Test strip for early screening of HPV (human papillomavirus) nucleic acid and preparation method of test strip

Abstract

The invention relates to the technical field of oil-control shampoo, and particularly discloses a test strip for early screening of HPV nucleic acid and a preparation method thereof.The test strip comprises a PVC backboard, a sample pad and a water absorption pad are arranged at the two ends of the top of the PVC backboard respectively, an NC film is arranged in the middle of the top of the PVC backboard, one side of the top of the NC film is in extrusion contact with the bottom of the water absorption pad, and the other side of the top of the NC film is in extrusion contact with the bottom of the water absorption pad; a combination pad is arranged on the other side of the top of the NC membrane in an extrusion contact manner, and one side of the top of the combination pad is in extrusion contact with the bottom of the sample pad. The invention provides a test strip for early screening of HPV (human papilloma virus), which aims at HPV nucleic acid and is developed on the basis of a colloidal gold technology. The test strip is used for solving the problems of complicated operation, dependence on professional instruments and personnel, high detection cost, incapability of meeting the requirement on convenience of early screening and the like in the existing HPV detection technology. Through the method, rapid and accurate detection of the HPV can be realized, and the method is suitable for large-scale early screening including scenes such as home self-inspection and the like.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a test strip for early screening of HPV nucleic acid and its preparation method. Background Technology

[0002] Human papillomavirus (HPV) is a double-stranded circular small DNA virus with epithelial tissue affinity. It primarily infects human skin and mucous membranes, inducing abnormal proliferation and is closely related to tumorigenesis. In clinical practice, HPV testing is crucial for the early screening and prevention of cervical cancer.

[0003] Currently, HPV testing technologies mainly cover the following categories: 1. Immunohistochemical detection is a commonly used clinical method. It is simple to operate and has a low cost, but it has problems such as difficulty in interpretation and susceptibility to human factors, which may lead to false positive results. 2. Nucleic acid detection methods are also widely used, including PCR, quantitative real-time PCR, and in situ hybridization (ISH). PCR uses universal primers to target and amplify HPV DNA sequences, followed by gel electrophoresis, restriction fragment length polymorphism (RFLP) analysis, or direct sequencing to identify genotypes, exhibiting good specificity and sensitivity. Quantitative real-time PCR is considered the gold standard for HPV detection due to its high sensitivity and specificity, but its cost is relatively high. ISH utilizes specific labeled probes to hybridize with DNA in tissue sections, allowing direct observation of targeted DNA within cells; however, it relies on surgically removed tissue samples and is an invasive procedure. Furthermore, methods that assess infection status by detecting HPV DNA or HPV E6 / L1 antibodies in a patient's peripheral blood also demonstrate good sensitivity and specificity.

[0004] However, the above detection methods generally have the following limitations: ① All of them require expensive testing instruments and rely on specific experimental environments and professional technicians to operate, making it difficult to achieve home testing, and the operation process is also quite complicated; ②Most mainstream HPV testing reagents are liquid reagents, which require specific temperature conditions for storage and transportation and have high requirements for cold chain logistics. This not only increases the complexity of storage and transportation, but also increases the risk of reagent loss. ③Most commercially available HPV test kits are geared towards laboratories. After sample lysis, they require complex nucleic acid purification steps and cannot be used directly for testing.

[0005] Therefore, there is an urgent need for an HPV testing method that is easy to operate, highly accurate, low in cost, and suitable for early screening. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a test strip for early HPV nucleic acid screening and its preparation method, thereby resolving the problems mentioned in the background section.

[0007] The present invention solves the technical problem by adopting the following technical solution: This invention provides a test strip for early HPV nucleic acid screening and its preparation method, comprising a PVC backing plate, a sample pad and an absorbent pad respectively disposed at both ends of the top of the PVC backing plate, an NC membrane disposed in the middle of the top of the PVC backing plate, one side of the top of the NC membrane being in pressure contact with the bottom of the absorbent pad, and a conjugate pad disposed in pressure contact with the other side of the top of the NC membrane, the top side of the conjugate pad being in pressure contact with the bottom of the sample pad; The conjugation pad is provided with colloidal gold and HPV thiol-probe 1; HPV-probe 2 and HPV-probe 3 are disposed on the NC membrane.

[0008] Preferably, the method for preparing the conjugate pad is as follows: S1: Using a 100ml solution limit, take 0.2g of Triton X-100 (polyethylene glycol octylphenyl ether) and 0.1xSSC (sodium sodium citrate hydrochloride: take 0.088g sodium chloride and 0.044g sodium citrate, dissolve in 100ml of ultrapure water) and mix them to form a pretreatment solution with a pH of 7.0. Soak the conjugate pad in the pretreatment solution for 10min and then dry it at 37℃ for 1 hour (humidity <30%). S2: Take 1g BSA (standard solution), 0.05g Tween-20 (polysorbate-20), 5g sucrose, and 0.01g... (Dissolve in 100ml PBS (phosphate buffer solution) and mix to form a protective solution. Place the colloidal gold in the protective solution and store it. S3: Dissolve HPV thiol-probe 1 in TE buffer (pH 8.0) and incubate in the dark for 30 min; S4: Add 0.01M drop by drop. Add the colloidal gold to the protective solution and adjust the pH to 8.5; S5: Mix colloidal gold: HPV thiol-probe 1 = 1 ml: 40 μL (100 μM) and incubate with shaking at 37°C for 1 h; S6: Add the solution of S2 to the solution of S5, centrifuge at 12000 rpm for 30 minutes, discard the supernatant, resuspend in the protective solution to achieve purification; S7: Drop the purified colloidal gold-coupled nucleic acid probe mixture onto the binding pad (nitrocellulose membrane) and dry it in a 37°C oven for 1 hour until the binding pad changes from red to purple. The prepared binding pad is then obtained.

[0009] Preferably, the method for preparing the NC membrane is as follows: S1: Soak the NC membrane in 100ml of 0.1xSSC (sodium citrate hydrochloride buffer: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) for 10 minutes, and then dry it at 37℃ for 1 hour. S2: Remove the NC membrane, spray HPV-probe 2 at 1μL / cm, and capture the T line; S3: Spray HPV-probe 3, 1μL / cm, to capture C-line; S4: Place the NC film coated with probe 2 and probe 3 at a height of 10cm under a UV lamp and irradiate for 15 minutes. S5: Dissolve 5g BSA (standard solution) and 0.1mg salmon sperm DNA in 100ml PBS to form a blocking solution. Soak the NC membrane obtained in S4 in the blocking solution for 30min, and then dry it at 37℃ for 1h.

[0010] Preferably, a PVC backing board is used as the base, with adhesive on the top of the PVC backing board. The NC film is placed in the middle of the top of the PVC backing board, an absorbent pad is pressed on one side of the NC film, a bonding pad is pressed on the other side of the NC film, and a sample pad is pressed on top of the bonding pad. Then, the components are joined together by using rollers to compact them. The sample pad is made of glass fiber membrane, and the absorbent pad is made of pure cotton fiber.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. Simple Operation: No complex instruments or equipment are required; only simple sampling tools, nucleic acid release reagents, isothermal amplification reagents, and colloidal gold immunochromatographic test strips are needed to complete the test. The entire testing process has few steps and does not require professional technicians, making it suitable for large-scale screening and home self-testing. 2. High Accuracy: The combined application of nucleic acid release, colloidal gold chromatography, and isothermal amplification technologies improves the sensitivity and specificity of the detection. Isothermal amplification specifically amplifies the target HPV nucleic acid, avoiding interference from other non-target nucleic acids, while exponentially increasing the target sequence to enhance detection sensitivity. The colloidal gold chromatography test strip captures the amplification products using a dual-nucleic acid probe sandwich method, further improving detection accuracy and reducing false positives and false negatives. Simultaneously, a quality control area is set up during the detection process to monitor quality and ensure the reliability of the results. 3. Low cost: The reagents and materials used in this invention are relatively inexpensive, and it does not require expensive instruments or complex experimental environments. The nucleic acid releasing agent can be transported and stored at room temperature, reducing transportation and storage costs. Furthermore, the detection method of this invention is simple to operate, has a short detection time, and can complete the detection of a large number of samples in a short period of time, improving detection efficiency and further reducing detection costs, making it suitable for large-scale application. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the structure of a test strip for early screening of HPV nucleic acid. Figure 2 This is a schematic diagram of the test results for a test strip used for early screening of HPV nucleic acid.

[0013] Explanation of reference numerals in the attached figures 1. PVC backing; 2. Sample pad; 3. HPV probe 2; 4. HPV probe 3; 5. Absorbent pad; 6. Conjugate pad; 7. NC membrane. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] This invention provides a test strip for early HPV nucleic acid screening and its preparation method, achieved through the following technical solution: (1) HPV thiol-probe 1 is an inverse complementary sequence designed for the 167-185bp nucleic acid sequence of HPV16E6. At the 3' end of the inverse complementary sequence to the target fragment, this scheme designs a random nucleic acid sequence that does not have homology with HPV or mammalian cells. The inverse complementary sequence of the target nucleic acid and the random nucleic acid sequence together constitute probe 1. Probe 1 is then modified with a 5' thiol group and chemically bonded to colloidal gold to form a colloidal gold-nucleic acid probe 1 complex, which is immobilized on the binding pad. When the target nucleic acid fragment is present in the sample to be tested, the inverse complementary sequence at the 5' end of probe 1 will specifically bind to the target fragment, forming a colloidal gold-nucleic acid probe 1-target nucleic acid complex.

[0016] (2) HPV-probe 2 is a reverse complementary sequence designed for the nucleic acid sequence of HPV16E6433-457bp. This probe is immobilized on the NC membrane by UV crosslinking and serves as a detection line. When the colloidal gold-nucleic acid probe-target nucleic acid complex passes through the detection line, probe 2 will specifically bind to the target fragment in the complex, thereby causing the colloidal gold to aggregate and develop color.

[0017] (3) HPV-probe 3 is a corresponding reverse complementary nucleic acid fragment designed for the random nucleic acid fragment at the 3' end of probe 1. It is fixed in the back half of the NC membrane by UV crosslinking, becoming the control line. When the colloidal gold-nucleic acid probe 1 complex is chromatographyd to the control line, probe 3 will recognize and specifically bind to the random nucleic acid fragment at the 3' end of nucleic acid probe 1 in the complex, thereby causing the colloidal gold to aggregate and develop color. The principle of color development is as follows: when the target HPV nucleic acid is present in the sample, after the nucleic acid release step, the nucleic acid product binds to the colloidal gold-labeled probe (complementary nucleic acid sequence) to form a complex. When the complex passes through the "detection line (T line)" of the test strip, it will be captured by the capture probe fixed on the T line and aggregate to form a visible band; the unbound colloidal gold probe is captured by the "control line (C line)" to form a visible band (used to verify the effectiveness of the test strip).

[0018] The optimal concentration ratio of probe 1, probe 2, and probe 3 was obtained through experiments in the following examples: Example 1 A test strip for early screening of HPV nucleic acid includes a PVC backing plate. A sample pad and an absorbent pad are respectively disposed at the top two ends of the PVC backing plate. An NC membrane is disposed in the middle of the top of the PVC backing plate. One side of the top of the NC membrane is in pressure contact with the bottom of the absorbent pad, and a conjugate pad is disposed in pressure contact with the other side of the top of the NC membrane. One side of the top of the conjugate pad is in pressure contact with the bottom of the sample pad. Colloidal gold and HPV thiol-probe 1 are disposed on the conjugate pad. HPV-probe 2 and HPV-probe 3 are disposed on the NC membrane.

[0019] The preparation method of the conjugate pad is as follows: S1: Using a 100ml solution limit, mix 0.2g Triton X-100 (polyethylene glycol octylphenyl ether) and 0.1x SSC (sodium sodium citrate hydrochloride: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) to form a pretreatment solution with a pH of 7.0. Soak the conjugation pad in this pretreatment solution for 10 minutes, then dry at 37℃ for 1 hour (humidity <30%). S2: Take 1g BSA (standard solution), 0.05g Tween-20 (polysorbate-20), 5g sucrose, and 0.01g... (Dissolve in 100ml PBS (phosphate buffer)) Mix and stir to form a protective solution, and store the colloidal gold in the protective solution; S3: Dissolve HPV thiol-probe 1 in TE buffer (pH 8.0) and incubate in the dark for 30 min; S4: Add 0.01M- dropwise. Add the colloidal gold to the protective solution and adjust the pH to 8.5; S5: Mix colloidal gold: HPV thiol-probe 1 = 1 ml: 40 μL (100 μM), add 1 / μL of HPV thiol-probe 1 and 200 / μL of colloidal gold, and incubate with shaking at 37°C for 1 h. S6: Add the solution of S2 to the solution of S5, centrifuge at 12000 rpm for 30 minutes, discard the supernatant, and resuspend in the protective solution to achieve purification; S7: Drop the purified colloidal gold-coupled nucleic acid probe mixture onto the conjugate pad (nitrocellulose membrane), and dry in an oven at 37℃ for 1 hour until the conjugate pad changes from red to purple to obtain the prepared conjugate pad.

[0020] The preparation method of NC membrane is as follows: S1: Soak the NC membrane in 100ml of 0.1xSSC (sodium citrate hydrochloride buffer: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) for 10 minutes, and then dry it at 37℃ for 1 hour. S2: Remove the NC membrane, spray HPV-probe 2 at 5μL / cm, and capture the T line; S3: Spray HPV-probe 3, 5μL / cm, to capture C-line; S4: Place the NC film coated with probe 2 and probe 3 at a height of 10cm under a UV lamp and irradiate for 15 minutes. S5: Dissolve 5g BSA (standard solution) and 0.1mg salmon sperm DNA in 100ml PBS to form a blocking solution. Soak the NC membrane obtained in S4 in the blocking solution for 30min, and then dry it at 37℃ for 1h.

[0021] Example 2 A test strip for early screening of HPV nucleic acid includes a PVC backing plate. A sample pad and an absorbent pad are respectively disposed at the top two ends of the PVC backing plate. An NC membrane is disposed in the middle of the top of the PVC backing plate. One side of the top of the NC membrane is in pressure contact with the bottom of the absorbent pad, and a conjugate pad is disposed in pressure contact with the other side of the top of the NC membrane. One side of the top of the conjugate pad is in pressure contact with the bottom of the sample pad. Colloidal gold and HPV thiol-probe 1 are disposed on the conjugate pad. HPV-probe 2 and HPV-probe 3 are disposed on the NC membrane.

[0022] The preparation method of the conjugate pad is as follows: S1: Using a 100ml solution limit, mix 0.2g Triton X-100 (polyethylene glycol octylphenyl ether) and 0.1x SSC (sodium sodium citrate hydrochloride: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) to form a pretreatment solution with a pH of 7.0. Soak the conjugation pad in this pretreatment solution for 10 minutes, then dry at 37℃ for 1 hour (humidity <30%). S2: Take 1g BSA (standard solution), 0.05g Tween-20 (polysorbate-20), 5g sucrose, and 0.01g... (Dissolve in 100ml PBS (phosphate buffer)) Mix and stir to form a protective solution, and store the colloidal gold in the protective solution; S3: Dissolve HPV thiol-probe 1 in TE buffer (pH 8.0) and incubate in the dark for 30 min; S4: Add 0.01M- dropwise. Add the colloidal gold to the protective solution and adjust the pH to 8.5; S5: Mix colloidal gold: HPV thiol-probe 1 = 1 ml: 40 μL (100 μM), add 2 μL of HPV thiol-probe 1 and 200 μL of colloidal gold, and incubate with shaking at 37°C for 1 h. S6: Add the solution of S2 to the solution of S5, centrifuge at 12000 rpm for 30 minutes, discard the supernatant, and resuspend in the protective solution to achieve purification; S7: Drop the purified colloidal gold-coupled nucleic acid probe mixture onto the conjugate pad (nitrocellulose membrane), and dry in an oven at 37℃ for 1 hour until the conjugate pad changes from red to purple to obtain the prepared conjugate pad.

[0023] The preparation method of NC membrane is as follows: S1: Soak the NC membrane in 100ml of 0.1xSSC (sodium citrate hydrochloride buffer: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) for 10 minutes, and then dry it at 37℃ for 1 hour. S2: Remove the NC membrane, spray HPV-probe 2 at 10 μL / cm, and capture the T line; S3: Spray HPV-probe 3 at 10 μL / cm to capture C-line; S4: Place the NC film coated with probe 2 and probe 3 at a height of 10cm under a UV lamp and irradiate for 15 minutes. S5: Dissolve 5g BSA (standard solution) and 0.1mg salmon sperm DNA in 100ml PBS to form a blocking solution. Soak the NC membrane obtained in S4 in the blocking solution for 30min, and then dry it at 37℃ for 1h.

[0024] Example 3 A test strip for early screening of HPV nucleic acid includes a PVC backing plate. A sample pad and an absorbent pad are respectively disposed at the top two ends of the PVC backing plate. An NC membrane is disposed in the middle of the top of the PVC backing plate. One side of the top of the NC membrane is in pressure contact with the bottom of the absorbent pad, and a conjugate pad is disposed in pressure contact with the other side of the top of the NC membrane. One side of the top of the conjugate pad is in pressure contact with the bottom of the sample pad. Colloidal gold and HPV thiol-probe 1 are disposed on the conjugate pad. HPV-probe 2 and HPV-probe 3 are disposed on the NC membrane.

[0025] The preparation method of the conjugate pad is as follows: S1: Using a 100ml solution limit, mix 0.2g Triton X-100 (polyethylene glycol octylphenyl ether) and 0.1x SSC (sodium sodium citrate hydrochloride: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) to form a pretreatment solution with a pH of 7.0. Soak the conjugation pad in this pretreatment solution for 10 minutes, then dry at 37℃ for 1 hour (humidity <30%). S2: Take 1g BSA (standard solution), 0.05g Tween-20 (polysorbate-20), 5g sucrose, and 0.01g... (Dissolve in 100ml PBS (phosphate buffer)) Mix and stir to form a protective solution, and store the colloidal gold in the protective solution; S3: Dissolve HPV thiol-probe 1 in TE buffer (pH 8.0) and incubate in the dark for 30 min; S4: Add 0.01M- dropwise. Add the colloidal gold to the protective solution and adjust the pH to 8.5; S5: Mix colloidal gold: HPV thiol-probe 1 = 1 ml: 40 μL (100 μM), add 3 μL of HPV thiol-probe 1 and 200 μL of colloidal gold, and incubate with shaking at 37°C for 1 h. S6: Add the solution of S2 to the solution of S5, centrifuge at 12000 rpm for 30 minutes, discard the supernatant, and resuspend in the protective solution to achieve purification; S7: Drop the purified colloidal gold-coupled nucleic acid probe mixture onto the conjugate pad (nitrocellulose membrane), and dry in an oven at 37℃ for 1 hour until the conjugate pad changes from red to purple to obtain the prepared conjugate pad.

[0026] The preparation method of NC membrane is as follows: S1: Soak the NC membrane in 100ml of 0.1xSSC (sodium citrate hydrochloride buffer: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) for 10 minutes, and then dry it at 37℃ for 1 hour. S2: Remove the NC membrane, spray HPV-probe 2 at 20 μL / cm, and capture the T line; S3: Spray HPV-probe 3 at 20 μL / cm to capture C-line; S4: Place the NC film coated with probe 2 and probe 3 at a height of 10cm under a UV lamp and irradiate for 15 minutes. S5: Dissolve 5g BSA (standard solution) and 0.1mg salmon sperm DNA in 100ml PBS to form a blocking solution. Soak the NC membrane obtained in S4 in the blocking solution for 30min, and then dry it at 37℃ for 1h.

[0027] Comparative Example 1: Unlike Examples 1-3, HPV thiol-probe 1 was added at 4 μL, HPV-probe 2 at 40 μL, and HPV-probe 3 at 40 μL. Comparative Example 2: Unlike Examples 1-3, HPV thiol-probe 1 was added at 5 μL, HPV-probe 2 at 60 μL, and HPV-probe 3 at 60 μL. Comparative Example 3: Unlike Examples 1-3, HPV thiol-probe 1 was added at 6 μL, HPV-probe 2 at 70 μL, and HPV-probe 3 at 70 μL. Comparative Example 4: Unlike Examples 1-3, HPV thiol-probe 1 was added at 7 μL, HPV-probe 2 at 80 μL, and HPV-probe 3 at 80 μL. Comparative Example 5: Unlike Examples 1-3, HPV thiol-probe 1 was added at 8 μL, HPV-probe 2 at 100 μL, and HPV-probe 3 at 100 μL.

[0028] 1. Optimal ratio of probe 1 to colloidal gold coupling Based on the data in the table above, and the analysis of Examples 1-3 and Comparative Examples 1-4, it is found that in the implementation of this invention, the optimal concentration of probe 1 is 8 μL of 0.1 mmol / L probe 1 coupled with 200 μL of colloidal gold at 37°C for 2 hours.

[0029] 2. Experiment on the optimal concentration of probe 2 Note: + indicates color development; - indicates no color development.

[0030] Based on the data in the table above, and the analysis of Examples 1-3 and Comparative Examples 1-4, it is found that the optimal concentration of probe 2 in the implementation of this invention is 80 μmmol / L.

[0031] 3. Experiment on the optimal concentration of probe 3 Note: + indicates color development; - indicates no color development.

[0032] Based on the data in the table above, and the analysis of Examples 1-3 and Comparative Examples 1-4, it is found that the optimal concentration of probe 3 in the implementation of this invention is 100 μmmol / L.

[0033] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0034] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A test strip for early screening of HPV nucleic acid, characterized in that, The device includes a PVC backing plate, with a sample pad and an absorbent pad respectively disposed at both ends of the top of the PVC backing plate. An NC membrane is disposed in the middle of the top of the PVC backing plate. One side of the top of the NC membrane is in pressure contact with the bottom of the absorbent pad, and a bonding pad is disposed in pressure contact with the other side of the top of the NC membrane. One side of the top of the bonding pad is in pressure contact with the bottom of the sample pad. The conjugation pad is provided with colloidal gold and HPV thiol-probe 1; HPV-probe 2 and HPV-probe 3 are disposed on the NC membrane.

2. The test strip for early HPV nucleic acid screening according to claim 1, characterized in that, The method for preparing the conjugate pad is as follows: S1: Using a 100ml solution limit, take 0.2g of Triton X-100 (polyethylene glycol octylphenyl ether) and 0.1xSSC (sodium sodium citrate hydrochloride: take 0.088g sodium chloride and 0.044g sodium citrate, dissolve in 100ml of ultrapure water) and mix them to form a pretreatment solution with a pH of 7.

0. Soak the conjugate pad in the pretreatment solution for 10min and then dry it at 37℃ for 1 hour (humidity <30%). S2: Take 1g BSA (standard solution), 0.05g Tween-20 (polysorbate-20), 5g sucrose, and 0.01g... (Dissolve in 100ml PBS (phosphate buffer solution) and mix to form a protective solution. Place the colloidal gold in the protective solution and store it. S3: Dissolve HPV thiol-probe 1 in TE buffer (pH 8.0) and incubate in the dark for 30 min; S4: Add 0.01M drop by drop. Add the colloidal gold to the protective solution and adjust the pH to 8.5; S5: Mix colloidal gold: HPV thiol-probe 1 = 1 ml: 40 μL (100 μM) and incubate with shaking at 37°C for 1 h; S6: Add the solution of S2 to the solution of S5, centrifuge at 12000 rpm for 30 minutes, discard the supernatant, resuspend in the protective solution to achieve purification; S7: Drop the purified colloidal gold-coupled nucleic acid probe mixture onto the binding pad (nitrocellulose membrane) and dry it in an oven at 37°C for 1 hour until the binding pad changes from red to purple. The prepared binding pad can then be obtained.

3. The test strip for early HPV nucleic acid screening according to claim 1, characterized in that, The method for preparing the NC membrane is as follows: S1: Soak the NC membrane in 100ml of 0.1xSSC (sodium citrate hydrochloride buffer: dissolve 0.088g sodium chloride and 0.044g sodium citrate in 100ml of ultrapure water) for 10 minutes, and then dry it at 37℃ for 1 hour. S2: Remove the NC membrane, spray HPV-probe 2 at 1μL / cm, and capture the T line; S3: Spray HPV-probe 3, 1μL / cm, to capture C-line; S4: Place the NC film coated with probe 2 and probe 3 at a height of 10cm under a UV lamp and irradiate for 15 minutes. S5: Dissolve 5g BSA (standard solution) and 0.1mg salmon sperm DNA in 100ml PBS to form a blocking solution. Soak the NC membrane obtained in S4 in the blocking solution for 30min, and then dry it at 37℃ for 1h.

4. A method for manufacturing a test strip for early HPV nucleic acid screening according to any one of claims 1-3, characterized in that, Using a PVC backing as the base, with adhesive on the top of the PVC backing, place the NC film in the middle of the top of the PVC backing, press an absorbent pad on one side of the NC film, press a bonding pad on the other side of the NC film, press a sample pad on top of the bonding pad, and then use rollers to compact the components together. The sample pad is made of glass fiber membrane, and the absorbent pad is made of pure cotton fiber.

5. The method of using a test strip for early HPV nucleic acid screening according to any one of claims 1-3, characterized in that, S1: Sample Collection: Use a specialized sampling tool, such as a cotton swab, to collect samples from suspected infection sites (such as the vaginal mucosa, cervix, etc.). Specifically, use a cotton swab to scrape the vaginal mucosa back and forth 6-10 times to obtain sufficient mucosal epithelial cells as a test sample. S2: Place the cotton swab containing the collected sample into the tube containing the nucleic acid release agent; The nucleic acid releasing agent is prepared as follows: 2 mmol Tris (tris(hydroxymethyl)aminomethane) (pH 8.0), 2.5 mmol NaCl (sodium chloride), 0.25 mmol EDTA (ethylenediaminetetraacetic acid), and 0.05 g SDS (sodium dodecyl sulfate) are mixed and stirred to form a lysis buffer. 0.05 g SDS refers to 0.05 g SDS dissolved in 100 ml of solution. Under the action of this lysis buffer, the cells in the sample are lysed, releasing the nucleic acid. Then, a filter paper is immersed in the lysis buffer to bind the nucleic acid. After 3 seconds, the filter paper is immersed in a washing buffer (100 ml, for example), which includes 1 mmol Tris (tris(hydroxymethyl)aminomethane) at pH 8.0 and 0.1 g Tween-20 (polysorbate-20). After washing for 3 seconds, the filter paper is placed in a room-temperature nucleic acid amplification tube. This step enables direct and rapid lysis of the sample at room temperature, requiring only one step for subsequent amplification steps, eliminating the need for complex nucleic acid purification processes. S3: Isothermal Amplification: In a room-temperature nucleic acid amplification tube, add a specific isothermal amplification reagent containing primers targeting high-risk HPV type 16 (HPV16E6-F1: 5'-GAGATTCACCTTCATTGCATGAAT-3'; HPV16E6-R1: 5'-TCCTAGATTATGGTATCTGAGATACA-3'), recombinase, single-stranded binding protein, stranded DNA polymerase, PCR mix, and double-distilled water. Place filter paper containing the sample nucleic acid into this amplification system and incubate at 37°C for 30 minutes to specifically amplify the target HPV nucleic acid fragment, obtaining the PCR-amplified test solution. S4: Add 80 μL of the processed solution after isothermal amplification to the sample pad of the test strip, and perform chromatography at room temperature for 10 minutes. The results are as follows: if both the T line (virus target) and the C line are colored, it is positive; if only the C line is colored, it is negative; if the C line is not colored, the test is invalid.

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