HPV16 E7 mRNA-specific recognition N-MSSRCA-CRISPR cascade amplification system, construction method and application

Abstract

The application relates to the technical field of biological detection, in particular to an N-MSSRCA-CRISPR cascade amplification system for specific recognition of HPV16 E7 mRNA, a construction method and application. Specific recognition is realized through target-dependent connection of Nick probes and Bind probes, a large number of RCA primers are generated through the 'extension-cutting-displacement' cycle of Bst DNA polymerase and nicking endonuclease, long-chain ssDNA products are generated through Phi29 polymerase-mediated MSSRCA rolling circle amplification, and finally secondary amplification of a fluorescence signal is realized through competitive crRNA-mediated CRISPR / LbCas12a cascade activation. The system exhibits a three-level cascade amplification effect, a double-specificity guarantee mechanism and a modular general design in HPV16 E7 mRNA detection, and has clinical transformation value for early screening of cervical cancer.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an N-MSSRCA-CRISPR cascade amplification system for HPV16 E7 mRNA specific recognition, its construction method, and its application. Background Technology

[0002] The occurrence of cervical cancer is closely related to persistent infection with high-risk human papillomavirus (HPV), with HPV types 16 and 18 being the most prevalent carcinogenic types, accounting for approximately 70% of cervical cancer cases worldwide. High-risk HPV interferes with the function of host cell tumor suppressor genes p53 and pRb through its oncogenes E6 and E7, leading to cell cycle dysregulation and malignant transformation. HPV E6 / E7 mRNA, as expression products of viral oncogenes, is a direct marker reflecting active viral replication and cell transformation status. It is more effective than HPV DNA detection in distinguishing between transient and persistent carcinogenic infections and has been widely recognized as an important molecular target for cervical cancer screening and early diagnosis.

[0003] Currently, commonly used clinical methods for HPV detection mainly include PCR (Polymerase Chain Reaction), reverse transcription PCR (RT-PCR), Hybrid Capture 2HC2, and Nucleic Acid Sequence-Based Amplification (NASBA). While PCR and its derivatives offer high sensitivity and specificity, they rely on sophisticated thermal cycling equipment and specialized operation, resulting in long testing cycles and hindering their widespread application in primary healthcare institutions. HC2 is relatively simple to operate but has lower sensitivity and cannot distinguish HPV types. NASBA is suitable for RNA detection, but its reaction system is complex and still requires expensive equipment. In summary, existing clinical methods generally suffer from strong equipment dependence, complex operation, long processing times, and high costs, making it difficult to meet the needs of on-site, real-time testing, especially lacking ultrasensitive and rapid detection methods for key biomarkers such as HPV E7 mRNA.

[0004] In recent years, isothermal nucleic acid amplification technology has gradually become a research hotspot due to its advantages such as not relying on thermal cycling equipment, mild reaction conditions, and suitability for point-of-care (POC) detection. Among them, rolling circle amplification (RCA) technology, based on circular DNA templates and strand substitution DNA polymerases, has the advantages of simple structure and high amplification efficiency. However, the circular templates of traditional RCA are prone to forming secondary structures, which hinder polymerase extension and lead to a decrease in amplification efficiency. To address this problem, Minimum Secondary Structure Rolling Circle Amplification (MSSRCA) significantly improves amplification efficiency and product linearity by rationally designing the circular template sequence to minimize the formation of secondary structures. To further improve detection speed and sensitivity, researchers introduced nicking endonucleases into MSSRCA to construct nicking enzyme-enhanced rolling circle amplification (N-MSSRCA). For example, Tao et al.'s N-MSSRCA technology, published in *Analytical Chemistry*, utilizes T4 DNA ligase for target-dependent probe ligation. Through the synergistic action of Bst DNA polymerase and the nicking endonuclease Nb.BbvCI, target recognition, primer generation, and signal amplification are completed in a single tube. Specific probes are designed for HPV16 E7 mRNA, which ligate to form a complete template in the presence of the target. Bst DNA polymerase extends this template to form a double-stranded ligation product containing the Nb.BbvCI recognition site. The nicking endonuclease cleaves the upper strand to create a nick, from which Bst DNA polymerase extends and displaces the downstream strand, cyclically generating numerous short DNA fragments as primers. These fragments hybridize with the MSSRCA circular template to initiate rolling circle amplification, generating a single-stranded DNA product containing repeating units. While N-MSSRCA achieves breakthroughs in isothermal amplification efficiency and speed, its product is a long single-stranded DNA. How to efficiently convert this long nucleic acid into a readable output signal remains crucial for improving overall detection performance.

[0005] Meanwhile, the CRISPR / Cas system, with its precise nucleic acid recognition and signal transduction capabilities, shows great promise in the field of molecular diagnostics. In particular, the Cas12a protein, after binding to specific crRNA to form a complex, can recognize target nucleic acids and activate non-specific single-stranded DNA trans-cleavage activity, cleaving fluorescent reporter molecules to generate a signal, thus converting the target recognition event into a significant fluorescent signal. Moon et al., in *Nature Communications*, proposed an asymmetric CRISPR cascade signal amplification strategy based on competitive crRNA, finding that intact crRNA and split crRNA competitively bind to Cas12a, with intact crRNA exhibiting stronger affinity and dominating. In the presence of the target, the intact crRNA / Cas12a complex recognizes the target and activates trans-cleavage to generate an initial signal; subsequently, split crRNA can replace the hybrid of intact crRNA and the target, reactivating Cas12a to generate a secondary cleavage, achieving cascade signal amplification. However, when the CRISPR / Cas system is used as a signal output unit, its sensitivity is highly dependent on the concentration of the upstream target nucleic acid. For extremely low abundances of HPV E7 mRNA, direct activation of Cas12a often fails to generate a sufficiently strong signal.

[0006] In view of the limitations of the prior art, this invention proposes an ultrasensitive detection method combining N-MSSRCA with the CRISPR / LbCas12a system. Utilizing the highly efficient isothermal amplification capability of N-MSSRCA, trace amounts of HPV E7 mRNA target are converted into a large amount of well-defined long single-stranded DNA amplification products. This long ssDNA product is then cleverly used as a specific activator for the CRISPR / LbCas12a system, and a competitive binding mechanism between intact and split crRNA is introduced to achieve secondary cascade amplification of the signal. First, N-MSSRCA technology specifically recognizes and efficiently amplifies HPV E7 mRNA. Through cyclic cleavage by nicking endonuclease and strand displacement amplification by Bst DNA polymerase, a large amount of long ssDNA products containing specific repetitive sequences are rapidly generated under isothermal conditions. This process solves the problem of "signal acquisition" for low-abundance targets. Second, the aforementioned long ssDNA product is used as a "key" to specifically activate the CRISPR / LbCas12a system. With the assistance of competitive crRNA, target-activated LbCas12a not only generates an initial signal but also achieves secondary cleavage activation by replacing intact crRNA with split crRNA, forming a cascaded signal amplification. This process solves the problem of "signal conversion and re-amplification" of amplified products. Through amplification cascade, ultra-high sensitivity detection of HPV E7 mRNA is achieved, with an overall signal-to-noise ratio and detection limit significantly superior to single technologies. Summary of the Invention

[0007] The purpose of this invention is to provide an N-MSSRCA-CRISPR cascade amplification system for HPV16 E7 mRNA specific recognition, its construction method, and its application.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: Step 1: Nucleic acid probe pretreatment: The synthesized Nick probe, Bind probe, MSSRCA circular template, short universal primer, complete crRNA, split crRNA handle, split crRNA spacer, DNA activator, fluorescent reporter molecule, and HPV16 E7 cDNA target probe lyophilized powder were prepared into 100µM stock solutions using DEPC water, aliquoted and stored at -20℃ for later use; all reagents and consumables involving RNA were inactivated by RNase. Step 2: Construction and annealing of the ligation reaction system: Nick probe, Bind probe, HPV16 E7 cDNA target, and short universal primer Ds primer were added to 1×T4 ligase buffer at a molar ratio of 1:1:1:1 and mixed thoroughly. The mixture was heated at 95°C for 5 min to fully denature and dissociate all nucleic acid double strands and open the secondary structures of the probes. Then, it was slowly cooled to room temperature to allow Nick and Bind probes to specifically hybridize with the target sequence, while the short universal primers complementarily bound to the repeat sequence at the 3' end of the Bind probe. After annealing, T4 DNA ligase was added directly to the system and incubated at room temperature for 10 min to complete the target-dependent probe ligation reaction. Step 3: Bst DNA polymerase-mediated double-strand formation and primer cycling generation: Bst DNA polymerase buffer, dNTPs mixture, MSSRCA circular template, Bst DNA polymerase, and Nb.BbvCI nicking endonuclease are directly added to the above ligation reaction system, and the reaction system is incubated at a constant temperature. During this process, Bst DNA polymerase extends short primers to form double-stranded ligation products, Nb.BbvCI recognizes and cleaves the recognition sites in the double strands, and the strand displacement activity of Bst DNA polymerase drives the "extension-cleavage-displacement" cycle, continuously generating a large number of short DNA fragments as primers for subsequent rolling circle amplification. Step 4: Phi29 polymerase-mediated rolling circle amplification: Phi29 DNA polymerase was added to the above reaction system, and the mixture was incubated at 37°C for 30 min. The short primers generated in Step 3 were hybridized with the MSSRCA circular template. Driven by the continuous synthesis capacity and strand displacement activity of Phi29 DNA polymerase, rolling circle amplification was performed along the circular template to generate a long single-stranded DNA product containing a large number of repeating units. After the reaction was completed, the enzyme was inactivated by heating at 80°C to obtain the long ssDNA product. Step 5: Competitive CRISPR / LbCas12a Cascade Signal Amplification and Fluorescence Detection of the N-MSSRCA-CRISPR Cascade Amplification System: Add intact crRNA, split crRNA handle and spacer, 1×NEBuffer, LbCas12a protein, activator, and fluorescent-quenched dual-labeled single-stranded DNA reporter molecules directly to the above N-MSSRCA amplification system. Make up the volume with DEPC water and mix thoroughly. Incubate the reaction system at 37°C for 30 min. During this process, the intact crRNA / LbCas12a complex preferentially recognizes the repetitive sequences in the N-MSSRCA product, forming a CRISPR / LbCas12a system based on intact crRNA, activating the first round of trans cleavage, which cleaves the fluorescent reporter molecule to generate the initial fluorescence signal. Subsequently, the split crRNA replaces the intact crRNA and rebinds with LbCas12a, forming a CRISPR / LbCas12a system based on split crRNA. After being activated by the DNA activator, it generates a second round of trans cleavage, achieving cascade signal amplification. After the reaction was completed, the LbCas12a protein was inactivated by heating at 85°C for 5 minutes.

[0009] Furthermore, the schematic diagram of the above experimental principle is as follows: Figure 1 As shown.

[0010] Furthermore, in step 3, the constant temperature incubation is 37°C, and the incubation time is 60 minutes.

[0011] Furthermore, in step 3, the working concentration of Bst DNA polymerase in the primer cycling generation step is 0.24 U / μL.

[0012] Furthermore, in step 4, the constant temperature incubation is 37°C, and the incubation time is 30 minutes.

[0013] Furthermore, in step 4, the reaction time for the rolling circle amplification stage is 60 minutes.

[0014] In step 5, the working concentration of LbCas12a protein in the CRISPR / LbCas12a system is 75 nM.

[0015] Furthermore, the endpoint fluorescence intensity of the N-MSSRCA-CRISPR cascade amplification system in step 5 was measured using an RF-6000 fluorescence spectrophotometer, with the excitation wavelength set to Ex=480nm and the emission wavelength set to Em=500-650nm.

[0016] Furthermore, the N-MSSRCA-CRISPR cascade amplification system specifically recognizes HPV16 E7 mRNA.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) High sensitivity: This invention achieves significant signal gain through a three-stage cascade amplification. The first stage is a "extension-cutting-displacement" cycle synergistically performed by Bst DNA polymerase and nicking enzyme, which converts single target recognition into a large number of RCA primers; the second stage is MSSRCA rolling circle amplification mediated by Phi29 polymerase, which converts the primers into long-chain ssDNA products containing a large number of repeating units; the third stage is a competitive crRNA-mediated CRISPR / LbCas12a cascade signal amplification, which achieves secondary signal amplification through two sequentially activated trans-cutting. The three-stage amplification mechanism can theoretically produce significant signal gain and is expected to enable the detection of targets at extremely low concentrations.

[0018] (2) High specificity: This invention employs a dual recognition mechanism to ensure specificity. The first layer consists of precise hybridization of the Nick and Bind probes with the target and the stringent ligation conditions of the T4 ligase; the second layer consists of the precise nucleic acid recognition capability of the CRISPR / LbCas12a system. Dual recognition can effectively distinguish non-target sequences, ensuring the accuracy of detection results.

[0019] (3) Low background signal: This invention suppresses background signal at the source. Ligation cannot be initiated without a target; primer cycling cannot proceed without a double-stranded structure; the reporter molecule remains intact when the CRISPR system is not activated; the fluorescence-quenching dual-label design ensures extremely low background fluorescence before cleavage. Multiple background suppression mechanisms ensure a high signal-to-noise ratio.

[0020] (4) Simple operation: The present invention is a one-tube isothermal reaction system, which does not require intermediate purification steps or precision thermal cycling equipment. It adopts endpoint fluorescence detection, simplifies the operation process, and is suitable for on-site POC detection.

[0021] (5) Platform universality: By redesigning the connection probe and crRNA sequence, it can be quickly adapted to the detection of other nucleic acid targets, and has good modular expansion potential.

[0022] (6) Cost advantage: It uses conventional commercial reagents, eliminates the need for expensive labeling, and the integrated design reduces the amount of consumables used. The cost per test is controllable and it is suitable for large-scale screening applications. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the experimental principle of the present invention; Figure 2 This is the result of the feasibility analysis of the probe assembly verification by electrophoresis in Experiment 1 of this invention; Figure 3 This is the result of the feasibility analysis of the system and target response fluorescence in Experiment 1 of this invention; Figure 4This is an analysis chart of the parameter optimization results in Experiment 2 of this invention; wherein, Figure 4 (a) Figure 4 (b) shows the results of the gradient optimization analysis of the reaction time for the formation and cleavage of the double-chain structure; Figure 4 (c) Figure 4 (d) shows the results of the RCA reaction time gradient optimization analysis; Figure 4 (e) Figure 4 (f) shows the results of the Bst DNA polymerase concentration optimization analysis; Figure 4 (g) Figure 4 (h) shows the results of the LbCas12a protein concentration optimization analysis; Figure 5 The figure shows the target specificity analysis results of the N-MSSRCA-CRISPR cascade amplification system in Experiment 3; where, Figure 5 (a) Fluorescence spectra of the N-MSSRCA-CRISPR cascade amplification system after incubation with different targets. Figure 5 (b) is a bar chart showing the maximum fluorescence intensity at 520 nm after incubation of the N-MSSRCA-CRISPR cascade amplification system with different targets; Figure 6 This is a graph showing the fluorescence sensitivity analysis results in Experiment 4 of this invention; Figure 6 (a) Fluorescence spectra of the N-MSSRCA-CRISPR cascade amplification system after incubation with different concentrations of target HPV16 E7 cDNA. Figure 6 (b) shows the maximum fluorescence intensity at 520 nm after incubation of the N-MSSRCA-CRISPR cascade amplification system with different concentrations of target HPV16 E7 cDNA. Figure 6 (c) is a linear relationship diagram between the N-MSSRCA-CRISPR cascade amplification system and different concentrations of target HPV16 E7 cDNA (25pM-500pM). Detailed Implementation

[0024] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0025] The probes involved in this invention were all purchased from Shanghai Sangon Biotech Co., Ltd., as shown in Table 1: Table 1 Probe Sequence

[0026] Example 1: Construction of an N-MSSRCA-CRISPR cascade amplification system Step 1: Nucleic acid probe pretreatment: The synthesized Nick probe, Bind probe, MSSRCA circular template, short universal primer, complete crRNA, split crRNA handle, split crRNA spacer, DNA activator, fluorescent reporter molecule, and HPV16 E7 cDNA target probe lyophilized powder were prepared into 100µM stock solutions using DEPC water, aliquoted and stored at -20℃ for later use; all reagents and consumables involving RNA were inactivated by RNase. Step 2: Construction and annealing of the ligation reaction system: Nick probe, Bind probe, HPV16 E7 cDNA target, and short universal primer Ds primer were added to 1×T4 ligase buffer at a molar ratio of 1:1:1:1 and mixed thoroughly. The mixture was heated at 95°C for 5 min to fully denature and dissociate all nucleic acid double strands and open the secondary structures of the probes. Then, it was slowly cooled to room temperature to allow Nick and Bind probes to specifically hybridize with the target sequence, while the short universal primers complementarily bound to the repeat sequence at the 3' end of the Bind probe. After annealing, T4 DNA ligase was added directly to the system and incubated at room temperature for 10 min to complete the target-dependent probe ligation reaction. Step 3: Bst DNA polymerase-mediated double-strand formation and primer cycling generation: Add Bst DNA polymerase buffer, dNTPs mixture, MSSRCA circular template, Bst DNA polymerase, and Nb.BbvCI nicking endonuclease directly to the above ligation reaction system, and incubate the reaction system at 37°C for 60 min. During this process, Bst DNA polymerase extends short primers to form double-stranded ligation products, Nb.BbvCI recognizes and cleaves the recognition sites in the double strands, and the strand displacement activity of Bst DNA polymerase drives the "extension-cleavage-displacement" cycle, continuously generating a large number of short DNA fragments as primers for subsequent rolling circle amplification. Step 4: Phi29 polymerase-mediated rolling circle amplification: Phi29 DNA polymerase was added to the above reaction system, and the mixture was incubated at 37°C for 30 min. The short primers generated in Step 3 were hybridized with the MSSRCA circular template. Driven by the continuous synthesis capacity and strand displacement activity of Phi29 DNA polymerase, rolling circle amplification was performed along the circular template to generate a long single-stranded DNA product containing a large number of repeating units. After the reaction was completed, the enzyme was inactivated by heating at 80°C to obtain the long ssDNA product. Step 5: Competitive CRISPR / LbCas12a cascade signal amplification and fluorescence detection of the N-MSSRCA-CRISPR cascade amplification system: Complete crRNA and split crRNA were directly added to the above N-MSSRCA amplification system. Handle and spacer, 1×NEBuffer, LbCas12a protein, activator, and fluorescent-quenched double-labeled single-stranded DNA reporter molecule were added to the container with DEPC water to make up the volume and mixed thoroughly. The reaction system was incubated at 37°C for 30 min. During this process, the intact crRNA / LbCas12a complex preferentially recognizes the repetitive sequence in the N-MSSRCA product, forming a CRISPR / LbCas12a system based on intact crRNA, activating the first round of trans cleavage, which cleaves the fluorescent reporter molecule to generate the initial fluorescent signal. Subsequently, split crRNA replaces intact crRNA and rebinds with LbCas12a, forming a CRISPR / LbCas12a system based on split crRNA. After activation by the DNA activator, a second round of trans cleavage occurs, thus successfully constructing the N-MSSRCA-CRISPR system, achieving cascade signal amplification and highly sensitive target detection. After the reaction, the LbCas12a protein was inactivated by heating at 85°C for 5 min. The endpoint fluorescence intensity of the N-MSSRCA-CRISPR cascade amplification system was measured using an RF-6000 fluorescence spectrophotometer. The excitation wavelength was set to Ex=480nm and the emission wavelength was set to Em=500-650nm.

[0027] Experiment 1: Feasibility test of N-MSSRCA-CRISPR cascade amplification system.

[0028] Feasibility analysis of electrophoretic reaction: The feasibility of the N-MSSRCA-CRISPR cascade amplification system was verified by 12% polyacrylamide gel electrophoresis, such as... Figure 2As shown. Lane M is the DNA marker; lane 1 is the Nick probe alone; lane 2 is the Bind probe alone; lane 3 is the HPV16 E7 cDNA target alone; lane 4 is the short universal primer alone (Ds primer); lane 5 is a mixture of the Bind probe and the short universal primer, showing a weak hybridization band due to sequence complementarity; lane 6 is a mixture of E7 cDNA, Nick probe, and Bind probe, with no specific hybridization complex band observed, indicating that the Bind probe and the short universal primer form a weak hybridization band due to sequence complementarity, suggesting that the three cannot ligate to form a complex without T4 ligase; lane 7 is a mixture of E7 cDNA, Nick probe, Bind probe, and the short universal primer, again showing no hybridization complex band; lane 8, after adding T4 DNA ligase to lane 7, shows a new band with a larger molecular weight, corresponding to the expected size of the ligation product, indicating that the Nick probe and Bind probe successfully ligated when the target was present, and the E7 target was clearly visible. The cDNA target was displaced; lane 9, which was based on lane 8 with only Bst DNA polymerase added but without Nb.BbvCI nicking endonuclease, showed no amplification product band, indicating that the nicking endonuclease is indispensable in primer cycling; lane 10, which was based on lane 8 with both Bst DNA polymerase and Nb.BbvCI nicking endonuclease added, showed an amplification product band, but the band was weak, indicating a low product concentration. Electrophoresis results show that the ligation reaction can be successfully activated in the presence of the target, demonstrating the feasibility of this design.

[0029] Fluorescence Feasibility Experiment Analysis: To verify the feasibility of the entire process of the N-MSSRCA-CRISPR cascade amplification system constructed in this experiment, from target ligation, primer cycling generation, rolling circle amplification to competitive CRISPR signal amplification, and to confirm the necessity of the core components in each key step, the following feasibility verification experiment was designed. HPV16 E7 cDNA was used as the target, and fluorescence intensity was measured using a fluorescence spectrophotometer. Three parallel controls were set up for each group. The results are as follows: Figure 3As shown, Group A is the complete experimental group, containing all key components: HPV16 E7 cDNA target, Nick probe, Bind probe, T4 DNA ligase, Bst DNA polymerase, Nb.BbvCI nicking endonuclease, MSSRCA circular template, Phi29 DNA polymerase, LbCas12a protein, intact crRNA, split crRNA (containing handle and spacer), DNA activator, and fluorescent-quenched dual-labeled reporter molecule. Group B is the traditional CRISPR control group, which, in addition to the complete experimental group, does not include split crRNA and DNA activator, and only uses intact crRNA for detection, used to compare the signal gain difference between competitive cascade amplification and the traditional mode. Group C lacked dNTPs to verify the dependence of DNA polymerase extension reaction on nucleotide substrate; Group D lacked Bst DNA polymerase to clarify the key role of this enzyme in the formation of double-strand ligation products and primer cycling; Group E lacked LbCas12a protein to confirm that signal generation originates from the trans-cleavage activity of LbCas12a; Group F lacked MSSRCA circular template to verify the dependence of rolling circle amplification on circular template; Group G lacked Bind probe to examine the necessity of Bind probe in the target recognition process; Group I served as the negative control group, with the remaining components identical to the complete experimental group, used to assess the background signal level of the system in the absence of a target. Each group underwent the following experimental steps sequentially: ligation reaction, Bst DNA polymerase-mediated double-strand formation and primer cycling, Phi29 polymerase-mediated rolling circle amplification, and competitive CRISPR / LbCas12a cascade signal amplification and fluorescence detection. The endpoint fluorescence intensity was measured after the reaction. The complete experimental group produced a significantly higher fluorescence signal than the negative control group, confirming the effective synergy of the entire process. While group B also produced a fluorescence signal, its intensity was significantly lower than that of group A, indicating that the cascade amplification mediated by split crRNA had a signal-gain effect. Groups C through H, lacking key components respectively, showed lower fluorescence signals, proving that the missing components were indispensable in their respective steps. The negative control group should have only low background fluorescence, indicating that the system is strictly dependent on the presence of the target. These results provide experimental evidence for the feasibility of the technical solution and the necessity of the key components of this invention.

[0030] Experiment 2: Given the feasibility of the electrophoresis and fluorescence experiments, the present invention optimized a series of experimental conditions to obtain the best detection results.

[0031] (i) To determine the optimal incubation time for the Bst DNA polymerase-mediated double-strand formation and cleavage cycle reaction in this experiment, a time gradient optimization experiment was set up.

[0032] In step 3, Bst DNA polymerase and Nb.BbvCI nicking endonuclease work synergistically to generate a large number of short DNA fragments through an "extension-cutting-displacement" cycle, which serve as primers for subsequent rolling circle amplification. The reaction time directly affects the primer generation efficiency and the final detection signal intensity. Using HPV16 E7 cDNA as the target, multiple identical reaction systems were established according to the optimized standard procedure of this invention. After sequential ligation, Bst DNA polymerase buffer, Nb.BbvCI buffer, dNTPs mixture, Bst DNA polymerase, and Nb.BbvCI nicking endonuclease were added, and the systems were incubated at 37°C. The corresponding reaction tubes were removed at 30 min, 45 min, 60 min, and 90 min, and immediately heated at 80°C for 10 min to inactivate all enzymes and terminate the reaction. Subsequently, MSSRCA circular template and Phi29 DNA polymerase were added to the reaction systems terminated at each time point, and incubation was continued at 37°C to complete rolling circle amplification. Finally, the amplification products at each time point were added to a competitive CRISPR / LbCas12a detection system for cascaded signal amplification and fluorescence detection. The endpoint fluorescence intensity was measured using a fluorescence spectrophotometer. A curve was plotted with reaction time on the x-axis and endpoint fluorescence intensity on the y-axis to compare the signal intensity at different incubation times.

[0033] Experimental results are as follows Figure 4 (a) Figure 4 As shown in (b), the fluorescence signal intensity gradually increases with increasing reaction time, reaching a plateau at 60 min, and showing no significant signal gain upon further extension to 90 min. Considering both detection efficiency and signal intensity, 60 min was determined to be the optimal incubation time for the Bst DNA polymerase-mediated double-strand formation and cleavage cycle. This optimization result provides experimental basis for establishing a rapid and sensitive detection procedure in this invention.

[0034] (ii) To determine the optimal reaction time for the rolling circle amplification phase mediated by Phi29 DNA polymerase in step 4, a time gradient optimization experiment was set up.

[0035] The rolling circle amplification (RoBA) reaction time directly affects the total amount of long single-stranded DNA product generated. If the reaction time is too short, the amplification will be insufficient, resulting in a low amount of ssDNA product, which will lead to low efficiency as an activator of the CRISPR / LbCas12a system, ultimately resulting in a weak fluorescence signal and affecting detection sensitivity. If the reaction time is too long, the amplification may enter a plateau phase, and the product growth will tend to slow down or even stop. This not only does not help improve the signal, but may also increase the risk of side reactions such as primer dimer formation, non-specific amplification, or enzyme activity decay due to prolonged incubation, leading to increased background signal or unstable results. Therefore, determining the optimal RoBA time is crucial for balancing detection sensitivity and reaction efficiency. Using HPV16 E7 cDNA as the target, the experiment followed the optimized standard procedure of this invention, sequentially performing the ligation reaction and Bst DNA polymerase-mediated double-strand formation and primer cycling generation (fixed at 60 min). Multiple identical RoBA reaction systems were then established, with MSSRCA circular template and Phi29 DNA polymerase added, and incubated at 37°C. The corresponding reaction tubes were removed at 15 min, 30 min, 45 min, 60 min, and 90 min of reaction initiation, and immediately heated at 80 °C for 10 min to inactivate all enzymes and terminate the rolling circle amplification reaction. The amplification products at each time point were then added to a competitive CRISPR / LbCas12a detection system for cascade signal amplification and fluorescence detection. The endpoint fluorescence intensity was measured using a fluorescence spectrophotometer. A reaction-time curve was plotted with the rolling circle amplification reaction time on the x-axis and the endpoint fluorescence intensity on the y-axis to compare the signal intensity at different incubation times.

[0036] Experimental results are as follows Figure 4 (c) Figure 4 As shown in (d), the fluorescence signal intensity gradually increases with the extension of the rolling circle amplification reaction time. Within the time range of 30 min to 60 min, the signal intensity shows a small but gradual increase, indicating that the amplification product accumulation has reached a high level. Considering both detection sensitivity and reaction efficiency, although a stronger signal output can be obtained at 60 min, considering the timeliness requirements of clinical testing and the risk of non-specific amplification due to prolonged incubation, 30 min was determined to be the optimal reaction time range for the Phi29 polymerase-mediated rolling circle amplification stage. Subsequent experiments selected 30 min as the standard reaction time. This time point ensures sufficient ssDNA product to effectively activate the subsequent CRISPR / LbCas12a system while achieving a good balance between detection efficiency and signal intensity, ensuring stable amplification efficiency and detection repeatability.

[0037] (III) To determine the optimal working concentration of Bst DNA polymerase in the primer cycling generation step of step 3, an enzyme concentration gradient optimization experiment was set up. Bst DNA polymerase is a key enzyme driving the "extension-cutting-displacement" cycle, and its concentration directly affects the generation efficiency of short-chain DNA primers, thereby affecting the product yield of subsequent rolling circle amplification and the final detection signal. If the enzyme concentration is insufficient, the primer generation efficiency will be low, resulting in insufficient amplification and weak downstream signals; if the enzyme concentration is too high, it may aggravate non-specific primer extension, triggering non-specific amplification when there is no target or trace contamination of the template, generating background signals, and reducing the specificity and signal-to-noise ratio of the detection. Therefore, determining the optimal Bst DNA polymerase concentration is crucial for balancing signal intensity and background suppression. The experiment used HPV16 E7 cDNA as the target, and a target-free negative control group was set up. After performing the ligation reaction sequentially according to the standard procedure optimized in this invention, multiple identical reaction systems were established, and different concentrations of Bst DNA polymerase were added to each system. Concentration gradients were set at 0, 0.08 U / μL, 0.16 U / μL, 0.24 U / μL, and 0.32 U / μL. Equal volumes of Nb.BbvCI nicking endonuclease, dNTPs mixture, and corresponding buffer were added to each system simultaneously, and the mixture was incubated at 37°C for 60 min to generate primers. After the reaction, all enzymes were inactivated by heating at 80°C for 10 min. Subsequently, MSSRCA circular template and Phi29 DNA polymerase were added to each system, and rolling circle amplification was completed at 37°C for 60 min. Finally, the amplification products from each concentration group were added to a competitive CRISPR / LbCas12a detection system for cascade signal amplification and fluorescence detection. The endpoint fluorescence intensity was measured using a fluorescence spectrophotometer. Fluorescence intensity curves for the target group and the negative control group without target were plotted with Bst DNA polymerase concentration as the x-axis, and the signal-to-noise ratio (the ratio of the signal in the target group to the signal in the negative control group) was calculated at each concentration.

[0038] Experimental results are as follows Figure 4 (e) Figure 4 As shown in (f), with the increase of Bst DNA polymerase concentration, the fluorescence signal intensity of the target group showed a trend of first increasing and then decreasing. When the enzyme concentration was 0, no signal was generated; as the concentration increased to 0.08 U / μL and 0.16 U / μL, the signal intensity gradually increased; the signal reached its highest value at 0.24 U / μL; when the concentration was further increased to 0.32 U / μL, the signal intensity decreased instead. Considering the amplification efficiency, 0.24 U / μL was determined to be the optimal working concentration of Bst DNA polymerase, and this concentration was selected as the standard reaction concentration in subsequent experiments to ensure efficient primer cycling. This optimization result provides key parameter basis for establishing a highly sensitive and specific detection system in this invention.

[0039] (iv) To determine the optimal working concentration of LbCas12a protein in the CRISPR / LbCas12a detection system in step 5, an enzyme concentration gradient optimization experiment was set up. LbCas12a protein is the core enzyme for signal conversion and amplification, and its concentration directly affects the formation efficiency of the complete crRNA / LBCas12a complex and subsequent trans-cleavage activity, thereby affecting the final fluorescence signal intensity. If the enzyme concentration is insufficient, the complex formation will be incomplete, resulting in low target recognition and signal conversion efficiency; if the enzyme concentration is too high, it will not only increase the experimental cost, but may also affect the stability of the reaction system due to the non-specific adsorption or aggregation of excessive enzyme protein, and may even lead to a decrease in the signal-to-noise ratio. Therefore, determining the optimal LbCas12a protein concentration is crucial for balancing detection sensitivity and cost-effectiveness. The experiment used HPV16 E7 cDNA as the target, and after performing the ligation reaction, Bst DNA polymerase-mediated primer cycling generation, and Phi29 polymerase-mediated rolling circle amplification according to the optimized standard procedure of this invention, equal amounts of N-MSSRCA amplification products were obtained as a unified activator. Equal volumes of amplified products were added to CRISPR detection systems containing different concentrations of LbCas12a protein. The final concentrations of LbCas12a were set to 0, 25 nM, 50 nM, 75 nM, and 100 nM. Fixed concentrations of complete crRNA, split crRNA and its handle and spacer, DNA activator, and fluorescence-quenched dual-labeled reporter molecule were added to each system simultaneously, and the volumes were brought up to the same level with 1×NE Buffer. Each reaction system was incubated at 37°C for 30 min for cascade signal amplification. After the reaction, the system was heated at 85°C for 5 min to inactivate LbCas12a protein. The endpoint fluorescence intensity was measured using a fluorescence spectrophotometer with an excitation wavelength of 480 nm and an emission wavelength of 500-650 nm. A curve was plotted with LbCas12a protein concentration on the x-axis and endpoint fluorescence intensity on the y-axis.

[0040] Experimental results are as follows Figure 4 (g) Figure 4 As shown in (h), the fluorescence signal intensity gradually increased with the increase of LbCas12a protein concentration from 0, reaching its highest value at 75 nM; however, the signal intensity decreased when the concentration was further increased to 100 nM. The signal-to-noise ratio (SNR) at each concentration was calculated, and the SNR at 75 nM was optimal. Considering both signal intensity and enzyme usage cost, 75 nM was determined to be the optimal working concentration of LbCas12a protein, and this concentration was selected as the standard reaction concentration for subsequent experiments. This optimization result ensures the high efficiency and stability of the signal conversion step in the detection system of this invention, providing key parameter basis for establishing a highly sensitive HPV16 E7 mRNA detection method.

[0041] Experiment 3: To investigate the specificity of the N-MSSRCA-CRISPR cascade amplification system constructed in this experiment for the detection of HPV16 E7 mRNA.

[0042] Enterovirus 71, Zika virus, random sequences, miR-200b, and miRNA-21 were selected as non-target controls and were used in parallel with the perfectly matched HPV16 E7 cDNA target. All non-target sequences were set to the same concentration as the target (10 nM). Following the optimized experimental procedures of this invention, the following reactions were performed sequentially: ligation reaction, Bst DNA polymerase-mediated double-strand formation and primer cycling, Phi29 polymerase-mediated rolling circle amplification, and competitive CRISPR / LbCas12a cascade signal amplification and fluorescence detection. The endpoint fluorescence intensity of each reaction system was measured.

[0043] Experimental results are as follows Figure 5 As shown, Figure 5 (a) Fluorescence spectra of the N-MSSRCA-CRISPR cascade amplification system after incubation with different targets. Figure 5 (b) is a bar chart showing the maximum fluorescence intensity at 520 nm after incubation of the N-MSSRCA-CRISPR cascade amplification system with different targets. In the presence of a perfectly matched HPV16 E7 cDNA target, the detection system produced a significant fluorescence signal, with fluorescence intensity much higher than the negative control group. However, the fluorescence signals of non-target control groups, such as enterovirus 71, Zika virus, miRNA-21, miR-200b, and random sequences, showed no significant difference from the negative control group, indicating that these non-target sequences could not effectively initiate the ligation reaction and subsequent cascade amplification process.

[0044] Experiment 4: Sensitivity of N-MSSRCA-CRISPR cascade amplification system for detecting HPV16 E7 mRNA and establishment of quantitative standard curve.

[0045] A serial dilution experiment was performed using synthesized HPV16 E7 cDNA standards. Target concentrations included a negative control group (0 pM) and ten concentration gradients: 10 pM, 25 pM, 100 pM, 250 pM, 500 pM, 1 nM, 2.5 nM, 5 nM, and 10 nM. Each concentration was used in triplicate, and the experiment was repeated three times to ensure reliability. Following the optimized experimental conditions of this invention, different concentrations of HPV16 E7 cDNA standards were added to the ligation reaction system, and the following reactions were performed sequentially: ligation, Bst DNA polymerase-mediated double-strand formation and primer cycling, and Phi29 polymerase-mediated rolling circle amplification. The amplified products were then added to a competitive CRISPR / LbCas12a detection system for cascade signal amplification and fluorescence detection. The endpoint fluorescence intensity of each reaction system was measured using a fluorescence spectrophotometer. The excitation wavelength was set to 480 nm, and the emission wavelength to 500-650 nm. The experimental results are shown below. Figure 6 As shown. Figure 6 (a) Fluorescence spectra of the N-MSSRCA-CRISPR cascade amplification system after incubation with different concentrations of target HPV16 E7 cDNA. Figure 6 (b) shows the maximum fluorescence intensity at 520 nm after incubation of the N-MSSRCA-CRISPR cascade amplification system with different concentrations of target HPV16 E7 cDNA. Figure 6 (c) is a linear relationship diagram between the N-MSSRCA-CRISPR cascade amplification system and different concentrations of target HPV16E7 cDNA (25pM-500pM).

[0046] A scatter plot was drawn with the logarithm of HPV16 E7 cDNA concentration on the x-axis and the corresponding endpoint fluorescence intensity on the y-axis. Linear regression fitting yielded the equation y = 3559.8x - 2299, with a correlation coefficient R² = 0.9922, indicating good linearity within the 25 pM to 500 pM range. The detection limit was calculated by adding three times the standard deviation to the mean fluorescence signal of the negative control group, yielding a detection limit of 0.18 pM for HPV16 E7 cDNA. This demonstrates that the N-MSSRCA-CRISPR cascade amplification system constructed in this invention possesses good sensitivity and quantitative detection capability.

[0047] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. For those skilled in the art, after learning the contents described in the present invention, several equivalent changes and substitutions can be made without departing from the principle of the present invention. These equivalent changes and substitutions should also be considered to fall within the protection scope of the present invention.

Claims

1. A method for constructing an N-MSSRCA-CRISPR cascade amplification system, characterized in that, Includes the following steps: Step 1: Prepare 100µM stock solutions of synthesized Nick probe, Bind probe, MSSRCA circular template, short universal primer, complete crRNA, split crRNA handle, split crRNA spacer, DNA activator, fluorescent reporter molecule and HPV16 E7 cDNA target lyophilized powder using DEPC water, aliquot and store at -20℃ for later use. Step 2: Add Nick probe, Bind probe, HPV16 E7 cDNA target, and short universal primer Ds primer to 1×T4 DNA ligase buffer at a molar ratio of 1:1:1:1 and mix well. Heat the mixture at 95°C for 5 min, then cool to allow Nick and Bind probes to specifically hybridize with the target sequence, while the short universal primer binds complementary to the repeat sequence at the 3' end of the Bind probe. After annealing, add T4 DNA ligase directly to the system and incubate for 10 min to complete the target-dependent probe ligation reaction. Step 3: Add Bst DNA polymerase buffer, dNTPs mixture, MSSRCA circular template, Bst DNA polymerase and Nb.BbvCI nicking endonuclease to the above ligation reaction system, and incubate the reaction system at a constant temperature to produce short DNA fragments; Step 4: Add Phi29 DNA polymerase to the above reaction system and incubate at a constant temperature for 30 min; use the short DNA fragment generated in Step 3 as a primer to hybridize with the MSSRCA circular template. Driven by the continuous synthesis capacity and strand displacement activity of Phi29 DNA polymerase, rolling circle amplification is performed along the circular template to generate a long single-stranded DNA product containing repeating units; after the reaction is completed, heat at 80℃ to inactivate the enzyme and obtain the long ssDNA product. Step 5: Directly add intact crRNA, split crRNA handle and spacer, 1×NEBuffer, LbCas12a protein, activator, fluorescent-quenched double-labeled single-stranded DNA reporter molecule, and DEPC water to the above N-MSSRCA amplification system and mix well; incubate the reaction system at 37℃ for 30 min to form a CRISPR / LbCas12a system, further activating the trans-cleavage active reporter molecule to achieve signal activation; after the reaction is completed, heat at 85℃ for 5 min to inactivate LbCas12a protein, obtaining the N-MSSRCA-CRISPR cascade amplification system.

2. The construction method according to claim 1, characterized in that: In step 3, the constant temperature incubation is 37℃ and the incubation time is 60 minutes.

3. The construction method according to claim 1, characterized in that: In step 3, the working concentration of Bst DNA polymerase in the primer cycling generation step is 0.24 U / μL.

4. The construction method according to claim 1, characterized in that: In step 4, the constant temperature incubation is 37℃, and the incubation time is 30 minutes.

5. The construction method according to claim 1, characterized in that: In step 4, the reaction time for the rolling circle amplification stage is 60 minutes.

6. The construction method according to claim 1, characterized in that: In step 5, the working concentration of LbCas12a protein in the CRISPR / LbCas12a system is 75 nM.

7. The N-MSSRCA-CRISPR cascade amplification system obtained by the construction method according to any one of claims 1-6.

8. An application of the N-MSSRCA-CRISPR cascade amplification system as described in claim 7, characterized in that: The N-MSSRCA-CRISPR cascade amplification system specifically recognizes HPV16 E7 mRNA.

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