Ribonucleotide site A ribonucleotide hyper acid can cause RNase H digestion specificity drop
Low, crossing can cause RNase H digesting efficiency to reduce at least.
Preferably, the ribonucleotide site B is made up of 1-6 continuous ribonucleotides, and more preferably 1-2 even
Continuous ribonucleotide；3,4,5 can also be selected.
Preferably, the DNAzyme restriction enzyme site is made up of 1-2 continuous ribonucleotides.
Preferably, single-chain nucleic acid detection kit as described above, the kit also include RNase H and/or reaction
Buffer solution；
In the reaction buffer at least containing RNase H and DNAzyme needed for confactor, usually from metal sun
Ion, can also containing pH buffer reagents, ATP and protection enzyme molecule on reproducibility group reducing agent in one kind or
A variety of, the conventional reagent of debita spissitudo, such as DTT (dithiothreitol (DTT)) can be selected in the reducing agent.
Preferably, the kit also includes reverse transcription reagents, such as stem ring primer, reverse transcriptase, dNTP, reverse transcription delay
One or more in fliud flushing.
Preferably, single-chain nucleic acid detection kit as described above, the RNA cutting-types DNAzyme include 10-23 types
Any of DNAzyme, 8-17 types DNAzyme and " gun type " DNAzyme.
Preferably, single-chain nucleic acid detection kit as described above, the fluorophor and the quenching group are located at institute
State the both ends or inside of molecular beacon, preferably both ends；
Preferably, the fluorophor include FITC, FAM, VIC, ROX, TET, Texas Red, HEX, TAMRA, Cy3,
One or more in Cy5, Rhodamine B and Perylene；
Preferably, the quenching group includes Dabcyl, NFQ, QYS-7, BHQ1, BHQ2, BHQ3, Anthraquinone
And the one or more in TAMRA.
Preferably, the sequence of the molecular beacon can only contain the complementary region, can also be the two of the complementary region
Extend extra sequence in end；It is furthermore preferred that extra sequence is extended at the both ends has complementary region；It is furthermore preferred that
It is DNA that extra sequence is extended at the both ends, and sequence number can select often to survey 1~14, or 2~12
It is individual, or 4~8 bases.
Preferably, single-chain nucleic acid detection kit as described above, the molecular beacon is including with loop-stem structure and not
Single stranded oligonucleotide with loop-stem structure.
Preferably, single-chain nucleic acid detection kit as described above, the preparation method of the ring-type DNAzyme include：
By linear DNAzyme-single-stranded effect in DNA ligase of complementary nucleic acid fragment oligonucleotides of 5 ' end phosphorylations
Lower connection cyclization；
Preferably, when connecting cyclic in the presence of DNA ligase, connected by the use of short chain DNA as template, or it is single with connection
The enzyme of chain DNA is directly connected to；It is furthermore preferred that the double stranded section at junction both ends is more than 5bp respectively.The system of ring-type DNAzyme
Standby principle schematic is as shown in Figure 2.
Preferably, the DNA ligase includes T4 DNA ligases, T3 DNA ligases, T7 DNA ligases, Taq
DNA ligase, E.Coli DNA ligases, 9 ° of NTM DNA ligases, CircLigaseTMssDNA Ligase、
CircLigaseTMAny of ssDNA Ligase II.
Preferably, single-chain nucleic acid detection kit as described above, the preparation method of the ring-type DNAzyme also include
Purification step, the method for the purifying include：With PAGE gel extractions, HPLC is purified, or removes not cyclized widow with excision enzyme
Nucleotide single-chain, phenol-chloroform extracting removing protein, then concentrated with ethanol precipitation or directly used glue reclaim, taken off with enriching and purifying ring-type
Oxygen ribozyme.
According to an aspect of the present invention, the invention further relates to a kind of single-chain nucleic acid detection method, including：
Measuring samples and ring-type DNAzyme as described above, molecular beacon, and RNase H, reaction buffer are mixed
After conjunction, appropriate time is reacted at a constant temperature and detects reaction system fluorescent value；By the fluorescent value and it is not added with measuring samples
The background fluorescence activity of control group be compared to judge that single-chain nucleic acid to be checked is present.
Optionally, when the single-chain nucleic acid to be checked is RNA, also include before each component is mixed：Will be described to be checked
Single-chain nucleic acid reverse transcription is cDNA.
Preferably, described steady temperature is 35 DEG C~40 DEG C；Described appropriate time is 30min~60min.
According to an aspect of the present invention, the invention further relates to kit as described above and method as described above to examine
The application surveyed in single-chain nucleic acid genomic viral or miRNA.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products of acquisition purchased in market can be passed through.
Embodiment 1
1st, the preparation and purifying of ring-type DNAzyme
It is single-stranded (containing DNAzyme sequence) that the oligonucleotides of 1 μM of 5 ' end phosphorylation modifications is added into ringed systems, 2
μM DNA profiling chain and 5U T4 DNA Ligase (being purchased from Thermo Scientific companies), and add 1 × T4 DNA
Ligase Buffer (Tris-HCl containing 40mM, 10mM MgCl2, 25 DEG C of 7.8@of 10mM DTT, 0.5mM ATP, pH), it is overall
It is for 100 μ L.In PCR instrument after 20 DEG C of isothermal reaction 12h, 65 DEG C of warm bath 10min are by enzyme-deactivating.
After cyclic product is concentrated into 10 μM or so, gel extraction, alcohol are carried out using 15% denaturing polyacrylamide gel
The product of heavy gel extraction, after aqua sterilisa dissolving, the concentration of ring-type DNAzyme is determined using Nanodrop2000.
2nd, signal iodine
200nM molecular beacons are added into signal amplification system, and (loop-stem structure, fluorophor FAM, quenching group are
Dabcyl), 100nM ring-types DNAzyme and 10pM-100nM target dna are (in the nonstructural gene NS1 of human parvovirus B19
One section of sequence), 1 × RNase H Reaction Buffer (75mM KCl, 50M Tris-HCl, 3mM MgCl2, 10mM
25 DEG C of 8.3@of DTT, pH), 17mM MgCl2(MgCl in system2Final concentration of 20mM), 1U RNase H (are purchased from Thermo
Scientific companies) and 8U RNase Inhibitor (Thermo Scientific), total system be 50 μ L.In PCR instrument
After 37 DEG C are reacted a period of time, 50 μ L 20mM EDTA solution are added into system and 65 DEG C of warm bath 20min are by enzyme-deactivating.
The negative control group for being added without target dna is set to be contrasted simultaneously.
100 μ L samples are added in 96 orifice plates, using the fluorescent value of ELIASA determination sample, excitation wavelength is set to
495nm, wavelength of fluorescence are set to 520nm.In this experiment, (F-F is usedB)÷(F0-FB) come represent the signal of molecular beacon amplify
Efficiency.Wherein F is that molecular beacon is deoxidized the fluorescent value after ribozyme digestion, F0Only to add molecular beacon, target dna is not added
Background fluorescence activity, FBThe fluorescent value of reaction buffer during to be added without molecular beacon.As (F-FB)÷(F0-FB)>When 1, explanation
The signal of molecular beacon is exaggerated, and (F-FB)÷(F0-FB) value show that signal amplification efficiency is higher more greatly.
Result in Fig. 3 shows that method for amplifying signal of the invention can realize the signal amplification to target dna, test limit
Up to 10pM.When the concentration of target dna is 100nM, (F-FB)÷(F0-FB) value up to 2.5.
The target dna, the DNAzyme oligonucleotides that are used in the embodiment 1 of table 1. be single-stranded and molecular beacon
Note：Overstriking and underscorePart is ribonucleotide, and remaining is deoxyribonucleotide
Embodiment 2
1st, the preparation and purifying of ring-type DNAzyme
Hold the oligonucleotides of phosphorylation modifications is single-stranded (to contain DNAzyme sequence in add 0.5 μM into ringed systems 5 '
Row), 100U CircLigaseTMSsDNA Ligase II (are purchased from Epicenter companies), 1 × CircLigase II
Reaction Buffer (Tris-Ac containing 33mM, 66mM KAc, 0.5mM DTT, pH 7.5), 2.5mM MnCl2With 1M beets
Alkali, total system are 100 μ L.In PCR instrument after 60 DEG C of isothermal reaction 16h, 80 DEG C of warm bath 10min are by enzyme-deactivating.
Added in ringed systems 2 μ L Exonulease I (being purchased from Thermo Scientific companies) and 1 ×
Reaction Buffer (glycine-KOH containing 67mM, 6.7mM MgCl2, 1mM DTT, pH 7.5), 37 DEG C of isothermal reaction 2h
Afterwards, 80 DEG C of warm bath 15min are by enzyme-deactivating.Cyclic product alcohol precipitation after digestion is purified to remove mononucleotide and ion therein
Deng impurity.Finally, after adding the DNA dissolvings that 20 μ L aqua sterilisas obtain alcohol precipitation, ring-type deoxidation is determined using Nanodrop2000
The concentration of ribozyme.
2nd, signal iodine
100nM molecular beacons are added into signal amplification system, and (loop-stem structure, fluorophor Perylene, is quenched base
Group is Anthraquinone), 50nM ring-types DNAzyme and 10pM-100nM target dna is (in Ebola viral G protein genes
One section of sequence), 1 × RNase H Reaction Buffer (75mM KCl, 50M Tris-HCl, 3mM MgCl2, 10mM
25 DEG C of 8.3@of DTT, pH), 37mM MgCl2(MgCl in system2Final concentration of 40mM), RNase H (Thermo
Scientific) and 8U RNase Inhibitor (Thermo Scientific), total system are 50 μ L.Through 37 in PCR instrument
After DEG C reaction a period of time, 50 μ L 40mM EDTA solution are added into system and 65 DEG C of warm bath 20min are by enzyme-deactivating.Simultaneously
The negative control group for being added without target dna is set to be contrasted.
100 μ L samples are added in 96 orifice plates, using the fluorescent value of ELIASA determination sample, excitation wavelength is set to
426nm, wavelength of fluorescence are set to 500nm.In this experiment, (F-F is usedB)÷(F0-FB) come represent the signal of molecular beacon amplify
Efficiency.Wherein F is molecular beacon by the fluorescent value after DNAzyme digestions, F0Only to add molecular beacon, target dna is not added
Or background fluorescence activity during linear DNA zyme, FBThe fluorescent value of reaction buffer during to be added without molecular beacon.As (F-FB)÷
(F0-FB)>When 1, illustrate that the signal of molecular beacon is exaggerated, and (F-FB)÷(F0-FB) value show signal amplification efficiency more greatly
It is higher.
Result in Fig. 4 shows that method for amplifying signal of the invention can realize the signal amplification to target dna, test limit
Up to 10pM.When the concentration of target dna is 100nM, (F-FB)÷(F0-FB) value up to 4.5.
The target dna, the DNAzyme oligonucleotides that are used in the embodiment 2 of table 2. be single-stranded and molecular beacon
Note：Overstriking and underscorePart is ribonucleotide, and remaining is deoxyribonucleotide
Embodiment 3
1st, the preparation and purifying of ring-type DNAzyme
It is single-stranded (containing DNAzyme sequence) that the oligonucleotides of 1 μM of 5 ' end phosphorylation modifications is added into ringed systems, 2
μM DNA profiling chain and 5U T4 DNA Ligase (being purchased from Thermo Scientific companies), and add 1 × T4 DNA
Ligase Buffer (Tris-HCl containing 40mM, 10mM MgCl2, 25 DEG C of 7.8@of 10mM DTT, 0.5mM ATP, pH), it is overall
It is for 100 μ L.In PCR instrument after 20 DEG C of isothermal reaction 12h, 65 DEG C of warm bath 10min are by enzyme-deactivating.
After cyclic product is concentrated into 10 μM or so, gel extraction, alcohol are carried out using 15% denaturing polyacrylamide gel
The product of heavy gel extraction, after aqua sterilisa dissolving, the concentration of ring-type DNAzyme is determined using Nanodrop2000.
2nd, stem ring primer method is to miRNA reverse transcriptions
(1) pretreatment of stem ring primer：Stem ring primer is diluted to 2 μM, 95 DEG C and then slowly drop are warming up in PCR instrument
Temperature (0.1 DEG C/s) is to 20 DEG C, so that primer can form preferable loop-stem structure.
(2) stem ring primer and miRNA anneal：The miRNA, 1 μ L 10mM of various concentrations are added into annealing system
DNTP, 12 μM of μ L stem ring primers, total system are 12.5 μ L.By the sample configured in 16 DEG C of warm bath 15min so that stem ring primer
Fully hybridize combination with miRNA.
(3) miRNA reverse transcription：10U RNase Inhibitor are added in (2)；200U Reverse
Transcriptase and 4 μ 5 × Reaction of L Buffer (250mM Tris-HCl, 250mM KCl, 20mM MgCl2,
25 DEG C of 8.3@of 50mM DTT, pH), then in PCR instrument 35 DEG C of reactions 1h, last 80 DEG C of warm bath 15min by enzyme-deactivating.
3rd, signal iodine
100nM molecular beacons are added into signal amplification system, and (non-loop-stem structure, fluorophor Perylene, is quenched
Group is Anthraquinone), 50nM ring-types DNAzyme and 10pM-100nM miRNA reverse transcriptions obtained cDNA, 1 ×
RNase H Reaction Buffer (75mM KCl, 50M Tris-HCl, 3mM MgCl2, the@25 of 10mM DTT, pH 8.3
DEG C), 37mM MgCl2(MgCl in system2Final concentration of 40mM), RNase H (Thermo Scientific) and 8U RNase
Inhibitor (Thermo Scientific), total system are 50 μ L.In PCR instrument after 37 DEG C are reacted a period of time, Xiang Ti
50 μ L 40mM EDTA solution are added in system and 65 DEG C of warm bath 20min are by enzyme-deactivating.The moon for being added without target dna is set simultaneously
Property control group is contrasted.
100 μ L samples are added in 96 orifice plates, using the fluorescent value of ELIASA determination sample, excitation wavelength is set to
426nm, wavelength of fluorescence are set to 500nm.Using in this experiment, using in this experiment, (F-F is usedB)÷(F0-FB) carry out table
Show the signal amplification efficiency of molecular beacon.Wherein F is molecular beacon by the fluorescent value after DNAzyme digestions, F0Only to add point
Sub- beacon, background fluorescence activity during targetDNA or linear DNA zyme, F are not addedBReacted during to be added without molecular beacon slow
The fluorescent value of fliud flushing.As (F-FB)÷(F0-FB)>When 1, illustrate that the signal of molecular beacon is exaggerated, and (F-FB)÷(F0-FB)
Value shows that more greatly signal amplification efficiency is higher.
Result in Fig. 5 shows that method for amplifying signal of the invention can realize that the cDNA obtained to reverse transcription signal is put
Greatly, test limit is up to 100pM.When cDNA concentration is 100nM, (F-FB)÷(F0-FB) value up to 3.5.
The miRNA, stem ring primer, the DNAzyme oligonucleotides that are used in the embodiment 3 of table 3. be single-stranded and molecular beacon
Note：Overstriking and underscorePart is ribonucleotide, and remaining is deoxyribonucleotide
The result of the above embodiments shows that the method for the present invention has preferable detection sensitivity, and signal amplification efficiency
It is higher.The fast and convenient specific detection of target dna can be realized by single stepping, therefore there is good popularization and application valency
Value.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. a kind of single-chain nucleic acid detection kit, it includes ring-type DNAzyme and molecular beacon；
The ring-type DNAzyme is connected to obtain from beginning to end single-stranded for sequence and the complementary nucleic acid fragment of RNA cutting-type DNAzymes
Ring-shaped sequence；
The complementary nucleic acid fragment can be complementary with Single-stranded DNA fragments to be checked, or obtains with RNA reverse transcriptions to be checked single-stranded
CDNA is complementary；The interlude of the complementary nucleic acid fragment is ribonucleotide site A, and remainder is deoxyribonucleotide,
The ribonucleotide site A is RNase H restriction enzyme site；After the ribonucleotide site A is digested, the complementation
The section of nucleic acid fragment splits the two-arm that part forms the DNAzyme；
The molecular beacon contains complementary region, and the interlude of the complementary region is ribonucleotide site B, on the molecular beacon
It is deoxyribonucleotide in addition to the ribonucleotide site B；Deoxidation core is reserved with the ribonucleotide site B
Enzyme restriction enzyme site, the complementary region sequence at the DNAzyme restriction enzyme site both ends are complementary with the two-arm of the DNAzyme respectively；
The molecular beacon also carries fluorophor and quenching group, and the fluorophor and quenching group are respectively positioned at described
The both sides of DNAzyme restriction enzyme site.
2. single-chain nucleic acid detection kit according to claim 1, it is characterised in that the ribonucleotide site A by
4-6 continuous ribonucleotide compositions；
Preferably, the ribonucleotide site B is made up of 1-6 continuous ribonucleotides, and more preferably 1-2 continuous
Ribonucleotide；
Preferably, the DNAzyme restriction enzyme site is made up of 1-2 continuous ribonucleotides.
3. single-chain nucleic acid detection kit according to claim 1, it is characterised in that the kit also includes RNase
H and/or reaction buffer；
Preferably, the kit also includes reverse transcription reagents, such as stem ring primer, reverse transcriptase, dNTP, RT Buffer
In one or more.
4. single-chain nucleic acid detection kit according to claim 1, it is characterised in that the RNA cutting-types DNAzyme
Including any of 10-23 types DNAzyme, 8-17 types DNAzyme and " gun type " DNAzyme.
5. single-chain nucleic acid detection kit according to claim 1, it is characterised in that the fluorophor and described be quenched
Group is located at the both ends or inside of the molecular beacon, preferably both ends；
Preferably, the fluorophor include FITC, FAM, VIC, ROX, TET, Texas Red, HEX, TAMRA, Cy3, Cy5,
One or more in Rhodamine B and Perylene；
Preferably, the quenching group include Dabcyl, NFQ, QYS-7, BHQ1, BHQ2, BHQ3, Anthraquinone and
One or more in TAMRA.
6. single-chain nucleic acid detection kit according to claim 1, it is characterised in that the molecular beacon includes having stem
Ring structure and the single stranded oligonucleotide without loop-stem structure.
7. single-chain nucleic acid detection kit according to claim 1, it is characterised in that the preparation of the ring-type DNAzyme
Method includes：
Hold linear DNAzyme-complementary nucleic acid fragment oligonucleotides of phosphorylations is single-stranded to connect in the presence of DNA ligase by 5 '
It is connected into ring；
Preferably, when connecting cyclic in the presence of DNA ligase, connected by the use of short chain DNA as template, or it is single-stranded with connecting
DNA enzyme is directly connected to；It is furthermore preferred that the double stranded section at junction both ends is more than 5bp respectively；
Preferably, the DNA ligase includes T4 DNA ligases, T3 DNA ligases, T7 DNA ligases, Taq DNA companies
Meet enzyme, E.Coli DNA ligases, 9oNTM DNA ligases, CircLigaseTMssDNA Ligase、
CircLigaseTMAny of ssDNA Ligase II.
8. single-chain nucleic acid detection kit according to claim 7, it is characterised in that the preparation of the ring-type DNAzyme
Method also includes purification step, and the method for the purifying includes：With PAGE gel extractions, HPLC is purified, or is removed with excision enzyme
Not cyclized oligonucleotides is single-stranded, phenol-chloroform extracting removing protein, then is concentrated with ethanol precipitation or directly used glue reclaim, with enrichment
Purified cyclic DNAzyme.
A kind of 9. single-chain nucleic acid detection method, it is characterised in that including：
By the ring-type DNAzyme described in measuring samples and claim any one of 1-8, molecular beacon, and it is RNase H, anti-
After answering buffer solution to mix, appropriate time is reacted at a constant temperature and detects reaction system fluorescent value, by the fluorescent value with not adding
The background fluorescence activity of the control group of measuring samples is added to be compared to judge that single-chain nucleic acid to be checked is present；
Optionally, when the single-chain nucleic acid to be checked is RNA, also include before each component is mixed：Will be described to be checked single-stranded
Nucleic acid reverse-transcription is cDNA；
Preferably, described steady temperature is 35 DEG C~40 DEG C；Described appropriate time is 30min~60min.
10. the method described in kit and claim 9 described in any one of claim 1~8 is in detection single-chain nucleic acid base
Because of the application in group virus or miRNA.
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