An ovarian cancer detection kit based on circRNA biomarkers

Abstract

The application provides an ovarian cancer detection kit based on a circRNA biomarker, and belongs to the technical field of biological detection. It is found that the expression of hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935 with nucleotide sequences shown in SEQ ID NO. 1-3 is obviously increased in the plasma of ovarian cancer patients, the 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. The application also provides an ovarian cancer detection kit based on the above-mentioned circRNA biomarker, which comprises a CRISPR / Cas12a detection system, the detection system comprises crRNA, Cas12a enzyme, ssDNA fluorescent probe, CRISPR buffer and other reagents, and the CRISPR detection system with multiple Cas12a enzymes and crRNA can realize one-step efficient detection of the circRNA biomarker, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to early detection of ovarian cancer, and in particular to a circRNA biomarker for ovarian cancer detection and application thereof. BACKGROUND

[0002] Ovarian cancer (OC) is a malignant tumor of the ovary, 90%-95% of which is primary ovarian cancer, and 5%-10% of which is metastasis of primary cancer in other parts to the ovary. Ovarian cancer has a high incidence rate, and its early symptoms are not obvious, and it is highly occult. Once it occurs, it is in the late stage, and therefore it is also known as the "silent killer". Therefore, discovering early diagnosis markers, finding new diagnostic tools, and improving the early diagnosis rate of ovarian cancer are key problems to be solved in the current treatment of ovarian cancer.

[0003] CircRNA was originally considered to be a splicing by-product and had no function. In recent years, with the continuous growth of circRNA-related biological information research, it has been found that circRNA is not a splicing by-product, has a wide source, 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, it can be quantitatively detected by qPCR, and it has strong operability. Therefore, using circRNA as a biomarker for disease diagnosis has clear advantages, and in recent years, 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 present in most bacteria and archaea, which uses guide RNA (crRNA) encoded in the CRISPR locus to guide CRISPR-associated endonuclease (Cas) to recognize and cut specific foreign nucleic acid sequences to protect them from viral infection. Researchers have found that some CRISPR / Cas systems, such as CRISPR / Cas12, can simultaneously target and cut target dsDNA (cis-cutting) and non-specifically and efficiently cut ssDNA in the system (trans-cutting). This property has prompted the rapid expansion of CRISPR technology into the field of gene detection.

[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-cutting activity and cleaving the quenched oligonucleotide probe to release a detectable fluorescent signal, achieving specific detection of the target gene. Currently, some studies have used the trans-cutting property of CRISPR / Cas system to construct nucleic acid diagnostic tools, but the discovery of unique circRNA nucleic acid markers for ovarian cancer and the development of diagnostic tools are still blank. SUMMARY

[0006] In view of the above technical problems existing in the prior art, the purpose of the present application is to provide a circRNA biomarker-based ovarian cancer detection kit, and it is found that the expression of hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935 with nucleotide sequences shown in SEQ ID NO. 1-3 is significantly increased in the plasma of ovarian cancer patients, and the 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 using a CRISPR detection system of multiple Cas12a enzymes and crRNA, the cleavage activity of Cas protein is enhanced, the relative high trans-cleavage activity is maintained, the fluorescence signal of the system is enhanced by several times, and one-step sensitive and efficient detection of circRNA biomarkers is realized. The detection kit of the present application is simple to operate, has high specificity and good sensitivity, and can detect multiple circRNA biomarkers in whole blood samples.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a circRNA biomarker for diagnosing ovarian cancer, wherein the circRNA biomarker comprises 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 nucleotide sequence of hsa_circ_0006935 is shown in SEQ ID NO. 3.

[0012] In a second aspect, the present application provides a construction method of the above-mentioned circRNA biomarker for diagnosing ovarian cancer, wherein a linear RNA template is obtained by in vitro transcription of a linear DNA template with a nucleotide sequence shown in SEQ ID NO. 1-3, and the linear RNA is circularized by using T4 RNA ligase 2 under the action of a DNA ligation primer, and the nucleotide sequence of the DNA ligation primer is shown in SEQ ID NO. 4-6.

[0013] According to the technical scheme, further, the construction method specifically includes the following steps: linear RNA templates are obtained by in vitro transcription of linear DNA templates with nucleotide sequences shown in SEQ ID NO. 1-3; the linear RNA templates are annealed with DNA ligation primers, incubated at 60-70℃ for 2-10 min, cooled to 10-15℃, and then T4 RNA ligase 2 and RNase inhibitor are added to the mixture, incubated at 35-39℃ for 30-120 min, incubated at 70-90℃ for 2-10 min, RNase R and RNase R buffer are added, incubated at 35-39℃ for 20-50 min, incubated at 60-80℃ for 5-30 min, DNase I is added, and incubated for 20-50 min to digest the excess DNA ligation primers, and the circRNA biomarker is obtained after purification.

[0014] In a third aspect, the present application further 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 application 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] According to the technical scheme, further, the Cas12a enzyme in the CRISPR / Cas12a detection system is at least one of LbCas12a and FnCas12a, and the crRNA is designed according to the two enzymes, 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, 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, and the sequence of FncrRNA3 is shown in SEQ ID NO. 13.

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

[0018] Based on the above technical scheme, further, the CRISPR buffer includes 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100ug / ml Recombinant Albumin(pH 7.9@25℃).

[0019] Based on the above technical scheme, further, the detection kit further includes at least one RNA enzyme inhibitor.

[0020] Based on the above technical scheme, further, the CRISPR reaction conditions are 35-38℃ for 30-120min, and the fluorescence signal is collected every 1-5min.

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

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

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

[0024] The present application finds that the expressions of hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935 are significantly increased in the plasma of ovarian cancer patients, and 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. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below.

[0026] Figure 1Figure 1 is the discovery and verification of circRNA biomarkers in Example 1 of the present application, wherein A is a heat map of differentially expressed circRNAs of NIHOVCAR3 vs HEK293T and SKOV3 vs HEK293T, B is a circRNA Wayne diagram of NIHOVCAR3 vs HEK293T and SKOV3 vs HEK293T, C-D are PPI protein interaction network analysis of interactions between mRNAs, screening of core genes, construction of a circRNA-miRNA-mRNA regulatory network, E is RNase R experiment to identify the presence of circRNA in cancer cells, and F is verification of the 3 circRNA biomarkers in blood samples of different stages of clinical cancer.

[0027] Figure 2 Figure 2 is the preparation and verification of circRNA standards in Examples 2-3 of the present application, wherein A is a schematic diagram of in vitro circularization of circRNA, B is RNase R verification of the synthesis of circRNA, and C is RCA amplification verification.

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

[0029] Figure 4 Figure 5 is the sensitivity detection results of the ovarian cancer circRNA detection method based on the kit in Example 5 of the present application, wherein A is the detection fluorescence kinetics and column chart of 4 crRNA combinations, B is a fluorescence intensity heat map of different numbers of crRNA combinations, and C is a slope sensitivity heat map of different numbers of crRNA combinations.

[0030] Figure 5 Figure 6 is the specificity detection results of the ovarian cancer circRNA detection method based on the kit in Example 6 of the present application, wherein A is the specific sequence and column chart of single base to 5 base differences, B is the fluorescence kinetics graph of single base to 5 base differences, C is the anti-interference ability verification fluorescence kinetics graph of linear RNA, circRNA, and different proportions of linear RNA and circRNA, D is the anti-interference ability verification column chart, E is the specificity fluorescence kinetics graph of the detection method verified by different source cancer cells, and F is the column chart of the detection method verified by different source cancer cells.

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

[0032] To make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0033] In the following examples, the experimental methods are conventional methods unless otherwise specified. The experiments are performed according to the literature or the description in the specification. The experimental materials used in the following examples can be obtained by commercial means unless otherwise specified. The quantitative tests in the following examples are set up with three repeated experiments unless otherwise specified, and the results are averaged.

[0034] The reporter DNA used in the following examples is a product of Guangzhou Meigene Bio-pharmaceutical Company. The enzymes in amplification and CRISRP / Cas are products of New Biolab Company.

[0035] Example 1 Discovery and verification of circRNA biomarkers

[0036] 1. Cell sequencing and analysis

[0037] The differences in gene expression levels obtained by sequencing of NIH OVCAR3 vs HEK293T and SKOV3 vs HEK293T were compared using the R package edgeR. The above sequencing data has been uploaded to the GEO database GSE271851. The screening conditions for differentially expressed circRNAs are: corrected p<0.05 and |log FC>1|, 637 differentially expressed circRNAs were obtained for NIH OVCAR3 vs 293T, of which 331 were up-regulated and 306 were down-regulated, and 616 differentially expressed circRNAs were obtained for SKOV3 vs 293T, of which 308 were up-regulated and 308 were down-regulated. The "ggplot2" and "pheatmap" heat maps were used to show the differential expression of circRNAs, as follows:Figure 1 A. The intersection of the differentially expressed circRNAs of NIHOVCAR3 vs 293T and SKOV3 vs 293T was obtained, and 244 differentially expressed circRNAs in ovarian cancer (OC) were obtained, denoted as candidate circRNAs, such as Figure 1 B.

[0038] According to the differentially expressed circRNAs, a circRNA-miRNA-mRNA regulatory network was constructed, and core genes were screened in the PPI protein network constructed according to the mRNA, and finally a circRNA-miRNA-mRNA regulatory network was reconstructed according to the core genes, such as Figure 1 C-D. Through 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 existence of circRNAs related to ovarian cancer in blood plasma, whether the expression level was detected in the RNase R(+) and RNase R(-) groups was used to prove that the detected molecule was circRNA. The total circRNA extracted from the ovarian cancer clinical blood samples from Liaoning Tumor Hospital was subjected to RNase R enzyme digestion experiment. The specific process is that 5 μg of total RNA from the blood of cancer patients is added with 3 U / μg RNase R and the corresponding 10x RNase R buffer, and the enzyme-free water is added to 20 μL. The samples treated with RNase and not treated with RNase are subjected to RT-qPCR detection, and the expression levels of three circRNAs (hsa_circ_0049101, hsa_circ_0006935 and hsa_circ_0007440) in the two groups of different treatment samples are verified.

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

[0042] To further investigate the correlation between these interesting circRNAs and ovarian cancer, 22 OC samples (early stage (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. As shown in Fig. F, hsa_circ_0049101, hsa_circ_0006935 and hsa_circ_0007440 were significantly up-regulated in OC patients. In addition, the three up-regulated circRNAs had relatively high significant differences in different cancer stages compared with the control group, and their expression increased with the increase of cancer stage grade. Figure 1 F. In addition, the three up-regulated circRNAs had relatively high significant differences in different cancer stages compared with the control group, and their expression increased with the increase of cancer stage grade.

[0043] Example 2 Preparation and verification of circRNA standard

[0044] Preparation of circRNA standard

[0045] Linear RNA was circularized by T4 RNA ligase 2, and linear RNA template was transcribed into linear RNA template in vitro using double-stranded linear DNA template. Linear RNA was circularized using T4 RNA ligase 2 on the DNA ligation primer.

[0046] Specifically, according to the number of reactions, the linear RNA template (1 μM) and the DNA ligation primer (5 μM) mixture was annealed, and the mixture was incubated at 65°C for 5 min, and cooled to 12°C at a rate of 1°C / min. The nucleotide sequences of the DNA ligation primers of the three circRNAs are 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 (2 U) and RNase inhibitor (0.5 U) were added to the mixture and incubated at 37°C for 90 min, and incubated at 80°C for 5 min. RNase R (1 U) and RNase R buffer were added and incubated at 37°C for 30 min, and incubated at 70°C for 10 min. After the reaction, DNase I (1 U) was used to digest the excess DNA ligation primer for 30 min. The reaction product was purified using RNA Cleanup Kit (50 μg, New England Biolabs), and 5 μL of supernatant was used for 1.0% agarose gel electrophoresis experiment verification. RNA Cleanup Kit (50 μg, New England Biolabs), and 5 μL of supernatant was used for 1.0% agarose gel electrophoresis experiment verification.

[0047] The specific process of circRNA synthesis is shown in Figure 2A. Specifically, the linear RNA template synthesized in vitro was annealed with the DNA ligation primer, and then the 5' and 3' of the linear RNA template were ligated with T4 RNA ligase 2. Subsequently, the excess DNA ligation primer was eliminated by DNase I, and RNase R can digest almost all linear RNA molecules, but cannot digest circular RNA. Therefore, the RNase R enzyme digestion was used to prove the feasibility of circRNA synthesis Figure 2 B). The results show that the circRNA has higher stability than the linear RNA, thereby indicating the successful synthesis of circRNA.

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

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

[0050] The results are shown in Figure 2 C gel electrophoresis, the first lane is the circular RNA template, the second lane is the RT-RCA primer, and the third lane to the fourth lane are the products of reverse transcription rolling circle amplification reaction for 30min, 1h and 1.5h, respectively. From the results, it can be seen that cDNA can be reverse transcribed at 30min, and when the reaction is carried out to 1h, it is the best, so it can be proved that the experiment is feasible and the optimal reaction condition is 1h.

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

[0052] 1. Various crRNA combination designs

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

[0054] LbCas12a protein of Trichoderma strain and FnCas12a protein of Francisella strain were studied. LbCas12a is more inclined to recognize PAM sequence of 5'-TTV-3', and FnCas12a recognizes PAM sequence which 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, LbCas12a and FnCas12a were selected to verify the CRISPR / Cas12a system in the present study.

[0055] 2. Screening and verification of crRNA

[0056] crRNA was designed according to LbCas12a and FnCas12a respectively, as shown in Figure 3 A, wherein 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] crRNA in vitro transcription gel electrophoresis verification experiment, according to the company to synthesize 69bp double-stranded DNA template with T7 promoter (1 μg), add T7 polymerase, dATP, dCTP, dGTP and dUTP each 100 nM, RNase inhibitor (1 U), T7 polymerase buffer, enzyme-free water to 20 μL. 1% agarose gel electrophoresis was used for verification. The results are shown in Figure 3 B, 69bp double-stranded DNA template was transcribed by T7 promoter in vitro to obtain 44nt single-stranded RNA, and the bands in B are crRNA1 and crRNA3 designed for LbCas12a, and crRNA1 and crRNA3 designed for FnCas12a.

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

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

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

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

[0062] The results are as follows Figure 3 As shown in Figures E and 3F, compared to the control, the fluorescence intensity of the LbCas12a / LbcrRNA1+FnCas12a / FncrRNA1+cDNA detection system, which combines two proteins, is significantly higher than that of the single-protein detection systems FnCas12a / FncrRNA1+cDNA and LbCas12a / LbcrRNA1+cDNA. The combined use of LbcrRNA1, LbcrRNA3, FncrRNA1, and FncrRNA3 exhibits the highest sensitivity. This suggests that combining Cas proteins targeting different PAMs has great potential to improve the detection sensitivity of circRNAs.

[0063] Example 5: Sensitivity detection of an ovarian cancer detection kit and standard based on circRNA biomarkers, as detailed below.

[0064] Sensitivity was tested by serially diluting the concentration of circRNA (hsa_circ_0049101) (2000pM to 0.5fM). The circRNA was then amplified by RT-RCA with the following composition: RT-RCA primer (SEQ ID NO.7) 480nM, Protoscript II reverse transcriptase 1.5U, 10×DTT, dNTP 400nM, and RNase inhibitor 1U. The mixture was incubated at 42°C for 1 hour to obtain the RT-RCA product.

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

[0066] The reaction mixture was incubated at 37°C for 2 hours, and the fluorescence emission spectrum was as follows: The assay was performed using a 200PRO microplate reader (Tecan, Switzerland), with an excitation wavelength of 525 nm. Fluorescence kinetics were performed on a Quant Studio3 RealTime system (Thermo Fisher Scientific, Waltham, MA).

[0067] CRISPR detection and fluorescence signal collection: Add the CRISPR reaction system prepared above into a PCR tube and place it in a real-time PCR instrument. Set the program to: 37℃, 30 cycles, and collect the fluorescence signal every 5 minutes.

[0068] The CRISPR-detected fluorescence signal value was greater than the average fluorescence signal of the negative control plus three times the standard deviation. These validation results indicate that when four crRNAs (LbcrRNA1 + LbcrRNA3 + FncrRNA1 + FncrRNA3) are combined, the detection sensitivity is further improved, being 4-11 times higher than that of single crRNAs and three crRNAs. The heatmap visually compares the endpoint fluorescence values ​​at different concentrations and analyzes the endpoint fluorescence values ​​and slopes for each different crRNA combination. The combination containing four crRNAs showed the highest sensitivity. Figure 4 BC).

[0069] Under the optimal experimental conditions, the sensitivity of detection of different concentrations of circRNA was detected. The fluorescence intensity curve F / F0 was proportional to the concentration of circRNA in the range of 2000 pM to 0.5 fM. The LOD was calculated as 0.5 fM. The correlation equation was y = 187.773 + 554.050 log C (R 2 = 0.993) Figure 4 ). This shows that this method is significantly better than other detection methods.

[0070] Example 6 Specific detection of the standard based on multiple circRNA biomarkers established by the present application

[0071] Five crRNAs with 3-base mismatches with LbcrRNA3 were designed, and the sequence of LbcrRNA3 was used to verify the ability of the detection method to distinguish single-base mismatches. The specific process was as follows: the concentration of circRNA (2000 pM), first, the circRNA was subjected to RT-RCA amplification, wherein the RT-RCA primer (SEQ ID NO. 7) was 480 nM, Protoscript II reverse transcriptase was 1.5 U, dNTP was 400 nM, RNase inhibitor was 1 U, and the mixture was incubated at 42°C for 1 hour to obtain the RT-RCA product. The CRISPR reaction system was prepared with the RT-RCA amplification product as the template, and the CRISPR reaction system was prepared. The reaction system (20 μL system) was as follows: the RT-RCA amplification product as the template, LbcrRNA1, LbcrRNA3, FncrRNA1 and FncrRNA3 were each 100 nM, LbCas12a was 200 nM, FnCas12a was 200 nM, ssDNA fluorescent probe was 400 nM (F-TTTTT-Q), DTT was 10 mM, and was added to the PCR tube, and a negative control with sterile water as the template was set. Among them, LbcrRNA1 was replaced with 1-base to 5-base mismatch sequences, and the specific mismatch sequences and positions were as follows: Figure 5 A, to verify the specificity of the established CRISPR / Cas detection circRNA method Figure 5 A-B), the results showed that the method had good single-base recognition ability.

[0072] Next, circRNA was mixed with linear RNA (the same as circRNA sequence) at a ratio of 1:5, 1:50 and 1:500, and the specific RT-RCA / CRISPR parameters and process were the same as above, the difference was that the detection template was replaced with different ratios of circRNA and linear RNA mixture, which was used to evaluate the anti-interference ability of the method Figure 5C-D). Mixing with high concentration of 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 is to verify the positive detection rate and accuracy of the method of the present application for detecting ovarian circRNA based on the CRISPR / Cas detection method established in the actual sample detection application. The whole blood samples of clinical ovarian cancer patients and healthy people were detected.

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

[0075] The samples detected according to the kit reagent instruction manual of KJ Biotech Co., Ltd. Blood & Tissue Kit instruction manual, after pretreatment of the sample, nucleic acid was extracted.

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

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

[0078] The PCR amplification curve and Cq value were analyzed to evaluate the quantity of circRNA in the clinical sample. For the RT-RCA / CRISPR / Cas12a detection system, the RT-RCA primer (SEQ ID NO. 7, 480 nM), Protoscript II reverse transcriptase (1.5 U), 10x DTT and dNTP (400 nM) were mixed with the RNA sample, incubated at 42°C for 60 min, and then 2 μL LbCas12a and FnCas12a protein (200 nM each), LbcrRNA1, LbcrRNA3, FnbcrRNA1, FncrRNA3 (100 nM each) and 1 μL ssDNA fluorescent probe F-TTTTT-Q (400 nM) were added. The mixture was incubated at 37°C for 40 min, and the measurement was performed using the Quant Studio 3 real-time system.

[0079] Results Figure 6 As shown in D-F, CA125, HE4 index was tested by the clinical standard ELISA detection method, and the ROMA index was obtained according to the results of CA125 and HE4. Double antibody sandwich ELISA method was used. Anti-human CA125 antibody was coated on the enzyme-labeled plate, and human CA125 in the sample (or standard) combined with the coated antibody during the experiment. Then biotinylated anti-human CA125 antibody and horseradish peroxidase labeled avidin were added in turn, the anti-human CA125 antibody combined with the human CA125 combined with the coated antibody, and the biotin and avidin specifically combined to form an immune complex, and the free components were washed away. Add color developing substrate (TMB), TMB presents blue color under the catalysis of horseradish peroxidase, and turns yellow after adding the stop solution. The OD value was measured at 450 nm wavelength by enzyme-labeled instrument, and the CA125 concentration was proportional to the OD450 value. The concentration of CA125 in the sample was calculated by drawing a standard curve, and the HE4 concentration detection method was the same as above, except that the antibody was replaced by anti-human HE4 antibody. The results are shown in Figure 6 As shown in D-F, CA125, HE4 index was tested by the clinical standard ELISA detection method, and the ROMA index was obtained according to the results of CA125 and HE4. Double antibody sandwich ELISA method was used. Anti-human CA125 antibody was coated on the enzyme-labeled plate, and human CA125 in the sample (or standard) combined with the coated antibody during the experiment. Then biotinylated anti-human CA125 antibody and horseradish peroxidase labeled avidin were added in turn, the anti-human CA125 antibody combined with the human CA125 combined with the coated antibody, and the biotin and avidin specifically combined to form an immune complex, and the free components were washed away. Add color developing substrate (TMB), TMB presents blue color under the catalysis of horseradish peroxidase, and turns yellow after adding the stop solution. The OD value was measured at 450 nm wavelength by enzyme-labeled instrument, and the CA125 concentration was proportional to the OD450 value. The concentration of CA125 in the sample was calculated by drawing a standard curve, and the HE4 concentration detection method was the same as above, except that the antibody was replaced by anti-human HE4 antibody. The results are shown in

[0080] As can be seen, the detection method constructed by the present application has relatively higher sensitivity and accuracy for ovarian cancer circRNA. Compared with the currently commonly used qPCR method, the detection method constructed by the present application greatly shortens the detection time, is more simple to operate and has higher sensitivity.

[0081] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A circRNA biomarker for diagnosing ovarian cancer stage III and IV, characterized in that, The circRNA markers are composed of hsa_circ_0049101, hsa_circ_0007440 and hsa_circ_0006935; The nucleotide sequence of hsa_circ_0049101 is shown as SEQ ID NO. 1, The nucleotide sequence of hsa_circ_0007440 is shown as SEQ ID NO. 2, The nucleotide sequence of hsa_circ_0006935 is shown as SEQ ID NO.

3.

2. The method of constructing circRNA biomarkers for the diagnosis of ovarian cancer in stages III and IV according to claim 1, characterized in that, The linear DNA templates with the nucleotide sequences shown as SEQ ID NO. 1-3 are transcribed in vitro to obtain linear RNA templates, which are circularized using T4 RNA ligase 2 under the action of DNA ligation primers; the nucleotide sequences of the DNA ligation primers are shown as SEQ ID NO. 4-6.

3. The use of the reagent for detecting the expression amount of the circRNA biomarker in claim 1 in the preparation of a kit for diagnosing stages III and IV of ovarian cancer.

4. Use according to claim 3, characterized in that, The kit comprises a CRISPR / Cas12a detection system, which comprises crRNA, Cas12a enzyme and ssDNA fluorescent probe, CRISPR buffer and DTT.

5. Use according to claim 4, characterized in that, The Cas12a enzyme in the CRISPR / Cas12a detection system 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 as SEQ ID NO. 8; the sequence of the LbcrRNA2 is shown as SEQ ID NO. 9; the sequence of the LbcrRNA3 is shown as SEQ ID NO. 10; the sequence of the FncrRNA1 is shown as SEQ ID NO. 11; the sequence of the FncrRNA2 is shown as SEQ ID NO. 12; and the sequence of the FncrRNA3 is shown as SEQ ID NO.

13.

6. Use according to claim 4, characterized in that, The structure of the ssDNA fluorescent probe is as follows: 5'-FAM-TTTTT-BHQ1-3'.

7. Use according to claim 4, characterized in that, The detection kit further comprises at least one RNAse inhibitor.

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