Urine high-risk papillomavirus rapid self-test kit

Abstract

The application discloses a urine papillomavirus rapid self-checking kit. The application provides a self-made composite magnetic bead, the magnetic bead has high DNA affinity, can rapidly enrich free DNA (cfDNA) in urine, and can detect 6 copies of HPV virus in each reaction by combining a CRISPR technology. The kit provided by the application is roughly divided into the following operation steps: collecting urine, mixing the sample, magnetic bead adsorption, resuspension and amplification of the sample, and the results show five steps. The whole operation is only needed to be carried out in an environment of about 37 DEG C, and the detection of each sample only needs less than 30 min, without the need of expensive equipment and professional personnel, and thus the home self-checking is truly realized.

Description

Technical Field

[0001] This invention relates to the field of point-of-care testing, and in particular to a rapid self-test kit for high-risk human papillomavirus in urine. Background Technology

[0002] Cervical cancer is a common malignant tumor that seriously threatens women's health and has become a major global public health issue. In 2020, the number of new cervical cancer cases in my country reached 109,000, and the number of deaths reached 59,000, accounting for 18.2% and 17.3% of the global incidence and mortality cases, respectively. The growth rate in rural areas is significantly higher than that in urban areas, and the age of onset is showing a trend towards younger ages.

[0003] In secondary prevention of cervical cancer, cervical cancer screening and triage of those with abnormal screening results are crucial management aspects. High-risk human papillomavirus (HPV) is the most significant causative factor of cervical cancer. There are 14 subtypes of high-risk HPV: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Compared to cytological examination, nucleic acid testing for high-risk HPV can detect more HSIL and higher-level lesions, while also having a higher negative predictive value and a lower false negative rate. In July 2023, experts from seven academic societies and associations, including the Colposcopy and Cervical Pathology Branch of the Chinese Association for Eugenics Science and the Gynecologic Oncology Branch of the Chinese Medical Association, jointly formulated the "Guidelines for Cervical Cancer Screening in China." The guidelines clearly state that high-risk HPV nucleic acid testing is the preferred screening method for cervical cancer.

[0004] my country has a large population and significant public health needs, but its medical resources are unevenly distributed. Cervical cancer incidence and mortality rates are particularly high in rural and resource-scarce areas, where access to HPV nucleic acid testing is limited. Currently, HPV screening in my country uses quantitative real-time PCR, a method that demands high levels of operator skill and equipment precision, and can only be performed in specialized institutions and platforms. Therefore, a simple, convenient, cost-effective, and accurate high-risk HPV self-testing product would not only effectively improve my country's cervical cancer screening level but also have a huge market potential. Summary of the Invention

[0005] The purpose of this invention is to provide a composite magnetic bead for real-time detection.

[0006] Another object of the present invention is to provide a method for preparing composite magnetic beads for real-time detection.

[0007] Another object of the present invention is to provide a kit for detecting high-risk human papillomavirus in urine.

[0008] Another object of the present invention is to provide a method for detecting high-risk human papillomavirus in urine.

[0009] To address the aforementioned technical problems, a first aspect of the present invention provides a composite magnetic bead for real-time detection, the composite magnetic bead comprising:

[0010] Magnetic Fe3O4 particles; a cationic polymer coating layer covering the magnetic Fe3O4 particles; and a nano-metal-organic framework UiO-66 covering the cationic polymer coating layer; wherein the cationic polymer coating layer may optionally be modified with organic functional groups.

[0011] In some embodiments, the cationic polymer is a DNA-high affinity cationic polymer.

[0012] In some embodiments, the DNA high-affinity cationic polymer is selected from at least one of polyethyleneimine (PEI), polyurethane (PBAE), chitosan, polyacrylamide (PAH), diethylaminoethyl dextran (DEAE-dextran), and polyurethane dendritic polymer (PAMAM), such as polyethyleneimine.

[0013] In some embodiments, the modification is any one or more combinations of carboxylation, hydroxylation, imidazoleization, and amination.

[0014] In some embodiments, the magnetic Fe3O4 particles are meso-2,3-dimercaptosuccinic acid nano-Fe3O4 particles.

[0015] In some embodiments, the diameter of the composite magnetic beads is in the range of 200-300 nm.

[0016] In another aspect, the present invention provides a method for preparing the above-mentioned composite magnetic beads, the method comprising the steps of:

[0017] S1: Coat the magnetic Fe3O4 particles with cationic polymer to obtain magnetic Fe3O4 particles coated with cationic polymer.

[0018] S2: Under heating conditions, the magnetic Fe3O4 particles coated with cationic polymer are treated with a solution containing ZrCl4 and 2-aminoterephthalic acid, and the nano-metal-organic framework UiO-66 is mounted on the magnetic Fe3O4 particles coated with cationic polymer to obtain the composite magnetic beads.

[0019] In some embodiments, step S1, after coating the magnetic Fe3O4 particles with the cationic polymer, further includes a step of modifying the cationic polymer-coated magnetic Fe3O4 particles. Preferably, the modification is a thiolization modification.

[0020] In some embodiments, in S1, the magnetic Fe3O4 particles are modified using a hot solvent method.

[0021] In some embodiments, in S2, the nano-metal-organic framework UiO-66 is mounted on the magnetic Fe3O4 particles coated with the cationic polymer using a hot solvent method. Preferably, the temperature of the hot solvent method is 100-140°C, for example, 120°C.

[0022] In some embodiments, in S2, the solution containing ZrCl4 and 2-aminoterephthalic acid is N,N-dimethylformamide containing ZrCl4 and 2-aminoterephthalic acid.

[0023] In some embodiments, in S2, ZrCl4 and 2-aminoterephthalic acid in a mass ratio of 1.3:1 are uniformly dispersed in N,N-dimethylformamide to obtain the solution containing ZrCl4 and 2-aminoterephthalic acid.

[0024] In another aspect, the present invention provides a kit for detecting high-risk human papillomavirus in urine, the kit comprising:

[0025] A first container containing a composite magnetic bead suspension;

[0026] The second container contains the sample resuspension solution;

[0027] The third container contains amplification reagents, which contain one or more primer sets, amplification reaction enzyme systems, positive controls, and negative controls for amplifying high-risk HPV viruses.

[0028] The fourth container contains detection reagents, including crRNA of one or more high-risk HPV viruses, a reporter probe, and CRISPR / Cas12a.

[0029] In some embodiments, the concentration of the composite magnetic beads in the composite magnetic bead suspension is 0.5 mg / ml to 15 mg / ml, for example, 10 mg / ml.

[0030] In some embodiments, the composite magnetic bead suspension comprises composite magnetic beads, sodium chloride, and water.

[0031] In some embodiments, the concentration of sodium chloride in the composite magnetic bead suspension is 0.2 mM to 2 mM, for example, 2 mM.

[0032] In some embodiments, the sample resuspension includes deionized water, Chelex-100, sodium citrate, lithium chloride, Tris-HCl, KCl, MgCl2, sodium sulfate, dNTPs, and rNTPs.

[0033] In some embodiments, the concentrations of the components of the sample resuspension are as follows: Chelex-100 mass-volume fraction of 2%-20%, sodium citrate concentration of 0.1mM-2mM, lithium chloride concentration of 1mM-10mM, Tris-HCl concentration of 2-200mM, MgCl2 concentration of 1-20mM, sodium sulfate concentration of 2-20mM, dNTP concentration of 1-10mM, and rNTP concentration of 1-10mM.

[0034] In some embodiments, the concentration of the primers is 1-10 μM.

[0035] In some implementations, the concentration of crRNA is 1-10 μM.

[0036] In some embodiments, the high-risk HPV virus is selected from at least one of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, preferably two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen.

[0037] In some implementations, the high-risk HPV virus is HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, or 68.

[0038] In some embodiments, the primer set for amplifying high-risk HPV viruses includes: a primer set for amplifying HPV16; a primer set for amplifying HPV18; a primer set for amplifying HPV31; a primer set for amplifying HPV33; a primer set for amplifying HPV35; a primer set for amplifying HPV39; a primer set for amplifying HPV45; a primer set for amplifying HPV51; a primer set for amplifying HPV52; a primer set for amplifying HPV56; a primer set for amplifying HPV58; a primer set for amplifying HPV59; a primer set for amplifying HPV66; and / or a primer set for amplifying HPV68.

[0039] In some embodiments, the primer set for amplifying HPV16 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.1 and the reverse primer is SEQ ID NO.2.

[0040] In some embodiments, the primer set for amplifying HPV18 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.1 and the reverse primer is SEQ ID NO.2.

[0041] In some embodiments, the primer set for amplifying HPV31 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.3 and the reverse primer is SEQ ID NO.4.

[0042] In some embodiments, the primer set for amplifying HPV33 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.3 and the reverse primer is SEQ ID NO.4.

[0043] In some embodiments, the primer set for amplifying HPV35 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.5 and the reverse primer is SEQ ID NO.6.

[0044] In some embodiments, the primer set for amplifying HPV39 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.5 and the reverse primer is SEQ ID NO.6.

[0045] In some embodiments, the primer set for amplifying HPV45 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.7 and the reverse primer is SEQ ID NO.8.

[0046] In some embodiments, the primer set for amplifying HPV51 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.9 and the reverse primer is SEQ ID NO.10.

[0047] In some embodiments, the primer set for amplifying HPV52 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.11 and the reverse primer is SEQ ID NO.12.

[0048] In some embodiments, the primer set for amplifying HPV56 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.11 and the reverse primer is SEQ ID NO.12.

[0049] In some embodiments, the primer set for amplifying HPV58 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.13 and the reverse primer is SEQ ID NO.14.

[0050] In some embodiments, the primer set for amplifying HPV59 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.13 and the reverse primer is SEQ ID NO.14.

[0051] In some embodiments, the primer set for amplifying HPV66 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.15 and the reverse primer is SEQ ID NO.16.

[0052] In some embodiments, the primer set for amplifying HPV68 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.17 and the reverse primer is SEQ ID NO.18.

[0053] In some embodiments, the crRNA of the high-risk HPV virus is selected from at least one of the following: HPV16 crRNA, HPV18 crRNA, HPV31 crRNA, HPV33 crRNA, HPV35 crRNA, HPV39 crRNA, HPV45 crRNA, HPV51 crRNA, HPV52 crRNA, HPV56 crRNA, HPV58 crRNA, HPV59 crRNA, HPV66 crRNA, and HPV68 crRNA.

[0054] In some embodiments, the HPV16 crRNA is selected from any one of SEQ ID NO.19-21.

[0055] In some embodiments, the HPV18 crRNA is selected from any one of SEQ ID NO.22-24.

[0056] In some embodiments, the HPV31 crRNA is selected from any one of SEQ ID NO.25-27.

[0057] In some embodiments, the HPV33 crRNA is selected from any one of SEQ ID NO.28-30.

[0058] In some embodiments, the HPV35 crRNA is selected from any one of SEQ ID NO. 31-33.

[0059] In some embodiments, the HPV39 crRNA is selected from any one of SEQ ID NO.34-36.

[0060] In some embodiments, the HPV45 crRNA is selected from any one of SEQ ID NO.37-39.

[0061] In some embodiments, the HPV51 crRNA is selected from any one of SEQ ID NO.40-42.

[0062] In some embodiments, the HPV52 crRNA is selected from any one of SEQ ID NO.43-45.

[0063] In some embodiments, the HPV56 crRNA is selected from any one of SEQ ID NO.46-48.

[0064] In some embodiments, the HPV58 crRNA is selected from any one of SEQ ID NO.49-51.

[0065] In some embodiments, the HPV59 crRNA is selected from any one of SEQ ID NO.52-54.

[0066] In some embodiments, the HPV66 crRNA is selected from any one of SEQ ID NO. 55-57.

[0067] In some embodiments, the HPV68 crRNA is selected from any one of SEQ ID NO.58-60.

[0068] In some embodiments, the reporting probe contains a biotin group and a FAM group.

[0069] In some implementations, the reporting probe sequence is as follows: Sequence: 56-FAM / TTATT / 3IABKFQ.

[0070] In some embodiments, the kit further includes a colloidal gold test strip containing a sample pad, a conjugate pad, and a detection pad. The detection pad has a T line and a C line drawn on it. The T line is coated with an anti-FAM antibody, and the C line is coated with an anti-Biotin group antibody.

[0071] In some embodiments, the amplification reaction enzyme system includes DNA recombinase, single-stranded conjugation enzyme, and Bst polymerase, wherein the mass ratio of the DNA recombinase, single-stranded conjugation enzyme, and Bst polymerase is one or any of the following ratios: 1:1:1, 1:2:3, or 3:2:1.

[0072] Compared with the prior art, the present invention has at least the following advantages:

[0073] (1) The present invention is derived from urine, and compared with conventional test kits, it truly achieves non-invasive testing.

[0074] (2) This invention provides a self-made composite magnetic bead with high DNA affinity, which can rapidly enrich cell-free DNA (cfDNA) in urine. Combined with CRISPR technology, this kit can detect at least 6 copies of HPV virus per reaction.

[0075] (3) The kit provided by this invention consists of five steps: urine collection, sample mixing, magnetic bead adsorption, resuspending, and sample amplification. The entire process can be carried out in an environment of about 37 degrees Celsius, and the test of each sample takes less than 30 minutes. It does not require expensive equipment or professional personnel, and can truly achieve home self-testing.

[0076] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0077] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0078] Figure 1 This is a schematic diagram of polyvinylamide-encapsulated Fe3O4-DMSA (PEI@Fe3O4-DMSA) mounted on a nano-metal-organic framework UiO-66 in specific embodiment 1 of the present invention;

[0079] Figure 2 is The zeta potential of PEI@Fe3O4-DMSA@UiO-66 in the dispersed phase in Specific Embodiment 1 of the present invention;

[0080] Figure 3 is A schematic diagram of the HPV detection principle in specific embodiment 2 of the present invention.

[0081] Figure 4 These are the detection results of HPV16 positive results, lowest detection value, and negative control samples in this invention.

[0082] Figure 5 These are the results of HPV31 positive results, lowest detection value, and negative control samples in this invention.

[0083] Figure 6 These are the results of the HPV45 positive result, the lowest detection value, and the negative control sample in this invention.

[0084] Figure 7 These are the results of HPV51 positive results, the lowest detection value, and the negative control sample in this invention.

[0085] Figure 8 These are the results of HPV52 positive results, the lowest detection value, and the negative control sample in this invention.

[0086] Figure 9 These are the results of the HPV56 positive result, the lowest detection value, and the negative control sample in this invention.

[0087] Figure 10 These are the results of the HPV58 positive result, the lowest detection value, and the negative control sample in this invention.

[0088] Figure 11 These are the results of HPV59 positive results, the lowest detection value, and the negative control sample in this invention.

[0089] Figure 12 These are the results of the HPV66 positive result, the lowest detection value, and the negative control sample in this invention. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0091] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0092] Unless otherwise specified, the term “or” means the term “and / or” and is used interchangeably with the term “and / or”.

[0093] As used herein, including the appended claims, unless the context clearly indicates otherwise, the singular forms of words such as “an,” “a,” and “the” include their respective plural referents.

[0094] Example 1: Preparation and characterization of PEI@Fe3O4-DMSA@UiO-66

[0095] (1) Preparation of PEI@Fe3O4-DMSA@UiO-66

[0096] See Figure 14.7 g FeCl3·6H2O and 15.86 g NaOA (sodium oleate) were dissolved in 200 mL of a mixed solvent (ultrapure water: anhydrous ethanol: n-hexane = 2:3:5), stirred at 80 °C for 4-6 hours, and the upper organic phase was collected. The mixture was then rotary evaporated at 33 °C and dried under vacuum at room temperature to obtain ferric oleate (Fe-OA). 3.6 g Fe-OA and 0.64 mL of oleic acid were slowly heated to 200 °C for half an hour in a N2 atmosphere, and then the temperature was increased to 300 °C at a rate of 4-6 °C / min and held at that temperature for 1 hour. After cooling to room temperature, the product was precipitated with 150 mL of anhydrous ethanol. The mixture was repeatedly washed, the precipitate was collected, and dried under vacuum at 60 °C overnight to obtain Fe3O4-OA. 100 mg Fe3O4-OA was dispersed in 20 mL of chloroform, 100 μL of triethylamine, and 50 mg DMSA in DMSO. The mixture was stirred at 60 °C for 12 h, centrifuged to collect the precipitate, and washed with anhydrous ethanol. The above product was dispersed in 100 mL of anhydrous ethanol and 50 μL of triethylamine, and 10 mL of DMSO containing 50 mg DMSA was added. The mixture was stirred at 60 °C overnight, centrifuged to collect the precipitate, and washed to obtain Fe3O4-DMSA. 1 mL of Fe3O4-DMSA (20 mg / mL) was added to 25 mg EDC·HCl, 57 mg sulfo-NHS, 1 g PEI-10000, and 2 mL of pure water, respectively. The mixture was stirred evenly for 48 h, centrifuged to collect the precipitate, and washed with ultrapure water to obtain PEI@Fe3O4-DMSA. The Fe3O4 nanoparticles were then functionalized with mercaptoacetic acid (MAA).

[0097] The above product was weighed and added to a mixture containing 10 mL of dimethylformamide (DMF), 0.12 g of ZrCl4, and 0.09 g of 2-aminoterephthalic acid. After uniform dispersion, the mixture was stirred at 120 °C for 24 h. After cooling to room temperature, PEI@Fe3O4-DMSA@UiO-66 was obtained under a magnetic field. The mixture was then dried at 60 °C for later use.

[0098] (2) Characterization of PEI@Fe3O4-DMSA@UiO-66

[0099] (a) Particle size detection

[0100] Tests showed that the diameter of the composite magnetic beads ranged from 200 to 300 nm.

[0101] (b) zeta potential detection

[0102] Magnetic nanoparticles and plasmid DNA were mixed at a 1:1 mass ratio and electrophoresed separately on 1% agarose gels at a constant voltage of 120V for 30 min. The DNA bands on the gel electrophoresis imager were then observed under UV light. The loading capacity was calculated by dividing the mass of the plasmid DNA by the sum of the masses of the plasmid DNA and the gene vector, thus assessing the gene vector's loading capacity. A schematic diagram of the measured zeta potentials can be found in [reference needed]. Figure 2.

[0103] (c) Magnetic test:

[0104] The physical property measurement system used was model PPMS-9 (Quantum Design, USA). Variable-temperature magnetic susceptibility curve testing conditions: Zero-Field-Cooled (ZFC) temperature 10K, Field-Cooled (FC) temperature 300K, external magnetic field strength 100Oe (1Oe = 79.6 A / m). Hysteresis loop testing conditions: External magnetic field strength –30000~30000Oe, constant temperature 290K. The test results show that PEI@Fe3O4-DMSA@UiO-66 retains more than 80% of its magnetism.

[0105] Example 2: Preparation of composite magnetic bead suspension

[0106] A composite magnetic bead suspension was obtained by dissolving the composite magnetic beads in an aqueous sodium chloride solution, wherein the sodium chloride concentration was 2 mM and the composite magnetic bead concentration was 10 mg / ml.

[0107] Example 3: Preparation of Sample Resuspension

[0108] Prepare the sample resuspension according to the composition shown in the table below:

[0109] content Chelex-100 quality 5％ Sodium citrate 2mM Lithium chloride 5mM Tris-HCl 1.5mM <![CDATA[MgCl2]]> 10mM Sodium sulfate 10mM dNTP 10mM rNTP 10mM <![CDATA[ddH2O]]> - PH 7.8

[0110] Example 4: Sample Amplification and Detection

[0111] (1) Kit components

[0112] The sample amplification was performed isothermally using an isothermal amplification kit. The isothermal amplification kit includes isothermal amplification reagents and detection reagents.

[0113] The isothermal amplification reagent composition is as follows:

[0114] a) Primer set: Contains forward and reverse primers for HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, as shown in Table 1. The primer concentration is 10 μM.

[0115] b) Positive control: The positive control is a plasmid solution containing a DNA fragment specific to the high-risk HPVL1 gene region, with a plasmid concentration of 10. 6 Copy / mL.

[0116] c) Negative control: Deionized water.

[0117] d) Amplification reaction enzyme system: DNA recombinase (UvsX), single-strand binding protein (Gp32), and DNA polymerase (Bst polymerase) are mixed in a 1:1:1 ratio.

[0118] The test reagent consists of the following components:

[0119] a) crRNA: 14 crRNAs of high-risk HPV, with nucleic acid sequences SEQ ID NO.19-NO.52, as shown in Table 2. The concentration of crRNA was 1-10 μM.

[0120] b) Reporter probe: The reporter probe is a single-stranded DNA probe, characterized by a biotin group labeled at the 5' end and a FAM group labeled at the 3' end. Probe sequence: 56-FAM / TTATT / 3IABKFQ.

[0121] c) Enzyme detection: CRISPR / Cas12a kit.

[0122] (2) Sample amplification and detection

[0123] Gently shake the collection tube to dislodge the magnetic bead suspension to the bottom. Open the cap and add approximately 4 ml of urine to the tube. Invert the tube several times to mix thoroughly and let it stand at room temperature for 3 minutes. Place the collection tube on a magnetic rack and allow it to adhere for 2 minutes. Then, empty the liquid from the tube completely, ensuring it remains on the magnetic rack throughout the process to prevent loss of the magnetic beads. Carefully add 500 μL of the sample resuspension (solution A) to the collection tube. Gently tap the bottom of the tube with your finger to resuspend the magnetic beads. After standing for 3 minutes, add the premixed amplification solution (Solution B) containing enzyme, primers, crRNA, and probe to the collection tube. Invert the tube 5-6 times to mix thoroughly, and start amplification at 37°C for 30 minutes. (Enzymes include UvsX (5U), Gp32 (5U), DNA polymerase (5U), and Cas12a (5U). Primers are detailed in the primer list; their working solution concentration is 10 μM. crRNA is detailed in the list; its working solution concentration is 10 μM. The probe concentration is 5 μM.) Finally, insert the colloidal gold lateral chromatography strip (AMP Future Biotech, catalog number: WLFS8206) into the amplification tube. The colloidal gold is coated with two antibodies, FAM and Biotin. FAM is coated on the T line as a control, and Biotin is coated on the C line as the test line. Because the probe contains two groups, Biotin and FAM, the biotin group can only be exposed to bind to the epitope of the antibody after the probe is cleaved by Cas12a. Therefore, when the reaction solution flows through the C line, FAM binds to the antibody, appearing as a red band. When the target band is amplified, the reaction solution flows through the T line, resulting in a positive signal. See details. Figure 3.

[0124] Table 1 Primer sequences for different HPV subtypes

[0125]

[0126] Table 2 crRNAs of different HPV subtypes

[0127]

[0128]

[0129]

[0130] See partial sensitivity test results. Figures 4-12 .

[0131] Example 5, Clinical Testing

[0132] Cervical smear samples and urine samples from 28 gynecological outpatients were collected and tested using the reaction system and procedure of Example 2, as well as a commercially available cervical sampling and urine HPV detection kit. The results are shown in Table 3.

[0133] Table 3. HPV test results of cervical smear specimens and urine samples from 28 gynecological outpatients.

[0134]

[0135]

[0136] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0137] In HPV testing of 28 patient samples, the detection rate and accuracy of the present invention for cervical and urine samples were 100%, and the detection results for cervical and urine samples were consistent. In contrast, a commercially available urine HPV test kit showed negative results in 3 urine samples. This demonstrates that the present invention has high accuracy in detecting high-risk HPV through urine.

[0138] The foregoing description illustrates and illustrates the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, which are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made in accordance with the scope of the patent and the description should still fall within the scope of the present invention. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0139] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A composite magnetic bead for real-time detection, characterized in that, The composite magnetic beads include: Magnetic Fe3O4 particles; a cationic polymer coating layer covering the magnetic Fe3O4 particles; and a nano-metal-organic framework UiO-66 covering the cationic polymer coating layer; wherein the cationic polymer coating layer may optionally be modified with organic functional groups.

2. The composite magnetic bead according to claim 1, characterized in that, The cationic polymer is a DNA high-affinity cationic polymer, which is selected from at least one of polyethyleneimine (PEI), polyurethane (PBAE), chitosan, polyacrylamide (PAH), diethylaminoethyl dextran (DEAE-dextran), and polyurethane dendritic polymer (PAMAM).

3. The composite magnetic bead according to claim 1, characterized in that, The modification is any one or more combinations of carboxylation, hydroxylation, imidazoleization, and amination.

4. The composite magnetic bead according to claim 1, characterized in that, The magnetic Fe3O4 particles are meso-2,3-dithiol succinate nano-Fe3O4 particles.

5. The composite magnetic bead according to claim 1, characterized in that, The diameter range of the composite magnetic beads is 200-300 nm.

6. A method for preparing the composite magnetic beads according to claim 1, characterized in that, The method includes the following steps: S1: Coat the magnetic Fe3O4 particles with cationic polymer to obtain magnetic Fe3O4 particles coated with cationic polymer. S2: Under heating conditions, the magnetic Fe3O4 particles coated with cationic polymer are treated with a solution containing ZrCl4 and 2-aminoterephthalic acid, and the nano-metal-organic framework UiO-66 is mounted on the magnetic Fe3O4 particles coated with cationic polymer to obtain the composite magnetic beads.

7. A kit for detecting high-risk human papillomavirus in urine, characterized in that, The kit includes: A first container containing a composite magnetic bead suspension; The second container contains the sample resuspension solution; The third container contains amplification reagents, which contain one or more primer sets, amplification reaction enzyme systems, positive controls, and negative controls for amplifying high-risk HPV viruses. The fourth container contains detection reagents, including crRNA of one or more high-risk HPV viruses, a reporter probe, and CRISPR / Cas12a.

8. The reagent kit according to claim 7, characterized in that, The high-risk HPV virus is selected from at least one of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

9. The reagent kit according to claim 7, characterized in that, The primer sets used for amplifying high-risk HPV viruses include: primer sets for amplifying HPV16; primer sets for amplifying HPV18; primer sets for amplifying HPV31; primer sets for amplifying HPV33; primer sets for amplifying HPV35; primer sets for amplifying HPV39; primer sets for amplifying HPV45; primer sets for amplifying HPV51; primer sets for amplifying HPV52; primer sets for amplifying HPV56; primer sets for amplifying HPV58; primer sets for amplifying HPV59; primer sets for amplifying HPV66; and / or primer sets for amplifying HPV68. The primer set for amplifying HPV16 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.1 and the reverse primer is SEQ ID NO.2; And / or, the primer set for amplifying HPV18 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.1 and the reverse primer is SEQ ID NO.2; And / or, the primer set for amplifying HPV31 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.3 and the reverse primer is SEQ ID NO.4; And / or, the primer set for amplifying HPV33 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.3 and the reverse primer is SEQ ID NO.4; And / or, the primer set for amplifying HPV35 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.5 and the reverse primer is SEQ ID NO.6; And / or, the primer set for amplifying HPV39 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.5 and the reverse primer is SEQ ID NO.6; And / or, the primer set for amplifying HPV45 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.7 and the reverse primer is SEQ ID NO.8; And / or, the primer set for amplifying HPV51 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.9 and the reverse primer is SEQ ID NO.10; And / or, the primer set for amplifying HPV52 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.11 and the reverse primer is SEQ ID NO.12; And / or, the primer set for amplifying HPV56 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.11 and the reverse primer is SEQ ID NO.12; And / or, the primer set for amplifying HPV58 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.13 and the reverse primer is SEQ ID NO.14; And / or, the primer set for amplifying HPV59 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.13 and the reverse primer is SEQ ID NO.14; And / or, the primer set for amplifying HPV66 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.15 and the reverse primer is SEQ ID NO.16; And / or, the primer set for amplifying HPV68 includes a forward primer and a reverse primer, wherein the forward primer is SEQ ID NO.17 and the reverse primer is SEQ ID NO.

18.

10. The reagent kit according to claim 9, characterized in that, The crRNA of the high-risk HPV virus is selected from at least one of the following: HPV16 crRNA, HPV18 crRNA, HPV31 crRNA, HPV33 crRNA, HPV35 crRNA, HPV39 crRNA, HPV45 crRNA, HPV51 crRNA, HPV52 crRNA, HPV56 crRNA, HPV58 crRNA, HPV59 crRNA, HPV66 crRNA, and HPV68 crRNA. The HPV16 crRNA is selected from any one of SEQ ID NO.19-21; And / or, the HPV18 crRNA is selected from any one of SEQ ID NO.22-24; And / or, the HPV31 crRNA is selected from any one of SEQ ID NO.25-27; And / or, the HPV33 crRNA is selected from any one of SEQ ID NO.28-30; And / or, the HPV35 crRNA is selected from any one of SEQ ID NO. 31-33; And / or, the HPV39 crRNA is selected from any one of SEQ ID NO.34-36; And / or, the HPV45 crRNA is selected from any one of SEQ ID NO. 37-39; And / or, the HPV51 crRNA is selected from any one of SEQ ID NO.40-42; And / or, the HPV52 crRNA is selected from any one of SEQ ID NO.43-45; And / or, the HPV56 crRNA is selected from any one of SEQ ID NO.46-48; And / or, the HPV58 crRNA is selected from any one of SEQ ID NO.49-51; And / or, the HPV59 crRNA is selected from any one of SEQ ID NO.52-54; And / or, the HPV66 crRNA is selected from any one of SEQ ID NO. 55-57; And / or, the HPV68 crRNA is selected from any one of SEQ ID NO.58-60.

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