CN109266784B - Closed DNA fluorescent biosensor and application thereof in detection of influenza A H1N1 virus - Google Patents
Closed DNA fluorescent biosensor and application thereof in detection of influenza A H1N1 virus Download PDFInfo
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Abstract
The invention discloses a closed DNA fluorescent biosensor and application thereof in detecting influenza A H1N1 virus. The sensor comprises an auxiliary probe, a signal probe and a fluorescent dye capable of specifically binding with the signal probe; wherein, the sequence of the signal probe is 5'-TCCTATTGTGACTTTGGGTAGGG CGGGTTGGG-3'; the sequence of the helper probe is 5'-GCCCTACCCGGGTGTATATTCTG-3'. The closed DNA fluorescent biosensor can quickly detect the concentration of the specific single-stranded DNA sequence of the influenza A H1N1 through the change of the fluorescent value, has the advantages of high sensitivity, good specificity, convenience in standardized operation, suitability for clinical screening and the like, provides certain theoretical guidance for the development of quick detection products of the influenza A H1N1, and has important significance for clinical monitoring of the influenza A virus.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis. More particularly, the invention relates to a closed DNA fluorescence biosensor and an application thereof in detecting H1N1 influenza A virus.
Background
H1N1 is an RNA virus of the orthomyxoviridae family, and through host variation and transmission of H1N1 virus, epidemic situation and large-area outbreak and transmission of human influenza can be caused. In 3 months in 2009, the new influenza a H1N1 virus was outbreak in the united states, mexico and other countries, and then spread rapidly worldwide, resulting in a large viral disaster with serious harm to humans and animals. In the later stage of epidemic of the H1N1 influenza virus, the influenza epidemic still continuously breaks out in different areas, and the influenza virus is possibly mutated and generates drug resistance, so that the influenza virus is changed into influenza virus and pathogenic bacteria which are easy to spread by mutation, and the health and public health safety of human beings and animals in the world are seriously influenced. Therefore, in order to screen suspected cases of influenza, control the spread of influenza virus and monitor the epidemic situation of the influenza virus in animals, the development of a rapid and effective detection method aiming at the novel influenza A H1N1 virus is urgently needed.
At present, a plurality of methods for detecting and diagnosing the influenza A H1N1 virus at home and abroad are available, and the methods mainly comprise the traditional virus separation and identification method, the immunofluorescence method, the real-time fluorescence quantitative PCR and other technical methods. The traditional separation culture detection technology has long culture time and relatively complex operation process, needs to be operated under aseptic condition, has higher requirements on related professional knowledge and operation technology of operators, and limits the application of the method in clinical and non-laboratory environments. The immunofluorescence is observed whether virus antigens exist or not through a fluorescence microscope so as to judge whether the viruses are infected or not, the sensitivity of the immunofluorescence is closely related to the quality of a specimen collected by an operator, the content of the specimen and related professional operation technologies, so that the sensitivity of the immunofluorescence is not as stable as expected, and further improvement and perfection are needed. The real-time fluorescent quantitative PCR detection has higher specificity and sensitivity, but also can generate false negative results, has higher technical requirements on operators, precise experimental instruments and integral laboratory facilities in the whole detection process, and is not suitable for basic large-scale screening detection.
Therefore, the development of a diagnostic method which is convenient, rapid, highly sensitive, highly specific and suitable for clinical screening is urgently needed to meet the detection requirement of the influenza A H1N1 virus.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a closed DNA fluorescence biosensor which has high specificity and sensitivity, good selectivity and high detection speed and can detect single-stranded DNA. The closed DNA fluorescence biosensor can quickly detect the concentration of a specific single-stranded DNA sequence of the influenza A H1N1 virus through the change of a fluorescence value, provides a certain theoretical guidance for the development of a quick detection product of the influenza A H1N1 virus, and has important significance for the clinical monitoring of the circulating virus.
The invention aims to provide a closed DNA fluorescence biosensor.
The second purpose of the invention is to provide the application of the blocked DNA fluorescent biosensor in detecting H1N1 influenza A virus.
The third purpose of the invention is to provide a method for detecting H1N1 influenza A virus.
The above purpose of the invention is realized by the following technical scheme:
a closed DNA fluorescence biosensor comprises an auxiliary probe, a signal probe and a fluorescent dye capable of being specifically combined with the signal probe;
the sequence of the signaling probe is 5'-TCCTATTGTGACTTTGGGTAGGGCGGGTTGGG-3';
the sequence of the helper probe is 5'-GCCCTACCCGGGTGTATATTCTG-3'.
The Influenza virus H1N1(Influenza A virus) nucleic acid sequence (A/Puerto Rico/8/1934(H1N1)) was obtained from the Gen Bank database (HE 802058.1). The invention carries out a large amount of analysis comparison and scientific verification on the influenza virus and other subtypes of influenza viruses to obtain the influenza virus H1N1 specific sequence segment with high specificity and high sensitivity, the specific sequence segment is composed of 27 basic groups, and the specific sequence segment is used as a target sequence of the closed DNA fluorescence biosensor.
In the sequence of the signaling probe, gene segment 5'-TCCTATTGTGACT-3' is a Target strand recognition sequence and can be complementary with a sequence segment of Target DNA; gene fragment 5'-GGGTAGGGCGGGTTG GG-3' is a G-quadruplex DNase sequence.
In the sequences of the auxiliary probes, a gene segment 5'-GGGTGTATATTCTG-3' is a Target strand recognition sequence and can be complementary with a sequence segment of Target DNA; the gene fragment 5'-GCCCTACCC-3' is 9 bases and can be complementary with the sequence fragment of the signal probe.
The invention takes H1N1 specific sequence fragments as target DNA, and designs an auxiliary probe and a signal probe with a G-quadruplex structure according to the complementary pairing principle of basic groups, wherein the auxiliary probe comprises a target chain recognition sequence and a sequence capable of recognizing the signal probe, the signal probe comprises a target chain recognition sequence and a G-quadruplex sequence, the G-quadruplex structure of the signal chain can be specifically combined with fluorescent dye to generate a fluorescent signal, so that the signal chain, the auxiliary chain and the target chain are hybridized to form a closed DNA double chain, thereby constructing the high-sensitivity DNA fluorescent biosensor for detecting the H1N1 related DNA sequence. The sensor can conveniently, quickly and sensitively detect the concentration of the H1N1 specific single-stranded DNA sequence through the change of the fluorescence value, and provides a new thought and a new method for detecting the influenza virus H1N 1.
Meanwhile, the invention can realize the detection of other virus single-stranded DNA sequences only by correspondingly changing the target strand recognition sequence according to the specific sequence of the detected virus, has great research and application significance in the field of virus detection, expands the application of the DNA fluorescence biosensor and has further promotion effect on the development of the detection of specific sequences such as future double-stranded DNA fragments, proteins, nucleic acids and the like.
Preferably, the number of complementary bases of the signaling probe and the auxiliary probe is 9. The number of bases in the complementary portions of the signaling probe and the helper probe directly affects the sensitivity of the entire experiment. When the complementary part of the signal probe and the auxiliary probe is too long, the number of complementary paired bases is large, the structure is relatively stable, so that the signal probe and the auxiliary probe can be hybridized when no target single strand exists, and the background is too large; when the complementary portion of the signal probe and the auxiliary probe is too short, the formed blocked double-stranded DNA is unstable, and the sensitivity is lowered.
Preferably, the molar ratio of the signal probe to the auxiliary probe is 0.6-1.6.
More preferably, the molar ratio of the signaling probe to the helper probe is 1.2.
Preferably, the molar ratio of the signaling probe to the fluorescent dye is 1: 1 to 6.
More preferably, the molar ratio of the signaling probe to the fluorescent dye is 1:4 to 6.
Most preferably, the molar ratio of the signaling probe to the fluorescent dye is 1: 4.
preferably, the fluorescent dye is N-methylporphyrin dipropionate IX (N-Methylesoporphyrin I X, NMM), 3, 6-dimethyl-2- (4-dimethylaminophenyl) -benzothiazole cation.
Accordingly, the application of the blocked DNA fluorescent biosensor in detecting H1N1 influenza A virus is also within the protection scope of the invention.
Preferably, the application refers to the application in detecting the single-stranded DNA sequence of the influenza A H1N1 virus.
The invention also provides a method for detecting H1N1 influenza A virus by using the closed DNA fluorescent biosensor, which comprises the following steps:
s1, adding a signal probe, an auxiliary probe and a sample to be detected into a buffer solution to serve as an experimental group, simultaneously setting a blank control group (taking the buffer solution without the sample to be detected and only adding the signal probe, the auxiliary probe and a potassium ion solution as the blank control group), respectively mixing uniformly, and reacting at a constant temperature of 80-90 ℃;
s2, cooling to room temperature, adding a fluorescent dye, uniformly mixing, and incubating at room temperature for 10-20 min;
and S3, measuring the fluorescence intensity difference between the experimental group and the blank control group to obtain the concentration of the specific single-stranded DNA sequence of the influenza A H1N1 virus in the sample to be detected.
Preferably, the influenza a H1N1 virus-specific single-stranded DNA sequence is 5'-CAGAATATACACCCAGTCACAATAGGA-3'.
Preferably, the influenza a H1N1 virus-specific single-stranded DNA sequence is 5'-CAGAATATA CACCCAGTCACAATAGGA-3'. Wherein, the gene segment 5'-CAGAATATACACCC-3' can be specifically recognized by the sequence segment of the auxiliary probe; gene fragment 5'-AGTCACAATAGGA-3' is specifically recognized by the sequence fragment of the signaling probe.
Preferably, in step S1, a potassium ion solution is further added to the buffer solution. K+The radius is larger, the two-layer G-quadruplex plane can enter the space between the two layers of G-quadruplex planes, the stability of the G-quadruplex structure is best, and the stability of the G-quadruplex structure can be obviously improved.
Preferably, the concentration of the potassium ions is 0.1-150 mM. The concentration refers to the final concentration of potassium ions in the reaction system.
More preferably, the concentration of the potassium ion is 50 to 150 mM.
Most preferably, the concentration of potassium ions is 50 mM.
Preferably, the potassium ion solution is a potassium chloride solution.
Preferably, the reaction time of step S1 is 5-15 min.
Preferably, step S3 is performed at room temperature for fluorescence detection.
Preferably, the excitation wavelength is 399nm and the emission wavelength scan range is: 580 to 650 nm.
Preferably, the room temperature is 20-28 ℃.
The concentration and the fluorescence signal value of the influenza A H1N1 target strand DNA are in good linear relation in the concentration range of 25nM to 700nM, the linear equation is that I is 0.0345C +90.67 (C: nM, I is the fluorescence signal value), the detection limit is 8nM, and the correlation coefficient is 0.9966.
The principle of the invention is as follows: when the system does not contain H1N1 virus target DNA, the fluorescent dye can be combined with the G-quadruplex structure of the signal chain to generate a fluorescent signal; when the system contains a target DNA sequence, the auxiliary strand and the signal strand can be specifically captured by the target strand due to the base complementary pairing principle of DNA, so that the signal strand, the auxiliary strand and the target strand are hybridized to form a Y-shaped DNA structure, the formation of a G-quadruplex is reduced, the fluorescence signal value is weakened, and the quantitative detection of the H1N1 specific single-stranded DNA sequence can be realized according to the change of the fluorescence intensity.
Compared with the prior art, the invention has the following beneficial effects:
the closed DNA fluorescence biosensor has the advantages of high sensitivity, good specificity, convenience for standardized operation, suitability for clinical screening and the like; the operation is simple and convenient, the detection period is short, and the carrying is easy; the process cost is low, and the method is suitable for the requirement of low price in industrialization; the preparation method is simple, stable in performance and good in repeatability.
The enclosed DNA fluorescent biosensor can realize quantitative detection of an H1N1 specific single-stranded DNA sequence according to the change of fluorescence intensity, plays an important role in the detection of influenza A H1N1 virus, has the maximum linear detection range of 25-700 nM, has a linear equation of I0.0345C +90.67 (C: nM and I is a fluorescence signal value), has the detection limit of 8nM and has a correlation coefficient of 0.9966.
Drawings
FIG. 1 is a detection schematic diagram of a closed DNA fluorescence biosensor.
FIG. 2 shows the ratio of signaling probe to auxiliary probe vs. Δ I (Δ I is defined as the difference in fluorescence intensityblank-Itarget；ItargetFluorescent signal indicating the presence of the target strand, IblankIndicating no fluorescent signal added to the target strand).
FIG. 3 is a graph showing the effect of the ratio of signaling probe to fluorescent dye on Δ I.
FIG. 4 is K+Effect of concentration on Δ I.
FIG. 5 is a graph of the effect of complementary base number on Δ I.
FIG. 6 is a fluorescence spectrum curve of the fluorescence signal value of the closed type DNA fluorescence biosensor according to the change of the concentration of the target strand DNA.
FIG. 7 is a graph showing the operation of the blocked DNA fluorescence biosensor.
FIG. 8 is a selectivity analysis of the method of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The sequences used in the examples are as follows:
oligo-1(Signal probe) is a Signal probe comprising 13 bases of the recognition sequence of the target strand and the G-quadruplex sequence, and the underlined part is the G-quadruplex DNase sequence comprising a sequence complementary to the auxiliary probe Oligo-2(Asisstant probe).
Oligo-2 is a helper probe comprising a target strand recognition sequence (14 bases, which is partially complementary to the sequence of the target sequence Oligo o-3) and a signaling probe recognition sequence (9 bases, which is partially complementary to the sequence of the signaling probe Oligo-1).
The Influenza virus H1N1(Influenza A virus) nucleic acid sequence (A/Puerto Rico/8/1934(H1N1)) was obtained from the Gen Bank database (HE 802058.1). After a large amount of analysis comparison and scientific verification are carried out on the influenza virus and other subtypes of influenza viruses, the influenza virus H1N1 specific sequence segment Oligo-3 with high specificity and high sensitivity is obtained, the influenza virus H1N1 specific sequence segment Oligo-3 is composed of 27 basic groups, and the specific sequence segment O Oligo-3 is used as a target sequence (target sequence) of the blocked DNA fluorescence biosensor.
Oligo-3 comprises 14 bases which are partially complementary to the sequence of the helper probe Oligo-2 and 13 bases which are partially complementary to the sequence of the signaling probe Oligo-1.
Oligo-4(M-1) is a single-base mismatched sequence of the target strand.
Oligo-5(M-2) and Oligo-6(M-3) are double-base mismatched sequences of the target strand.
Oligo-7, Oligo-8, Oligo-9, Oligo-10, Oligo-11 are test sequences for helper probe optimization experiments.
Example 1 detection of H1N1
(1) Adding 50 μ L of Oligo-1(12 μ M), 50 μ L of Oligo-2(10 μ M), 50 μ L of KCl (500mM) and 50 μ L of target single-stranded DNA (Oligo-3) with different series concentrations or a sample to be tested into 250 μ L of Tris-HCl buffer solution (20mM Tris-HCl, pH 7.2,400mM Na Cl), setting a blank control group (taking buffer solution without the sample to be tested and only adding a signal probe, an auxiliary probe and a potassium ion solution as the blank control group), uniformly mixing, and heating to 88 ℃ for 10 min;
(2) gradually cooling to room temperature to form closed double-stranded DNA; then adding 50 mu L NMM (48 mu M) into the solution, and incubating for 15min at room temperature (20-28 ℃);
(3) transferring the solution to a fluorescence detector, and measuring the fluorescence intensity difference between the experimental group and the blank control group at room temperature; calculating a regression equation according to a standard curve of the concentration-fluorescence intensity of the target single-stranded DNA; calculating the concentration of the influenza A H1N1 virus single-stranded DNA sequence in the sample to be detected according to the fluorescence intensity of the sample to be detected; wherein, the fluorescence spectrum measurement parameters are set as follows: the excitation wavelength was 399nm (slit width 5nm), and the emission wavelength scan range was: 580 to 650nm (slit width of 5 nm).
The experimental schematic diagram is shown in fig. 1. The closed DNA fluorescent biosensor for detecting the influenza A H1N1 mainly comprises two probes: a signaling probe and an auxiliary probe. The signal probe (Oligo-1) is a single-stranded DNA sequence rich in G, and mainly comprises a target strand (Oligo-3) recognition sequence and a G-quadruplex sequence. Oligo-2 is used as an auxiliary probe and simultaneously has a target strand and an Oligo-1 recognition sequence; when the target strand is not added, Oligo-1 and Oligo-2 do not hybridize with each other, and K is+In the presence of the fluorescent probe, the signal chain can form a G-quadruplex structure, and is combined with NMM to emit a fluorescent signal. However, when the target strand is added, Oligo-1, Oligo-2 and the target strand are hybridized with each other, so that the signal strand cannot form a G-quadruplex structure, and the fluorescence signal is weakened, thereby realizing the quantitative detection of the H1N1 specific single-stranded DNA sequence according to the change of the fluorescence intensity.
The fluorescence spectrum and the working curve of the invention are shown in FIG. 6 and FIG. 7, respectively. The concentration of H1N1 target strand DNA and the fluorescence signal value are in good linear relation in the concentration range of 25-700 nM, the linear equation is that I is 0.0345C +90.67 (C: nM, I is the fluorescence signal value), the detection limit is 8nM, and the correlation coefficient is 0.9966.
By observing and comparing a fluorescence spectrum curve and a working curve, the fluorescence signal value is increased along with the increase of the concentration of the target strand DNA when the concentration of the target strand DNA of H1N1 is in the range of 25-700 nM, and a good linear relation is presented, which shows that the closed DNA fluorescence biosensor has the advantages of high sensitivity, good specificity and good selectivity in the detection of H1N 1.
EXAMPLE 2 variation of fluorescence intensity with the ratio of Oligo-1 to Oligo-2
1. Method of producing a composite material
Based on example 1, the molar ratio of Oligo-1 to Oligo-2 was used as a one-factor variable, and Δ I was used as an index (Δ I was defined as the difference in fluorescence intensity, and Δ I was defined as Δ I ═ Iblank-Itarget，ItargetFluorescent signal indicating the presence of the target strand, IblankIndicating that no fluorescent signal of the target strand was added), the effect of the ratio of different Oligo-1 and Oligo-2 on the detection effect of the H1N1 virus-specific single-stranded DNA sequence was examined.
2. Results
As shown in FIG. 2, the auxiliary strand (Oligo-2) can hybridize with Oligo-1 and the Target strand (Target DNA) to inhibit the formation of G-quadruplex, and the change in the ratio of Oligo-1 to Oligo-2 significantly affects the intensity of fluorescence intensity detected. As can be seen from FIG. 2, the detection effect is better when the molar ratio of the signal probe to the auxiliary probe is 0.6-1.6; initially, Δ I values increase with increasing Oligo-1/Oligo-2 molar ratio; Δ I reaches a maximum when the Oligo-1/Oligo-2 molar ratio reaches 1.2; when the Oligo-1/Oligo-2 molar ratio exceeds 1.2, Δ I begins to gradually decrease, indicating that when the concentration of the helper probe is too high, the background signal increases, resulting in a decrease in Δ I.
EXAMPLE 3 variation of fluorescence intensity with the ratio of Oligo-1 to NMM
1. Method of producing a composite material
On the basis of example 1, the influence of the ratios of Oligo-1 and NMM on the detection effect of the H1N1 virus-specific single-stranded DNA sequence is examined by taking the ratio of Oligo-1 and NMM as a single-factor variable and taking Delta I as an index.
2. Results
As shown in FIG. 3, the fluorescent dye NMM is bound to the G-quadruplex in a certain ratio to reach the maximum fluorescence signal value. As can be seen from fig. 3, the molar ratio of the signaling probe to the fluorescent dye is 1: 1-6 hours, the detection effect is good; the fluorescence intensity of the detection system is gradually increased along with the increase of the ratio of Oligo-1 to NMM, and when the molar ratio of G-quadruplex to NMM is 1:4, the net increase delta I of the fluorescence signal value of the detection system reaches the maximum value; and when the molar ratio of the signal probe to the fluorescent dye is 1: 4-6, the fluorescence intensity is basically unchanged.
Example 4 fluorescence intensity with K+Variation of concentration
1. Method of producing a composite material
Based on example 1, with K in the reaction system+Concentration is a single factor variable, and different Ks are examined by taking Delta I as an index+Effect of concentration on the detection effect of the H1N1 virus-specific single-stranded DNA sequence.
2. Results
The results of the experiment are shown in FIG. 4, when K is present in the reaction system+When the concentration is changed within the range of 0-50 mM, the delta I value is gradually increased; when K is+When the concentration is changed within the range of 50-150 mM, the fluorescence intensity is stronger, but the change of the delta I value is not large. As can be seen from FIG. 4, K+The addition of the fluorescent dye can obviously improve the stability of a G-quadruplex structure, is beneficial to the combination of the fluorescent dye NMM and the G-quadruplex, can improve the stability of the G-quadruplex structure when the concentration of potassium ions is 0.1-150 mM, and has better detection effect; when K is+When the concentration is 50-150 mM, the fluorescence intensity is stronger, and the detection effect is better.
EXAMPLE 5 variation of fluorescence intensity according to the number of complementary bases of Signal Probe and auxiliary Probe
1. Method of producing a composite material
In addition to example 1, oligodeoxyribonucleotide sequences 5'-CTACCCGGGTGTATATTCTG-3', 5'-CCT ACCCGGGTGTATATTCTG-3', 5'-CCCTACCCGGGTGTATATTCTG-3', 5'-GCCC TACCCGGGTGTATATTCTG-3' (helper probes Olige-2), 5'-CGCCCTACCCGGGT GTATATTCTG-3' and 5'-CCGCCCTACCCGGGTGTATATTCTG-3', which have the number of bases complementary to the signaling probe of 6, 7, 8, 9, 10 and 11, respectively, were selected as helper probes and added to the detection system of example 1, and the effect of the different numbers of bases complementary to the signaling probe and the helper probe on the detection effect of the H1N1 virus-specific single-stranded DNA sequence was examined using Δ I as an index.
2. Results
As shown in FIG. 5, Δ I reaches a maximum value when the number of complementary bases is increased to 9 (i.e., when the helper probe Olige-2 is used). As the complementary bases continue to increase, the Δ I value decreases. As can be seen from FIG. 5, when the complementary portion of the signaling probe and the auxiliary probe is too long, the number of complementary paired bases is large, the structure is relatively stable, so that the signaling probe and the auxiliary probe can hybridize without a target single strand, and the background is too large; when the complementary portion of the signal probe and the auxiliary probe is too short, the formed blocked double-stranded DNA is unstable, and the sensitivity is lowered.
EXAMPLE 6 Selectivity of detection method
1. Method of producing a composite material
In order to verify the selectivity of the closed DNA fluorescence biosensor and the detection method thereof, on the basis of the embodiment 1, base mismatch sequences M-1(Oligo-4), M-2(Oligo-5) and M-3(Oligo-6) with the same concentration are selected to replace a completely complementary sequence (Target DNA) and added into a reaction system for hybridization reaction.
2. Results
As shown in FIG. 8, the fluorescence signal value of the mismatch sequence is far lower than that of the complete complementary sequence, thereby indicating that the blocked DNA fluorescence biosensor and the detection method thereof applied to the H1N1 specific single-stranded DN A sequence have good selectivity.
Example 7
The influenza A H1N1 virus and the following different types of influenza viruses are respectively used as samples to be tested: influenza a H1N1, influenza a H0N1, influenza a H2N2, influenza a H3N2, Victoria b, Yamagata; the closed DNA fluorescence biosensor is adopted to detect the sample to be detected.
The detection results are shown in table 1, and compared with other different types of influenza viruses, the detection results are positive only in a sample to be tested of the influenza A H1N1 virus. The result shows that the closed DNA fluorescence biosensor for carrying out fluorescence detection on the influenza A H1N1 virus has good specificity and good reaction system specificity.
TABLE 1 test results
Note: (+) indicates that H1N1 influenza A virus was detected, and (-) indicates that H1N1 influenza A virus was not detected.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
<110> Guangdong ocean university
<120> closed DNA fluorescent biosensor and application thereof in detection of influenza A H1N1 virus
<140> 2018108436341
<141> 2018-07-27
<160> 11
<170> SIPOSequenceListing 1.0
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<213> M-3(Oligo-6)
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ccctacccgg gtgtatattc tg 22
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Claims (9)
1. A closed DNA fluorescence biosensor is characterized by comprising an auxiliary probe, a signal probe and a fluorescent dye capable of being specifically combined with the signal probe;
the sequence of the signaling probe is 5'-TCCTATTGTGACTTTGGGTAGGGCGGGTTGGG-3';
the sequence of the auxiliary probe is 5'-GCCCTACCCGGGTGTATATTCTG-3';
wherein the molar ratio of the signal probe to the auxiliary probe is 1.0-1.6;
the number of the complementary bases of the signal probe and the auxiliary probe is 9.
2. The closed DNA fluorescence biosensor of claim 1, wherein the molar ratio of the signal probe to the fluorescent dye is 1:4 to 6.
3. The closed DNA fluorescence biosensor of claim 1, wherein the fluorescent dye is N-methyl porphyrin dipropionate IX or 3, 6-dimethyl-2- (4-dimethylamino-benzene) -benzothiazole cation.
4. Use of the blocked DNA fluorescence biosensor according to any one of claims 1 to 3 for the detection of H1N1 influenza a virus for non-diagnostic purposes.
5. The use according to claim 4, wherein the use is for detecting a single-stranded DNA sequence of an influenza A H1N1 virus.
6. A method for detecting H1N1 influenza A virus for non-diagnostic purposes, which comprises detecting the influenza A virus using the blocked DNA fluorescent biosensor according to any one of claims 1 to 3.
7. The method of claim 6, comprising the steps of:
s1, adding a signal probe, an auxiliary probe and a sample to be detected into the buffer solution to serve as an experimental group, setting a blank control group, respectively mixing uniformly, and reacting at a constant temperature of 80-90 ℃;
s2, cooling to room temperature, adding a fluorescent dye, uniformly mixing, and incubating at room temperature for 10-20 min;
s3, determining the difference of the fluorescence intensity of the experimental group and the blank control group to obtain the concentration of the influenza A H1N1 virus specific single-stranded DNA sequence in the sample to be detected.
8. The method of claim 7, wherein the influenza A H1N1 specific single stranded DNA sequence is 5'-CAGAATATACACCCAGTCACAATAGGA-3'.
9. The method according to claim 7, wherein in step S1, a potassium ion solution is further added to the buffer solution.
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