CN109266784A - A kind of enclosed type DNA biological sensor and its application in detection H1N1virus - Google Patents

A kind of enclosed type DNA biological sensor and its application in detection H1N1virus Download PDF

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CN109266784A
CN109266784A CN201810843634.1A CN201810843634A CN109266784A CN 109266784 A CN109266784 A CN 109266784A CN 201810843634 A CN201810843634 A CN 201810843634A CN 109266784 A CN109266784 A CN 109266784A
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CN109266784B (en
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李宇彬
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Guangdong Ocean University
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Abstract

The invention discloses a kind of enclosed type DNA biological sensor and its applications in detection H1N1virus.The sensor includes assist probes, signal probe and the fluorescent dye that can be specifically bound with signal probe；Wherein, the sequence of signal probe is 5'-TCCTATTGTGACTTTGGGTAGGGCGGGTTGGG-3'；The sequence of assist probes is 5'-GCCCTACCCGGGTGTATATTCTG-3'.The variation that enclosed type DNA biological sensor of the invention passes through fluorescent value, the specific single-chain DNA sequence dna concentration of H1N1virus can quickly be detected, have many advantages, such as that high sensitivity, specificity are good, is convenient for normalizing operation, is suitable for Clinical screening, certain theoretical direction is provided for the exploitation of the quick testing product of H1N1virus, the clinical monitoring of right pop virus is of great significance.

Description

A kind of enclosed type DNA biological sensor and its in detection Influenza A H1N1 disease Application in poison
Technical field
The invention belongs to biochemical analysis technical fields.It is sensed more particularly, to a kind of enclosed type DNA biological Device and its application in detection H1N1virus.
Background technique
H1N1 is a kind of RNA virus of Orthomyxoviridae family, the variation and propagation of the host by H1N1 virus, it will lead Cause the outburst and propagation of epidemic situation and the influenza large area of the mankind.This has been broken out in countries such as the U.S., Mexico in March, 2009 Kind novel H1N1virus, then quickly spreads in world wide, results in a large-scale viral disaster, to people and The harm of animal is all extremely serious.In the later period of H1N1 influenza virus epidemic situation prevalence, Influenza epidemic situation still different regions not between Disconnected outburst, and influenza virus may greatly make a variation and the matter that develops drug resistance, and variation can allow it to become more to be easy to propagate Influenza virus and pathogenic bacteria, drastically influence the health and public health security of global human and animal.It therefore is screening Influenza doubtful case controls the sprawling of influenza virus, and monitors popularity of this virus in animal simultaneously, there is an urgent need to Exploitation is directed to the quickly and effectively detection method of this novel H1N1virus.
Currently, there are many method of detection and diagnosis H1N1virus both at home and abroad, it mainly include traditional virus point From technical methods such as identification method, immunofluorescence technique, real-time fluorescence quantitative PCRs.It is traditional when being separately cultured detection technique culture Between it is longer, operating process is relative complex, aseptically to operate, and to the relevant professional knowledge and operating technology of operator It is required that it is relatively high, this method is limited in clinical and non-lab environment application.Immunofluorescence passes through fluorescence microscopy Mirror sees whether that with the presence of viral antigen so as to judge whether to be infected, sensitivity and operator acquire and mark This quality, the content of sample and relevant speciality operating technology has substantial connection, so the sensitivity of immunofluorescence is not As it is expected that stablize, require further improvement with it is perfect.Real-time fluorescence quantitative PCR detection specificity with higher and sensitivity Property, but also will appear false negative result, in entire detection process, to operator, accurate laboratory apparatus and integral laboratory The requirement of facility has higher technical requirements, is not suitable for the detection of base's Large-scale Screening.
Therefore, there is an urgent need to develop convenient and efficient, highly sensitive, high specific and be suitable for the diagnostic method of Clinical screening To meet the detection needs of H1N1virus.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiency, provide a species specificity and High sensitivity, selectivity are good, detect the fireballing enclosed type DNA biological sensor for being able to detect single stranded DNA.The closing Type DNA biological sensor can quickly detect the specific single-chain of H1N1virus by the variation of fluorescent value DNA sequence dna concentration provides certain theoretical direction, right pop disease for the exploitation of the quick testing product of H1N1virus The clinical monitoring of poison is of great significance.
The object of the present invention is to provide a kind of enclosed type DNA biological sensors.
A second object of the present invention is to provide above-mentioned enclosed type DNA biological sensors in detection Influenza A H1N1 Application in virus.
Third object of the present invention is to provide a kind of methods for detecting H1N1virus.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of enclosed type DNA biological sensor, including assist probes, signal probe and can be carried out with signal probe The fluorescent dye of specific binding；
The sequence of signal probe is 5'-TCCTATTGTGACTTTGGGTAGGGCGGGTTGGG-3'；
The sequence of assist probes is 5'-GCCCTACCCGGGTGTATATTCTG-3'.
Influenza virus H1N1 (Influenza A virus) nucleic acid sequence (A/Puerto Rico/8/1934 (H1N1)) from (HE802058.1) is obtained in Gen Bank database.The present invention is largely analyzed the influenza virus of itself and other hypotypes Comparison and scientific validation obtain and have both high specific and highly sensitive influenza virus H1N1 specific sequence segment, by 27 Base is constituted, using the specific sequence segment as the target sequence of inventive closure type DNA biological sensor.
In the sequence of the signal probe, genetic fragment 5'-TCCTATTGTGACT-3' is object chain identification sequence, can It is complementary with the sequence fragment of Target DNA；Genetic fragment 5'-GGGTAGGGCGGGTTG GG-3' is tetra- serobila DNA enzymatic sequence of G- Column.
In the sequence of the assist probes, genetic fragment 5'-GGGTGTATATTCTG-3' is object chain identification sequence, energy It is enough complementary with the sequence fragment of Target DNA；Genetic fragment 5'-GCCCTACCC-3' is 9 bases, can be with signal probe Sequence fragment is complementary.
The present invention is designed using H1N1 specific sequence segment as target dna further according to the complementary pairing principle of base Assist probes and signal probe with tetra- stranded structure of G-, assist probes include that object chain identification sequence and discernible signal are visited The sequence of needle, signal probe include object chain identification sequence and tetra- serobila sequence of G-, tetra- stranded structures of G- of signal chains can with it is glimmering Photoinitiator dye is specifically bound, and generates fluorescence signal, and signal chains, auxiliary chain and object chain hybridization is made to form a kind of enclosed type DNA double chain, to construct a kind of highly sensitive DNA biological sensor of detection H1N1 associated dna sequence.The biography Sensor convenient, fast, sensitively can detect H1N1 specific single-chain DNA sequence dna concentration by the variation of fluorescent value, for inspection Flow measurement Influenza Virus H1N1 provides new approaches and new method.
Meanwhile the present invention only needs accordingly to change object chain identification sequence according to the specific sequence of detection virus, so that it may To realize the detection to other viral single-stranded DNA sequences, there is great research and application value in field of virus detection, expand The application of DNA biological sensor, development to the detection of the particular sequences such as the following double chain DNA fragment, protein, nucleic acid There is further impetus.
Preferably, the complementary base radix of signal probe and assist probes is 9.Signal probe and assist probes complementary portion Base number will have a direct impact on the sensitivity entirely tested.When signal probe and assist probes complementary portion are too long, complementary pairing Base number is more, and structure is more stable, causes signal probe and assist probes that can hybridize when no target is single-stranded, background mistake Greatly；When signal probe and assist probes complementary portion are too short, the enclosed type double-stranded DNA of formation is unstable, makes sensitivity decrease.
Preferably, the molar ratio of the signal probe and assist probes is 0.6~1.6.
It is highly preferred that the molar ratio of the signal probe and assist probes is 1.2.
Preferably, the molar ratio of the signal probe and fluorescent dye is 1:1~6.
It is highly preferred that the molar ratio of the signal probe and fluorescent dye is 1:4~6.
Most preferably, the molar ratio of the signal probe and fluorescent dye is 1:4.
Preferably, the fluorescent dye be N- methyl porphyrin dipropionic acid IX (N-methylmesoporphyrin I X, NMM), 3,6- dimethyl -2- (4- dimethylamino benzene)-benzene thiazolium cation.
Correspondingly, application of the above-mentioned enclosed type DNA biological sensor in detection H1N1virus, also exists Within protection scope of the present invention.
Preferably, the application refers to the application in the single-stranded DNA sequence of detection H1N1virus.
H1N1virus is detected using above-mentioned enclosed type DNA biological sensor the present invention also provides a kind of Method, comprising the following steps:
S1. signal probe, assist probes and sample to be tested are added into buffer solution as experimental group, while blank is set Control group (using not plus sample to be tested, only be added signal probe, assist probes and potassium ion solution buffer solution as blank pair According to group), 80~90 DEG C of isothermal reactions after mixing respectively；
S2. after being cooled to room temperature, fluorescent dye is added, 10~20min is incubated at room temperature after mixing；
S3. the fluorescence intensity difference of measurement experiment group and blank control group obtains Influenza A H1N1 disease in sample to be tested The concentration of malicious specific single-chain DNA sequence dna.
Preferably, the influenza A H 1 N 1 virus specific single-stranded DNA sequence is 5'- CAGAATATACACCCAGTCACAATAGGA-3'。
Preferably, the influenza A H 1 N 1 virus specific single-stranded DNA sequence is 5'-CAGAATATA CACCCAGTCACAATAGGA-3'.Wherein, genetic fragment 5'-CAGAATATACACCC-3' can be by the tract of assist probes Section specific recognition；Genetic fragment 5'-AGTCACAATAGGA-3' can be by the sequence fragment specific recognition of signal probe.
Preferably, in step S1, potassium ion solution has been additionally added in buffer solution.K+Radius is larger, is able to enter two layers It is best to the stabilizing power of tetra- stranded structure of G- in space among G- tetrad plane, it can significantly improve tetra- serobila of G- The stability of structure.
Preferably, the concentration of the potassium ion is 0.1~150mM.The concentration refers to that potassium ion is most in reaction system Final concentration.
It is highly preferred that the concentration of the potassium ion is 50~150mM.
Most preferably, the concentration of the potassium ion is 50mM.
Preferably, the potassium ion solution is Klorvess Liquid.
Preferably, the reaction time of step S1 is 5~15min.
Preferably, step S3 carries out fluorescence detection in room temperature.
Preferably, excitation wavelength 399nm, launch wavelength scanning range are as follows: 580~650nm.
Preferably, room temperature of the present invention is 20~28 DEG C.
The concentration and fluorescence signal value of H1N1virus object chain DNA is in 25nM to 700nM concentration range Good linear relationship, linear equation are I=0.0345C+90.67 (C:nM, I are fluorescence signal value), and detection is limited to 8nM, phase Relationship number is 0.9966.
The principle of the present invention is: when not containing H1N1 hiv target DNA in system, fluorescent dye can be with the G- of signal chains Four stranded structures combine, and generate fluorescence signal；When containing target dna sequence in system, since the base pair complementarity of DNA is former Then, auxiliary chain can be captured with signal chains by object chain specificity, and signal chains, auxiliary chain and object chain hybridization is made to form one kind The DNA structure of Y type, to reduce the formation of tetra- serobila of G-, fluorescence signal value weakens, so as to according to the change of fluorescence intensity Change the quantitative detection to realize H1N1 specific single-chain DNA sequence dna.
Compared with prior art, the invention has the following advantages:
Enclosed type DNA biological sensor of the invention have high sensitivity, specificity it is good, convenient for normalizing operation, The advantages that suitable for Clinical screening；It is easy to operate, detection cycle is short, portable；Process costs are low, inexpensive suitable for industrialization Requirement；Preparation method is simple, and performance is stablized, reproducible.
Enclosed type DNA biological sensor of the invention can realize H1N1 specificity according to the variation of fluorescence intensity The quantitative detection of single-stranded DNA sequence plays a significant role in the detection of H1N1virus, and maximum linear detects model It encloses for 25~700nM, linear equation is I=0.0345C+90.67 (C:nM, I are fluorescence signal value), and detection is limited to 8nM, related Coefficient is 0.9966.
Detailed description of the invention
Fig. 1 is the detection principle diagram of enclosed type DNA biological sensor.
Δ I (is defined as the difference of fluorescence intensity to Δ I for the ratio of signal probe and assist probes by Fig. 2.Δ I's determines Adopted formula is Δ I=Iblank-Itarget；ItargetIndicate that there are the fluorescence signal of object chain, IblankIt indicates that object chain is not added Fluorescence signal) influence.
Fig. 3 is influence of the ratio of signal probe and fluorescent dye to Δ I.
Fig. 4 is K+Influence of the concentration to Δ I.
Fig. 5 is influence of the complementary base radix to Δ I.
Fig. 6 is that the fluorescence spectrum that enclosed type DNA biological sensor fluorescence signal value changes with object chain DNA concentration is bent Line.
Fig. 7 is the working curve of enclosed type DNA biological sensor.
Fig. 8 is that method choice of the invention is analyzed.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
It is as follows using sequence in embodiment:
Oligo-1 (Signal probe) is signal probe, tetra- chain of 13 bases and G- including object chain identification sequence Body sequence, underscore part are tetra- serobila DNA enzymatic sequence of G-, including mutual with assist probes Oligo-2 (Asisstant probe) Complementary series.
Oligo-2 is assist probes, including (14 bases, can be with target sequence Olig o-3's for object chain identification sequence Sequence is complementary) and signal probe identification sequence (9 bases, can be complementary with the Sequence of signal probe Oligo-1).
Influenza virus H1N1 (Influenza A virus) nucleic acid sequence (A/Puerto Rico/8/1934 (H1N1)) from (HE802058.1) is obtained in Gen Bank database.The present invention is largely analyzed the influenza virus of itself and other hypotypes It compares with after scientific validation, obtains and have both high specific and highly sensitive influenza virus H1N1 specific sequence segment Oligo- 3, it is made of 27 bases, using specific sequence segment O ligo-3 as inventive closure type DNA biological sensor Target sequence (target sequence).
Oligo-3 include can be complementary with the Sequence of assist probes Oligo-2 14 bases and can be with signal probe Complementary 13 bases of the Sequence of Oligo-1.
Oligo-4 (M-1) is the single base mismatch sequence of object chain.
Oligo-5 (M-2) and Oligo-6 (M-3) are the double alkali yl mismatch of object chain.
Oligo-7, Oligo-8, Oligo-9, Oligo-10, Oligo-11 are the test sequence for assisting probe optimization experiment Column.
The detection of 1 H1N1 of embodiment
(1) in 250 μ L Tris-HCl buffer solutions (20mM Tris-HCl, pH=7.2,400mM Na Cl), respectively 50 μ L Oligo-1 (12 μM), 50 μ L Oligo-2 (10 μM), 50 μ L KCl (500mM) and 50 μ L different series concentration are added Target single stranded DNA (Oligo-3) or sample to be tested, while blank control group is set (sample to be tested, signal is added not plus only The buffer solution of probe, assist probes and potassium ion solution is as blank control group), 88 DEG C of holding 10min are heated to after mixing；
(2) it is gradually cooling to room temperature, to form case type double-stranded DNA；50 μ L NMM (48 μM) are added in above-mentioned molten In liquid, and in (20~28 DEG C) incubation 15min of room temperature；
(3) above-mentioned solution is transferred in fluorescence detector, at room temperature the fluorescence of measurement experiment group and blank control group Strength difference；According to target Single stranded DNA concentration-fluorescence intensity standard curve, regression equation is calculated；According to the glimmering of sample to be tested Luminous intensity calculates the concentration of H1N1virus single-stranded DNA sequence in sample to be tested；Wherein, fluorescence spectrometry parameter It is provided that excitation wavelength is 399nm (slit width 5nm), launch wavelength scanning range are as follows: (slit is wide by 580~650nm Degree is 5nm).
Experimental principle figure is as shown in Figure 1.The enclosed type DNA biological sensor master of the detection H1N1virus It to include two kinds of probes: signal probe and assist probes.Signal probe (Oligo-1) is the single-stranded DNA sequence for being rich in G, It mainly include object chain (Oligo-3) identification sequence and tetra- serobila sequence of G-.Oligo-2 has mesh as assist probes It marks chain and Oligo-1 identifies sequence；When object chain is not added, phase mutual cross does not occur for Oligo-1 and Oligo-2, in K+In the presence of, Signal chains are capable of forming tetra- stranded structure of G-, in conjunction with NMM, issue fluorescence signal.However, after object chain is added, Oligo- 1, Oligo-2 and object chain phase mutual cross, so that signal chains can not form tetra- stranded structure of G-, fluorescence signal weakens, so as to To realize the quantitative detection of H1N1 specific single-chain DNA sequence dna according to the variation of fluorescence intensity.
Fluorescence spectra of the present invention and working curve difference are as shown in Figure 6 and Figure 7.The concentration of H1N1 object chain DNA and glimmering Optical signal value in 25~700nM concentration range be in good linear relationship, linear equation be I=0.0345C+90.67 (C: NM, I are fluorescence signal value), detection is limited to 8nM, related coefficient 0.9966.
By observation comparison fluorescent spectrum curve and working curve, can show with being apparent when H1N1 object chain DNA is dense When degree is in 25~700nM concentration range, fluorescence signal value increases with the increase of object chain DNA concentration, and shows Good linear relationship illustrates that enclosed type DNA biological sensor of the invention has high sensitivity, spy to the detection of H1N1 Anisotropic advantage good, selectivity is good.
2 fluorescence intensity of embodiment with Oligo-1 and Oligo-2 ratio variation
1, method
On the basis of embodiment 1, using the molar ratio of Oligo-1 and Oligo-2 as single-factor variable, using Δ I as index (Δ I is defined as to the difference of fluorescence intensity, the definition of Δ I is Δ I=Iblank-Itarget, ItargetIndicate that there are object chains Fluorescence signal, IblankIndicate the fluorescence signal that object chain is not added), investigate the ratio pair of different Oligo-1 and Oligo-2 The influence of H1N1 virus-specific single-stranded DNA sequence detection effect.
2, result
Experimental result is as shown in Fig. 2, auxiliary chain (Oligo-2) can be miscellaneous with Oligo-1, object chain (Target DNA) It hands over, inhibits the formation of tetra- serobila of G-, being changed significantly for Oligo-1 and Oligo-2 ratio influences the power of fluorescence intensity.From As can be seen that detection effect is preferable when the molar ratio of signal probe and assist probes is 0.6~1.6 in Fig. 2；When initial, Δ I Value increases with the increase of Oligo-1/Oligo-2 molar ratio；When the molar ratio of Oligo-1/Oligo-2 reaches 1.2, Δ I reaches maximum value；When the molar ratio of Oligo-1/Oligo-2 is more than 1.2, Δ I starts to be gradually reduced, and illustrates to work as assist probes When concentration is excessive, background signal be will increase, and Δ I is caused to decline.
3 fluorescence intensity of embodiment with Oligo-1 and NMM ratio variation
1, method
On the basis of embodiment 1, it using the ratio of Oligo-1 and NMM as single-factor variable, using Δ I as index, investigates not With influence of the ratio to H1N1 virus-specific single-stranded DNA sequence detection effect of Oligo-1 and NMM.
2, result
Experimental result is as shown in figure 3, fluorescent dye NMM will can be only achieved according to a certain percentage in conjunction with tetra- serobila of G- Maximum fluorescence signal value.From the figure 3, it may be seen that detection effect is preferable when the molar ratio of signal probe and fluorescent dye is 1:1~6； The fluorescence intensity of detection architecture is gradually increased as the ratio of Oligo-1 and NMM increases, when mole of G- tetra- serobilas and NMM When than for 1:4, the net increment Delta I of the fluorescence signal value of detection architecture reaches maximum value；And when signal probe and fluorescent dye When molar ratio is 1:4~6, fluorescence intensity is basically unchanged.
4 fluorescence intensity of embodiment is with K+The variation of concentration
1, method
On the basis of embodiment 1, with K in reaction system+Concentration is single-factor variable, using Δ I as index, is investigated different K+Influence of the concentration to H1N1 virus-specific single-stranded DNA sequence detection effect.
2, result
Experimental result is as shown in figure 4, as K in reaction system+In 0~50mM range, Δ I value gradually increases concentration Greatly；Work as K+For concentration in 50~150mM range, fluorescence intensity is stronger, but the variation of Δ I value is little.As shown in Figure 4, K+Plus Enter, the stability of tetra- stranded structure of G- can be significantly improved, be conducive to the combination of tetra- serobila of fluorescent dye NMM and G-, when potassium from When the concentration of son is 0.1~150mM, the stability of tetra- stranded structure of G- can be improved, detection effect is preferable；Work as K+Concentration is 50 When~150mM, fluorescence intensity is stronger, and detection effect is more preferable.
5 fluorescence intensity of embodiment with signal probe and assist probes complementary base radix variation
1, method
On the basis of embodiment 1, it selects respectively and signal probe complementary base radix is respectively 6,7,8,9,10,11 widow Poly- deoxyribonucleotide sequence 5'-CTACCCGGGTGTATATTCTG-3', 5'-CCT ACCCGGGTGTATATTCTG-3', 5'-CCCTACCCGGGTGTATATTCTG-3', 5'-GCCC TACCCGGGTGTATATTCTG-3'(assist probes Olige-2), 5'-CGCCCTACCCGGGT GTATATTCTG-3' and 5'-CCGCCCTACCCGGGTGTATATTCTG-3' as assist probes, It is added in the detection architecture of embodiment 1, using Δ I as index, investigates signal probe complementary base radix different from assist probes Influence to H1N1 virus-specific single-stranded DNA sequence detection effect.
2, result
As shown in figure 5, when complementary base radix increases to 9 (when as assist probes Olige-2), Δ I reaches experimental result To maximum value.When complementary base continues to increase, Δ I value is reduced.As shown in Figure 5, when signal probe and assist probes complementary portion Too long, the base number of complementary pairing is more, and structure is more stable, cause signal probe and assist probes when no target is single-stranded just It can hybridize, background is excessive；When signal probe and assist probes complementary portion are too short, the enclosed type double-stranded DNA of formation is unstable, Make sensitivity decrease.
The selectivity of 6 detection method of embodiment
1, method
In order to verify the selectivity of inventive closure type DNA biological sensor and its detection method, in embodiment 1 On the basis of, select the base mispairing sequence M-1 (Oligo-4), M-2 (Oligo-5) and M-3 (Oligo-6) of same concentrations to replace The sequence (Target DNA) of complete complementary, which is added in reaction system, carries out hybridization reaction.
2, result
As shown in figure 8, fluorescence signal of the fluorescence signal value of mismatch well below fully-complementary sequence, to show Enclosed type DNA biological sensor of the invention and its detection method applied to H1N1 specific single-chain DN A sequence have Good selectivity.
Embodiment 7
Respectively using H1N1virus and following different types of influenza virus as sample to be tested: H1N1 stream Influenza Virus, A type H0N1 influenza virus, A type H2N2 influenza virus, A type H3N2 influenza virus, B-mode Victoria influenza disease Malicious, B-mode Yamagata influenza virus；Above-mentioned sample to be tested is carried out using enclosed type DNA biological sensor of the invention Detection.
Testing result is as shown in table 1, compared with other different types of influenza viruses, only in H1N1virus Sample to be tested, testing result are positive.As a result illustrate, the present invention is used to carry out fluorescence detection to H1N1virus The specificity of enclosed type DNA biological sensor is good, and reaction system specificity is good.
1 testing result of table
Note: (+) represents detection H1N1virus, and (-) represents and H1N1virus is not detected.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (10)

1. a kind of enclosed type DNA biological sensor, which is characterized in that including assist probes, signal probe and can be with letter The fluorescent dye that number probe is specifically bound；
The sequence of signal probe is 5'-TCCTATTGTGACTTTGGGTAGGGCGGGTTGGG-3'；
The sequence of assist probes is 5'-GCCCTACCCGGGTGTATATTCTG-3'.
2. enclosed type DNA biological sensor according to claim 1, which is characterized in that the signal probe with it is auxiliary The molar ratio for helping probe is 0.6~1.6.
3. enclosed type DNA biological sensor according to claim 1, which is characterized in that the signal probe with it is glimmering The molar ratio of photoinitiator dye is 1:1~6.
4. enclosed type DNA biological sensor according to claim 1, which is characterized in that the fluorescent dye is N- Methyl porphyrin dipropionic acid IX or 3,6- dimethyl -2-(4- dimethylamino benzene)-benzene thiazolium cation.
5. any enclosed type DNA biological sensor of Claims 1 to 4 is in detection H1N1virus Using.
6. application according to claim 5, which is characterized in that the application refers in detection H1N1virus Application in single-stranded DNA sequence.
7. a kind of method for detecting H1N1virus, which is characterized in that use any envelope of Claims 1 to 44 Closed form DNA biological sensor is detected.
8. the method according to the description of claim 7 is characterized in that the following steps are included:
S1. signal probe, assist probes and sample to be tested are added into buffer solution as experimental group, while blank pair is set According to group, 80~90 DEG C of isothermal reactions after mixing respectively；
S2. after being cooled to room temperature, fluorescent dye is added, 10~20 min are incubated at room temperature after mixing；
S3. it is special to obtain H1N1virus in sample to be tested for the fluorescence intensity difference of measurement experiment group and blank control group The concentration of bispecific single-chain DNA sequence dna.
9. according to the method described in claim 8, it is characterized in that, the influenza A H 1 N 1 virus specific single stranded DNA sequence It is classified as 5'-CAGAATATACACCCAGTCACAATAGGA-3'.
10. according to the method described in claim 8, it is characterized in that, to be additionally added potassium ion in step S1, in buffer solution molten Liquid.
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