Human papilloma virus detection kit and detection method thereof

Abstract

The invention provides a human papilloma virus (HPV) detection kit and a detection method thereof, and the HPV detection method provided by the invention ensures the accuracy and reliability of detection through the steps of systematic sample treatment, PCR amplification, hybridization operation and quality control. The whole detection process is reasonable in design, clear in steps and simple to operate. The whole process from sample treatment to final result interpretation is relatively rapid, especially in PCR amplification and hybridization operation, optimized temperature and time setting is adopted, the detection period is shortened, and the method is suitable for large-scale screening.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and specifically relates to a human papillomavirus detection kit and a detection method thereof. Background Art

[0002] Human papillomavirus (HPV) is a group of DNA viruses belonging to the papillomaviridae family that can infect human skin and mucous membranes. There are more than 200 different types of HPV, which are divided into high-risk and low-risk types according to their carcinogenic potential. Certain HPV types are closely associated with the occurrence of various cancers and are called high-risk HPV. Common high-risk HPV types include 16, 18, 31, 33, 45, 52 and 58. Among them, HPV16 and HPV18 are the two most common high-risk types, and about 70% of cervical cancer cases are related to these two types. High-risk HPV is also associated with cancers such as vulvar cancer, vaginal cancer, anal cancer, and oropharyngeal cancer. Low-risk HPV: Low-risk HPV usually causes benign lesions, such as genital warts (condyloma acuminatum). Common low-risk HPV types include types 6 and 11, which account for about 90% of genital warts cases.

[0003] Although HPV testing technology has made significant progress, there are still some challenges and difficulties in practical application. HPV testing usually requires the collection of cell samples from the cervix, vulva, anus or mouth. The quality of the sample directly affects the accuracy of the test results. If the sample is not collected properly (such as insufficient sampling, contamination or poor storage conditions), false negative or false positive results may result. Different types of samples (such as cervical smears, self-collected vaginal swabs, anal swabs, etc.) have different effects on the test results. Some sample types may not be suitable for the detection of all HPV types, or there are differences in sensitivity and specificity. Sample transportation and storage: HPV is a DNA virus that is sensitive to environmental conditions. If the sample is affected by factors such as temperature and humidity during transportation or storage, it may cause the viral DNA to degrade, thereby affecting the test results.

[0004] There are more than 200 different types of HPV, of which about 40 can infect the reproductive tract. Different types of HPV have different carcinogenic potential and clinical manifestations, so detection methods that can distinguish between high-risk and low-risk types are needed. However, existing detection technologies may not cover all types, especially rare types. Many patients are infected with multiple HPV types at the same time, which increases the complexity of detection. Multi-type infection can make it difficult to interpret test results, especially when using reagents that can only detect a few common types, which may miss other important types.

[0005] There are currently a variety of HPV detection methods available, including PCR (polymerase chain reaction), hybrid capture method, gene chip, sequencing, etc. Each method has its own advantages and disadvantages in terms of sensitivity, specificity, cost and operational complexity. Choosing the right detection method is crucial to ensure accurate diagnosis. There are currently embodiments that use nucleic acid test strips to interpret the results, but the test strips used therein will produce serious non-specific color development, which makes it extremely difficult to interpret the results. Summary of the invention

[0006] The purpose of the present invention is to provide a human papillomavirus detection kit and a detection method thereof.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: This embodiment provides a method for detecting human papillomavirus, comprising the following steps: 1. Sample processing and DNA extraction: number the samples and put them into storage tubes; prepare 1.5-3mL centrifuge tubes with the same number; vortex the sample storage tubes for 10-30 seconds, transfer 1-1.2mL of storage solution to the corresponding numbered centrifuge tubes: centrifuge the numbered samples at 10000-12000rpm for 5-10 minutes, and discard the supernatant; add 1mL of sterile saline to the sample precipitate, wash the precipitate, centrifuge at 10000-12000rpm for 5-10 minutes, and discard the supernatant; take the pretreated sample cell precipitate, add 50-60 microliters of nucleic acid extraction or purification reagent, and oscillate to mix; lyse at 100℃ for 10-12 minutes; centrifuge at 10000-12000rpm for 5-7 minutes, take the supernatant for use, and store at -20℃; 2. PCR amplification: Take a PCR reaction tube, add 20 μL of the extracted sample DNA and HPV positive and negative quality control DNA respectively, centrifuge at 6000-6300rpm, transfer the transfer window of each reaction tube to the gene amplification room, and then put the reaction tube into the PCR instrument, and amplify it on the machine according to the following conditions: 50℃, 3 minutes, 95℃ pre-denaturation for 15 minutes, and then amplify 40 cycles in the order of 94℃, 40 seconds; 55℃, 40 seconds; 72℃, 40 seconds, and finally extend at 72℃ for 7 minutes; 3. Hybridization operation: (1) Prepare the reagents for hybridization operation; denature the PCR amplification product at 98℃ in a thermal cycler for 8-10 minutes, and then immediately place it in an ice-water mixture for 2-5 minutes; take a 5-15mL plastic centrifuge tube, put it into the HPV typing hybridization membrane strip marked with the sample number to be tested, add 4mL hybridization solution I and 40-50μL of the denatured PCR product corresponding to the membrane strip number, tighten the tube cap, write the corresponding sample number on the tube cap, and place the centrifuge tube flat in a 42℃ water bath shaker, rotate at a low speed of 100-150rpm, and hybridize for 1-1.2 hours; put hybridization solution II in a water bath shaker to preheat to 42℃ and set aside; (2) Binding: Take out the HPV typing hybridization membrane strips, wash them with clean water for 1-2 minutes, then transfer 5 membrane strips / tube into a 50mL centrifuge tube containing 20mL preheated hybridization solution II, add 20μL solution I, shake in a 42-45℃ water bath, and rotate at low speed for 10-12 minutes; (3) Washing the membrane: Take out the HPV typing hybridization membrane strip and transfer it into a 50 mL centrifuge tube filled with preheated hybridization solution II. Wash the membrane at a low speed in a 42°C water bath shaker for 10-12 minutes. (4) Color development: Prepare the required color development solution according to the number of samples and the preparation method of the color development solution in the above table, put it into the washed HPV typing hybridization membrane strip, and color it in the dark at 20-25°C for 3-10 minutes. Take out the membrane strip and put it into 20 mL of solution II for 2-3 minutes to stop the color development. Discard solution II, add 20 mL of pure water, shake it gently for 2-3 minutes, and take it out; use absorbent paper to absorb the water on the surface of the membrane strip; (5) Preservation of membrane strip results; 4. Quality control: (1) CC point: If it is a blue dot with clear edges visible to the naked eye, the binding and color development process is normal; otherwise, the binding and color development process needs to be rechecked; (2) PC sites: (1) A blue dot with clear edges visible to the naked eye indicates that the sampling, DNA extraction, PCR, and hybridization processes are normal; (2) No blue dot with clear edges visible to the naked eye, ① The type point is colored and is a blue dot with clear edges, the experiment is unsuccessful and the sample should be resampled or re-extracted; ② The type point is not colored and appears as an individual case in the entire batch of experiments, indicating that there are problems in one or more of the sampling, DNA extraction, PCR, or hybridization processes, and the experiment is unsuccessful. The sample should be resampled or re-extracted; (3) HPV positive quality control: CC point, PC point, and HPV18 typing site should be blue dots with clear edges visible to the naked eye; other sites should not show color; (4) Negative control: CC point should be a blue dot with clear edges visible to the naked eye; other sites should not show color; 5. Analysis of test results: The membrane strips that meet the quality control requirements will proceed to the next step of HPV type color point result interpretation, which can be divided into: 1. Blank; 2. Faintly visible blue dots + type; 3. Lighter but clear-edged blue dots + type; 4. Blue dots + type with clear edges; Very dark blue dots + type with clear edges.

[0008] The present invention ensures the specificity of the test results by setting up strict quality control measures in multiple links, including the use of positive and negative quality control products, the inspection of CC points and PC sites, etc. In particular, the use of HPV positive and negative quality control products can effectively verify whether each link in the experimental process is carried out normally. By coloring the specific sites on the hybridization membrane strip, different types of HPV, especially high-risk and low-risk types, can be clearly distinguished. Not only can common HPV types be detected, but other rare types can also be identified, thereby improving the comprehensiveness and accuracy of the detection. The combination of PCR amplification and hybridization operations ensures that only the target HPV DNA sequence can specifically bind to the probe on the hybridization membrane strip, reducing the possibility of nonspecific binding, thereby improving the specificity of the detection.

[0009] Furthermore, the hybridization solution I is prepared by diluting 50 mL of hybridization solution I concentrate with 200 mL of purified water.

[0010] Furthermore, the hybridization solution II is prepared by diluting 30 mL of the hybridization solution II concentrate with 120 mL of purified water.

[0011] Further, solution II was prepared by diluting 25 mL of solution II concentrate with 100 mL of purified water; Further, the solution IV was diluted 10 times by adding 900 μL of purified water to 100 μL of solution IV.

[0012] Furthermore, the color developing solution includes 3 mL of solution II+0.3 mL of solution III+1.5 µl of solution IV.

[0013] Furthermore, for each tube of 10 mL of hybridization solution II, add 10 μL of solution I, and 2-3 membrane strips can be placed in it for combination; for each tube of 20 mL of hybridization solution II, add 20 μL of solution I, and 5 membrane strips can be placed in it for combination.

[0014] Furthermore, each tube of 20 mL of hybridization solution II can wash 2-3 membrane strips; each tube of 30 mL of hybridization solution II can wash 5 membrane strips, and each tube can wash a maximum of 5 membrane strips at a time.

[0015] Furthermore, the method for preserving the membrane strip results is as follows: stick the membrane strips on the experimental process record sheet according to the corresponding numbers, read the results and take photos and save them in the result folder, cover the membrane strips with transparent tape, and then put the record sheet into a sealed bag and store it away from light.

[0016] The detection method of the present invention ensures high specificity, high sensitivity and good repeatability of the detection through optimized sample processing, PCR amplification, hybridization operation and strict quality control. The characteristics of simple operation and intuitive results make it very suitable for primary medical institutions and large-scale screening projects. At the same time, the wide applicability and cost-effectiveness of the method also provide strong support for the popularization of HPV detection. In this way, HPV-related diseases can be more effectively prevented and managed to protect public health.

[0017] The present invention also provides a human papillomavirus detection kit, which includes hybridization solution I, hybridization solution II, solution II, solution IV diluted 10 times and a color developing solution; the hybridization solution I is 50 mL of hybridization solution I concentrated solution diluted with 200 mL of purified water; the hybridization solution II is 30 mL of hybridization solution II concentrated solution diluted with 120 mL of purified water; the solution II is 25 mL of solution II concentrated solution diluted with 100 mL of purified water; the solution IV diluted 10 times is 100 μL of solution IV diluted with 900 μL of purified water; the color developing solution includes 3 mL of solution II+0.3 mL of solution III+1.5 μl of solution IV.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The HPV detection method of the present invention ensures the accuracy and reliability of the detection through systematic sample processing, PCR amplification, hybridization operation and quality control steps. The entire detection process is reasonably designed, with clear steps and easy operation. For example, conventional laboratory equipment such as centrifugation and oscillation are used in the sample processing process, which reduces complex manual operations and reduces human errors. Each step has clear time and condition requirements (such as centrifugation speed, temperature, time, etc.) to ensure the consistency and repeatability of the operation. This not only improves the standardization level of the experiment, but also facilitates the training of technicians. From sample processing to final result interpretation, the whole process is relatively fast, especially in PCR amplification and hybridization operations, optimized temperature and time settings are used to shorten the detection cycle, which is suitable for large-scale screening.

[0019] 2. In the PCR amplification process, the present invention adopts the strategy of pre-denaturation and multi-cycle amplification to ensure the effective amplification of the target HPV DNA. The temperature settings of 94°C, 55°C and 72°C, and the number of amplifications of 40 cycles ensure that even samples with low viral loads can be detected.

[0020] 3. The present invention can develop color through the blue dots on the hybridization membrane strip, and the test results are intuitive and easy to read, and can be interpreted without complex instruments or professional skills. This enables primary medical institutions and laboratories with fewer technical personnel to easily perform HPV testing. According to the color depth and edge clarity of the color development points, it is divided into different interpretation levels (blank, vaguely visible, lighter but clear, clear edges, very dark), which provides clinicians with a detailed reference basis and helps to more accurately assess the patient's infection status. It is not only suitable for cervical smear samples, but also can be used for a variety of sample types such as self-collected vaginal swabs and anal swabs. This flexibility allows the detection method to be applied to different clinical scenarios to meet diverse detection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of test tubes prepared for hybridization reagents; Figure 2 Schematic diagram of membrane washing. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1 See also Figure 1-2 This embodiment provides a method for detecting human papillomavirus, comprising the following steps: 1. Sample processing and DNA extraction 1.1. Number the samples (each batch of samples is tested with positive and negative in-house quality control products, and the quality control products are treated in the same way as the samples), put them into storage tubes; and prepare 1.5mL centrifuge tubes with the same numbers; 1.2. Use a vortex oscillator to shake the sample storage tube for 10 seconds (to allow the cells to fully fall into the liquid), transfer 1mL of storage solution to the corresponding numbered 1.5mL centrifuge tube (do not remove the brush, and pour the sample directly into the centrifuge tube to effectively prevent the influence of mucus on sample extraction): 1.3. Centrifuge the numbered samples at 10,000 rpm for 5 minutes and discard the supernatant; 1.4. Add 1 mL of sterile saline to the sample precipitate, wash the precipitate, centrifuge at 10,000 rpm for 5 minutes, and discard the supernatant; 1.5. Take the pretreated sample cell pellet, add 50 μl of nucleic acid extraction or purification reagent (there are granules in the nucleic acid extraction or purification reagent, which needs to be fully mixed before use), and shake to mix; 1.6. Lyse at 100℃ for 10 minutes. Centrifuge at 10,000 rpm for 5 minutes, take the supernatant and store at -20℃.

[0024] 2. PCR amplification (set the PCR cycle program on the instrument according to the following conditions): Take a PCR reaction tube, add 20 μL of the extracted sample DNA and HPV positive and negative quality control DNA respectively, centrifuge at 6000rpm for 5 seconds, transfer the transfer window of each reaction tube to the gene amplification room, and then put the reaction tube into the PCR instrument, and amplify it under the following conditions: 50℃, 3 minutes, pre-denaturation at 95℃ for 15 minutes, and then amplify for 40 cycles in the order of 94℃, 40 seconds; 55℃, 40 seconds; 72℃, 40 seconds, and finally extend at 72℃ for 7 minutes.

[0025] 3. Hybridization operation 3.1 Preparation of hybridization reagents, see Table 1 for details.

[0026] Table 1 Actual hybridization preparation

[0027] 3.2 Hybridization process: (1) Hybridization Denature the PCR amplification product at 98°C for 8 minutes in a thermal cycler, and then immediately place it in an ice-water mixture for more than 2 minutes (make sure the paraffin oil in the PCR tube solidifies in about 10 seconds, otherwise it will affect subsequent experimental results); Take a 5mL (or 15mL plastic tube) plastic centrifuge tube, put it into the HPV typing hybridization membrane strip marked with the sample number to be tested, add 4mL hybridization solution I and 40μL of denatured PCR product corresponding to the membrane strip number, tighten the tube cap, number the corresponding sample number on the tube cap, and place the centrifuge tube flat in a 42℃ water bath shaker, rotate at a low speed (100rpm), and hybridize for 1 hour; put hybridization solution II in a water bath shaker and preheat it to 42℃ for use.

[0028] (2) Combination Take out the HPV typing hybridization membrane strips, wash them with clean water for 1 minute, then transfer 5 membrane strips / tube into a 50mL centrifuge tube containing 20mL preheated hybridization solution II, add 20μL solution I, and shake in a 42℃ water bath at low speed (100rpm) for 10 minutes (each tube has 10mL hybridization solution II, add 10μL solution I, and 3 membrane strips can be placed for combination; each tube has 20mL hybridization solution II, add 20μL solution I, and 5 membrane strips can be placed for combination). Note: Hybridization solution II must be preheated to 42℃ first, and then solution I is added after the membrane strips are placed. A maximum of 5 membrane strips can be combined at a time in each tube.

[0029] (3) Membrane washing Take out the HPV typing hybridization membrane strips, transfer them into a 50mL centrifuge tube filled with preheated hybridization solution II, and wash the membrane at low speed (100rpm) for 10 minutes in a 42℃ water bath shaker (20mL hybridization solution II per tube can wash 3 membrane strips; 30mL hybridization solution II per tube can wash 5 membrane strips, and a maximum of 5 membrane strips can be washed in each tube at one time).

[0030] (4) Color rendering According to the number of samples, prepare the required color development solution according to the preparation method of color development solution in Table 1, put it into the washed HPV typing hybridization membrane strip, color it at 20℃ in the dark for 3 minutes, take out the membrane strip and put it into 20mL solution II to stop color development for 2 minutes, discard solution II, add 20mL pure water, shake it gently for 2 minutes and take it out. Use absorbent paper to absorb the water on the surface of the membrane strip.

[0031] (5) Save the results of the membrane strip Stick the membrane strips on the experimental process record sheet according to the corresponding numbers, read the results and take a photo to save in the result folder, and cover the membrane strips with transparent tape (to slow down the fading process of the blue color of the membrane strips), then put the record sheet into a sealed bag and store it away from light.

[0032] 4. Quality Control (1) CC point: If it is a blue dot with clear edges visible to the naked eye, the binding and color development process is normal. Otherwise, the binding and color development process needs to be rechecked.

[0033] (2) PC sites: (1) A blue dot with clear edges visible to the naked eye indicates that the sampling, DNA extraction, PCR, and hybridization processes are normal. (2) No blue dot with clear edges visible to the naked eye, ① The type point is colored and is a blue dot with clear edges, the experiment is unsuccessful, and the sample should be resampled or re-extracted; ② The type point is not colored and appears as an individual case in the entire batch of experiments, indicating that there are problems in one or more of the sampling, DNA extraction, PCR, or hybridization processes, and the experiment is unsuccessful. The sample should be resampled or re-extracted.

[0034] (3) HPV-positive quality control product: CC point, PC point, and HPV18 typing site should be blue dots with clear edges visible to the naked eye; other sites should not show any color.

[0035] (4) Negative control: CC point should be a blue dot with clear edges visible to the naked eye; other sites should not show any color.

[0036] 5. Analysis of test results 1. Interpretation of results The membrane strips that meet the quality control requirements will be read for the next step of HPV type color spot results, which can be divided into: 1. Blank; 2. Faintly visible blue dots + type; 3. Lighter but clear-edged blue dots + type; 4. Clear-edged blue dots + type; Very dark blue dots + type with clear edges; Among them, readings 1 and 2 are negative results. If there is any objection to result 2, it is recommended to repeat the sample, and the amount of extracted samples and the amount of sample added should be increased. This result often occurs in mixed infections; readings 3, 4, and 5 are positive results.

[0037] The present invention can develop color through the blue dots on the hybridization membrane strip, and the test results are intuitive and easy to read, and can be interpreted without complex instruments or professional skills. This enables primary medical institutions and laboratories with fewer technical personnel to easily perform HPV testing. According to the color depth and edge clarity of the color development points, it is divided into different interpretation levels (blank, vaguely visible, lighter but clear, clear edges, very dark), which provides clinicians with a detailed reference basis and helps to more accurately assess the patient's infection status. It is not only suitable for cervical smear samples, but also can be used for self-collected vaginal swabs, anal swabs and other sample types. This flexibility allows the detection method to be applied to different clinical scenarios to meet diverse detection needs.

[0038] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting human papillomavirus, characterized in that: The following steps are involved:

1. Sample processing and DNA extraction: number the samples and put them into storage tubes; prepare 1.5-3mL centrifuge tubes with the same numbers; vortex the sample storage tubes for 10-30 seconds, transfer 1-1.2mL of storage solution to the corresponding numbered centrifuge tubes: centrifuge the numbered samples at 10000-12000rpm for 5-10 minutes, and discard the supernatant; add 1mL of sterile saline to the sample precipitate, wash the precipitate, centrifuge at 10000-12000rpm for 5-10 minutes, and discard the supernatant; take the pretreated sample cell precipitate, add 50-60 microliters of nucleic acid extraction or purification reagent, and oscillate to mix; lyse at 100℃ for 10-12 minutes; centrifuge at 10000-12000rpm for 5-7 minutes, take the supernatant for use, and store at -20℃; 2. PCR amplification: Take a PCR reaction tube, add 20 μL of the extracted sample DNA and HPV positive and negative quality control DNA respectively, centrifuge at 6000-6300rpm, transfer the transfer window of each reaction tube to the gene amplification room, and then put the reaction tube into the PCR instrument, and amplify it on the machine according to the following conditions: 50℃, 3 minutes, 95℃ pre-denaturation for 15 minutes, and then amplify 40 cycles in the order of 94℃, 40 seconds; 55℃, 40 seconds; 72℃, 40 seconds, and finally extend at 72℃ for 7 minutes; 3. Hybridization operation: (1) Prepare the reagents for hybridization operation; denature the PCR amplification product at 98℃ in a thermal cycler for 8-10 minutes, and then immediately place it in an ice-water mixture for 2-5 minutes; take a 5-15mL plastic centrifuge tube, put it into the HPV typing hybridization membrane strip marked with the sample number to be tested, add 4mL hybridization solution I and 40-50μL of the denatured PCR product corresponding to the membrane strip number, tighten the tube cap, write the corresponding sample number on the tube cap, and place the centrifuge tube flat in a 42℃ water bath shaker, rotate at a low speed of 100-150rpm, and hybridize for 1-1.2 hours; put hybridization solution II in a water bath shaker to preheat to 42℃ and set aside; (2) Binding: Take out the HPV typing hybridization membrane strips, wash them with clean water for 1-2 minutes, then transfer 5 membrane strips / tube into a 50mL centrifuge tube containing 20mL preheated hybridization solution II, add 20μL solution I, shake in a 42-45℃ water bath, and rotate at low speed for 10-12 minutes; (3) Washing the membrane: Take out the HPV typing hybridization membrane strip and transfer it into a 50 mL centrifuge tube filled with preheated hybridization solution II. Wash the membrane in a 42°C water bath shaker at low speed for 10-12 minutes. (4) Color development: Prepare the required color development solution according to the number of samples and the preparation method of the color development solution in the above table, put it into the washed HPV typing hybridization membrane strip, and color it in the dark at 20-25°C for 3-10 minutes. Take out the membrane strip and put it into 20 mL of solution II for 2-3 minutes to stop the color development. Discard solution II, add 20 mL of pure water, shake it gently for 2-3 minutes, and take it out; use absorbent paper to absorb the water on the surface of the membrane strip; (5) Preservation of membrane strip results; 4. Quality Control: (1) CC point: If it is a blue dot with clear edges visible to the naked eye, the binding and color development process is normal; otherwise, the binding and color development process needs to be rechecked; (2) PC sites: (1) A blue dot with clear edges visible to the naked eye indicates that the sampling, DNA extraction, PCR, and hybridization processes are normal; (2) No blue dot with clear edges visible to the naked eye, ① The type point is colored and is a blue dot with clear edges, the experiment is unsuccessful and the sample should be resampled or re-extracted; ② The type point is not colored and appears as an individual case in the entire batch of experiments, indicating that there are problems in one or more of the sampling, DNA extraction, PCR, or hybridization processes, and the experiment is unsuccessful. The sample should be resampled or re-extracted; (3) HPV positive quality control: CC point, PC point, and HPV18 typing site should be blue dots with clear edges visible to the naked eye; other sites should not show color; (4) Negative control: CC point should be a blue dot with clear edges visible to the naked eye; other sites should not show color; 5. Analysis of test results: The membrane strips that meet the quality control requirements will proceed to the next step of HPV type color point result interpretation, which can be divided into:

1. Blank; 2. Faintly visible blue dots + type; 3. Lighter but clear-edged blue dots + type; 4. Blue dots + type with clear edges; Very dark blue dots + type with clear edges.

2. The method for detecting human papillomavirus according to claim 1, characterized in that: The hybridization solution I is prepared by diluting 50 mL of hybridization solution I concentrate with 200 mL of purified water.

3. The method for detecting human papillomavirus according to claim 1, characterized in that: Hybridization solution II is prepared by diluting 30 mL of hybridization solution II concentrate with 120 mL of purified water.

4. The method for detecting human papillomavirus according to claim 1, characterized in that: Solution II is prepared by diluting 25 mL of Solution II concentrate with 100 mL of purified water.

5. The method for detecting human papillomavirus according to claim 1, characterized in that: The solution IV diluted 10 times is diluted by adding 900 μL of purified water to 100 μL of solution IV.

6. The method for detecting human papillomavirus according to claim 1, characterized in that: The color developing solution includes 3mL solution II+0.3mL solution III+1.5µl solution IV.

7. The method for detecting human papillomavirus according to claim 1, characterized in that: For each tube with 10mL of hybridization solution II, add 10μL of solution I, which can be used to combine 2-3 membrane strips; for each tube with 20mL of hybridization solution II, add 20μL of solution I, which can be used to combine 5 membrane strips.

8. The method for detecting human papillomavirus according to claim 1, characterized in that: Each tube of 20mL hybridization solution II can wash 2-3 membrane strips; each tube of 30mL hybridization solution II can wash 5 membrane strips. Each tube can wash a maximum of 5 membrane strips at a time.

9. The method for detecting human papillomavirus according to claim 1, characterized in that: The method for preserving the membrane strip results is as follows: stick the membrane strips on the experimental process record sheet according to the corresponding numbers, read the results and take a photo to save them in the result folder, cover the membrane strips with transparent tape, and then put the record sheet into a sealed bag and store it away from light.

10. A human papillomavirus detection kit, characterized in that: The kit comprises hybridization solution I, hybridization solution II, solution II, solution IV diluted 10 times and color developing solution; the hybridization solution I is prepared by diluting 50 mL of hybridization solution I concentrate with 200 mL of purified water; the hybridization solution II is prepared by diluting 30 mL of hybridization solution II concentrate with 120 mL of purified water; the solution II is prepared by diluting 25 mL of solution II concentrate with 100 mL of purified water; the solution IV diluted 10 times is prepared by diluting 100 μL of solution IV with 900 μL of purified water; the color developing solution comprises 3 mL of solution II+0.3 mL of solution III+1.5 μl of solution IV.

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