HPV rapid detection method based on optimized CRISPR-Cas13a platform

Abstract

The invention relates to the technical field of HPV (human papillomavirus) detection, and discloses an HPV rapid detection method based on an optimized CRISPR-Cas13a platform, which comprises the following steps: step S1, splitting and purifying a sample to obtain an HPV DNA (deoxyribonucleic acid) template; s2, performing amplification and transcription on the HPV target nucleic acid to obtain an amplification product; step S3, mixing an amplification product with a detection system of CRISPR-Cas13a protein, crRNA and reporter molecules, and then carrying out a cleavage reaction; and step S4, detecting the state change of the reporter molecule through a lateral flow chromatography test strip so as to judge whether the HPV exists in the sample or not. According to the invention, the recombinase polymerase is combined with CRISPR-Cas13a, so that the process from nucleic acid amplification to signal detection is completed at a single and mild constant temperature, the operation complexity and environmental requirements are remarkably reduced, and the HPV can be rapidly detected.

Description

Technical Field

[0001] This invention relates to the field of HPV detection technology, specifically to a rapid HPV detection method based on an optimized CRISPR-Cas13a platform. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors among women worldwide, with consistently high incidence and mortality rates. Modern medical research has confirmed that persistent infection with high-risk human papillomavirus (HPV) is the main cause of cervical cancer and its precancerous lesions. Among the many high-risk HPV types, HPV16 and HPV18 are particularly critical, directly associated with over 70% of cervical cancer cases. Therefore, early, rapid, and accurate screening and detection of high-risk HPV, especially HPV16 and HPV18, is of vital clinical significance for the prevention, timely intervention, and effective treatment of cervical cancer.

[0003] Currently, the main molecular diagnostic methods used clinically for HPV detection include polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and gene sequencing. While these methods offer high sensitivity and specificity, their applications also have significant limitations. First, these techniques are relatively complex, typically involving multiple steps such as nucleic acid extraction, thermal cycling amplification, and signal detection. The entire process is time-consuming, usually requiring several hours to obtain results. Secondly, the aforementioned traditional methods heavily rely on sophisticated and expensive laboratory equipment, such as PCR thermal cyclers and fluorescence detectors. This not only results in high equipment costs but also demands a high level of expertise from the operating environment and technical personnel. These limitations make it difficult to popularize and implement such testing methods in primary healthcare institutions, resource-scarce areas, or point-of-care testing (POCT) environments where immediate results are required, thus restricting their application in screening a wider population. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a rapid HPV detection method based on an optimized CRISPR-Cas13a platform, which solves the problems of cumbersome procedures, reliance on expensive equipment, long detection cycles, and difficulty in meeting the needs of on-site real-time detection in traditional molecular diagnostic technologies for HPV detection.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a rapid HPV detection method based on an optimized CRISPR-Cas13a platform, comprising: Step S1: Lyse and purify the sample to be tested to obtain a template containing HPV DNA; Step S2: Under isothermal conditions, the HPV target nucleic acid is amplified and transcribed in vitro to obtain the amplification product; Step S3: Mix the amplification product with a detection system containing Cas13a protein, crRNA and reporter molecule, and perform a cleavage reaction at the same temperature as described in Step S2. Step S4: Detect the state changes of the reporter molecules using a lateral flow chromatography test strip to determine whether HPV is present in the sample.

[0006] First, the target nucleic acid sequence was rapidly enriched at an isothermal temperature through in vitro amplification and transcription steps. Then, at the same temperature, the CRISPR-Cas13a system was used for recognition and signal transduction. When the target sequence was present in the amplification product, the Cas13a protein was specifically activated, triggering the cleavage of the reporter molecule in the system.

[0007] Preferably, the pyrolysis process in step S1 specifically includes: The test sample was treated with a lysis buffer containing ethylene glycol blue coprecipitant, and then purified using a polyethersulfone membrane. The sample to be tested was a cervical swab sample.

[0008] By employing a lysis buffer containing ethylene glycol blue coprecipitant, cell structure can be disrupted, facilitating the precipitation and enrichment of target nucleic acids from complex sample matrices. Subsequently, physical filtration purification using a polyethersulfone membrane replaces the cumbersome centrifugation or magnetic bead adsorption steps of traditional methods, simplifying the process to lysis-enrichment-filtration, significantly reducing sample pretreatment time and increasing the speed of the detection workflow.

[0009] Preferably, in step 2, the amplification is recombinase polymerase amplification.

[0010] By amplifying the recombinase polymerase, a high-temperature denaturation step is eliminated, enabling exponential amplification of the DNA template at a single, mild temperature. Furthermore, the Cas13a enzyme exhibits excellent activity at this temperature, thus eliminating the need for complex temperature control and enhancing the flexibility of this method.

[0011] Preferably, the constant temperature condition is 37°C.

[0012] Preferably, the target sequence of the HPV is located in the L1 gene of high-risk HPV.

[0013] Preferably, the high-risk HPV type includes HPV16 or HPV18, or a combination of HPV16 and HPV18.

[0014] The L1 gene is a conserved sequence encoding the main capsid protein of the virus. Using it as a target can ensure the specificity of the detection and effectively distinguish HPV from other microorganisms. This invention focuses on HPV types 16 and 18. By utilizing crRNAs that specifically target HPV types 16 and 18, this detection method can accurately identify the virus types that pose the greatest clinical threat.

[0015] Preferably, in step S3, the Cas13a protein is the LwCas13a protein.

[0016] By utilizing the bypass cleavage activity of the orthogonal enzyme of CRISPR-Cas13a, it can be activated by the target sequence, efficiently and massively cleaving free reporter molecules in the system, thereby generating a strong intensity signal in a short time, thus improving detection sensitivity and shortening reaction time.

[0017] Preferably, in step S2, the amplification time is 15-25 minutes, and in step S3, the cleavage reaction time is 10-20 minutes.

[0018] Preferably, the lateral flow chromatography test strip includes a detection line for indicating a positive result and a control line for indicating the validity of the test strip.

[0019] Preferably, the reporter molecule is a FAM-labeled nucleic acid probe; the test strip surface is provided with a reaction area and a detection line, the reaction area is coated with anti-FAM particles, and the detection line is immobilized with FAM molecules; when the sample is positive, the reporter molecule is cleaved, and the anti-FAM particles bind to the FAM molecules on the detection line to produce color.

[0020] The test uses a lateral flow chromatography strip, with the reporter molecule being a FAM-labeled nucleic acid probe. The reaction zone of the strip is pre-coated with movable anti-FAM particles, and the detection line immobilizes the FAM molecules. When the sample is negative, the Cas13a protein is not activated, the reporter molecule remains intact, and the flowing anti-FAM particles cannot remain on the detection line. When the sample is positive, the activated Cas13a protein cleaves a large number of FAM-labeled reporter probes, releasing the anti-FAM particles that were originally bound to the reporter probes. These released anti-FAM particles then flow with the liquid and are captured by the FAM molecules upon reaching the detection line, thus aggregating and developing color, forming a visible band.

[0021] This invention provides a rapid HPV detection method based on an optimized CRISPR-Cas13a platform. It has the following beneficial effects: 1. This invention achieves the process from nucleic acid amplification to signal detection by combining recombinase polymerase and CRISPR-Cas13a at a single, mild, and constant temperature. Therefore, it can significantly reduce the complexity of operation and environmental requirements by eliminating the reliance on precision and complex thermal cycling equipment such as traditional PCR instruments, thus enabling rapid detection of HPV.

[0022] 2. This invention cleaves CRISPR-Cas13a protein, crRNA, and reporter molecules, and then converts them into visible band signals using a lateral flow chromatography test strip. This eliminates the need for expensive fluorescence reading equipment to interpret the test results, allowing for easy observation to determine whether the result is negative or positive, greatly improving the convenience and universality of the test. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the overall technical route of the present invention; Figure 2 This is a schematic diagram of the lateral effluent results reading of the present invention. In the figure, the blue band is the streptavidin-biotin binding band, which is the control. The red band is the FAM binding band, which indicates that HPV16 and / or HPV18 are positive. Only the blue band indicates that it is negative. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] To better understand the present invention, the above content will be described in detail below with reference to specific embodiments.

[0026] Please see the attached Figure 1 and attached Figure 2 This invention provides a rapid HPV detection method based on an optimized CRISPR-Cas13a platform, specifically including the following steps: Step S1: Lyse and purify the sample to be tested to obtain a template containing HPV DNA; In this embodiment, a cervical swab sample is first taken and placed into a centrifuge tube containing optimized lysis buffer. The lysis buffer consists of ethylene glycol blue co-precipitant, which efficiently disrupts the cell and nuclear membranes, releasing HPV DNA, and simultaneously precipitates the target nucleic acid. The swab and lysis buffer are thoroughly mixed and vortexed, and the lysis process is carried out at room temperature for 5 minutes. Then, the lysed mixture is transferred to a purification device equipped with a polyethersulfone (PES) membrane. Under centrifugal force or positive pressure, the lysis buffer and cell debris pass through the filter membrane, while the HPV DNA is retained on the membrane surface. The PES membrane is then rinsed with ethanol solutions of different concentrations to remove residual impurities and inhibitors. Finally, the purified HPV DNA template is eluted from the membrane with elution buffer to obtain a pure template solution that can be directly used for subsequent amplification.

[0027] Step S2: Under isothermal conditions, the HPV target nucleic acid is amplified and transcribed in vitro to obtain the amplification product; In this step, the HPV DNA template obtained in step S1 is added to a pre-configured recombinase polymerase amplification (RPA) reaction system. The RPA reaction system includes commercially available RPA reaction beads, forward and reverse primers specifically designed for the L1 gene of high-risk HPV16 and HPV18, reaction buffer, and T7 RNA polymerase. All components are then thoroughly mixed, and the reaction tube is placed in a 37°C incubator for amplification. Under the synergistic action of the recombinase, single-strand binding protein, and strand displacement polymerase, the target sequence is amplified. Simultaneously, because the primers contain the T7 promoter sequence, the amplified DNA product is transcribed into RNA under the action of T7 RNA polymerase. The entire amplification and transcription process is carried out at 37°C for 20 minutes, finally yielding an amplified product containing the target RNA sequence.

[0028] Step S3: Mix the amplification product with a detection system containing CRISPR-Cas13a protein, crRNA and reporter molecule, and then perform a cleavage reaction at the same temperature as described in step S2. In this step, the amplification product obtained in step S2 is directly added to the prepared CRISPR-Cas13a detection system. This detection system contains the core LwCas13a protein, pre-designed crRNAs (guide RNAs) targeting HPV16 and HPV18 amplification products respectively, and FAM (carboxyfluorescein)-labeled single-stranded nucleic acid probes as signal reporter molecules. After mixing the amplification product with the detection system, the reaction tube is placed in a 37°C incubator for 15 minutes for the cleavage reaction. If the amplification product contains the target HPV RNA sequence, the crRNA will recognize this sequence and guide the LwCas13a protein to bind to it, thereby activating the bypass cleavage activity of the LwCas13a protein. Finally, the activated LwCas13a protein will non-specifically and extensively cleave the free FAM-labeled nucleic acid probes in the system.

[0029] Step S4: Detect the state changes of the reporter molecules using a lateral flow chromatography test strip to determine whether HPV is present in the sample.

[0030] In this step, the liquid from step S3, after the reaction is complete, is added dropwise to the sample pad of the lateral flow chromatography test strip. The reaction area of ​​the test strip is pre-coated with movable anti-FAM antibody particles, and FAM molecules are immobilized on the detection line (T line). A control line (C line) is also provided to verify the effectiveness of the test strip. When the liquid sample flows through the test strip under capillary action: if the sample is positive (i.e., the FAM-labeled reporter probe is largely cleaved), the free anti-FAM antibody particles will flow with the liquid and be captured by FAM molecules upon reaching the detection line, thus aggregating to form a visible red band. If the sample is negative (the reporter probe remains intact), the anti-FAM antibody particles will bind to the intact reporter probe to form a large complex, which cannot be captured by the FAM molecules on the detection line; in this case, the T line will not show color. Therefore, regardless of whether the sample is positive or negative, the control line should show color; and by visually observing whether a red band appears on the detection line, the presence of HPV infection in the sample can be determined.

[0031] In another specific embodiment; 1. Optimize the SHERLOCK platform and establish an HPV detection method. The DNA extraction method, simultaneous detection of HPV16 and HPV18, and result reading method in the SHERLOCK platform were optimized to achieve rapid, sensitive, and convenient detection.

[0032] Specifically, it includes: 1.1 HPV Degradation A cell lysis method was used to replace the previous multi-step extraction. Cervical swabs were mixed with lysis buffer containing ethylene glycol blue co-precipitant and placed on a polyethersulfone (PES) membrane to extract HPV DNA. The DNA was purified by washing with ethanol of different concentrations. A lysis time of 5 minutes was selected to minimize the overall analysis time. The concentration was measured at 260 nm using a spectrophotometer and compared with the method of a DNA extraction kit.

[0033] 1.2 RPA Methods The extracted DNA was amplified using the RPA method. The RPA reaction used commercially available RPA beads, primers, and buffer solution, which were mixed with the extracted HPV DNA and incubated at 37°C for 20 minutes.

[0034] 1.3. CRISP / Cas13a Detection A CRISP / Cas13a method for detecting HPV was established. DNA amplified by RPA was mixed with 1 µL of each of the two crRNAs, 1 µL of LwCas13a (Cas13a derived from ciliates), 0.2–2 µL of T7 RNA polymerase mixture, 1–10 µL of NTP mix, and 1 µL of fluorescently labeled ssDNA. The mixture was then incubated at 37 °C for 15 minutes, and the results were read.

[0035] 1.4. Preparation and Result Interpretation of Colorimetric Test Strips Color development paper strips, such as Figure 2 As shown, the reaction zone is coated with anti-FAM particles, the control band is coated with streptavidin A, and the positive band represents FAM. When Cas13a / crRNA binds to the target sequence, the ssDNA labeled with FAM in the non-target sequence is cleaved, and the anti-FAM in the reaction zone is released. It can then bind to the FAM in the positive band, resulting in a red band. This can detect one or both of HPV16 and HPV18 co-infection.

[0036] 2. Evaluation of the CRISP / Cas13a method for HPV detection Mainly includes: 2.1 Assessing Sensitivity Sensitivity was evaluated using the limit of detection (LOD), with HPV16 concentrations ranging from 1×10⁻⁶. 4 Samples of copies / µL were serially diluted 1:10 to 1 copy / µL for the above detection. Each reaction was repeated twice, and results were read using both fluorescence signal and lateral efflux readings to determine the limit of detection (LOD).

[0037] 2.2 Specificity evaluation This method was used to detect 11 other HPV genotypes (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and common reproductive tract pathogens, using plasmids containing conserved regions of the aforementioned fungi, viruses, and bacteria at a concentration of 1×10⁻⁶. 4 The assay is performed using copies / µL. If the result shows no fluorescence signal or the lateral effluent result is negative, it confirms that this method has good specificity.

[0038] 2.3 Linear range and negative predictive value, positive predictive value The known quantity is 1×10 7 HPV16 virus samples were serially diluted to 1 g copy / µL and mixed into healthy samples for the above-mentioned detection. The fluorescence intensity was read using a fluorescence reading device. A standard curve was constructed with 1 g copy / µL as the x-axis and the fluorescence intensity of the detection signal as the y-axis. The regression equation (1×10⁷ copies / µL) and the correlation coefficient (r value) were calculated to determine the linear range of the detection.

[0039] For testing clinical specimens, the negative predictive value is calculated using the following formulas: Positive predictive value = True positive / (True positive + False positive), which is the ratio of correctly predicted positive specimens to all positive specimens; Negative predictive value = True negative / (True negative + False negative), which is the ratio of correctly predicted negative samples to all negative samples.

[0040] 2.4. RT-qPCR method Clinical specimens were tested according to the established method, and simultaneous detection was performed using an RT-qPCR kit (Thermo-Fisher). Follow the instructions. Compare the results of the two methods to evaluate the concordance rate between the established method and RT-qPCR detection results, as well as differences in detection limits, linear range, and specificity compared to RT-qPCR.

[0041] 3. Statistical Analysis Statistical data were analyzed using STATA-13.1. All graphs were plotted using GraphPadPrism 6.0 software. Statistical differences between the two groups were assessed using t-tests. Pearson correlation analysis was used to assess the correlation between variables; a p-value < 0.05 was considered statistically significant.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A rapid HPV detection method based on an optimized CRISPR-Cas13a platform, characterized in that, The following steps are involved: Step S1: Lyse and purify the sample to be tested to obtain a template containing HPV DNA; Step S2: Under isothermal conditions, the HPV target nucleic acid is amplified and transcribed in vitro to obtain the amplification product; Step S3: Mix the amplification product with a detection system containing CRISPR-Cas13a protein, crRNA and reporter molecule, and then perform a cleavage reaction at the same temperature as described in step S2. Step S4: Detect the state changes of the reporter molecules using a lateral flow chromatography test strip to determine whether HPV is present in the sample.

2. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, The pyrolysis process in step S1 specifically includes: The test sample was treated with a lysis buffer containing ethylene glycol blue coprecipitant, and then purified using a polyethersulfone membrane. The sample to be tested was a cervical swab sample.

3. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, In step 2, the amplification is recombinase polymerase amplification.

4. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, The constant temperature condition is 37°C.

5. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, The target sequence of the HPV is located in the L1 gene of high-risk HPV.

6. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 5, characterized in that, The high-risk HPV types include HPV16 or HPV18, or a combination of HPV16 and HPV18.

7. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, In step S3, the CRISPR-Cas13a protein is the LwCas13a protein.

8. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, In step S2, the amplification time is 15-25 minutes, and in step S3, the cleavage reaction time is 10-20 minutes.

9. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, The lateral flow chromatography test strip includes a detection line for indicating a positive result and a control line for indicating the validity of the test strip.

10. The rapid HPV detection method based on the optimized CRISPR-Cas13a platform according to claim 1, characterized in that, The reporter molecule is a FAM-labeled nucleic acid probe; the test strip surface is provided with a reaction area and a detection line, the reaction area is coated with anti-FAM particles, and the detection line is immobilized with FAM molecules; when the sample is positive, the reporter molecule is cleaved, and the anti-FAM particles bind to the FAM molecules on the detection line to produce color.

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