Ovarian cancer detection kit based on circRNA biomarker

Abstract

The invention provides an ovarian cancer detection kit based on a circRNA biomarker, and belongs to the technical field of biological detection. According to the present invention, the expression of hsacirc0049101, hsacirc0007440 and hsacirc0006935 respectively represented by nucleotide sequences such as SEQ ID NO.1-3 in the plasma of an ovarian cancer patient is significantly increased, and the three circRNA biomarkers have high specificity and high sensitivity on the diagnosis of ovarian cancer, can be used as new biomarkers for the detection of OC, and can be used for the detection of ovarian cancer, such as ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis, ovarian cancer diagnosis and ovarian cancer diagnosis. The invention further provides an ovarian cancer detection kit based on the circRNA biomarker, the detection kit comprises a CRISPR / Cas12a detection system, the detection system comprises crRNA, Cas12a enzyme, an ssDNA fluorescent probe, a CRISPR buffer solution and other reagents, one-step efficient detection of the circRNA biomarker can be achieved by adopting the CRISPR detection system of various Cas12a enzymes and crRNA, and the circRNA biomarker has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to early detection of ovarian cancer, and specifically provides a circRNA biomarker for ovarian cancer detection and application thereof. Background Art

[0002] Ovarian cancer (OC) is a malignant tumor among ovarian tumors, of which 90%-95% are primary ovarian cancers, and the other 5%-10% are primary cancers from other parts of the body that have metastasized to the ovaries. Ovarian cancer is also known as the "silent killer" because its early symptoms are not obvious and it is highly hidden. Once it appears, it is in the late stage. Therefore, discovering early diagnostic markers, finding new diagnostic tools, and improving the early diagnosis rate of ovarian cancer are key issues that need to be urgently addressed in the current treatment of ovarian cancer.

[0003] Circular RNA was previously considered a byproduct of splicing and had no function. In recent years, with the continuous growth of circRNA-related biological information research, it has been found that circular RNA is not a byproduct of splicing. It has a wide range of sources, is stable, has tissue specificity, and plays a variety of functional roles in the growth and development of organisms. The length of circRNA is mostly between 200-400 bases, and can be quantitatively detected by qPCR, which is highly operable. Therefore, using circRNA as a biomolecular marker for disease diagnosis has clear advantages. Recently, circRNAs with early diagnostic value have been found in gynecological tumors such as ovarian cancer and breast cancer.

[0004] The CRISPR / Cas system is an adaptive immune system found in most bacteria and archaea, which uses guide RNA (crRNA) encoded in the CRISPR locus to guide CRISPR-associated endonucleases (Cas) to recognize and cut specific exogenous nucleic acid sequences to protect them from viral infection. Researchers have found that some CRISPR / Cas systems, such as CRISPR / Cas12, can efficiently cut ssDNA in the system (trans-cutting) while targeting the target dsDNA (cis-cutting). This feature has led to the rapid expansion of CRISPR technology into the field of genetic testing.

[0005] In a typical CRISPR nucleic acid detection application, the crRNA / Cas complex is used to specifically recognize the target amplicon, thereby activating its trans-cleavage activity and cleaving the quenched oligonucleotide probe, releasing a detectable fluorescent signal, and achieving specific detection of the target gene. Currently, some studies have used the trans-cleavage characteristics of the CRISPR / Cas system to construct nucleic acid diagnostic tools, but the discovery and development of diagnostic tools for unique circRNA nucleic acid markers for ovarian cancer is still blank. Summary of the invention

[0006] In view of the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide an ovarian cancer detection kit based on circRNA biomarkers. The present invention finds that the nucleotide sequences in the plasma of ovarian cancer patients are respectively as shown in SEQ ID NO.1-3. The expression of hsa_circ_0049101, hsa_circ_0007440, and hsa_circ_0006935 is significantly increased. These three circRNA biomarkers have high specificity and sensitivity for the diagnosis of ovarian cancer, and can be used as new biomarkers for OC detection. By using a variety of Cas12a enzymes and crRNA CRISPR detection systems, the cleavage activity of Cas proteins is enhanced, and relatively high trans-cleavage activity is maintained, so that the fluorescence signal of the system is enhanced several times, and one-step sensitive and efficient detection of circRNA biomarkers is achieved. The detection kit of the present invention is easy to operate, has high specificity, good sensitivity, and can detect a variety of circRNA biomarkers in whole blood samples.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a circRNA biomarker for diagnosing ovarian cancer, wherein the circRNA marker includes hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935;

[0009] The nucleotide sequence of hsa_circ_0049101 is shown in SEQ ID NO.1.

[0010] The nucleotide sequence of hsa_circ_0007440 is shown in SEQ ID NO.2.

[0011] The hsa_circ_0006935 nucleotide sequence is shown in SEQ ID NO.3.

[0012] In a second aspect, the present invention provides a method for constructing the above-mentioned circRNA biomarker for diagnosing ovarian cancer, wherein a linear DNA template with a nucleotide sequence as shown in SEQ ID NO.1-3 is transcribed in vitro to obtain a linear RNA template, and the linear RNA is cyclized using T4 RNA ligase 2 under the action of a DNA ligation primer; the nucleotide sequence of the DNA ligation primer is as shown in SEQ ID NO.4-6.

[0013] Based on the above technical scheme, further, the specific process of the construction method is as follows: a linear DNA template with a nucleotide sequence as shown in SEQ ID NO.1-3 is transcribed in vitro to obtain a linear RNA template, the linear RNA template is annealed with a DNA primer, incubated at 60-70°C for 2-10 minutes, cooled to 10-15°C, T4 RNA ligase 2 and RNase inhibitor are added to the mixture and incubated at 35-39°C for 30-120 minutes, incubated at 70-90°C for 2-10 minutes, RNase R and RNase R buffer are added and incubated at 35-39°C for 20-50 minutes, incubated at 60-80°C for 5-30 minutes, DNase I is added, incubated for 20-50 minutes to digest excess DNA primers, and circRNA biomarkers are obtained after purification.

[0014] In a third aspect, the present invention also provides the use of the above-mentioned circRNA biomarker in the preparation of a kit for diagnosing ovarian cancer.

[0015] In a fourth aspect, the present invention provides an ovarian cancer detection kit based on the above-mentioned circRNA biomarker, wherein the detection kit comprises a CRISPR / Cas12a detection system, and the detection system comprises crRNA, Cas12a enzyme and ssDNA fluorescent probe, CRISPR buffer and DTT.

[0016] Based on the above technical solution, further, in the CRISPR / Cas12a detection system, the Cas12a enzyme is at least one of LbCas12a and FnCas12a, and the crRNA is designed according to the two enzymes LbCas12a and FnCas12a, wherein the crRNA designed for LbCas12a is LbcrRNA1, LbcrRNA2, and LbcrRNA3, and the crRNA designed for FnCas12a is FncrRNA1, FncrRNA2, and FncrRNA3; the crRNA is at least one of LbcrRNA1, LbcrRNA2, LbcrRNA3, FncrRNA1, FncrRNA2, and FncrRNA3, and the sequence of LbcrRNA1 is shown in SEQ ID NO.8; the sequence of LbcrRNA2 is shown in SEQ ID NO.9; the sequence of LbcrRNA3 is shown in SEQ ID NO.10; the sequence of FncrRNA1 is shown in SEQ ID NO.11; the sequence of FncrRNA2 is shown in SEQ ID NO.12; the sequence of the FncrRNA3 is shown in SEQ ID NO.13.

[0017] Based on the above technical solution, further, the structure of the ssDNA fluorescent probe is as follows: 5'-FAM-NNNNN-BHQ1-3', where N represents any base sequence among A, T, C, and G.

[0018] Based on the above technical solution, further, the CRISPR buffer includes 50mM NaCl, 10mM Tris-HCl, 10mM MgCl 2 , 100μg / ml Recombinant Albumin (pH 7.9@25℃).

[0019] Based on the above technical solution, further, the detection kit also includes at least one RNase inhibitor.

[0020] Based on the above technical solution, further, the conditions of the CRISPR reaction are to react at 35-38° C. for 30-120 min, and collect the fluorescence signal every 1-5 min.

[0021] Based on the above technical solution, the sample to be detected is further obtained by lysing, extracting and concentrating the body fluid sample to be detected to obtain an RNA sample to be detected.

[0022] Based on the above technical solution, further, the body fluid sample includes any one of blood, saliva, and oral swab.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention found that the expression of hsa_circ_0049101, hsa_circ_0007440, and hsa_circ_0006935 in the plasma of ovarian cancer patients was significantly increased. These three circRNA biomarkers have high specificity and sensitivity for the diagnosis of ovarian cancer and can be used as new biomarkers for the detection of OC. By adopting circRNA biomarkers, at least one crRNA, and at least one Cas protein, while maintaining relatively high trans-cleavage activity, the detection efficiency is improved, and one-step efficient CRISPR detection of circRNA biomarkers is achieved, which provides a reference for the diagnosis of OC and has good application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0026] Figure 1The invention discloses the discovery and verification of circRNA biomarkers in Example 1 of the present invention, wherein A is a heat map of differentially expressed circRNAs of NIHOVCAR3 vs HEK293T and SKOV3 vs HEK293T, B is a Venn diagram of differentially expressed circRNAs of NIHOVCAR3 vs HEK293T and SKOV3 vs HEK293T, CD is a PPI protein interaction network to analyze the interaction between mRNAs, screen out core genes, and construct a circRNA-miRNA-mRNA regulatory network, E is an RNase R experiment of candidate circRNA biomarkers to identify the presence of circRNAs in cancer cells, and F is a verification of three circRNA biomarkers in blood samples of different clinical stages.

[0027] Figure 2 The preparation and verification of the circRNA standard in Example 2-3 of the present invention, wherein A is a schematic diagram of circRNA in vitro cyclization, B is RNase R verification of circRNA synthesis, and C is RCA amplification verification.

[0028] Figure 3 It is the crRNA screening and verification result in Example 4 of the present invention, wherein A is the crRNA sequence for LbCas12a and FnCas12a, B is the crRNA in vitro transcription gel electrophoresis verification, C and D are the sensitivity comparisons of each crRNA for LbCas12a and FnCas12a, and E and F are the feasibility verifications of CRISPR / Cas12a detection.

[0029] Figure 4 It is the sensitivity detection result of the method for detecting ovarian cancer circRNA based on the kit in Example 5 of the present invention, wherein A is the detection fluorescence kinetics and bar graph of the combination of four crRNAs, B is the fluorescence intensity heat map of different numbers of crRNA combinations, and C is the slope sensitivity heat map of different numbers of crRNA combinations.

[0030] Figure 5 Specific detection results of the method for detecting ovarian cancer circRNA based on the kit in Example 6 of the present invention, A is the specific sequence and bar graph of single base to 5 base differences, B is the fluorescence kinetic graph of single base to 5 base differences, C is the fluorescence kinetic graph for verifying the anti-interference ability of linear RNA, circRNA and different proportions of mixed linear RNA and circRNA, D is the bar graph for verifying the anti-interference ability, E is the specific fluorescence kinetic graph for verifying the detection method of cancer cells from different sources, and F is the bar graph for verifying the detection method of cancer cells from different sources.

[0031] Figure 6The blood sample test results of ovarian cancer patients in Example 7 of the present invention, wherein A is a schematic diagram of clinical blood RT-qPCR and CRISPR method detection, B is a comparative bar graph of circRNA detection by RT-qPCR and CRISPR methods, C is a correlation analysis of RT-qPCR and CRISPR methods, D is a CA125 ELISA graph, E is a HE4 ELISA graph, F is a ROMA index graph, G is the result of the DCMC-CRISPR method of this method, and H is a ROC curve comparing the sensitivity and specificity of CA125, HE4, ROMA and DCMC-CRIPSR of this method. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experiments were performed according to the literature or the description in the specification. The experimental materials used in the following examples, unless otherwise specified, can be purchased through commercial channels. Unless otherwise specified, the quantitative tests in the following examples were repeated three times, and the results were averaged.

[0034] The reporter DNA used in the following examples is a product of Guangzhou Meige Biotech Co., Ltd. The enzymes used in amplification and CRISRP / Cas are products of New Biolab Co., Ltd.

[0035] Example 1 Discovery and verification of circRNA biomarkers

[0036] 1. Cell Sequencing and Analysis

[0037] The R package edgeR was used to compare the differences in gene expression levels obtained by sequencing NIHOVCAR3 vs HEK293T and SKOV3 vs HEK293T. The above sequencing data have been uploaded to the GEO database GSE271851. The screening conditions for differentially expressed circRNAs were: corrected p<0.05 and |log FC>1|. NIHOVCAR3 vs 293T obtained 637 differential circRNAs, of which 331 were upregulated and 306 were downregulated. SKOV3 vs 293T obtained 616 differential circRNAs, of which 308 were upregulated and 308 were downregulated. Use "ggplot2" and "pheatmap" heat maps to display the differential expression of circRNAs, such as Figure 1 A. The differential circRNAs of NIHOVCAR3 vs 293T and SKOV3 vs 293T were intersected to obtain 244 circRNAs differentially expressed in ovarian cancer (OC), which were recorded as candidate circRNAs, such as Figure 1 B.

[0038] According to the above differentially expressed circRNAs, the circRNA-miRNA-mRNA regulatory network was constructed, and the core genes were screened out from the PPI protein network constructed based on mRNA. Finally, the circRNA-miRNA-mRNA regulatory network was reconstructed based on the core genes, such as Figure 1 CD. After verification, three circRNAs were finally obtained: hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935. The nucleotide sequence of hsa_circ_0049101 is shown in SEQ ID NO.1, the nucleotide sequence of hsa_circ_0007440 is shown in SEQ ID NO.2, and the nucleotide sequence of hsa_circ_0006935 is shown in SEQ ID NO.3.

[0039] 2. Blood verification of three circRNAs

[0040] In order to verify the presence of circRNAs associated with ovarian cancer in plasma, the detected molecule was proved to be circRNA according to whether the expression level was detected in the RNase R (+) and RNase R (-) groups. The total circRNA from the clinical blood samples of ovarian cancer from Liaoning Cancer Hospital was extracted and subjected to RNase R enzymatic hydrolysis experiment. The specific process is that 5 μg of total RNA from the blood of cancer patients was taken, 3U / μg RNase R and the corresponding 10×RNase R buffer were added respectively, and enzyme-free water was added to 20 μL. RT-qPCR was performed on the samples treated with RNase and without RNase treatment to verify the expression levels of three cricRNAs (hsa_circ_0049101, hsa_circ_0006935 and hsa_circ_0007440) in the two groups of different treated samples.

[0041] The results are as follows Figure 1 As shown in E, the expression of OC-related circRNAs was detected in both the RNase R (+) and RNase R (-) experimental groups, and all three circRNAs were resistant to RNase R treatment. These results also confirmed their presence in blood samples.

[0042] To further investigate the relevance of these circRNAs of interest to ovarian cancer, 22 OC samples (early stage (I and II) = 5, stage III = 9, stage IV = 8) and 28 normal plasma samples were used to detect the relative expression of circRNAs by qRT-PCR. Figure 1 As shown in Figure F, hsa_circ_0049101, hsa_circ_0006935, and hsa_circ_0007440 were significantly upregulated in OC patients. In addition, these three upregulated circRNAs had relatively high significant differences in different cancer stages compared with the control group, and their expressions increased with the increase of cancer stage grade.

[0043] Example 2 Preparation and Verification of circRNA Standards

[0044] Preparation of circRNA standards

[0045] Linear RNA circularization was achieved using T4 RNA ligase 2. Double-stranded linear DNA template was in vitro transcribed into a linear RNA template, and T4 RNA ligase 2 was used on the DNA ligation primer to circularize the linear RNA.

[0046] Specifically, according to the number of reactions, a mixture of a linear RNA template (1 μM) and a DNA primer (5 μM) was prepared according to the reaction system and annealed. The mixture was incubated at 65°C for 5 minutes and cooled to 12°C at a rate of 1°C / min. The nucleotide sequences of the DNA primers of the three circRNAs were SEQ ID NO.4 (hsa_circ_0049101), SEQ ID NO.5 (hsa_circ_0006935), and SEQ ID NO.6 (hsa_circ_0007440). Subsequently, T4 RNA ligase 2 (2U) and RNase inhibitor (0.5U) were added to the mixture and incubated at 37°C for 90 minutes and 80°C for 5 minutes. RNase R (1U) and RNase R buffer were added and incubated at 37°C for 30 minutes and 70°C for 10 minutes. After the reaction, DNase I (1U) was used to digest the excess DNA primer. The reaction product was used The RNA was purified using RNA Cleanup Kit (50 μg, New England Biolabs), and 5 μL of the supernatant was used for 1.0% agarose gel electrophoresis.

[0047] The detailed process of circRNA synthesis can be found in Figure 2A. Specifically, the linear RNA template synthesized by in vitro transcription was annealed with a DNA ligase primer, and then the 5' and 3' ends of the linear RNA template were ligated using T4 RNA ligase 2. Subsequently, the excess DNA ligase primer was eliminated by DNase I, and RNase R can digest almost all linear RNA molecules, but not circular RNA. Therefore, RNase R digestion was used to demonstrate the feasibility of circRNA synthesis ( Figure 2 B). The results showed that the circRNA had higher stability than linear RNA, indicating the successful synthesis of circRNA.

[0048] Example 3 Verification results of circRNA-based RT-RCA

[0049] The experimental process of RT-RCA amplification verification, such as Figure 2 As shown in C, the synthesized circRNA template (hsa_circ_0049101), 480nM reverse transcription primer (SEQ ID NO.7), Protoscript II reverse transcriptase (1.5U), 10×DTT, dNTP (400nM), enzyme-free water were added to 20μL, and incubated at 42°C for 30min, 1h and 1.5h.

[0050] The results are as follows Figure 2 As shown in C gel electrophoresis, the first lane is the circular RNA template, the second lane is the RT-RCA primer, and the third to fourth lanes are the product characterizations of reverse transcription rolling circle amplification reactions for 30 minutes, 1 hour, and 1.5 hours, respectively. From the results, it can be seen that cDNA can be reverse transcribed after 30 minutes of reaction, and the optimal time is when the reaction is carried out for 1 hour, so it can be proved that the feasibility of the experiment and the optimal reaction conditions are 1 hour.

[0051] Example 4: Screening and verification results of crRNA based on CRISPR / Cas system

[0052] 1. Multiple crRNA combination design

[0053] Previous studies have shown that Cas12a does not require the assistance of PAM sequences when detecting ssDNA. However, in this CRISPR experiment, it was found that the efficiency of crRNA recognition reaction based on PAM sites was higher than that of crRNA recognition reaction without PAM sequence assistance.

[0054] The LbCas12a protein of the Trichophyton strain and the FnCas12a protein of the Francisella strain were studied. LbCas12a prefers to recognize the PAM sequence of 5'-TTV-3', and the PAM sequence recognized by FnCas12a can be extended to 5'-TTV-3'. The introduction of FnCas12a can greatly increase the targeting range of Cas12a and the sensitivity of CRISPR detection. Therefore, this study selected LbCas12a and FnCas12a to verify the CRISPR / Cas12a system.

[0055] 2. Screening and verification of crRNA

[0056] The crRNAs were designed based on LbCas12a and FnCas12a enzymes, respectively. Figure 3 As shown in A, the crRNA designed for LbCas12 is LbcrRNA1 (as shown in SEQ ID NO.8), LbcrRNA2 (as shown in SEQ ID NO.9), LbcrRNA3 (as shown in SEQ ID NO.10), and the crRNA designed for FnCas12 is FncrRNA1 (as shown in SEQ ID NO.11), FncrRNA2 (as shown in SEQ ID NO.12) and FncrRNA3 (as shown in SEQ ID NO.13).

[0057] In the gel electrophoresis verification experiment of crRNA in vitro transcription, according to the company's synthesis of 69bp double-stranded DNA template with T7 promoter (1μg), T7 polymerase, dATP, dCTP, dGTP and dUTP 100nM each, RNase inhibitor (1U), T7 polymerase buffer, enzyme-free water to 20μL. 1% agarose gel electrophoresis was used for verification. The results are as follows Figure 3 As shown in Figure B, a 69 bp double-stranded DNA template was transcribed in vitro by the T7 promoter to obtain a 44 nt single-stranded RNA. The bands in Figure B are crRNA1 and crRNA3 designed for LbCas12a, and crRNA1 and crRNA3 designed for FnCas12a.

[0058] The detection sensitivity of the three crRNAs designed by LbCas12a and the three crRNAs designed by FnCas12a were verified respectively. LbCas12a and FnCas12a were combined with six crRNAs to detect circRNAs. The trans-cleavage activity of each cirRNA was determined by fluorescence kinetics and endpoint fluorescence values, and the sensitivity of the six crRNAs was compared. The specific experimental process is as follows: the reaction system includes: circRNA (hsa_circ_0049101) concentration is 50pM, after Protoscript II reverse transcription, 200nM LbCas12a and 200nM FnCas12a proteins are added, 100nM of each crRNA (LbcrRNA1, LbcrRNA2, LbcrRNA3, FncrRNA1, FncrRNA2, FncrRNA3), 400nM ssDNA fluorescent probe (5'-FAM-TTTTT-BHQ1-3') mixture is incubated at 37°C, and the fluorescence intensity value is measured every 5min using real-time fluorescence quantification. The reaction test is 120min, and the end point fluorescence intensity value is collected.

[0059] The results are as follows Figure 3 As shown in C and 3D, LbCas12a / LbcrRNA1 and FnCas12a / FncrRNA1 have the highest sensitivity in detecting circRNA. When the concentration of circRNA is 50pM, the detection sensitivity of these crRNAs is: LbcrRNA1>FncrRNA1>LbcrRNA3>LbcrRNA3>LbcrRNA2>FncrRNA2.

[0060] The detection activity and sensitivity of the above-mentioned single crRNA were verified, and the feasibility of the CRISPR / Cas12a system and the combination of FnCas12a and LbCas12a were further verified.

[0061] like Figure 3As shown in E and F, FnCas12a / FncrRNA1, LbCas12a / LbcrRNA1 and LbCas12a / LbcrRNA1+FnCas12a / FncrRNA1 are control systems without the addition of the detection target, FnCas12a / FncrRNA1+cDNA and LbCas12a / LbcrRNA1+cDNA are single protein detection systems with the addition of the detection target, followed by the LbCas12a / LbcrRNA1+FnCas12a / FncrRNA1+cDNA detection system combining two proteins. The specific experiment was as follows: the concentration of circRNA (hsa_circ_0049101) was 50 pM, and after reverse transcription, it was cDNA. 200 nM LbCas12a or 200 nM FnCas12a protein was added, of which each crRNA was 100 nM (LbcrRNA1, LbcrRNA2, LbcrRNA3, FncrRNA1, FncrRNA2, FncrRNA3), and 400 nM ssDNA fluorescent probe (5'-FAM-TTTTT-BHQ1-3') The mixture was incubated at 37 ° C, and the fluorescence intensity value was measured every 5 min using real-time fluorescence quantification. The reaction test was 120 min, and the endpoint fluorescence intensity value was collected.

[0062] The results are as follows Figure 3 As shown in E and 3F, compared with the control, the fluorescence intensity of the LbCas12a / LbcrRNA1+FnCas12a / FncrRNA1+cDNA detection system combined with two proteins was significantly higher than that of the single protein detection system FnCas12a / FncrRNA1+cDNA and LbCas12a / LbcrRNA1+cDNA. Among them, the combined use of LbcrRNA1, LbcrRNA3, FncrRNA1 and FncrRNA3 had the highest sensitivity. This suggests that the combination of Cas proteins targeting different PAMs has great potential to improve the detection sensitivity of circRNA.

[0063] Example 5 A sensitivity test of an ovarian cancer detection kit and standard based on circRNA biomarkers is described as follows.

[0064] The concentration of circRNA (hsa_circ_0049101) was diluted stepwise (2000pM~0.5fM) for sensitivity detection, and circRNA was amplified by RT-RCA, wherein RT-RCA primer (SEQ ID NO.7) 480nM, Protoscript II reverse transcriptase 1.5U, 10×DTT, dNTP 400nM, RNase inhibitor 1U, the mixture was incubated at 42°C for 1 hour to obtain RT-RCA product.

[0065] Preparation of CRISPR reaction system Using the RT-RCA amplification product as a template, the CRISPR reaction system was prepared. The reaction system (20 μL system) was: RT-RCA amplification product as a template, 100 nM each of LbcrRNA1, LbcrRNA3, FncrRNA1 and FncrRNA3, 200 nM LbCas12a, 200 nM FnCas12a, 400 nM ssDNA fluorescent probe (F-TTTTT-Q), and 10 mM DTT were added to the PCR tube, and a negative control with sterile enzyme-free water as a template was set.

[0066] The reaction mixture was incubated at 37°C for 2 h, and the fluorescence emission spectra were The fluorescence kinetics were performed on a Quant Studio 3 RealTime system (Thermo Fisher Scientific, Waltham, MA).

[0067] CRISPR detection and fluorescence signal collection: Add the above-configured CRISPR reaction system into a PCR tube and place it in a fluorescence quantitative PCR instrument. Set the program as follows: 37°C, 30 cycles, and collect fluorescence signals every 5 minutes.

[0068] The fluorescence signal value of CRISPR detection is greater than the average value of the negative control fluorescence signal plus 3 times the standard deviation. The above verification results show that when 4 crRNAs (LbcrRNA1+LbcrRNA3+FncrRNA1+FncrRNA3) are combined, the detection sensitivity is further improved, which is 4-11 times higher than the detection sensitivity of single crRNA and 3 crRNAs. The heat map intuitively compares the endpoint fluorescence values ​​at different concentrations and analyzes the endpoint fluorescence values ​​and slopes of each different crRNA combination. The combination containing 4 crRNAs has the highest sensitivity ( Figure 4 BC).

[0069] Under the optimal experimental conditions, the sensitivity of circRNA detection was tested by measuring different concentrations. F / F of the fluorescence intensity curve 0 Proportional to the circRNA concentration in the range of 2000pM to 0.5fM. LOD was calculated to be 0.5fM. The related equation is y=187.773+554.050logC(R 2 =0.993)( Figure 4 ). This shows that this method is significantly better than other detection methods.

[0070] Example 6 Specific detection of multiple circRNA biomarker detection standards established by the present invention

[0071] Five crRNAs with base mismatches with LbcrRNA3 were designed, and the LbcrRNA3 sequence was used to verify the ability of the detection method to distinguish single base mismatches. The specific process is: the concentration of circRNA (2000pM), first RT-RCA amplification of circRNA, where RT-RCA primer (SEQ ID NO.7) 480nM, Protoscript II reverse transcriptase 1.5U, dNTP 400nM, RNase inhibitor 1U, the mixture was incubated at 42°C for 1 hour to obtain RT-RCA product. Preparation of CRISPR reaction system Using RT-RCA amplification product as template, prepare CRISPR reaction system, the reaction system (20μL system) is: RT-RCA amplification product as template, LbcrRNA1, LbcrRNA3, FncrRNA1 and FncrRNA3 100nM each, LbCas12a200nM, FnCas12a200nM, ssDNA fluorescent probe 400nM (F-TTTTT-Q), DTT 10mM added to the PCR tube, and set up a negative control with sterile enzyme-free water as template. Among them, LbcrRNA1 is replaced with a 1-5 base mismatch sequence. The specific mismatch sequence and position are shown in Figure 5 A, to verify the specificity of the established CRISPR / Cas method for detecting circRNA ( Figure 5 AB), the results showed that this method has good single-base recognition ability.

[0072] Next, circRNA was mixed with homologous linear RNA (same as circRNA sequence) at a ratio of 1:5, 1:50 and 1:500. The specific RT-RCA / CRISPR parameters and process were the same as above, except that the detection template was replaced with a mixture of circRNA and linear RNA in different ratios to evaluate the anti-interference ability of this method ( Figure 5CD). Mixing with high concentration linear RNA did not affect the detection efficiency of circRNA. There was no statistical difference between the mixture of circRNA and linear RNA.

[0073] Example 7 To verify the positive detection rate and accuracy of the CRISPR / Cas-based ovarian circRNA detection method established in the present invention in actual sample detection applications, whole blood samples from clinical ovarian cancer patients and healthy controls were used for detection.

[0074] Based on the detection method established by the present invention, the samples to be tested include: blood samples from 22 ovarian cancer patients and 28 healthy blood donors were provided by Liaoning Cancer Hospital Research Institute (Shenyang, Liaoning, China). All patients were diagnosed with OC by pathological examination and did not receive any preoperative anticancer treatment. All cases were confirmed by clinical pathology. In addition, informed consent was obtained from all participating subjects. RT-qPCR and the established RT-RCA / CRISPR / Cas12a method were used to detect circRNA in clinical samples.

[0075] The samples tested were based on the Blood&Tissue Kit instructions, extract nucleic acid after pre-treatment of samples.

[0076] Whole blood samples: RNA was isolated from blood samples using the miRNeasy Micro Kit (Qiagen, Germany); approximately 1 μg of RNA was extracted in 20 μL of solution.

[0077] Reverse transcription was performed using the Go reverse transcription kit. Then 0.4 μL of 10 μM forward primer, 0.4 μL of 10 μM reverse primer, 6 μL of reverse transcription solution, and 10 μL of qPCR SYBR Green Master Mix were used for PCR reaction. The PCR process included 95°C pre-denaturation for 10 min, followed by 40 cycles of 95°C denaturation for 15 s, 60°C annealing for 60 s, and 72°C extension for 20 s.

[0078] PCR amplification curves and Cq values ​​were analyzed to evaluate the number of circRNAs in clinical samples. For the RT-RCA / CRISPR / Cas12a detection system, RT-RCA primers (SEQ ID NO.7, 480nM), Protoscript II reverse transcriptase (1.5U), 10×DTT and dNTP (400nM) were mixed with RNA samples and incubated at 42°C for 60min, followed by the addition of 2μL LbCas12a and FnCas12a proteins (200nM each), LbcrRNA1, LbcrRNA3, FnbcrRNA1, FncrRNA3 (100nM each) and 1μL ssDNA fluorescent probe F-TTTTT-Q (400nM). The mixture was incubated at 37°C for 40min and measured using the Quant Studio 3 real-time system.

[0079] result Figure 6 As shown in DF, CA125 and HE4 indexes are tested by clinical standard ELISA detection methods, and ROMA index is obtained by constructing a model based on the results of CA125 and HE4. Double antibody sandwich ELISA method is used. Anti-human CA125 antibody is coated on the enzyme labeling plate. During the experiment, human CA125 in the sample (or standard) will bind to the coated antibody. Then biotinylated anti-human CA125 antibody and horseradish peroxidase-labeled avidin are added in sequence. The anti-human CA125 antibody binds to the human CA125 bound to the coated antibody, and biotin specifically binds to avidin to form an immune complex, and the free components are washed away. A colorimetric substrate (TMB) is added, and TMB appears blue under the catalysis of horseradish peroxidase, and turns yellow after adding the stop solution. The OD value was measured at a wavelength of 450nm using an enzyme marker. The CA125 concentration was proportional to the OD450 value. The CA125 concentration in the sample was calculated by drawing a standard curve. The HE4 concentration detection method was the same as above, except that the antibody was changed to anti-human HE4 antibody. The results are shown in Figure 2. Figure 6 As shown, the kit established by the present invention can detect ovarian cancer-related circRNA in whole blood samples. The detection method constructed by the present invention is superior to clinical CA125 and HE4 detection indicators, and is similar to the gold standard RT-qPCR detection method.

[0080] It can be seen that the detection method constructed by the present invention has relatively higher sensitivity and accuracy for ovarian cancer circRNA. Compared with the currently commonly used detection methods such as qPCR, the detection method constructed by the present invention greatly shortens the detection time, is easier to operate and has higher sensitivity.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A circRNA biomarker for diagnosing ovarian cancer, characterized in that: The circRNA markers include hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935; The nucleotide sequence of hsa_circ_0049101 is shown in SEQ ID NO.

1. The nucleotide sequence of hsa_circ_0007440 is shown in SEQ ID NO.

2. The hsa_circ_0006935 nucleotide sequence is shown in SEQ ID NO.

3.

2. The method for constructing a circRNA biomarker for diagnosing ovarian cancer according to claim 1, characterized in that: The linear DNA template with nucleotide sequence as shown in SEQ ID NO.1-3 is transcribed in vitro to obtain a linear RNA template, and the linear RNA is circularized using T4 RNA ligase 2 under the action of DNA ligation primers; the nucleotide sequence of the DNA ligation primers is shown in SEQ ID NO.4-6.

3. Use of the circRNA biomarker according to claim 1 in the preparation of a kit for diagnosing ovarian cancer.

4. An ovarian cancer detection kit based on the circRNA biomarker of claim 1, characterized in that: The detection kit includes a CRISPR / Cas12a detection system, which includes crRNA, Cas12a enzyme and ssDNA fluorescent probe, CRISPR buffer and DTT.

5. The detection kit according to claim 4, characterized in that In the CRISPR / Cas12a detection system, the Cas12a enzyme is at least one of LbCas12a and FnCas12a, the crRNA is at least one of LbcrRNA1, LbcrRNA2, LbcrRNA3, FncrRNA1, FncrRNA2 and FncrRNA3, the sequence of the LbcrRNA1 is shown in SEQ ID NO.8; the sequence of the LbcrRNA2 is shown in SEQ ID NO.9; the sequence of the LbcrRNA3 is shown in SEQ ID NO.10; the sequence of the FncrRNA1 is shown in SEQ ID NO.11; the sequence of the FncrRNA2 is shown in SEQ ID NO.12; the sequence of the FncrRNA3 is shown in SEQ ID NO.

13.

6. The detection kit according to claim 4, characterized in that The structure of the ssDNA fluorescent probe is as follows: 5'-FAM-NNNNN-BHQ1-3', where N represents any base sequence among A, T, C and G.

7. The detection kit according to claim 4, characterized in that The CRISPR buffer includes 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml Recombinant Albumin (pH 7.9@25°C).

8. The detection kit according to claim 4, characterized in that The detection kit also includes at least one RNase inhibitor.

9. The detection kit according to claim 4, characterized in that The CRISPR reaction conditions are as follows: react at 35-38° C. for 30-120 min, and collect the fluorescence signal every 1-5 min.

10. The detection kit according to claim 4, characterized in that The sample to be detected is obtained by lysing, extracting and concentrating the body fluid sample to be detected to obtain the RNA sample to be detected; the body fluid sample includes any one of blood, saliva and oral swab.

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