Primer probe group for specifically recognizing fourteen high-risk HPVs and rapidly detecting based on RPA isothermal amplification method, kit and application

Abstract

The invention belongs to the technical field of HPV (human papillomavirus) detection, and particularly relates to a primer probe group and a kit for specific recognition and rapid detection of fourteen high-risk HPVs based on an RPA (recombinase polymerase amplification) isothermal amplification method, and application of the primer probe group and the kit. The primer probe group comprises a primer group and a probe group, wherein the primer group comprises a peripheral upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1, a peripheral downstream primer with a nucleotide sequence as shown in SEQ ID NO: 2 and a peripheral downstream primer with a nucleotide sequence as shown in SEQ ID NO: 3; the nucleotide sequence of the peripheral downstream primer is as shown in SEQ ID NO: 2. The primer probe set and the kit have high sensitivity and high specificity, the detection sensitivity of the primer probe set and the kit to HPV high-risk viruses can reach 100 copies / mL, and the performance is superior to that of existing detection products in the market; the design of the specific probe can accurately detect fourteen high-risk HPV types, and does not generate cross reaction with low-risk HPV types or other viruses. The whole detection process is carried out at room temperature, laboratory operation is not needed, the detection time is about 30 minutes, and the detection can be completed in one detection rod.

Description

Technical Field

[0001] This invention belongs to the field of HPV virus detection technology. Specifically, it relates to a primer and probe set, reagent kit, and application for rapid detection of fourteen high-risk HPV types based on RPA isothermal amplification method. Background Technology

[0002] Cervical cancer is the second most common malignant tumor among women, after breast cancer. Statistics show that in 2020, there were 604,000 new cases of cervical cancer and 342,000 deaths worldwide, with 80% occurring in low-resource countries. This means that every minute a woman is diagnosed with cervical cancer, and every two minutes a woman dies from it. Therefore, rapid, convenient, and accessible cervical cancer screening is crucial for reducing its incidence and mortality rates. In North America, Australia, and Europe, well-established screening systems have significantly reduced cervical cancer incidence and mortality rates. However, in many developing countries, cervical cancer incidence and mortality rates have not improved significantly. As the world's largest developing country, China's implementation of universal cervical cancer screening is of great significance for improving public health. Most cervical cancers are caused by human papillomavirus (HPV), which is classified into high-risk and low-risk types based on its carcinogenic potential. The vast majority (99.7%) of cervical cancer cases are caused by persistent or recurrent infection with high-risk HPV types. Therefore, HPV nucleic acid testing plays a crucial role in the early screening of cervical cancer and can significantly improve the detection rate of cervical cancer and its precancerous lesions.

[0003] Currently, common methods for HPV testing include DNA hybridization capture (HC-II) and PCR fluorescence technology. HC-II utilizes molecular hybridization and chemiluminescence signal amplification to detect multiple high-risk HPV types. While this method has high sensitivity, it is complex, time-consuming, and costly. Another method is PCR fluorescence technology, which detects HPV through temperature cycling and fluorescence signal collection, allowing for the simultaneous detection of multiple HPV types. Although relatively simple to operate, the entire amplification process takes 1-2 hours, is time-consuming, requires expensive PCR equipment and professional personnel, and is susceptible to cross-contamination, limiting its application in point-of-care testing and home testing.

[0004] Therefore, there is an urgent need to develop a new method for rapid, simple and accurate HPV nucleic acid testing. Summary of the Invention

[0005] The purpose of this invention is to provide a primer and probe set, kit, and application for rapid detection of fourteen high-risk HPV types based on RPA isothermal amplification method, so as to achieve high sensitivity, high specificity, and accurate detection of fourteen high-risk HPV types.

[0006] Therefore, the present invention provides the following technical solution.

[0007] The first aspect of this invention provides a primer-probe set for rapid detection of fourteen high-risk HPV types based on RPA isothermal amplification, the primer-probe set comprising a primer set and a probe set, the primer set comprising: The nucleotide sequence is shown in the peripheral upstream primer of SEQ ID NO: 1; The nucleotide sequence is shown in SEQ ID NO: 2. The peripheral downstream primer is as shown.

[0008] In a preferred embodiment of the present invention, the 5' end of the peripheral upstream primer is labeled with biotin.

[0009] In a preferred embodiment of the present invention, the probe set includes probes that specifically identify fourteen high-risk HPV types, the nucleotide sequences of which are shown in SEQ ID NO: 3-SEQ ID NO: 16.

[0010] In a preferred embodiment of the present invention, the 5' end of the probe is marked with digoxigenin, the 3' end is closed by a C3 spacer, and the 16th base at the 3' end is replaced with tetrahydrofuran.

[0011] A second aspect of the present invention provides a kit for the specific identification of fourteen high-risk HPV types based on RPA isothermal amplification, the kit comprising the primer and probe set as described above.

[0012] In a preferred embodiment of the present invention, the concentrations of the primers and probes in the kit are each independently 10 μM.

[0013] In a preferred embodiment of the present invention, the kit further includes magnesium acetate and RPA Mix.

[0014] In a preferred embodiment of the present invention, the kit further includes: a colloidal gold immunochromatographic reagent card.

[0015] A third aspect of the invention provides the use of the primer-probe set or kit as described above in the preparation of reagents for detecting fourteen high-risk HPV types.

[0016] In a preferred embodiment of the present invention, the fourteen high-risk HPV types include one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68.

[0017] By employing the above technical solution, the present invention has at least the following advantages: 1) This invention is the first to use multiplex RPA isothermal amplification technology to achieve rapid detection of fourteen high-risk HPV types, making up for the shortcomings of existing HPV detection technologies and inventing an HPV rapid test (HPV-POCT) product (POCT, point-of-care testing) that does not require laboratory testing, with a testing time of 30 minutes.

[0018] 2) This invention provides a primer set and probe combination capable of rapidly detecting the nucleic acids of fourteen high-risk HPV types, as well as a detection kit and analytical method containing these primer sets and probes. This invention uses a primer set capable of amplifying 14 high-risk HPV types as primers, combined with 14 specific probes, and uses an RPA rapid amplification system to amplify the template. The results are then determined using an immunochromatographic test strip to identify whether the sample is infected with one or more of the fourteen high-risk HPV types.

[0019] 3) The primer-probe set and kit of this invention have high sensitivity and high specificity. Its detection sensitivity for high-risk HPV types can reach 100 copies / mL, a performance superior to many commercially available detection products. The specific probe design can accurately detect one or more of the fourteen high-risk HPV types without cross-reacting with low-risk HPV types or other viruses.

[0020] 4) The kit and method of the present invention are simple to operate, have low requirements for equipment and personnel, short detection time, and do not require professional personnel or equipment throughout the process. They can be carried out at room temperature of 25-42℃, have high sensitivity and high specificity, are suitable for home self-testing and community screening, and have broad application prospects.

[0021] 5) This invention performs quantity quality control on the detected exfoliated cells.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0023] Figure 1 The schematic diagram of the detection principle of the nucleic acid detection kit (isothermal amplification method) of the present invention is shown; Figure 2 A structural diagram of the colloidal gold immunochromatographic reagent card in the nucleic acid detection kit of the present invention is shown; Figure 3 The results discrimination diagram of the colloidal gold immunochromatographic test strip of the present invention is shown; Figure 4The results of the kit of the present invention for detecting 14 high-risk HPV types are shown in the figure; Figure 5 The results of the kit of the present invention in detecting 14 low-risk HPV types are shown in the figure. Figure 6 The results of the kit of the present invention in detecting five common pathogens of the reproductive tract are shown in the figure. Figure 7 A flowchart illustrating the preparation process of the freeze-dried microspheres of the present invention is shown; Figure 8 The image shows a gel electrophoresis diagram of the products obtained after amplification of 14 high-risk HPV types by the lyophilized microspheres of the present invention. Figure 9 The diagram shows the detection results of the freeze-dried microspheres of the present invention for high-risk HPV, low-risk HPV, and other pathogens. Detailed Implementation

[0024] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] Example 1: Design and screening of primers and probes First, sequence data of 14 high-risk human papillomavirus (HPV) types were retrieved and downloaded from the GeneBank database. Next, DNAMAN software was used to perform alignment analysis on these sequences to identify conserved sequences in the L1 region of the genome. Based on this, a universal pair of peripheral upstream and downstream primers was designed for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Considering the high tolerance of recombinase polymerase amplification (RPA) technology to base mismatches, a small number of base mismatches were allowed in the primers. The resulting set of specific RPA primer sequences is shown below, where the 5' end of the peripheral upstream primer F is biotin-labeled: The nucleotide sequence of the upstream primer F is shown in SEQ ID NO: 1: 5'-biotin-GCNCARGGHYWHAAYAATGG-3'; The nucleotide sequence of the downstream primer R is shown in SEQ ID NO: 2: 5'-AAHAYAAMYTGYAVDTCAWAYTCYTC-3'; For probe design, the focus was on the most specific sequence regions of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, which exhibit high specificity compared to other HPV types. Specific probes were used for 14 high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). For these 14 specific probes, the 5' end was labeled with digoxigenin, and the 3' end was blocked by a C3 spacer; the 16 bases at the 3' end were substituted with tetrahydrofuran. The specific probe sequences and their labeling methods are as follows: The nucleotide sequence of the probe targeting HPV16 is shown in SEQ ID NO: 3: Dig-AGTTAGTATTTTTATATGTAGTTTCTGAAG / THF / AgATATggCAgCACA-C3 Spacer The nucleotide sequence of the probe targeting HPV18 is shown in SEQ ID NO: 4: Dig-ATTTGGTAGCATCATATTGCCCAGGTACAGGAGA / THF / TGTGTAGAAGCACAT-C3 Spacer The nucleotide sequence of the probe targeting HPV31 is shown in SEQ ID NO: 5: Dig-AATTACTACTTTTAAATGTAGTATCACTGT / THF / TGCAATTGCAGCACA-C3 Spacer The nucleotide sequence of the probe targeting HPV33 is shown in SEQ ID NO: 6: Dig-AATTTTCATTTTTATATGTACTGTCACTAG / THF / TACTTGTGTGCATAA-C3 Spacer The nucleotide sequence of the probe targeting HPV35 is shown in SEQ ID NO: 7: Dig-AATTGTCATTTTTATATGTACTGTCACTAG / THF / AGACACAGCAGAACA-C3 Spacer The nucleotide sequence of the probe targeting HPV39 is shown in SEQ ID NO: 8: Dig-ACTTAGAAGGATCATATGTAGAAGGTATGG / THF / AGACTCTATAGAGGT-C3 Spacer The nucleotide sequence of the probe targeting HPV45 is shown in SEQ ID NO: 9: Dig-ACTTAGTAGGGTCATATGTACTTGGCACAG / THF / ATTTTGTGTAGAGGC-C3 Spacer The nucleotide sequence of the probe targeting HPV51 is shown in SEQ ID NO: 10: Dig-AGTTACTTGGAGTAAATGTTGGGGAAAACCG / THF / AGCAGTGGCAGTGCT-C3 Spacer The nucleotide sequence of the probe targeting HPV52 is shown in SEQ ID NO: 11: Dig-AATTTTCATTTTTATATGTGCTTTCCTTTT / THF / AACCTCAGCACATAA-C3 Spacer The nucleotide sequence of the probe targeting HPV56 is shown in SEQ ID NO: 12: Dig-TTTTTCGTGCATCATATTTACTTAACTGTT / THF / TGTAGCAGTACTAAT-C3 Spacer The nucleotide sequence of the probe targeting HPV58 is shown in SEQ ID NO: 13: Dig-AATTATCATTTTTATATGTACCTTCCTTAG / THF / TACTTCAGTGCATAA-C3 Spacer The nucleotide sequence of the probe targeting HPV59 is shown in SEQ ID NO: 14: Dig-AACTGGTAGGTGTGTATACATTAGGAATAG / THF / AGAAGTAGTAGAAGC-C3 Spacer The nucleotide sequence of the probe targeting HPV66 is shown in SEQ ID NO: 15: Dig-TTTCACGGGCATCATATTTAGTTAATGTGC / THF / TTTAGCTGCATTAAT-C3 Spacer The nucleotide sequence of the probe targeting HPV68 is shown in SEQ ID NO: 16: Dig-ATTTATTAGAATCATACACAGCTGGTACAG / THF / AGAGTCTGTAGTAGT-C3 Spacer Example 2: Quality control of exfoliated cell count A 0.4% trypan blue solution (prepared with a pH 7.2-7.3 buffered isotonic saline solution) was used to prepare the exfoliated cell preservation solution. Exfoliated cells from the sample were collected. When the number of exfoliated cells in the collection bottle reached 5000 cells / mL (1000 cells per 0.2 mL of cell preservation solution in this device), the cell preservation solution in the collection bottle would turn blue. The mixture of exfoliated cells meeting the cell count and the cell preservation solution was stored for later use.

[0026] Example 3: A kit for detecting fourteen high-risk HPV types Through multiple optimizations and screenings, this invention determined that the optimal concentrations of primers and probes in the RPA amplification reaction are each 10 μM. Simultaneously, a rapid detection kit for fourteen high-risk HPV types was designed, comprising: an RPA amplification reaction system and a colloidal gold immunochromatographic reagent card. The colloidal gold immunochromatographic reagent card consists of a colloidal gold immunochromatographic test strip and an aerosol-resistant casing.

[0027] (1) The 200 μL RPA amplification reaction system in the kit consists of: 2.5 μL of 280 mM magnesium acetate, 150 μL of template, 38.9 μL of RPA Mix, and a total volume of primers and probes of 8.6 μL, of which 1.5 μL each of universal peripheral upstream primer (concentration of 10 μM) and universal peripheral downstream primer (concentration of 10 μM) and 5.6 μL of probe (concentration of 10 μM).

[0028] (2) The preparation process of the colloidal gold immunochromatographic test strip in the kit is as follows: a) Sample pad treatment: Treat the glass fiber sample pad with a special treatment solution (0.02 mol / L PBS, 0.5% BSA, 0.05% Tween-20, 0.2% Proclin 300), and then dry it at 37°C for 18-24 hours to ensure that the sample to be tested can be uniformly released into the label pad.

[0029] b) Buffer pad preparation: The glass fiber buffer pads were treated with a special treatment solution (0.02 mol / L PBS, 0.5% BSA, 0.05% Tween-20, 0.2% Proclin 300) and dried at 37°C for 18-24 hours. This ensures that the sample can uniformly ascend to the nitrocellulose filter membrane (NC membrane) after being added.

[0030] c) Preparation of labeled pads: Polyester fiber labeled pads were sealed with a pretreatment solution, and then digoxin antibody-colloidal gold labeled solution was uniformly sprayed onto the polyester membrane (4 μL / cm) and dried at 45°C for 18-24 hours.

[0031] d) Preparation of coating membrane: Fix the absorbent pad and NC membrane on a PVC plastic sheet, and use the coating solution to coat the NC membrane (1 mm wide) with streptavidin (1.5 mg / mL) and internal control antibody (1 mg / mL) to form the detection line (T line) and the control line (C line), and dry at 37°C for 18-24 hours.

[0032] e) Assembly and Packaging: Following the assembly principle of chromatography test strips, the sample pad, marker pad, buffer pad, NC membrane and absorbent pad are combined together, cut into 3.48 mm wide test strips, placed in aerosol-proof card cases, and desiccant is added to prepare colloidal gold immunochromatographic reagent cards, which are then sealed and stored.

[0033] The detection principle of the colloidal gold immunochromatographic reagent cards for detecting fourteen high-risk HPV types provided by this invention is as follows: Figure 1 As shown. β-actin is a protein widely distributed in cells and is part of the cytoskeleton. It consists of 375 amino acids, has a molecular weight of approximately 42-43 kDa, and is expressed in very high amounts, accounting for about 50% of all cellular proteins. β-actin is found in cervical cells and cancer cells.

[0034] Figure 2 A structural diagram of the colloidal gold immunochromatographic reagent card in the nucleic acid detection kit of the present invention is shown. Figure 2 As shown, the reagent card consists of an aerosol-proof casing and colloidal gold immunochromatographic test strips. The aerosol-proof casing design has undergone three iterations to meet requirements for aerosol protection and ease of manufacturing. It mainly consists of three parts: a sample application port, an incubation chamber, and a chromatography chamber, which together detect fourteen high-risk HPV types. Specifically, the aerosol-proof casing comprises: a sealing cap, an incubation chamber, a biological substrate, a top cover, and a base (see...). Figure 2The colloidal gold immunochromatographic test strip consists of two C lines and one T line. The two C lines detect β-actin to confirm whether a sufficient number of cells were obtained from the sample, while the other C line verifies the validity and completeness of the test process.

[0035] This embodiment also provides a detection method for the above-mentioned reagent kit, which specifically includes the following steps: Step a: Nucleic acid release: Immerse the swab head containing the collected sample into the elution tube containing the nucleic acid release agent and rotate to mix 10 times for at least 30 seconds. At the same time, squeeze the swab head through the outer wall of the elution tube at least 5 times to ensure that the sample is fully eluted and mixed to obtain a nucleic acid solution.

[0036] Step b: RPA amplification: Using the nucleic acid solution obtained in step a as a template, the RPA isothermal amplification method was employed. The 200 μL RPA amplification reaction system included: 150 μL template, 38.9 μL RPA Mix, 8.6 μL primers and probes, and 2.5 μL 280 mM magnesium acetate. The RPA isothermal amplification reaction was carried out between 25-42℃ for 10-20 minutes to obtain the RPA amplification product.

[0037] Step c: Colloidal gold immunochromatographic assay: Add 80 μL of RPA amplification product to the sample well of the colloidal gold immunochromatographic assay card, let stand for 10 minutes, and then read the results. The interpretation of the results is as follows: Figure 3 As shown.

[0038] Example 4: Sensitivity and specificity of the kit for HPV detection 1. Sensitivity detection To test the sensitivity of the kit in Example 3, this example used HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 reference samples from the human papillomavirus L1 genotyping reference materials (sequence data of high-risk human papillomavirus (HPV) types were retrieved and downloaded from the GeneBank database). The reference samples were diluted to 10 with nucleic acid release agent. 2 The concentration was 10 μL / mL, using a diluted reference sample as a template. The RPA amplification reaction system was prepared according to the description in Example 3. Magnesium acetate was added to initiate the RPA amplification reaction, which was carried out at 25-42°C for 10-20 minutes to obtain the RPA amplification product. 80 μL of the obtained RPA amplification product was added to a colloidal gold chromatography reagent card for testing. The results were checked after 10 minutes. The results are shown in [Figure Number]. Figure 4 .like Figure 4As shown, all fourteen high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) tested positive.

[0039] 2. Specific detection To verify the specificity of the kit in Example 2 for detecting low-risk HPV, this example used low-risk HPV types (HPV6, HPV11, HPV40, HPV42, HPV44, HPV54, HPV61, HPV67, HPV69, HPV70, HPV71, HPV72, HPV81, HPV83) from the human papillomavirus L1 genotyping reference (obtained by retrieving and downloading sequence data of high-risk human papillomavirus (HPV) types from the GeneBank database). The reference was diluted to 10... 5 The copie / mL was tested using the same procedure as in step 1 above, and the results are shown below. Figure 5 .like Figure 5 As shown, all fourteen low-risk HPV types (HPV6, 11, 40, 42, 44, 54, 61, 67, 69, 70, 71, 72, 81, 83) tested negative, with no cross-contamination with high-risk HPV types, demonstrating good specificity.

[0040] In addition, this embodiment also uses cytomegalovirus (CMV), herpes simplex virus type II (HSV-II), herpes simplex virus type I (HSV-I), group B streptococci (GBS), and ureaplasma urealyticum (UU) from the human papillomavirus whole genome typing reference as templates (concentration of 10). 2 (copie / mL), the test was performed using the same procedure as in step 1 above, and the results are shown below. Figure 6 .like Figure 6 As shown, the kit of the present invention showed negative results for five common reproductive tract pathogens (cytomegalovirus (CMV), herpes simplex virus type II (HSV II), herpes simplex virus type I (HSV I), group B streptococcus (GBS), and ureaplasma urealyticum (UU)), with no cross-contamination with high-risk HPV types, demonstrating good specificity.

[0041] Example 5: Lyophilized microspheres for detecting high-risk HPV types This embodiment also prepared a lyophilized microsphere for the detection of high-risk HPV viruses. Specifically, the RPA amplification system components were lyophilized and divided into two types: lyophilized microspheres A and B. Microspheres B were made of Mg... 2+ Magnesium acetate (A-spheres) are other components of the RPA amplification system, including enzymes, ATP, primers, and probes. The preparation process of these lyophilized microspheres is described below. Figure 7 The specific method includes the following steps: Mg2+ Mix with a protective agent, freeze in liquid nitrogen to form microspheres, and then freeze-dry to obtain microsphere B; mix enzyme, ATP, primers, and probes with a protective agent, freeze in liquid nitrogen to form microspheres, and then freeze-dry to obtain microsphere A.

[0042] The detection method for the lyophilized microspheres is as follows: A-spheres and B-spheres are added to the sample to be tested and mixed, and then the RPA isothermal amplification reaction is performed to specifically identify and detect whether there are fourteen high-risk HPV types in the sample to be tested.

[0043] The freeze-dried microspheres were used to detect 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) according to the above-described detection method. The results are shown below. Figure 8 .like Figure 8 As shown, the freeze-dried microspheres were tested for 14 high-risk HPV types using the above-mentioned detection method, and the viral load was only 10. 2 Even at a concentration of 1000 mg / mL, the result was still positive.

[0044] The freeze-dried microspheres were used to detect 14 low-risk HPV types (6, 11, 40, 42, 44, 54, 61, 67, 69, 70, 71, 72, 81, 83) and cytomegalovirus (CMV), herpes simplex virus type II (HSV-II), herpes simplex virus type I (HSV-I), group B streptococci (GBS), and ureaplasma urealyticum (UU) using the same method described above. The results are shown in the figure. Figure 9 .like Figure 9 As shown, the freeze-dried microspheres of this invention can detect all 14 high-risk HPV types with a detection sensitivity of 100 copies / mL. There is no cross-contamination with low-risk types. There is no cross-contamination with cytomegalovirus, herpes simplex virus types 1 / 2, group B streptococci, and ureaplasma urealyticum, and the specificity meets the requirements.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A primer-probe set for rapid detection of fourteen high-risk HPV types based on RPA isothermal amplification, characterized in that, The primer-probe set includes a primer set and a probe set, wherein the primer set includes: The nucleotide sequence is shown in the peripheral upstream primer of SEQ ID NO: 1; The nucleotide sequence is shown in SEQ ID NO:

2. The peripheral downstream primer is as shown in SEQ ID NO:

2.

2. The primer-probe set according to claim 1, characterized in that, The 5' end of the peripheral upstream primer is labeled with biotin.

3. The primer-probe set according to claim 1, characterized in that, The probe set includes probes that specifically recognize fourteen high-risk HPV types, and the nucleotide sequences of the probes are shown in SEQ ID NO: 3-SEQ ID NO:

16.

4. The primer-probe set according to claim 3, characterized in that, The probe is labeled with digoxigenin at its 5' end, and its 3' end is closed by a C3 spacer. The 16th base at the 3' end is replaced with tetrahydrofuran.

5. A kit for specifically identifying fourteen high-risk HPV types based on RPA isothermal amplification, characterized in that, The kit comprises the primer and probe set as described in any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that, The concentrations of the primers and probes in the kit are each independently 10 μM.

7. The reagent kit according to claim 5, characterized in that, The kit also includes magnesium acetate and RPA Mix.

8. The reagent kit according to claim 5, characterized in that, The kit also includes: a colloidal gold immunochromatographic reagent card.

9. Use of the primer and probe set according to any one of claims 1-4, or the kit according to any one of claims 5-8, in the preparation of reagents for detecting fourteen high-risk HPV types.

10. The use according to claim 9, characterized in that, The fourteen high-risk HPV types include one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68.

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