CN112795627A

CN112795627A - Method for improving sensitivity of detecting circular RNA and detection kit

Info

Publication number: CN112795627A
Application number: CN202110193824.5A
Authority: CN
Inventors: 王芙艳; 郭灿; 熊炜; 曲红科; 曾朝阳; 黄河; 伍旭; 葛军尚; 刘凌云; 石磊; 张文玲; 李桂源
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2021-02-21
Filing date: 2021-02-21
Publication date: 2021-05-14

Abstract

The invention provides a method for improving the sensitivity of detecting circular RNA and a detection kit. The method comprises the steps of adding a sample to be detected, a molecular beacon probe and T7 exonuclease into a detection system for reaction, detecting fluorescence intensity, and obtaining the concentration of circular RNA through a standard curve prepared in advance. The circRNA can be hybridized with the molecular beacon probe, T7Exo can carry out enzyme digestion on the hybridized compound, the released fluorophore can enhance the fluorescent signal, and the circRNA can enter into the continuous hybridization with MB, so that the cycle process of hybridization, enzyme digestion and release is realized, the fluorescent signal of the system is enhanced by multiple times, and the aim of sensitive detection of the circRNA is fulfilled. The method is simple and convenient to operate, does not need a complex instrument, has high specificity and good sensitivity, has the lower detection limit as low as 0.31pM, and can be applied to the detection of the circRNA in a cell lysate. The method has good application prospect.

Description

Method for improving sensitivity of detecting circular RNA and detection kit

Technical Field

The invention belongs to the technical field of circular RNA detection, and particularly relates to a method for improving the sensitivity of circular RNA detection and a detection kit.

Background

The CircRNA is a novel non-coding RNA and widely exists in various organisms. It is formed by reverse splicing of precursor RNA (pre-RNA), some of which introns or exons are formed into circular structures by reverse splicing. Recent studies have shown that circular RNA is more stable and conserved than linear RNA because the 3 'and 5' ends are not exposed. The study of their expression profiles also showed that their specificity was high. Circular RNA has a variety of biological functions, including acting as a miRNAs sponge, binding to RNA binding proteins, regulating transcription or splicing, encoding small peptides, regulating epigenetics, and the like. In recent years, more and more reports show that the expression profile of circRNA is changed in various diseases, such as the change of tumor, the regulation and control of metabolic diseases, the change of nervous system diseases and the like. Based on the changes in the expression profile of circRNA, we believe that circRNA can be an effective diagnostic and therapeutic target for a variety of diseases. Therefore, it is important to establish a sensitive and selective detection method for circRNA.

The traditional circRNA detection methods such as northern blotting, real-time fluorescent polymerase chain reaction (qRT-PCR), microarray analysis, next-generation sequencing and the like have certain limitations. Multiple Northern blotting steps, long hybridization time, large sample amount and low sensitivity; qRT-PCR requires a necessary reverse transcription step to obtain cDNA followed by an amplification step, which increases the cost of the experiment and complexity of design, and may also have false positive results; the microarray technology and the second generation sequencing detection have the disadvantages of complicated steps, high cost and the like, which limit the wide application. Therefore, it is important to develop a new detection method with high sensitivity, high specificity, low cost, high efficiency and simplicity.

The fluorescent sensor based on T7Exo auxiliary circulation signal amplification realizes the sensitive detection of the circular RNA for the first time, the detection limit can reach 0.31pM, and the method can be applied to the detection of cell lysate samples, and has great application value.

Disclosure of Invention

The invention designs a molecular beacon probe (MB) capable of being used for circular RNA detection, wherein the 5' end of the molecular beacon probe (MB) is provided with a quenching group BHQ1, the 3' end of the molecular beacon probe is provided with a fluorescent group FAM, the fluorescence is firstly quenched due to the existence of a hairpin structure, and 2-6 unhybridized bases are additionally designed at the 5' end to protect the MB from being digested by T7 exonuclease (T7 Exo). T7Exo was able to degrade RNA or DNA on the RNA/DNA hybrid duplex in the 5'→ 3' direction, but was unable to degrade double-stranded or single-stranded RNA. Circbart2.2 was chosen as a model to validate the detection system proposed by the present invention. In a reaction system, MB can perform hybridization reaction with a target circular RNA and be cut by T7Exo enzyme, complete circRNA and fluorophore can be released, the circRNA can continue to perform hybridization reaction, the cycle process of hybridization, enzyme cutting and fluorophore release of one circRNA and a plurality of MB is realized, a fluorescence signal is obviously amplified, and the rapid and sensitive detection of the circRNA can be realized by recording the fluorescence signal of the system.

The primary object of the present invention is to provide a method for improving the sensitivity of detecting circular RNA based on the above reaction mechanism. The method comprises the following steps: adding a sample to be detected, a molecular beacon probe and T7 exonuclease into a detection system for reaction, detecting the fluorescence intensity, and obtaining the concentration of the circular RNA through a standard curve.

Further, the molecular beacon probe is a double-stranded stem-loop structure, the 5 'end of the molecular beacon probe is marked with a quenching group, the 3' end of the molecular beacon probe is marked with a fluorescent group, and 2-6 unhybridized bases, preferably 3 unhybridized bases are added to the 5 'end of the molecular beacon probe than to the 3' end of the molecular beacon probe; the molecular beacon probe with the double-stranded stem-loop structure cannot be cut by T7 exonuclease.

Further, the 5' end of the molecular beacon probe comprises 6 to 20T bases, preferably 6T bases, in order to make the molecular beacon probe more stable.

Further, when the target circular RNA exists, the target circular RNA is hybridized with the molecular beacon probe to form a DNA/RNA double-helix structure, so that the stem part of the molecular beacon probe is opened, the molecular beacon probe can be cut by T7 exonuclease, the circular RNA is separated from the molecular beacon probe, a fluorophore and the circular RNA are released, the hybridization part of the molecular beacon probe is cut into single bases, and the fluorophore is free in the reaction system and keeps the inherent fluorescence property; the released circular RNA hybridizes with another molecular beacon probe, triggering the next hybridization and cleavage cycle, thereby releasing more fluorophore.

Further, the concentration of the molecular beacon probe is 100-300nM, preferably 200nM, and the concentration of the T7 exonuclease is 100-200U/ul, preferably 100U/ul.

Furthermore, in the reaction system, when the reaction temperature is 35-40 ℃, preferably 37 ℃, the reaction time is 3-5h, preferably 3h,

furthermore, buffer, RNase inhibitor and enzyme-free water are required to be added into the reaction system.

Further, when circbart2.2 is detected, the designed molecular beacon probe sequence is as follows: BHQ1-ACG CCG GAC CTT GCC CGT TTT TTT GTC CGG-FAM.

The second object of the present invention is to provide a kit for detecting the above-mentioned method, comprising: molecular beacon probes and T7 exonuclease.

The kit further comprises: buffer, RNase inhibitor, enzyme free water.

The method of the invention excludes the use for diagnostic purposes.

The third purpose of the invention is to provide the application of the molecular beacon probe in preparing the preparation for detecting the circular RNA.

The molecular beacon probe is of a double-chain stem-loop structure, the 5 'end of the molecular beacon probe is marked with a quenching group, the 3' end of the molecular beacon probe is marked with a fluorescent group, and 2-6 unhybridized basic groups are added to the 5 'end of the molecular beacon probe than to the 3' end of the molecular beacon probe; the molecular beacon probe with the double-chain stem-loop structure cannot be cut by T7 exonuclease; the ring part of the molecular beacon probe comprises 6-20T bases; the target circular RNA can be hybridized with the molecular beacon probe to form a DNA/RNA double helix structure, so that the stem part of the molecular beacon probe is opened.

In conclusion, the invention designs a fluorescent sensor by utilizing T7 exonuclease auxiliary target circulation, which is used for rapidly and sensitively detecting circRNA. The target circRNA is quantitatively detected by fluorescence enhancement by utilizing the enzymatic activity of T7Exo and the unique structure of MB, so that high sensitivity and selectivity are achieved. The method directly detects the circRNA, does not need to carry out external modification on the circRNA, does not damage the structure of the circRNA in the reaction, does not need complicated steps and complex instruments, and is simple, convenient, high in sensitivity and good in specificity; the amplification of a fluorescent signal can be realized by a trace amount of target, and the detection of pM level can be realized; this is difficult to achieve by the circRNA detection technology reported at present. The invention not only detects the synthesized sample, but also successfully detects the expression condition of the circRNA in different cell lines in cell lysate, and has very good practicability.

In the invention, the detection of the expression level is carried out by taking circBART2.2 coded by EBV as an example, and a detection means can be provided for early diagnosis of diseases related to EBV infection. The method can also be applied to the detection of other circRNA, and has potential application value in clinical and biomedical research.

Drawings

FIG. 1 is a schematic diagram illustrating the principle of the method for detecting circRNA according to the present invention;

FIG. 2 shows the feasibility results of detecting linear DNA in the examples of the present invention;

FIG. 3 shows the results of varying the reaction conditions in detecting linear DNA in the examples of the present invention;

wherein A is the reaction temperature, B is the reaction time, C is the concentration of MB, and D is the concentration of T7 Exo;

FIG. 4A shows fluorescence intensities of linear DNAs at different concentrations in the reaction system according to the example of the present invention;

FIG. 4B is a graph showing the relationship between the linear DNA concentration and the fluorescence intensity according to the present invention;

FIG. 5A is a schematic diagram of the synthesis of circDNA according to the invention;

FIG. 5B shows the efficiency of circDNA synthesis using polypropylene gel electrophoresis in accordance with the present invention;

FIG. 5C feasibility results of circDNA detection in the examples of the present invention;

FIG. 5D shows fluorescence intensity of circDNA at different concentrations in the reaction system in the example of the present invention;

FIG. 5E shows the specific relationship between the concentration of circDNA and the fluorescence intensity according to the present invention;

FIG. 6A is a schematic diagram of the present invention for the synthesis of circRNA;

FIG. 6B shows the efficiency of circRNA synthesis detected by polypropylene gel electrophoresis according to the present invention;

FIG. 6C feasibility results of circRNA detection in the examples of the present invention;

FIG. 6D shows the fluorescence intensity of circRNA at different concentrations in the reaction system in the example of the present invention;

FIG. 6E shows the specific relationship between the concentration of circRNA and the fluorescence intensity of the present invention;

FIG. 7A shows the results of fluorescence detection of circRNA separately transfected with the nasopharyngeal carcinoma cell lines HONE1 into the overexpression vectors circBART2.2, circRNF13, circRILPL1, circPVT1, and circADARB 1;

FIG. 7B shows the results of fluorescence detection of RNA extracted from C666-1, Akata, HONE1-EBV (+), HONE1, HNE2 and CNE2 cell lines.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

Example (b):

1. materials and methods

1.1 materials

T7 exonuclease, T4 DNA ligase, T4 RNA ligase 2 purchased from New England Biolabs, RNase R purchased from Genesed Biotech Co., Ltd, exonuclease I purchased from Takara, RNase inhibitor purchased from Solambio, SuperGelRed purchased from US EVERBRIGHT INC; molecular beacon probes (MB), linear DNA1(LD1), linear DNA2(LD2), linear RNA1(LR1), Guide DNA (GD), DEPC water were purchased from sangon biotech (shanghai, china), the sequences of which are shown in table 1. enzyme-free water (18.2M Ω. cm) and enzyme-free tip box EP tubes were used in the experiments to avoid the effect of ribonuclease on the detection of circRNA.

TABLE 1

1.2 instruments

The fluorescence spectrum is collected by a fluorescence spectrometer (SHIMADZU RF-6000Japan), the excitation wavelength is 496nm, the emission wavelength is 510-650nm, and the slit value is 5 nm. Each set of samples was repeated in three groups, each sample having a total volume of 100 ul.

1.3 Synthesis of circular DNA

The circular DNA is formed by circularizing the linear DNA2 by T4 DNA ligase in the presence of guide DNA. mu.L of 50. mu.M guide DNA and 1. mu.L of 50. mu.M linear DNA2 were first incubated at 65 ℃ for 5min and then slowly cooled to 25 ℃ (1 ℃ for one minute). Then 1. mu.L (40U/. mu.L) of T4 DNA ligase and 1. mu.L of 10XT4 DNA ligase buffer were added, reacted at 37 ℃ for 1h, and then incubated at 80 ℃ for 5 min; then 0.5. mu.L (5U/. mu.L) of exonuclease I and 1. mu.L of 10x exonuclease I buffer were added and incubated at 37 ℃ for 30min, and finally the enzyme activity was inactivated at 70 ℃ for 10 min.

1.4 Synthesis of circular RNA

The circular RNA is formed by circularization of linear RNA1 by T4 RNA ligase 2 in the presence of guide DNA. mu.L of 50. mu.M guide DNA and 1. mu.L of 50. mu.M linear RNA were first incubated at 65 ℃ for 5min and then slowly cooled to 25 ℃ (1 ℃ for one minute). Then 2. mu.L (10U/. mu.L) of

T4 RNA ligase

2 and 1. mu.L of 10XT4 RNA ligase 2 buffer are added, 2ul of ribonuclease inhibitor is reacted for 3h at 37 ℃, and the incubation is carried out for 5min at 80 ℃; then 0.5. mu.L (5U/. mu.l) exonuclease I, 1. mu.L ribonuclease R (20U/. mu.l) and 1ul10x exonuclease I, 1ul10x ribonuclease R buffer are added for reaction at 37 ℃ for 30min, and finally the enzyme activity is inactivated by incubation at 70 ℃ for 10 min.

1.5 PAGE electrophoretic analysis

20% denaturing PAGE was used to verify the effect of the synthesis of circDNA and circRNA, gel electrophoresis was performed in 1XTBE buffer at a constant pressure of 150V for 90min at room temperature, followed by bubble staining for 30min using SuperGelRed on a slow shaking shaker, and the gel was developed using a UV gel imager ((Bio-Rad, USA)).

1.6T 7Exo method for detecting circular DNA or circular RNA

mu.L of 10. mu.M MB, 10. mu.L of 10 XNE buffer 4, 1.0. mu. L T7 exonuclease, and 1. mu.L ribonuclease inhibitor were mixed with 85. mu.L of enzyme-free water. Adding 1 mu L of circDNA or circRNA with different concentrations into the solution, reacting for 3h at 37 ℃, and collecting fluorescence spectra by using a fluorescence spectrometer with an excitation wavelength of 496 nm.

1.7 specificity test

To investigate the specificity of this method for the detection of circRNA, several other different circrnas were artificially overexpressed for comparative analysis. The overexpression vector is constructed by amplifying and cloning a target fragment into a pcDNA3.1(+) CircRNA small vector, and tandem repeat sequences are contained at two ends of an insertion sequence to help the circularization of the CircRNA, so that the overexpression of the CircRNA is realized. In vitro culture of human nasopharyngeal carcinoma cell line HONE1 cells were cultured in a 37 ℃ incubator (95% air and 5% CO) using a 1640 medium containing 10% fetal bovine serum and 1% diabody₂) And (4) incubating. After growth to a certain stage, the overexpression vector circbart2.2, circRNF13, circRILPL1, circPVT1, circadrb 1 vector (from the institute of tumor research, university of south and middle university, circular RNA sequence see SEQ ID No.6-10) was transiently transfected into cells, 36h later total cellular RNA was extracted, total RNA was extracted using Trizol kit (Invitrogen, CA, USA) and linear RNA was digested using ribonuclease R, and the concentration of circRNA was determined using Nanodrop spectrophotometer (Thermo, USA), respectively. RNA was stored at-80 ℃.

1.8 analysis of actual samples and preparation of cell lysates

In order to verify the practical application of the circRNA detection method provided by the invention, the expression of circBART2.2 in different cell lines, including C666-1, Akata, HONE1-EBV (+), HONE1, HNE2 and CNE2 cell lines (from the institute of tumor research at university of Central and south China) was analyzed. Cell culture and total RNA extraction the procedure was as mentioned above.

2. Results

2.1circRNA detection principle

The principle of the fluorescent sensor for detecting circRNA based on the T7 exonuclease-assisted cycling enzyme amplification is shown in FIG. 1. Circbart2.2 was chosen as a model to demonstrate the sensor proposed by the present invention. The invention designs a molecular beacon probe (MB) as a fluorescent probe, wherein a part (a') can be hybridized with a characteristic sequence of circBART2.2(a), and the MB forms an intermolecular hairpin structure so that a quencher and a fluorophore are close to each other. Thus, fluorescence is first quenched by the hairpin structure. Three more unhybridized bases are present at the 5 'end than at the 3' end. This stem structure protected the MB from digestion by T7Exo when there was no circRNA in the system, since T7Exo only initiated enzymatic digestion of the 5' end of double stranded DNA or DNA/RNA hybridization. In the presence of the target circRNA, the red region of circRNA (a) hybridizes to the red region of MB (a') to form a perfectly matched DNA/RNA duplex, opening the hairpin structure of MB and restoring FAM fluorescence. In addition, T7Exo in the system started to digest the 5' -end DNA/RNA duplex structure of the MB probe, released the target circRNA, and released FAM-labeled single-stranded DNA (signal DNA product: SDP). The released target circRNA triggers more cycles of hybridization to MB and T7 Exo-assisted digestion, thereby generating more SDPs. Through the amplification process, not only the circRNA can be specifically identified by MB, but also a target circRNA can generate a large amount of SDPs, so that the fluorescence intensity of the sensor is greatly increased, and the rapid and sensitive detection of the circRNA is realized.

2.2 detection of Linear DNA

Since DNA is more stable than RNA and is not easily degraded, a linear DNA matched to MB was first synthesized for feasibility and optimization studies. In a feasibility experiment, as shown in fig. 2, when the target linear DNA (LD1) LD1 was not present, the MB showed a weak background fluorescence signal, since fluorescence was quenched due to the proximity of the fluorophore and quencher; on the basis, when T7Exo is added, the fluorescence value is not obviously increased, because the special stem structure of MB makes the MB not a substrate of T7 Exo; when LD1 and MB were added, but no T7Exo was added, some increase in fluorescence was observed, because LD1 was able to open the MB hairpin loop structure, separating the fluorophore and quencher, and showing a fluorescent signal, but the increase in fluorescence signal was limited due to the lack of further cycling reactions. When the MB, the LD1 and the T7Exo coexist in the reaction system, the fluorescence signal can be obviously increased, because the MB can perform hybridization reaction with the LD1 to open a hairpin ring structure, so that the fluorophore and the quenching group are separated, the T7Exo enzyme-cleaved hybrid compound enables the MB to be cleaved into a single base, the LD1 and the fluorophore are released, and the series of reactions of hybridization, enzyme cleavage and release can be cyclically performed in the system, so that the fluorescence signal is obviously changed.

In order to achieve the best performance of the detection system, a series of reaction conditions such as reaction temperature, reaction time, MB concentration, T7Exo concentration and the like are optimized. First, the temperature of the reaction system was optimized, as shown in fig. 3A, the samples with and without LD1 and LD1 were tested at different temperatures (25 ℃,30 ℃,37 ℃,45 ℃,50 ℃), and the fluorescence intensity ratio of the detection system was used as the ordinate to plot the fluorescence intensity maps at different temperatures, and as a result, it was found that the fluorescence intensity ratio increased with the increase of the reaction temperature and reached the plateau after 37 ℃. Therefore, in the following experiments, 37 ℃ was selected as the optimum reaction temperature. The reaction times (1h, 2h, 3h, 4h, 5h) were then optimized, as shown in FIG. 3B, the fluorescence intensity ratio increased with increasing reaction time, reaching a plateau at 3h, since the reaction time not only affected the hybridization efficiency but also the cleavage activity of T7 Exo. Therefore, the optimal reaction time for this experiment was 3 h. Thereafter, MB concentrations (50nM,100nM,200nM,300nM,400nM) and T7Exo concentrations (0.03U/ul, 0.07U/ul, 0.1U/ul, 0.13U/ul, 0.17U/ul) were optimized in the same manner to obtain the highest fluorescence intensity ratios, as shown in FIGS. 3C and 3D, which were highest at 200nM and 0.1U/. mu.L, respectively.

Under the best experimental conditions, LD1 was quantitatively detected. The fluorescence spectra of the detection system were measured in the presence of different concentrations of LD 1. As shown in FIG. 4A, the fluorescence intensity of the detection system increased and gradually saturated as the concentration of LD1 increased from 0nM to 5 nM. As can be seen in FIG. 4B, the specific relationship between LD1 concentration and fluorescence intensity, when LD1 concentration was increased from 0 to 1nM, fluorescence intensity was observedIncrease, plateau after 1 nM. As shown in FIG. 4B, the detection system has a good linearity range between 100fM and 100pM, and the correlation calibration equation is Y-57.64 x +1368 (R)²0.9815) (Y is fluorescence intensity and X is LD1 concentration). The limit of detection (LOD) of LD1 by this assay system was calculated to be 0.44pM (S/N-3).

2.3 detection of circDNA

The above experimental results demonstrate that the detection of linear DNA by the detection system proposed by the present invention is feasible, and then the present invention detects circDNA synthesized from a ligated linear DNA (LD 2). circDNA was first synthesized with the aid of Guide DNA using T4 DNA ligase (fig. 5A). The efficiency of circDNA synthesis was then analyzed using polypropylene gel electrophoresis, and as shown in fig. 5B, LD2 was digested after addition of Exo I treatment, whereas the synthesized circDNA was not digested, indicating successful synthesis of circDNA.

The circDNA sample synthesized was tested using the optimal conditions of example 1. As shown in FIG. 5C, in the absence of addition of circDNA, MB shows a weak background fluorescence signal due to quenching by fluorescence energy resonance transfer; even with the addition of T7Exo, there was no change in fluorescence intensity because the overhanging 5' end was not a substrate for E7 Exo; after the MB and the circDNA are mixed, the fluorescence intensity of the MB is increased by 2.4 times, because the MB and the target circDNA can generate hybridization reaction, a fluorophore and a quenching group are separated, and a certain fluorescence signal is generated; when the MB, the circDNA and the T7Exo are mixed, the fluorescence intensity of the system is improved by 7.5 times, because one circDNA can perform hybridization, enzyme digestion and release cycling reaction with a plurality of MBs, the fluorescence signal of the system is greatly enhanced.

Then, under the optimal experimental conditions, the circDNA is quantitatively detected, the fluorescence intensity of the circDNA with different concentrations is measured, and a fluorescence spectrum graph and a linear graph are drawn. As shown in FIG. 5D, the fluorescence intensity of the reaction system increased with increasing concentration of circDNA from 0 to 5 nM. The change in the peak fluorescence signal of circDNA at different concentrations can be seen in FIG. 5E, with an increase in fluorescence intensity of nearly 6.4 fold after addition of 100pM of circDNA. As shown in FIG. 5E, in the range of 1-100pMIn addition, the fluorescence intensity of the detection system and the circDNA concentration have a good linear relation. The calculation formula is that Y is 55.22x +1068 (R)²0.9804) (Y is fluorescence intensity and X is circDNA concentration). The limit of detection (LOD) of the detection system for circDNA was calculated to be 0.24pM from 3 times the standard deviation of the blank sample.

2.4 detection of circRNA

Circular RNA was synthesized by ligation of a linear RNA (LR 1). FIG. 6A shows the synthesis of circRNA1 by ligation of T4 RNA ligase 2 with the aid of Guide DNA. The efficiency of circRNA synthesis was then analyzed using polypropylene gel electrophoresis. As shown in FIG. 6B, the linear RNA was rapidly digested by RNase R, while the synthesized circRNA was intact after RNase R digestion, indicating that the circRNA was successfully prepared.

The samples of synthetic circRNA1 were tested using the optimal conditions and methods of example 1. The results are shown in FIG. 6C, where MB or the mixture of T7Exo added has a weaker fluorescence intensity, indicating that the background signal of the system is lower. When 5nM circRNA1 was mixed with MB, the fluorescence intensity of MB increased by 2.5-fold. In the presence of T7Exo, the fluorescence intensity of the system is improved by 7.3 times, which indicates the amplification process of the enzyme in the detection system.

Subsequently, the sensitivity of the circRNA was detected using the proposed detection system. As shown in FIG. 6D, the fluorescence intensity of the reaction system can be increased continuously as the concentration of circRNA1 is increased from 0 to 5 nM. In FIG. 6E it can be seen that the concentration of circRNA1 ranged from 1pM to 100pM, and the fluorescence intensity of the system was well linearly related to its concentration by the equation Y52.63X +1061 (R)²0.9868) (Y is the fluorescence value for the corresponding circRNA1 concentration and X is the concentration of circRNA 1). The lower limit of detection (LOD) for circRNA1 was calculated to be 0.31pM (S/N-3). The detection effect of the detection method designed by the invention has obvious effect, and the detection method is simple to operate and low in cost.

In order to verify the specificity of the proposed detection system for detecting circRNA, several other circrnas were selected for analysis and comparison in the present invention. The overexpression vectors of circBART2.2, circRNF13, circRILPL1, circPVT1 and circADARB1 are transfected in a nasopharyngeal carcinoma HONE1 cell line, and full-length sequencing proves that the overexpression is really the circular RNAs.

Among these circular RNAs, only circBART2.2 has an RNA sequence complementary to the designed MB and is considered as the target circular RNA. Total RNA was extracted and treated with RNase R to obtain purified circular RNA. As shown in FIG. 7A, only the extract containing circRNA1 and overexpressed circBART2.2 was found to have stronger fluorescence intensity compared to the other groups, and circRNA1 was artificially synthesized based on the sequence of circBART2.2, which was identical to the sequence to which the probe hybridized. In the presence of circRNA1 and circBART2.2, the fluorescence intensity of the detection system was increased by 6.1-fold and 4.6-fold, respectively. In contrast, the fluorescence intensity of the detection system with the addition of other circRNA did not change significantly compared to the control. Therefore, the detection system has higher specificity for the detection of the target circRNA.

2.5 actual sample detection

To verify the practical application of this method, circbart2.2 was quantified in different cell lines. As circBART2.2 is encoded by EBV, which plays an important role in nasopharyngeal carcinoma, multiple nasopharyngeal carcinoma cell lines such as C666-1, Akata, HONE1-EBV (+), HONE1, HNE2, CNE2 and the like were selected for research. As shown in fig. 7B, different expression levels of circbart2.2 were observed in different cell lines. C666-1, Akata and HONE1-EBV (+) are EBV positive cell lines and can encode circBART 2.2. Thus, the fluorescence intensity of the test lines was increased in these EBV positive cell lines, indicating that circbart2.2 was overexpressed. While in EBV negative cell lines, including HONE1, HNE2, CNE2 cells, the expression level of circbart2.2 was lower. Therefore, lower fluorescence intensities of the detection system were observed in these EBV negative cell lines. In conclusion, the results show that the detection system provided by the invention can be successfully applied to the detection of cell lysate samples, and has reliability in the sensitive detection of the target circRNA in real samples.

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gcgacgagau ccgcgccaag gacagggagc ugggccugaa aaaugaggac guugaggcuu 120

uacagcagca gcagacacgg cugaugaaga ucaaccauga ccuucggcac cgggucacgg 180

ugguggaggc ccaggggaaa gcccugaucg aacagaaggu ggagcuggag gcagaccugc 240

agaccaagga gcaggagaug ggcagccugc gagcagagcu ggggaaguug cgagagaggc 300

ugcaggggga gcacagccag aauggggagg aggagccuga g 341

<210> 9

<211> 410

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 9

gccugaucuu uuggccagaa ggagauuaaa aagaugcccc ucaagauggc ugugccuguc 60

agcugcaugg agcuucguuc aaguauuuuc ugagccugau ggauuuacag ugaucuucag 120

uggucugggg aauaacgcug guggaaccau gcacuggaau gacacacgcc cggcacauuu 180

caggauacua aaagugguuu uaagggaggc uguggcugaa ugccucaugg auucuuacag 240

cuuggauguc caugggggac gaaggacugc agcuggcuga gaggguugag aucucuguuu 300

acuuagaucu cugccaacuu ccuuuggguc ucccuaugga auguaagacc ccgacucuuc 360

cuggugaagc aucugaugca cguuccaucc ggcgcucagc ugggcuugag 410

<210> 10

<211> 290

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 10

aguggagccu uucaggcugg cauggagagc uuaaggggca acugaaggag acacacuggc 60

caagcgcgga guucugcuua cuucaguccu gcugagauac ucucucaguc cgcucgcacc 120

gaaggaagcu gccuugggau cagagcagac auaaagcuag aaaaauuuca agaugaaauu 180

gaaauggcca augagcacac ccucauucau ccagcgagca uugaggaccc ccuagaggcc 240

aggcccauga gugaugaaga ucccgaggau gaagacgccu ugccagccag 290

Claims

1. A method for improving the sensitivity of detecting circular RNA, which is characterized by comprising the following steps: adding a sample to be detected, a molecular beacon probe and T7 exonuclease into a detection system for reaction, detecting the fluorescence intensity, and obtaining the concentration of the circular RNA through a standard curve.

2. The method of claim 1, wherein the molecular beacon probe is a double-stranded stem-loop structure, wherein the 5 'end is labeled with a quenching group, the 3' end is labeled with a fluorescent group, and 2-6 unhybridized bases are added to the 5 'end compared with the 3' end; the molecular beacon probe with the double-stranded stem-loop structure cannot be cut by T7 exonuclease.

3. The method of claim 2, wherein the molecular beacon probe loop comprises 6-20T bases.

4. The method of claim 1, wherein when the target circular RNA exists, the target circular RNA hybridizes with the molecular beacon probe to form a DNA/RNA double helix structure, so that the stem part of the molecular beacon probe is opened, the molecular beacon probe can be cut by T7 exonuclease, the circular RNA is separated from the molecular beacon probe, the fluorophore and the circular RNA are released, the hybridization part of the molecular beacon probe is cut into a single base, and the fluorophore maintains the inherent fluorescence property when being free in a reaction system; the released circular RNA hybridizes with another molecular beacon probe, triggering the next hybridization and cleavage cycle, thereby releasing more fluorophore.

5. The method of claim 1, wherein the molecular beacon probe is at a concentration of 100-300nM and the T7 exonuclease is at a concentration of 100-200U/ul.

6. The process according to claim 1, wherein the reaction time is 3 to 5 hours at a reaction temperature of 35 to 40 ℃.

7. The kit for use in the method of any one of claims 1 to 6, comprising: molecular beacon probes and T7 exonuclease.

8. The kit of claim 7, further comprising: buffer, RNase inhibitor, enzyme free water.

9. The application of the molecular beacon probe in preparing a preparation for detecting circular RNA.

10. The use of claim 9, wherein the molecular beacon probe is a double-stranded stem-loop structure, wherein the 5 'end is labeled with a quenching group, the 3' end is labeled with a fluorescent group, and 2-6 unhybridized bases are added to the 5 'end compared with the 3' end; the molecular beacon probe with the double-chain stem-loop structure cannot be cut by T7 exonuclease; the ring part of the molecular beacon probe comprises 6-20T bases; the target circular RNA can be hybridized with the molecular beacon probe to form a DNA/RNA double helix structure, so that the stem part of the molecular beacon probe is opened.