CN109957607A - A detection probe and its application - Google Patents

Abstract

The present invention provides a detection probe and an application thereof. The probe includes a probe A labeled with luciferase and a probe B labeled with quantum dots; wherein, one end of the probe A is connected to the target nucleic acid. One end of the partial sequence is complementary, one end of the probe B is complementary to the other end partial sequence of the target nucleic acid, and the other end of the probe A and the other end of the probe B have a partially complementary sequence; The needle B and the target nucleic acid are hybridized to form a stable T-shaped structure, and the luciferase and the quantum dots can be paired with each other for bioluminescence resonance energy transfer; the present invention cleverly combines the probe and the nucleic acid to form a stable T-shaped structure by designing a specific probe. The structure, using the principle of bioluminescence energy resonance transfer, optimizes the reaction conditions, can accurately distinguish single base mutations, effectively detect target nucleic acid, simple operation, short time required, convenient and fast, and low cost.

Description

A detection probe and its application

technical field

The invention relates to the field of biotechnology, in particular to a detection probe and its application.

Background technique

Bioluminescence resonance energy transfer (BRET) is a non-radiative energy transfer that occurs between bioluminescent proteins such as luciferase (donor) and a fluorescent substance (acceptor). Because BRET does not require exogenous excitation, has low background and high sensitivity, it has been widely used as an optical ruler with small distances in biological analysis related fields such as protein interaction, live imaging, nucleic acid analysis, protease detection and high-throughput screening. BRET technology is based on the phenomenon of resonance energy transfer in some marine animals (such as Renilla luciferase) and results in non-radioactive energy transfer between energy donors and energy acceptors. In BRET, the energy donor is a luminescent luciferase, which emits a spectrum of the corresponding wavelength in the presence of the corresponding substrate. The energy acceptor is usually a fluorescent protein or fluorescent material (small molecule dye or nanomaterial), as long as it can be excited. Light is emitted at long wavelengths by absorbing energy in the spectral range. One of the keys to the formation of BRET is that the donor emission spectrum and acceptor absorption spectrum must overlap for energy transfer to occur. Different substrates have different wavelength ranges that can be excited, and the selected acceptor material also needs to be adjusted accordingly.

Quantum dot (QD) is a kind of semiconductor nanomaterial, because of its high quantum yield, tunable emission wavelength and narrow emission peak, it is suitable for multi-component fluorescence detection of biomarkers, which can shorten the time required for analysis. , saving detection reagents, reducing analysis costs, and becoming an important frontier in the development of fluorescent biosensors. QDs can be excited by a single light source, which is different from other fluorescent dyes (such as fluorescein isothiocyanate (FITC) and rhodamine), which greatly simplifies the dependence of multicolor fluorescence detection on multiple light sources. QDs can also be completely independent of external excitation light sources. In the detection mode established by this invention, the QD, as an acceptor, obtains energy by way of vibrational energy transfer from the luciferase (donor) immobilized on its surface to emit light. An important difference between bioluminescence and general fluorescence detection methods is that it does not require an excitation light source. Their luminescence relies on the oxidation process of fluorescein, and the bioluminescence energy is transferred to the quantum dots in a nonradiative resonance manner to cause them to emit light. Luciferase catalyzes the oxidation of substrates in the presence of oxygen, causing them to emit light. Luciferase can be viewed as a fragment of two linked proteins. These two fragments have the same emission spectral characteristics as the intact luciferase, but their respective catalytic luminescence intensities are only 1/50 and 1/100 of the intact luciferase, respectively. This difference in catalytic luminescence ability is an important basis for luciferase fragment complementation to control bioluminescence and to transfer energy to quantum dots while maintaining low background radiation.

At present, the detection methods of microRNA mainly include Northern blot analysis, microarray analysis and real-time fluorescence quantitative polymerase chain reaction. Northern blot analysis is a common method for detecting RNA based on hybridization. It is one of the earliest methods for microRNA analysis. This method is simple and easy to operate, and can be operated by most laboratories without additional capital investment and Device update. Microarray analysis is also based on the principle of hybridization to detect microRNAs. It analyzes the expression regulation mechanism of microRNAs and the expression of genes regulated by microRNAs by measuring the expression levels of microRNAs in a specific process. Microarrays use high-density fluorescently labeled probes to hybridize with RNA samples, obtain expression maps through fluorescence scanning, and perform miRNA expression analysis with the help of corresponding software. Since all available miRNA sequences can be included when designing probes, microarrays can be used for high-throughput miRNA analysis. Real-time fluorescence quantitative polymerase chain reaction through amplification technology refers to adding fluorescent groups to the polymerase chain reaction system, using the accumulation of fluorescent signals to monitor the entire reaction process in real time, and finally quantitatively analyzing the unknown template through the standard curve. . The higher the copy number of the starting target nucleic acid, the sooner a significant increase in fluorescence intensity is observed.

CN105807064A provides a luciferase complementary quantum dot biosensor and its construction method and application. The luciferase complementary quantum dot biosensor comprises quantum dots, luciferase amino-terminal fragment, luciferase carboxyl-terminal fragment, luciferase A probe that specifically recognizes the analyte and a substrate that can undergo a bioluminescence reaction with the luciferase; the invention combines the optical advantages of the quantum dot sensor, by realizing the amino-terminal fragment and carboxyl-terminal fragment of the luciferase on the surface of the quantum dot Induced complementation, reconstruction of catalytic function, and construction of a new type of high-sensitivity biosensor, which can be applied to the highly targeted detection of various biomarkers, and is suitable for accurate quantification of target detection substances in a homogeneous system, but the steps of this method are Complicated and time-consuming.

To sum up, the rapid development of the national economy has greatly increased the people's attention to health. In order to achieve a high standard of health while achieving a low cost of health, it is urgent to develop a new detection kit based on bioluminescence resonance energy transfer and its Detection method to achieve the purpose of efficiently detecting nucleic acid.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art and actual needs, the present invention provides a detection probe and its application. The present invention designs two probes with specificity of luciferase and quantum dots according to the target nucleic acid, and skillfully combines the The probe and the target nucleic acid form a T-shaped structure. Using the principle of bioluminescence energy resonance transfer, the reaction conditions are optimized, and the target nucleic acid is accurately and efficiently detected, which saves time and cost and has important application value.

For this purpose, the present invention adopts the following technical solutions:

In one aspect, the present invention provides a detection probe, which is designed based on a target nucleic acid, and the probe includes a probe A labeled with luciferase and a probe B labeled with quantum dots;

One end of the probe A is complementary to the partial sequence of one end of the target nucleic acid, one end of the probe B is complementary to the partial sequence of the other end of the target nucleic acid, and the other end of the probe A is complementary to the other end of the probe B There are partially complementary sequences;

The probe A, the probe B and the target nucleic acid are hybridized to form a stable T-shaped structure, and the luciferase and the quantum dots can be paired with each other to perform bioluminescence resonance energy transfer.

In the present invention, in the process of long-term experimental practice, the inventor fully understands the principle and advantages of bioluminescence energy resonance transfer, combines it with the method for detecting nucleic acid by PCR, and ingeniously designs a There are two specific probes of luciferase and quantum dots, which can hybridize with the target nucleic acid to form a stable T-shaped structure, so that luciferase and quantum dots can undergo bioluminescence energy resonance transfer, optimize reaction conditions, and adjust reaction temperature and time. and concentration, accurate and efficient detection of nucleic acid can distinguish single base mutations, saving time and convenient operation.

Bioluminescence resonance energy transfer (BRET) is based on the phenomenon of resonance energy transfer in some marine animals (such as Renilla luciferase) and results in non-radioactive energy transfer between energy donors and energy acceptors. In BRET, the energy donor is a luminescent luciferase, which emits a spectrum of the corresponding wavelength in the presence of the corresponding substrate. The energy acceptor is usually a fluorescent protein or fluorescent material (small molecule dye or nanomaterial), as long as it can be excited. Light is emitted at long wavelengths by absorbing energy in the spectral range. One of the keys to the formation of BRET is that the donor emission spectrum and acceptor absorption spectrum must overlap for energy transfer to occur. Different substrates have different wavelength ranges that can be excited, and the selected acceptor material also needs to be adjusted accordingly.

Preferably, the luciferase includes any one of Gaussia luciferase, Renilla luciferase, sea luciferase or firefly luciferase, preferably Gaussia luciferase.

Preferably, the emission wavelength of the luciferase is 400-600 nm, such as 400 nm, 430 nm, 460 nm, 480 nm, 500 nm, 530 nm or 580 nm, preferably 450-550 nm.

Gaussia luciferase is the latest luciferase discovered, with a molecular weight of 19.9kDa, which is the smallest luciferase (Fluc: 62kDa; Rluc: 36kDa) discovered so far, and has a higher molecular weight than firefly luciferase and Renilla luciferase Ultra-high luminous efficiency (1000 times), extremely high thermal stability (95 ℃ for 30 minutes can still maintain 65% of the bioluminescence capacity). Gaussia luciferase catalyzes the oxidation of Colenterazine in the presence of oxygen, generates its oxidation products and carbon dioxide and emits light. Dots form BRET pairings.

Preferably, the quantum dots include any one of CdSe/ZnS quantum dots, CdTe quantum dots, CdTe/CdS quantum dots, CdTe/CdS/ZnS quantum dots, ZnTe quantum dots, InP/ZnS quantum dots or ZnSe quantum dots .

Preferably, the emission wavelength of the quantum dots is 400-750 nm, such as 400 nm, 430 nm, 460 nm, 500 nm, 550 nm, 600 nm, 655 nm, 680 nm, 700 nm or 750 nm, preferably 600-700 nm.

Preferably, the distance between the luciferase and the quantum dots is 5-10 nm, such as 5 nm, 6 nm, 7 nm, 8 nm, 9 nm or 10 nm.

Preferably, the luciferase is linked to the thiol end of the probe A through SMCC activation.

The SMCC is succinimide-4-(N-maleimide)cyclohexane-1-1 hydroxy acid ester.

Preferably, the quantum dots are linked to the biotin-labeled end of the probe B through streptavidin modification.

Preferably, the length of the partially complementary sequence of probe A and probe B is 4-9 bases, for example, it can be 4, 5, 6, 7, 8 or 9, preferably 5 -8 bases.

In the present invention, when there is no target nucleic acid, probe A and probe B form an unstable double strand, and dissociation occurs when the temperature reaches 37°C, so there is no resonance energy transfer phenomenon. In the case of no excitation light source The quantum dots will not be excited either, but when the length of the complementary sequence between probe A and probe B is greater than 9 bases, a stable duplex will be formed without the presence of the target, which will affect the experimental results.

In the present invention, two nucleic acid probes are designed according to the target nucleic acid, respectively labeling luciferase and 655 quantum dots (luciferase is activated and labeled by SMCC on the nucleic acid with sulfhydryl group, and the quantum dots modified by SA are selected to be labeled to biotin labeled nucleic acid above), in which the emission spectrum of luciferase can excite quantum dots at 655 nm, and in the presence of target RNA, the three can be hybridized to form a stable T-type hybrid structure, resulting in the interaction between luciferase and quantum dots. The distance is shortened so that fluorescence resonance energy transfer can occur. Under the action of the substrate, luciferase catalyzes the oxidation of the substrate to generate its oxidation product and carbon dioxide, and the emission wavelength is 480nm. The efficiency is related to the concentration of the target substance, and the quantitative detection of the target substance is carried out with the fluorescence signal ratio of QD655/Gluc480 as the response value. In the absence of the target RNA, the two nucleic acid probes only have 7 bases complementary to each other, forming an unstable double strand, which will dissociate when the temperature reaches 37 °C, so there is no resonance energy transfer phenomenon. Quantum dots are also not excited without an excitation light source. Among them, after optimizing the reaction time and the ion concentration of the buffer solution in the reaction system, 10 mM magnesium ion concentration for half an hour was selected as the optimal condition. The method can effectively discriminate highly similar RNA families even if only two bases are changed, and can be used to detect DNA, and can detect single-base mutations in DNA.

In a second aspect, the present invention provides a kit comprising the detection probe according to the first aspect.

Preferably, the kit further comprises a luciferase catalytic substrate, a magnesium ion solution and a Tris-HCl buffer;

The concentration of the Tris-HCl buffer was 50 mM, pH=7.4.

In a third aspect, the present invention provides a kit according to the second aspect for detecting relevant RNA biomarkers in blood.

In a fourth aspect, the present invention provides a method for detecting nucleic acid, using the kit described in the second aspect, comprising the following steps:

(1) adding probe A, probe B and luciferase substrate to the magnesium ion solution to obtain a mixed solution;

(2) adding the nucleic acid to be tested to the mixture obtained in step (1), and performing hybridization;

(3) After the hybridization reaction, the fluorescence intensity was detected, and the fluorescence intensity ratio of luciferase and quantum dots was calculated.

Preferably, the concentration of probe A and probe B is 50-200nM, for example, it can be 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 120nM, 140nM, 150nM, 160nM, 180nM or 200nM, preferably 80- 150nM.

Preferably, the concentration of the luciferase substrate is 0.2-1 μg/μL, such as 0.2 μg/μL, 0.3 μg/μL, 0.5 μg/μL, 0.7 μg/μL, 0.9 μg/μL or 1 μg/μL , preferably 0.5 μg/μL;

Preferably, the concentration of the magnesium ion solution is 5-15 mM, such as 5 mM, 7 mM, 9 mM, 11 mM, 13 mM or 15 mM, preferably 9-13 mM.

Preferably, the concentration of the nucleic acid to be detected is 5-100 nM, such as 5 nM, 20 nM, 40 nM, 80 nM or 100 nM, preferably 20-80 nM.

Preferably, the temperature of the hybridization is 30-40°C, such as 30°C, 33°C, 35°C, 38°C or 40°C, preferably 35-38°C.

Preferably, the reaction time of the hybridization is 20-40min, such as 20min, 23min, 25min, 28min, 30min, 35min or 40min, preferably 25-35min.

As a preferred technical solution, a method for detecting nucleic acid specifically includes the following steps:

(1) Add probe A and probe B with a concentration of 50-200 nM and a luciferase substrate with a concentration of 0.2-1 μg/μL into a magnesium ion solution with a concentration of 5-15 nM to obtain a mixed solution;

(2) adding the nucleic acid to be tested into the mixture obtained in step (1), and performing hybridization reaction at 30-40°C for 20-40min;

(3) After the hybridization reaction, the fluorescence intensity was detected, and the fluorescence intensity ratio of luciferase and quantum dots was calculated.

Compared with the prior art, the present invention has the following beneficial effects:

The detection probe provided by the present invention utilizes the principle of bioluminescence energy resonance transfer, and designs probes with specificities of luciferase and quantum dots according to the target nucleic acid. The probe and the nucleic acid can hybridize to form a stable T-shaped structure. Detecting the intensity of fluorescence emitted by luciferase and quantum dots is more accurate and efficient than detecting nucleic acids. It only needs one step of hybridization. The operation is simple. Laboratory conditions are less demanding.

Description of drawings

Fig. 1 is the primary structure distribution diagram of Gaussia luciferase in the specification of the present invention;

Fig. 2 is the bioluminescence principle diagram of Gaussia luciferase in the specification of the present invention;

Fig. 3 is the emission spectrum characteristic diagram of Gaussia luciferase in the specification of the present invention

Fig. 4 is the experimental principle diagram of the present invention;

5 is a fluorescence spectrum response diagram of the present invention, the dark curve is the response diagram of the non-target nucleic acid miR208a, and the light-colored curve is the response diagram of the target nucleic acid miR208a;

Fig. 6 is the homologous RNA detection result figure of the present invention;

FIG. 7 is a diagram showing the results of DNA single-base mutation discrimination according to the present invention.

Detailed ways

In order to further illustrate the technical means adopted by the present invention and its effects, the technical solutions of the present invention are further described below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the scope of the embodiments.

Example 1 Assembling the kit

Design probe A and probe B according to the target series miR208a, wherein the 5' end of probe A is modified with Gaussia luciferase, the 3' end of probe B is modified by quantum dot QD655, target nucleic acid, probe A and probe The sequence of B is shown in Table 1:

Table 1

The primary structure distribution of Gaussia luciferase is shown in Figure 1. The amino acid sequences 27-97 (hGL-27-97) and 98-168 (hGL-98/168) constitute two independent enzyme catalytic structures; Gaussia fluorescence The schematic diagram of bioluminescence of luciferase is shown in Figure 2; the emission spectrum characteristic diagram of Gaussia luciferase is shown in Figure 3.

Other conventional reagents include: 50 mM Tris-HCl buffer pH=7.4.

Probe A and probe B at a concentration of 100 nM, luciferase substrate at a concentration of 0.5 μg/μL, magnesium ion solution at a concentration of 10 mM, other conventional reagents and instructions for use were assembled into a kit.

Embodiment 2 Experimental detection

(1) Add probe A and probe B with a concentration of 100 nM and a luciferase substrate with a concentration of 0.5 μg/μL into a magnesium ion solution with a concentration of 10 nM to obtain a mixed solution;

(2) adding the nucleic acid to be tested to the mixture obtained in step (1), and performing hybridization reaction at 37°C for 30 min;

(3) Detect the fluorescence intensity after the hybridization reaction, and calculate the fluorescence intensity ratio of luciferase and quantum dots;

The sequence of the nucleic acid miR208b to be tested is shown in SEQ ID NO: 4:

SEQ ID NO: 4AUA AGA CGA ACA AAA GGU UUG U

Example 3

Compared with Example 2, except that the nucleic acid to be tested is changed to miR-155, other conditions are the same as in Example 2;

The sequence of MiR-155 is shown in SEQ ID NO:5:

SEQ ID NO: 5UUA AUG CUA AUC GUG AUAGGG GU

Example 4

Compared with Example 2, except that the nucleic acid to be tested is changed to miR-183, other conditions are the same as in Example 2;

The sequence of mi-R183 is shown in SEQ ID NO:6:

SEQ ID NO: 6GUG AAU UAC CGA AGG GCC AUA A

Example 5

Compared with Example 2, except that the nucleic acid to be tested is changed to snp1, other conditions are the same as those of Example 2;

The sequence of snp1 is shown in SEQ ID NO:7:

SEQ ID NO: 7ATA ATA CGA GCA AAA AGC TTG T

Example 6

Compared with Example 2, except that the nucleic acid to be tested is changed to miR-108a, other conditions are the same as in Example 2;

The sequence is shown in SEQ ID NO:1:

SEQ ID NO: 1AUA AGA CGA GCA AAA AGC UUG U

Example 7

Compared with Example 2, other conditions are the same as Example 2, except that the nucleic acid to be tested is a mixture of all mRNAs (including miR-155, miR-185, miR-208a, and miR-208b).

Example 8

Compared with Example 2, other conditions were the same as Example 2 except that the nucleic acid to be tested was changed to a blank control.

result detection

A black 96-well microplate (VICTOR X4, Waltham, MA, PerkinElmer, City, USA) was used to detect the fluorescence of Examples 2-8, and perform statistical analysis;

The principle of the experimental reaction is shown in Figure 4; the fluorescence spectrum response diagrams of miR208a without target nucleic acid and miR208a with target nucleic acid added are shown in Figure 5; miR208a and miR208b are homologous RNAs, and miR-155 and miR-183 are homologous RNAs. The detection result of homologous RNA is shown in Figure 6, snp1 is the single-base mutation DNA of miR208a, and the results of DNA single-base mutation distinction are shown in Figure 7.

To sum up, the present invention provides a detection probe and its application. Through the probes with specificities of luciferase and quantum dots designed according to the target nucleic acid, the probe and the nucleic acid are skillfully formed into a stable T structure. , using the principle of bioluminescence energy resonance transfer, optimizing the reaction conditions, can accurately distinguish single base mutations, effectively detect target nucleic acid, simple operation, short time required, convenient and fast, and low cost.

The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Claims (10)

1. a detection probe, designed based on target nucleic acid, is characterized in that, described probe comprises the probe A that is labeled with luciferase and the probe B that is labeled with quantum dots; One end of the probe A is complementary to the partial sequence of one end of the target nucleic acid, one end of the probe B is complementary to the partial sequence of the other end of the target nucleic acid, and the other end of the probe A is complementary to the other end of the probe B There are partially complementary sequences; The probe A, the probe B and the target nucleic acid are hybridized to form a stable T-shaped structure, and the luciferase and the quantum dots can be paired with each other for bioluminescence resonance energy transfer. 2. probe according to claim 1, is characterized in that, described luciferase comprises any one in Gaussia luciferase, Renilla luciferase, sea luciferase or firefly luciferase, preferably is Gaussia luciferase; Preferably, the emission wavelength of the luciferase is 400-600 nm, preferably 450-550 nm; Preferably, the quantum dots include any one of CdSe/ZnS quantum dots, CdTe quantum dots, CdTe/CdS quantum dots, CdTe/CdS/ZnS quantum dots, ZnTe quantum dots, InP/ZnS quantum dots or ZnSe quantum dots ; Preferably, the emission wavelength of the quantum dots is 400-750 nm, preferably 600-700 nm; Preferably, the distance between the luciferase and the quantum dots is 5-10 nm. 3. probe according to claim 1 and 2, is characterized in that, described luciferase is connected with the sulfhydryl end of described probe A through SMCC activation; Preferably, the quantum dots are linked to the biotin-labeled end of the probe B through streptavidin modification. 4. The probe according to any one of claims 1-3, wherein the length of the partially complementary sequence of the probe A and the probe B is 4-9 bases, preferably 5-8 base. 5. A test kit, characterized in that, comprising the detection probe as described in any one of claims 1-4; Preferably, the kit further includes a luciferase catalytic substrate, a magnesium ion solution and a Tris-HCl buffer. 6. A kit as claimed in claim is used to detect relevant RNA biomarkers in blood. 7. a method for detecting nucleic acid, is characterized in that, adopts the test kit as claimed in claim 5, comprises the steps: (1) adding probe A, probe B and luciferase substrate to the magnesium ion solution to obtain a mixed solution; (2) adding the nucleic acid to be tested to the mixture obtained in step (1), and performing hybridization; (3) After the hybridization reaction, the fluorescence intensity was detected, and the fluorescence intensity ratio of luciferase and quantum dots was calculated. 8. The method according to claim 7, wherein the concentration of probe A and probe B is 50-200nM, preferably 80-150nM; Preferably, the concentration of the luciferase substrate is 0.2-1 μg/μL, preferably 0.5 μg/μL; Preferably, the concentration of the magnesium ion solution is 5-15mM, preferably 9-13mM; Preferably, the concentration of the nucleic acid to be detected is 5-100 nM, preferably 20-80 nM. 9. The method according to claim 7 or 8, wherein the temperature of the hybridization is 30-40°C, preferably 35-38°C; Preferably, the reaction time of the hybridization is 20-40 min, preferably 25-35 min. 10. The method according to any one of claims 7-9, characterized in that, it specifically comprises the steps: (1) Add probe A and probe B with a concentration of 50-200 nM and a luciferase substrate with a concentration of 0.2-1 μg/μL into a magnesium ion solution with a concentration of 5-15 nM to obtain a mixed solution; (2) adding the nucleic acid to be tested to the mixture obtained in step (1), and performing hybridization reaction at 30-40°C for 20-40min; (3) After the hybridization reaction, the fluorescence intensity was detected, and the fluorescence intensity ratio of luciferase and quantum dots was calculated.