Fluorescent sensing probe system and method for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction

Abstract

The invention provides a fluorescent sensing probe system and method for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction, and belongs to the technical field of biological analysis and detection.According to the scheme, target miRNA and hairpin specific recognition-mediated bidirectional primer exchange reaction technology is creatively utilized, and through a double-primer system and a bidirectional strand displacement mechanism, the ovarian cancer miRNA can be detected through the fluorescent sensing probe system. The tandem G-rich repetitive sequence can provide hundreds of tandem binding sites for a signal molecule ThT, and a G tetramer-ThT tandem activation signal amplification system is constructed. According to the system, specific recognition of target miRNA and a hairpin and effective combination of a double-primer system and a two-way strand displacement mechanism mediated nucleic acid amplification technology are fully utilized to develop a serum-based'one-tube 'ovarian cancer specific miRNA accurate quantitative detection technology, and efficient, accurate and rapid detection of a novel ovarian cancer biomarker miRNA can be realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological analysis detection, and particularly relates to a fluorescence sensing probe system and method for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction. BACKGROUND

[0002] Ovarian cancer (OC) is one of the common malignant tumors of the female reproductive system, which seriously threatens the life and health of women. Its incidence rate is high, ranking second in the world, and its mortality rate ranks fourth. There are about 530,000 cases worldwide each year, of which about 275,000 cases die. With the progression of the disease, patients may have abdominal distension or abdominal enlargement, eating difficulties or early satiety, pelvic or abdominal pain, urinary frequency or urgency, indigestion, constipation, etc. These symptoms are often misdiagnosed as irritable bowel syndrome, functional indigestion or urinary tract infection, leading to delayed diagnosis. Studies have shown that if patients can be diagnosed early, the 5-year survival rate can reach 80-90%. Therefore, early screening and diagnosis of ovarian cancer is the key to reducing the death of OC patients.

[0003] The low specificity and high false positive rate of ovarian cancer have always been a problem in screening. Transvaginal ultrasonography (TVS) and pelvic examination are currently effective methods for diagnosing ovarian tumors, which can display pelvic organ images and improve diagnostic results, but they are highly dependent on physician experience and have high false positive rates, but lack sensitivity. Serum biomarker tests have been used for early diagnosis of ovarian cancer, such as ovarian cancer tumor markers CA-125, human epididymis protein (HE4), alpha-fetoprotein, kallikrein, prostaglandin, beta human chorionic gonadotropin, etc. Currently, CA-125 is the most commonly used biomarker for differentiating benign and malignant ovarian cancer, but it has low specificity and can also be elevated in benign diseases such as endometriosis and ovarian cysts; its sensitivity is also low, with about 50% of early patients not expressing CA-125. Despite the discovery of these tumor biomarkers, an ideal biomarker with high specificity and sensitivity remains a challenge.

[0004] MicroRNA (miRNA) is a class of endogenous, non-coding RNA molecules with a length of about 19-25 nucleotides, which regulates the expression of target genes mRNA or inhibits the translation process by binding to the 3' non-coding region (3'UTR) of the mRNA. Studies have shown that the expression of dysfunctional miRNA plays an important role in the mechanism of ovarian cancer progression, and is an important regulatory factor in the process of ovarian cancer. miRNA is closely related to the occurrence and development of ovarian cancer, and is simple, convenient, and less painful for patients when used for detection. Its expression is stable and can be expressed and occurred in different environments. Compared with CA-125, miRNA has higher sensitivity and specificity, and is considered to be a suitable research marker for several human cancers including ovarian cancer, showing great research potential. Therefore, the use of abnormal miRNA levels in tissues, serum, plasma, and urine as a new biomarker for OC diagnosis is expected to achieve non-invasive diagnosis of ovarian cancer through liquid biopsy technology, and its detection results have more clinical significance than traditional diagnostic targets.

[0005] Traditional miRNA detection methods include Northern blotting, PCR (Polymerase chain reaction), RT-qPCR (Real time quantitative PCR), and sequencing technology. Northern blotting is the earliest attempt to analyze miRNA expression. This method based on probe hybridization technology has low specificity and sensitivity, is time-consuming and requires a large amount of RNA sample; PCR and RT-qPCR have the advantages of mature technology, high sensitivity, and have become the gold standard for nucleic acid detection, but have limitations such as non-specific amplification, expensive equipment, the need for specialized PCR laboratories and skilled personnel, high detection cost, long detection time, etc. Sequencing technology also requires a long time and depends on sequencing instruments. At present, the demand for high sensitivity, rapid, and home-based detection is particularly urgent to solve the problems of shortage of professional personnel, long waiting time for detection results, and low detection reliability.

[0006] Primer Exchange Reaction (PER) is a signal amplification technology that can autonomously synthesize and assemble single-stranded DNA molecules according to a preset reaction path and sequence. The core of PER is programmable catalytic hairpin assembly and step-by-step primer extension, which can autonomously synthesize long single-stranded DNA in an isothermal, programmable and site-responsive pathway with the help of DNA polymerase, short primers and hairpins. These DNA sequences can be designed to bind dyes to generate fluorescent signals, or as efficient reporter molecules or pre-amplifiers for subsequent detection. However, the traditional PER has only one hairpin primer unidirectional extension, which has the disadvantages of slow reaction rate and low amplification performance. Bidirectional Primer Exchange Reaction (B-PER) allows two primers to extend simultaneously towards the middle, and finally connect into a complete double strand, which has the advantages of doubling the reaction speed (bidirectional synthesis) and higher product purity (reducing non-specific by-products), effectively solving the shortcomings of traditional PER. B-PER has broad application prospects in biological analysis due to its stronger programmability and easy control. SUMMARY

[0007] To solve the above technical problems, the present scheme provides a fluorescence sensing probe system for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction, as well as a preparation method and application thereof. The specific recognition of the target and the hairpin mediates the bidirectional primer exchange reaction (B-PER) technology, and through the double primer system and the bidirectional strand displacement mechanism, a primer sequence that can be bidirectionally combined is obtained to solve the problem of non-specific adsorption and false positive of primer. On this basis, DNA polymerase extension is combined to obtain a DNA long chain sequence with G-Quadruplex array, which provides more binding structures for the "lighting" of ThT signal molecules, and establishes a rapid, label-free nucleic acid detection technology platform.

[0008] To achieve the above purpose, the present scheme first provides a fluorescence sensing probe system for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction, which comprises a hairpin probe Y1, a DAB arch bridge, a Bst DNA polymerase and a K + In the presence of the target miRNA, the target miRNA specifically binds to the hairpin probe Y1, triggering the bidirectional primer amplification of the DAB arch bridge to detect the target miRNA; The sequence of the hairpin probe Y1 is shown in SEQ ID NO. 1; The DAB arch bridge is self-assembled by base complementary pairing of probe F1 and probe F2, the sequence of the probe F1 is shown in SEQ ID NO. 2; and the sequence of the probe F2 is shown in SEQ ID NO. 3; The target miRNA is miRNA-21, and the sequence of miRNA-21 is shown as SEQ ID NO. 4.

[0009] Based on the overall inventive concept, the scheme also provides a method for detecting ovarian cancer miRNA by using a fluorescent sensing probe system for non-diagnostic purposes, comprising the following steps: S1, probe pretreatment: the synthesized hairpin probe Y1, probe F1 and F2 lyophilized powder are respectively configured into 100 µM stock solution with DEPC water, and stored at 4 ℃ for standby; S2, annealing of hairpin probe Y1: after annealing the hairpin probe Y1 at 95 ℃ for 5 min, slowly cool it to room temperature, so that it self-assembles into a hairpin structure through base complementary pairing, and is stored in a 4 ℃ refrigerator for standby; S3, DAB arch bridge construction: mix probes F1 and F2 in PBS buffer at a molar ratio of 1:1, anneal at 95 ℃ for 5 min, then slowly cool to room temperature, so that it self-assembles into an arch bridge structure DAB through base complementary pairing, and is stored in a 4 ℃ refrigerator for standby; S4, target activated bidirectional primer exchange reaction: incubate the miRNA to be tested with 1 µM hairpin probe Y1 in PBS buffer at 37 ℃ for 30 min, add Bst DNA polymerase, reaction raw materials dATP, dTTP, dCTP and 1× Bst Reaction Buffer required for primer exchange reaction, and carry out bidirectional primer exchange reaction at 60 ℃ for 120 min, then incubate at 80 ℃ for 2 min to inactivate the enzyme and terminate the reaction; S5, G-quadruplex structure formation and ThT signal activation and fluorescence detection: add 40 µM ThT to the system prepared in S4, react in PBS buffer at 37 ℃ for 30 min, and collect the fluorescence spectrum of the miRNA to be tested activating the PER reaction by RF-6000 fluorescence spectrophotometer As a preferred, the PBS buffer in step S3 has a pH of 7.4 and contains 5 mM Mg 2+ .

[0010] As a preferred, the PBS buffer in step S4 has a pH of 7.4 and contains 5 mM Mg 2+ .

[0011] As a preferred, the 1× Bst Reaction Buffer in step S4 has a pH of 8.8 and includes 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, and 0.1% Triton X-100.

[0012] As preferred, the PBS buffer in step S5 has a pH of 7.4, and includes 50 mM KCl.

[0013] As preferred, the fluorescence spectrophotometer in step S5 is set to an excitation wavelength Ex = 450 nm and an emission wavelength Em = 465-600 nm.

[0014] The mechanism of the detection system provided in the present solution for detecting the target miRNA-21 is as follows: The functional hairpin structure Y1 (the primer sequence T is closed in the hairpin structure) and the DNA arch (DAB) structure with bidirectional primer amplification potential, when encountering the target miRNA, the miRNA first specifically binds to Y1 through the base complementary pairing mediated by the sticky end, the hairpin structure is opened, thereby exposing the primer sequence T (Y1-T) which can be complementary to the b domain on the right side of the DAB arch. At this time, Y1-T binds to the b domain on the right side of the DAB through base complementary pairing, providing a site for the first PER reaction to occur. The primer sequence T amplifies a new primer sequence along the b domain on the right end of the DAB by using the 3'-5' extension property of the Bst DNA polymerase, forms a Y1-T-b-a structure, and separates itself from the DAB arch; at this time, the a domain in the Y1-T-b-a structure can be used as a new primer sequence to bind to the a domain on the left side of the DAB through base complementary pairing, further extending the a-a sequence under the action of the Bst DNA polymerase, and spontaneously separating from the DAB to release into the system, i.e., triggering the second PER reaction.

[0015] The a domain sequence is composed of the telomere sequence TTAGGG, and under the mediation of the double-primer system and the bidirectional strand displacement mechanism, a repeated tandem TTAGGG sequence [TTAGGG] n , under the assistance of K + , a G-quadruplex array can be formed, providing hundreds of binding sites for the embedding of ThT signal molecules, and the fluorescence is significantly enhanced, thereby realizing the specific and accurate quantitative detection of miRNA in the serum of ovarian cancer patients.

[0016] The detection of the present probe system is crucial in that: (1) Design of the functional hairpin probe Y1: the key primer sequence T is closed by the hairpin structure in the initial state, and only when the target miRNA precisely pairs through the sticky end, the hairpin structure can be opened, thereby realizing target-triggered initiation and significantly improving the specificity.

[0017] (2) Bidirectional primer function of the DNA arch structure (DAB): The right b domain: complementary to the primer T released by Y1, initiating the first PER extension; Left a domain: contains telomere repeat sequence (TTAGGG), which is complementary to the a domain in the first extension product, triggering the second PER extension and exponential amplification.

[0018] (3) Dual cascade PER amplification mechanism: First stage amplification: miRNA opens Y1→ exposes primer T→ binds DAB-b domain→ extension generates Y1-T-b-a; Second stage amplification: a domain in the product binds DAB-a domain→ extension generates a-a double strand→ new product can again bind other DAB as primer, forming a self-circulating amplification, achieving exponential signal amplification.

[0019] (4) G-quadruplex array signal output system: Amplification product contains [TTAGGG] n repeat units→ self-assemble into large G-quadruplex arrays in the presence of K + +→ embed ThT dye to produce ultra-high intensity fluorescence (single array binds hundreds / thousands of dye molecules).

[0020] (5) Integrated detection at constant temperature: Throughout the process relies on Bst DNA polymerase (with both strand displacement activity and 3'→5' extension ability), to complete target recognition, dual amplification and signal output under a single constant temperature condition.

[0021] The high specificity and sensitivity of the probe system of this scheme are embodied in the following points: (1) High specificity: Dual recognition mechanism: detection initiation depends on the precise base pairing of the target miRNA and the sticky end of the Y1 hairpin to open the hairpin. The subsequent cascade reaction depends on the precise base pairing of Y1-T and DAB b domain, and the a domain in the extension product and DAB a domain.

[0022] Hairpin structure closure: in the initial state, the key primer sequence T is closed in the hairpin structure, and can only be exposed and involved in the reaction when the correct target exists, effectively reducing the generation of non-specific background signal.

[0023] (2) Ultra-high sensitivity: Dual cascade signal amplification: First stage amplification (PER1): a single miRNA molecule triggers Y1 and DAB binding, Bst polymerase extension generates Y1-T-b-a structure (contains an a domain).

[0024] Second-stage amplification (PER2 + tandem amplification): the a domain in Y1-T-b-a serves as a new primer to bind with the a domain of DAB, and again extension produces a-a structure. Most importantly, this a-a structure (TTAGGG) can also serve as a primer to bind with the a domain of another DAB molecule, initiating the next round of PER2 reaction. This forms an exponential chain displacement amplification cycle, constantly producing more products containing [TTAGGG]n repeat units.

[0025] G-quadruplex array super-amplification: the produced [TTAGGG]n long chain forms a huge G-quadruplex array in the presence of K+. One such array can bind hundreds of ThT fluorescent molecules, producing an extremely strong fluorescence signal. This dual amplification mechanism consisting of enzymatic amplification (PER) and nanostructure self-assembly (G-quadruplex) converts a single miRNA event into an extremely bright fluorescence signal, achieving ultra-sensitive detection.

[0026] (3) Simple and fast operation: Constant temperature reaction: the entire detection process only relies on Bst DNA polymerase (with strand displacement activity) and is carried out at a single constant temperature (usually 60-65 ℃). No complex PCR thermal cycler is needed, simplifying equipment requirements, shortening detection time, and being more suitable for on-site or resource-limited environments.

[0027] (4) Stable signal output and low background: "Turn-On" fluorescence detection: ThT dye has very weak fluorescence in the free state, and only when it is embedded in the G-quadruplex structure does the fluorescence significantly increase. This feature makes the background signal extremely low, the signal-to-noise ratio high, and the detection result more reliable and intuitive.

[0028] Solid-state fluorescence enhancement: the formation of G-quadruplex array provides a solid or quasi-solid fluorescence enhancement platform, with better stability than single molecule labeling in solution.

[0029] (5) Ingenious design, no need for complex labeling: The core of the entire system is the ingeniously designed DNA structure (Y1 hairpin and DAB arch) and sequence (telomere repeat sequence TTAGGG). Signal generation relies on enzymatic reaction and nucleic acid self-assembly, without the need for expensive fluorescent / quenching group labeling of probes or targets, reducing costs.

[0030] Compared with the prior art, the present application has the following beneficial effects: (1) The scheme can realize the target activation-B-PER amplification-signal transduction "one-pot" cascade reaction, which can effectively avoid the defects of traditional PER detection, improve the sensitivity, and reduce the non-specific reaction. The nucleic acid detection platform technology is used to develop a "one-pot" blue super specific miRNA cascade detection method.

[0031] (2) The DNA arch structure (DAB) two-way primer function, double cascade PER amplification mechanism and G-quadruplex array signal output system are innovatively combined to construct a low-background, label-free, constant-temperature integrated system for precise detection of ovarian cancer target miRNA, which has the characteristics of strong specificity, high sensitivity, simple operation and the like.

[0032] (3) The scheme overcomes the problems of high cost, low sensitivity and specificity, and radiation of existing clinical imaging diagnosis of OC, solves the problems of complex operation, expensive equipment and high learning cost of existing nucleic acid detection technology, and the slow reaction rate and low amplification performance of traditional PER, and provides a promising solution for the field of ovarian cancer detection, which has important significance for improving the accuracy of ovarian cancer diagnosis, early detection and treatment.

[0033] (4) The scheme innovatively uses the target miRNA and hairpin specific recognition mediated two-way primer exchange reaction (B-PER) technology to obtain a series of G-rich repeat sequences through a double primer system and a two-way strand displacement mechanism, which can provide hundreds of thousands of series of binding sites for the signal molecule ThT, and construct a G-quadruplex-ThT series of activated signal amplification system.

[0034] (5) The system fully utilizes the specific recognition of target miRNA and hairpin and the nucleic acid amplification technology mediated by double primer system and two-way strand displacement mechanism, and develops a "one-pot" ovarian cancer specific miRNA precise quantitative detection technology based on serum, which can realize efficient, precise and rapid detection of new biomarker miRNA of ovarian cancer. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0036] Figure 1 The probe system detection principle diagram in embodiment 1 is shown in Figure 1. Figure 2 The DNA probe assembly and target activation feasibility analysis in embodiment 2 are shown in Figure 2. Figure 3 For the B-PER cycle feasibility analysis in Example 3; Figure 4 For the B-PER reaction analysis initiated by the target miR-21 in Example 3; Figure 5 For the performance analysis of the PER and the target in Example 4, a is a fluorescence intensity graph of the B-PER system and different concentrations of miRNA-21, and b is a linear relationship graph of the B-PER system and different concentrations of miRNA-21; Figure 6 For the target specificity analysis of the PER system in Example 5, a is a fluorescence intensity graph, and b is a fluorescence intensity quantitative graph. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and advantages to be solved by the present application clearer, specific embodiments will be described in detail below with reference to the drawings.

[0038] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0039] If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art; if not specifically indicated, the reagents used in the examples are commercially available.

[0040] The probes involved in the present application are all purchased from Shanghai Sangon Biological Engineering Co., Ltd., as shown in Table 1:

[0041] Example 1 Detection of ovarian cancer miRNA based on a fluorescence sensing probe system of bidirectional primer exchange reaction.

[0042] (1) Probe pretreatment: The synthesized probes Y1, F1, F2 and miRNAs were respectively configured into 100 µM stock solutions with DEPC water, and stored at 4 ℃ for standby.

[0043] (2) Annealing of hairpin probe: After annealing Y1 hairpin probe at 95 ℃ for 5 min, it was slowly cooled to room temperature to self-assemble into a hairpin structure through base complementary pairing, and stored in a 4 ℃ refrigerator for standby.

[0044] (3) DAB arch bridge construction: Probes F1 and F2 were added to 5 mM Mg 2+The DNA probes were mixed uniformly in pH 7.4 PBS buffer, annealed at 95 ℃ for 5 min, and then slowly cooled to room temperature to self-assemble into an arch structure by base complementary pairing, and stored in a refrigerator at 4 ℃ for standby.

[0045] (4) Target-activated B-PER reaction: first, the miRNA to be tested was incubated with 1 µM Y1 probe in pH 7.4 PBS (5 mM Mg 2+ ) at 37 ℃ for 30 min. If the miRNA to be tested was the target miRNA-21, it was fully combined with Y1 to expose the primer sequence T (Y1-T) complementary to the right b domain of DAB. At this time, Y1-T was combined with the right b domain of DAB through base complementary pairing (37 ℃ reaction for 30 min) to provide a site for the first PER reaction, and the Bst enzyme, reaction raw materials dATP, dTTP, dCTP, and 1× Bst Reaction Buffer (pH 8.8, including 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100) required for the PER reaction were added. At this time, the a domain in the Y1-T-b-a structure can act as a new primer sequence and combine with the a domain on the left side of DAB through base complementary pairing, and further extend the a-a sequence under the action of Bst DNA polymerase and spontaneously separate from DAB to release into the system, triggering the second PER reaction. In this way, multiple cycles can be performed to produce a large amount of G-rich DNA sequence (65 ℃ reaction for 120 min). Subsequently, the enzyme can be inactivated by incubation at 80 ℃ for 2 min, and finally the target activation feasibility can be verified by polyacrylamide gel electrophoresis.

[0046] (5) G-quadruplex structure formation and ThT signal activation and fluorescence detection: 40 µM ThT was added to the above system, and the reaction was carried out in PBS buffer (pH 7.4, 50 mM KCl) at 37 ℃ for 30 min. The fluorescence spectrum of the target miRNA-activated PER reaction was collected using an RF-6000 fluorescence spectrophotometer with an excitation wavelength Ex = 450 nm and an emission wavelength Em = 465-600 nm.

[0047] The detection principle is shown in Figure 1 .

[0048] Example 2 DNA probe assembly and target activation feasibility analysis.

[0049] The DNA probe assembly and target miRNA-21 activation feasibility analysis were investigated by 12% polyacrylamide gel electrophoresis (PAGE), as shown inFigure 2 As shown, lane 1 is a DNA marker, lane 2 is the miRNA-21 chain, lane 3 is the Y1 sequence, lane 4 is the F1 chain, and lane 5 is the F2 chain. These serve as basic elements, and the band positions when they exist alone reflect the molecular mass characteristics of each oligonucleotide chain. With the gradual addition of different DNA complementary sequences, the electrophoresis bands change regularly. In lane 6, after the Y1 chain and miRNA-21 chain are mixed, the appearance of new bands indicates that the two have achieved initial assembly through complementary base pairing; in lane 7, the F1 chain and F2 chain combine to form characteristic bands, indicating the formation of a DAB bridge; lane 8 shows the Y1 plus miRNA-21 after water bath annealing and the F1 plus F2 after water bath annealing, taking the same volume and incubating at 37°C for 30 min, a band higher than the DAB bridge and Y1 plus miRNA-21 appeared in the lane, indicating that the two were paired; Lane 9 was a mixture of F1, F2 and Y1, and a band of F1 plus F2 pairing appeared, but no three-chain binding product appeared, indicating that the T sequence in the Y1 structure could not bind to the DAB bridge when the target was not present; Lane 10 was the electrophoresis band after the DAB bridge after water bath annealing reacted with Y1 annealed in a water bath; The bands in lanes 9 and 10 were compared with the results in lane 8, which showed that after annealing the two, in the absence of the target (miRNA-21), Y1 would not react with the DAB bridge and would not enter the next B-PER cycle reaction; Lane 11 mixed the four chains Y1, miRNA-21, F1 and F2, further confirming the feasibility of multi-chain assembly; The electrophoresis experiment results showed that this study could respond to the target miRNA-21.

[0050] Example 3 B-PER cycle feasibility analysis.

[0051] To verify whether PER cycle products were generated, 12% PAGE was used for further investigation. Figure 3 As shown, lane 1 is a DNA marker, lane 2 shows the bands after the reaction of Y1 with miRNA-21, and lane 3 shows the paired binding of F1 and F2. Lane 4 shows the annealed F1 and F2 followed by the addition of Y1. Only the binding product of F1 and F2 is observed, with no bands indicating three-strand binding, consistent with the expected results. Lane 5 shows the assembly of four strands, Y1, miRNA-21, F1, and F2. A higher band is observed in this lane, indicating the presence of a binding product. Lane 6 shows the PER reaction in which Y1 is added to the target (miRNA-21), DAB, enzyme, and dNTPs at 65°C for 60 minutes. Bands appear at different positions in this lane, which, compared to lanes 2, 3, and 5, indicates the formation of a PER product. Lane 7 is a control reaction without the addition of target. The absence of a higher band in lane 7 indicates that the PER reaction cannot proceed to the next step without the addition of target.

[0052] Subsequently, the target miRNA-21 initiated B-PER reaction was investigated by fluorescence spectrophotometer. The fluorescence of ThT was significantly enhanced after the interaction of ThT with the DNA G-quadruplex activated by B-PER product. The label-free fluorescent signal output mode of G-quadruplex “turn on” ThT (G-Quadruplex-ThT) was constructed, and the results are shown in Figure 4 As there was no target miRNA-21 in the control group, Y1 and DAB remained the initial structure, no G-quadruplex was formed, and ThT could not be embedded. However, there was still a certain background fluorescence signal when ThT was added. When the target miRNA-21 was present, Y1 and miRNA-21 and DAB bridge could undergo PER cycle, constantly producing telomere sequences of TTAGGG, which could form G-quadruplex and produce strong fluorescence signal after binding with ThT, indicating that the sensing probe could be used for the analysis and detection of miRNA-21.

[0053] Example 4 Analysis of the response performance of PER to the target.

[0054] To evaluate the response ability of the B-PER system to the target miRNA-21 molecule in detail, we investigated the fluorescence response behavior of different concentrations of miRNA-21 (0 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM) to the B-PER system. The results are shown in Figure 5 As the concentration of miRNA-21 increased, the fluorescence signal gradually increased. In the range from 0 pM to 10 nM, it was found that there was an excellent linear relationship between the fluorescence intensity and the logarithm (Log) of the concentration of miRNA-21, and the linear regression equation was y = 276.99x + 579.05 (x represented the logarithm of the concentration of miRNA-21), and the linear correlation coefficient R 2 was 0.9645. The lowest detection limit (LOD) was calculated by 3 times the standard deviation method to be 150 fM, indicating that the B-PER system could realize the sensitive detection of the target miRNA-21.

[0055] Example 5 Analysis of the specificity of the B-PER system to the target.

[0056] At present, more than 1000 kinds of human miRNA have been found, and distinguishing different miRNA can provide new methods and means for early diagnosis and treatment of cancer. In order to study the specificity of the B-PER system in the design, different miRNA were used as controls, including miRNA-155 (SEQ ID NO. 5), miRNA-24 (SEQ ID NO. 6), and miRNA-200b (SEQ ID NO. 7), which were measured at the same volume and concentration, and the fluorescence intensity was as shown in Figure 6 The fluorescence intensity of miRNA-21 was the highest, and the results showed that the B-PER system had good specificity and could accurately detect the target miRNA-21.

[0057] The above is only the preferred embodiment of the present application, and the protection scope of the present application is not limited to the above examples. Improvements and changes obtained by those skilled in the art without departing from the technical concept of the present application should also be considered as the protection scope of the present application.

Claims

1. A fluorescent sensing probe system for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction, characterized in that, The system comprises a hairpin probe Y1, a DAB arch bridge, a Bst DNA polymerase and K + In the presence of the target miRNA, the target miRNA specifically binds to the hairpin probe Y1, triggering bidirectional primer amplification of the DAB arch bridge to realize detection of the target miRNA. The sequence of the hairpin probe Y1 is shown as SEQ ID NO.

1. The DAB arch bridge is formed by self-assembly of base complementary pairing of the probe F1 and the probe F2, the sequence of the probe F1 is shown as SEQ ID NO. 2; the sequence of the probe F2 is shown as SEQ ID NO.

3. The target miRNA is miRNA-21, and the sequence of miRNA-21 is shown as SEQ ID NO.

4.

2. A method for detecting ovarian cancer miRNA using the fluorescent sensing probe system according to claim 1 for non-diagnostic purposes, characterized by, The method comprises the following steps: S1, probe pretreatment: the synthesized hairpin probe Y1, probes F1 and F2 are configured into 100 µM stock solution with DEPC water respectively, and stored at 4 ℃ for standby; S2, hairpin probe Y1 annealing: after annealing the hairpin probe Y1 at 95 ℃ for 5 min, it is slowly cooled to room temperature, so that it is self-assembled into a hairpin structure by base complementary pairing, and stored in a 4 ℃ refrigerator for standby; S3, DAB arch bridge construction: the probes F1 and F2 are mixed uniformly in PBS buffer according to a molar ratio of 1:1, annealed at 95 ℃ for 5 min, and then slowly cooled to room temperature, so that they are self-assembled into an arch bridge structure DAB by base complementary pairing, and stored in a 4 ℃ refrigerator for standby; S4, target activated bidirectional primer exchange reaction: the miRNA to be tested is incubated with 1 µM hairpin probe Y1 in PBS buffer at 37 ℃ for 30 min, and the Bst DNA polymerase, reaction raw materials dATP, dTTP, dCTP and 1×Bst Reaction Buffer required for primer exchange reaction are added to occur bidirectional primer exchange reaction at 60 ℃ for 120 min, and then incubated at 80 ℃ for 2 min to deactivate the enzyme and terminate the reaction; S5, G-quadruplex structure formation and thioflavine T signal activation and fluorescence detection: 40 µM thioflavine T is added to the system prepared in S4, and reacted in PBS buffer at 37 ℃ for 30 min, and the fluorescence spectrum of the miRNA to be tested activating the PER reaction is collected by RF-6000 fluorescence spectrophotometer.

3. The method of claim 2, wherein, The PBS buffer in step S3 has a pH of 7.4 and contains 5 mM Mg 2+ .

4. The method of claim 2, wherein, The PBS buffer in step S4 has a pH of 7.4 and contains 5 mM Mg 2+ .

5. The method of claim 2, wherein, The pH of the 1× Bst Reaction Buffer in step S4 is 8.8, which comprises 20 mM Tris-HCl, 10 mM(NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100.

6. The method of claim 2, wherein, The pH of the PBS buffer in step S5 is 7.4, which comprises 50 mM KCl.

7. The method of claim 2, wherein, The fluorescence spectrophotometer in step S5 is set to an excitation wavelength Ex=450 nm and an emission wavelength Em=465-600 nm.

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