HPV (human papillomavirus) household self-test kit based on CRISPR / Cas12 and LAMP (loop-mediated isothermal amplification) technology

Abstract

The invention relates to the technical field of nucleic acid detection, and discloses an HPV (human papillomavirus) home self-test kit based on CRISPR / Cas12 and LAMP (loop-mediated isothermal amplification) technologies, comprising: a nucleic acid extraction module; the specific amplification module comprises an isothermal amplification reagent and an HPV16 specific LAMP (Loop-Mediated Isothermal Amplification) primer group; an HPV18 specific LAMP (loop-mediated isothermal amplification) primer group; the signal activation module comprises a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas12a / crRNA ( An ssDNA reporter probe; and the detection module comprises a lateral chromatography test strip. The HPV home self-test kit based on CRISPR / Cas12a and LAMP technology provided by the invention does not need complex instruments, has high sensitivity, has the remarkable advantages of extremely simple operation, efficient amplification, visual direct reading and strong interference resistance, and provides a full-chain solution with high integration level for on-site real-time detection.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection technology, and in particular to an HPV home self-testing kit based on CRISPR / Cas12 and LAMP technology. Background Art

[0002] Persistent infection with high-risk human papillomavirus (HPV) is a necessary cause of cervical cancer, with HPV16 and HPV18 accounting for 50% and 20% of cervical cancer cases, respectively.

[0003] HPV DNA testing currently includes PCR-based detection technology, which has the following limitations: (1) High dependence on equipment: Precision temperature control equipment (thermal cycler) and fluorescence detection module are required, and the equipment purchase cost exceeds US$50,000; (2) High operational complexity: Involving multiple steps such as nucleic acid extraction, amplification, electrophoresis / fluorescence detection, the entire process takes 3-5 hours and requires professional training; (3) Cross-contamination risk: Open tube operation is prone to aerosol contamination, and the laboratory environment must reach BSL-2 level; (4) Limited typing ability: Multiplex PCR can distinguish up to 12 high-risk types, but primer cross-reaction leads to decreased specificity. Loop-mediated isothermal amplification (LAMP) technology has the following limitations: (1) Nonspecific amplification: Complex primer design (requires 6 specific regions), easy to form primer dimers or non-target amplification, and the false positive rate can reach 15%; (2) Sensitivity fluctuation: Inhibitors in clinical samples (such as hemoglobin and mucin) are easy to interfere Bst DNA polymerase activity, resulting in a 10-100-fold increase in the detection limit; (3) Subjective interpretation of results: Relying on turbidity changes or fluorescent dye visual inspection, low-concentration samples are prone to false negatives. CRISPR / Cas diagnostic systems have obvious shortcomings: (1) Equipment bundling: Most systems rely on fluorescent reporter molecules (such as FAM / HEX) and require a fluorescent plate reader or microscopic imaging device; (2) Difficulty in multiplex detection: The coexistence of multiple crRNAs in a single reaction system may trigger nonspecific activation, resulting in false positive signals. Lateral flow test strips face fundamental limitations in HPV detection: (1) Insufficient sensitivity: The detection limit of direct detection of HPV DNA is usually >1 μM, far below clinical needs; (2) Inability to distinguish subtypes: Antibody-capture-based detection modes have difficulty achieving specific recognition of HPV16 / 18; (3) Signal interference: Sample matrices (such as mucin in cervical exfoliated cells) are prone to nonspecific binding, with background color development rates as high as 25%.

[0004] In summary, existing detection technologies face severe challenges in practical applications: (1) Imbalance in resource accessibility: Traditional laboratory detection methods (such as PCR and hybridization capture) rely on high-end instruments and professional technicians, resulting in screening coverage rates of less than 30% in low-income areas; (2) Timeliness bottleneck: It usually takes 3-7 days from sample collection to report acquisition, delaying the opportunity for early intervention; (3) Typing detection needs: The risk of cancer in HPV16 / 18-infected people is significantly higher than that of other subtypes, but most existing technologies cannot achieve accurate typing; (4) Cost control pressure: The cost of a single test of a commercial HPV detection kit is as high as US$20-50, which is difficult to meet the needs of large-scale screening.

[0005] Therefore, it is urgent to develop an HPV home self-test kit that is rapid, highly sensitive, accurately typing and low-cost, so as to solve the impossible triangle dilemma of "sensitivity-specificity-convenience" that HPV detection technology has always faced. Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] Another object of the present invention is to provide an HPV home self-test kit based on CRISPR / Cas12 and LAMP technology, which does not require complex instruments, has high sensitivity, and has the significant advantages of extremely simple operation, efficient amplification, visual direct reading, and strong anti-interference, providing a highly integrated full-chain solution for on-site instant detection.

[0008] Another object of the present invention is to provide a home self-test for HPV based on CRISPR / Cas12 and LAMP technology. Kit, including: Nucleic acid extraction module; A specific amplification module, comprising a constant temperature amplification reagent, an HPV16-specific LAMP primer set, whose polynucleotide sequences are shown in SEQ ID NO. 1 to SEQ ID NO. 5; and an HPV18-specific LAMP primer set, whose polynucleotide sequences are shown in SEQ ID NO. 6 to SEQ ID NO. 11; A signal activation module comprising a CRISPR / Cas12a / crRNA complex system, wherein the polynucleotide sequence of a crRNA specific for HPV16 is shown in SEQ ID NO. 12; the polynucleotide sequence of a crRNA specific for HPV18 is shown in SEQ ID NO. 13; and the polynucleotide sequence of an ssDNA reporter probe specifically modified with digoxigenin and biotin at both ends is shown in SEQ ID NO. 14; and The detection module includes a lateral flow test strip, which includes a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad arranged in sequence on a PVC base plate, wherein the conjugation pad is sprayed with gold nanoparticles modified with digoxigenin antibodies, and the nitrocellulose membrane is sequentially coated with a quality control line sprayed with streptavidin and a detection line sprayed with a heterologous secondary antibody.

[0009] Preferably, when isothermal amplification is performed using a specific module, an HPV16-specific LAMP primer premix and an HPV18-specific LAMP primer premix are prepared separately and set aside; The HPV16-specific LAMP primer premix includes 16 μM inner primer FIP with a polynucleotide sequence as shown in SEQ ID NO.3, 16 μM inner primer BIP with a polynucleotide sequence as shown in SEQ ID NO.4, 4 μM loop primer LB with a polynucleotide sequence as shown in SEQ ID NO.5, 2 μM outer primer F3 with a polynucleotide sequence as shown in SEQ ID NO.1, 2 μM outer primer B3 with a polynucleotide sequence as shown in SEQ ID NO.2, and 5 μM nuclease-free water. The HPV18-specific LAMP primer premix includes 16 μM inner primer FIP with a polynucleotide sequence as shown in SEQ ID NO.8, 16 μM inner primer BIP with a polynucleotide sequence as shown in SEQ ID NO.9, 4 μM loop primer LB with a polynucleotide sequence as shown in SEQ ID NO.11, 2 μM outer primer F3 with a polynucleotide sequence as shown in SEQ ID NO.6, 2 μM outer primer B3 with a polynucleotide sequence as shown in SEQ ID NO.7, and nuclease-free water.

[0010] Preferably, the method further comprises: the nucleic acid extraction module comprises silanol-modified magnetic beads, virus lysis solution, and a magnetic stand or magnet.

[0011] Preferably, the nucleic acid extraction process using the nucleic acid extraction module is as follows: 1) Add 30 μL of Si-OH MBs and viral lysis buffer (LB) to a 1.5 mL EP tube and mix thoroughly. The viral lysis buffer (LB) contains 2.5 mM EDTA-Na2, 20 mM Tris, 4 M GuSCN, and 2 M NaCl, pH 6.5. 2) Add the sample to be tested into the above EP tube; 3) Vortex or invert to mix thoroughly and incubate at room temperature for 10 minutes to ensure that the virus particles in the sample are fully lysed; 4) Place the EP tube on a magnetic stand and wait for 15 seconds, or place a magnet at the bottom of the EP tube to allow the Si-OH MBs to move and aggregate at the bottom of the EP tube for magnetic separation. Afterwards, discard the supernatant. 5) Add 800-1000 μL of 80% ethanol to the EP tube from step 4) to wash away any residual LB or other impurities on the surface of the Si-OH MBs. Leave for 5 s and repeat step 4) to obtain a no-elution nucleic acid extract. Air-dry the extract at room temperature for later use.

[0012] Preferably, the minimum detection limit of the lateral flow test strip is 100 nM.

[0013] The present invention has at least the following beneficial effects: The HPV home self-test kit based on CRISPR / Cas12 and LAMP technology provided by the present invention is used. The CRISPR / Cas12-LAMP combination technology is used, combined with the elution-free characteristics of magnetic beads for efficient nucleic acid capture, fully leveraging the LAMP isothermal amplification and CRISPR / Cas12a cascade signal amplification mechanism, and achieving visual interpretation through colloidal gold test strips. The entire process does not require complex instruments, and on-site rapid testing can be completed within 1.5 hours. It also solves the false positive problem caused by the non-specific amplification of traditional LAMP; achieves specific differentiation detection of HPV16 / 18 dual subtypes; and increases the detection sensitivity of lateral flow test strips to 100 nM level.

[0014] In summary, the HPV home self-test kit based on CRISPR / Cas12 and LAMP technology provided by the present invention does not require complex instruments, has high sensitivity, and has the significant advantages of extremely simple operation, efficient amplification, visual direct reading, and strong anti-interference, providing a highly integrated full-chain solution for on-site instant detection.

[0015] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the structure of the lateral flow test strip in Example 1 of the present invention, wherein a positive test result is shown; Figure 2 Schematic diagram of the structure of the lateral flow test strip in Example 1 of the present invention, wherein a negative test result is shown; Figure 3 Schematic diagram of the structure of the lateral flow test strip in Example 2 of the present invention, wherein a positive test result is shown. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0018] It should be understood that terms such as “having,” “including,” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0019] Example 1 The specific steps for HPV16 specific testing are as follows: Step 1: Use the nucleic acid extraction module to perform Si-OH MBs-based elution-free nucleic acid enrichment Viral nucleic acid extraction is a critical step in the entire detection process. The Si-OH MBs method is a simple, low-cost, and rapid method for viral extraction. Si-OH MBs have a high specific surface area and can effectively adsorb nucleic acid molecules, achieving efficient enrichment. Compared to other nucleic acid extraction methods, this method can efficiently enrich nucleic acid molecules in low-concentration samples. The specific steps are as follows: (1) Add 30 μL of Si-OH MBs and virus lysis buffer (Lysis buffer, LB, containing 2.5 mM EDTA-Na2, 20 mM Tris, 4 M GuSCN and 2 M NaCl, pH 6.5) to a 1.5 mL EP tube and mix well.

[0020] (2) Add the sample to be tested into the above EP tube.

[0021] (3) Vortex or invert to mix well and incubate at room temperature for 10 min to ensure that the viral particles are fully lysed.

[0022] (4) Place the EP tube on a magnetic stand and wait for 15 seconds, or use a magnet to assist magnetic aggregation, to allow the Si-OH MBs to migrate and aggregate to the bottom of the EP tube. The solution gradually becomes clear, achieving magnetic separation, and the supernatant is slowly removed by aspiration.

[0023] (5) Add 800–1000 μL of freshly prepared 80% ethanol (aq) to the EP tube to wash away any LB or other impurities remaining on the surface of the Si-OH MBs. Leave for 5 s, repeat step (4) to achieve magnetic separation, and slowly remove the supernatant by aspiration.

[0024] (6) Open the lid and dry the Si-OH MBs at room temperature to ensure that the residual ethanol washing solution is removed; (7) Close the EP tube cap tightly and remove it from the magnetic stand for subsequent detection and analysis.

[0025] Step 2: Use a specific amplification module to perform LAMP reaction: (1) Prepare 10× LAMP primer premix: including 16 μM inner primer (FIP / BIP), 4 μM loop primer LB, 2 μM outer primers (F3 / B3), and nuclease-free water.

[0026] (2) Prepare LAMP amplification reaction solution (2×WarmStart LAMP reaction solution (with UDG): 12.5 μL, Mix 2.5 μL of 10× primer premix and 5 μL of nuclease-free water to obtain the LAMP amplification reaction solution. Seal the tube with an equal volume of mineral oil to eliminate bubbles.

[0027] (3) Add 40 μL of LAMP amplification reaction solution to the EP tube in step 1 (7), and amplify at 65°C for 30 min to obtain the LAMP amplification product.

[0028] Step 3: Use the signal activation module to activate the signal: (1) Mixed system: Add 2 μL of LAMP amplification product obtained in step 2 to a reaction tube containing the following components: 0.5 μL Cas12a enzyme (250 nM), 0.5 μL guide RNA (crRNA, 500 nM), 1 μL ssDNA reporter probe (8 nt, 500 nM), 15 μL Buffer 3.1 (reaction buffer), and 1 μL RNase inhibitor (to prevent RNA degradation).

[0029] Incubate the reaction at 47°C for 20 min to trigger the CRISPR / Cas12a reaction. (2) Signal activation module when target is present: Target recognition: crRNA specifically binds to the target sequence in the LAMP amplicon, activating the nuclease activity of Cas12a. Trans-cleavage activation: The activated Cas12a enzyme produces nonspecific cleavage activity, randomly cleaving the single-stranded DNA reporter in the system. Signal generation: The cleaved reporter is released. Core principle: LAMP amplification → CRISPR / Cas12a recognizes LAMP amplicon → trans-cleaves ssDNA reporter probe → fluorescence or LFA method detects signal.

[0030] Step 4: Use the detection module to perform typing detection: The sample activated by the signal obtained in step 3 is dropped onto the lateral flow test strip and the test result is positive, T line 6 is colored, while C line 7 is not colored. Figure 1As shown, the lateral flow test strip comprises a sample pad 2, a conjugate pad 3, a nitrocellulose membrane 4 and a water-absorbing pad 5 which are sequentially arranged on a PVC base plate 1.

[0031] Negative control experiment: The method and steps are the same as above. When the target is not present in the sample to be tested, the trans-cleavage activity of Cas12a is not activated, and the ssDNA reporter probe cannot be non-specifically cleaved. The uncut ssDNA reporter probe, after its digoxigenin-modified end binds to the gold nanoparticles modified with digoxigenin antibodies, flows to line C. At the same time, the biotin end of the ssDNA reporter probe binds to the streptavidin at line C, causing line C to develop color. However, since Cas12a is not activated and there is no cleavage product, the digoxigenin-antibody-modified gold nanoparticle complex cannot be captured at line T, so line T does not develop color or only shows weak color (the color depth is relatively lower than line C).

[0032] The specific principles are: When there is no target, the ssDNA reporter probe is not cut off. The ssDNA reporter probe combines with the gold nanoparticles modified with digoxigenin antibody to form a complex and flows to the C line. The biotin end of the ssDNA reporter probe binds to the streptavidin at the C line to develop color. That is, the ssDNA reporter probe is intercepted by the C line and no longer flows to the T line, so the T line does not develop color. Figure 2 shown.

[0033] When there is a target, the trans-cleavage activity of Cas12a in the sample to be tested is activated, non-specifically cleaving the ssDNA reporter probe. The digoxigenin modified on the ssDNA reporter probe binds to the gold nanoparticles modified with the digoxigenin antibody and flows through the C line together. When it continues to flow to the T line, it is captured by the heterologous secondary antibody and colored, while the C line does not show color or shows weak color (the color depth is relatively lower than the T line). Note: If part of the ssDNA reporter probe is not cut, the digoxigenin-modified end of the uncut ssDNA reporter probe binds to the gold nanoparticles modified with the digoxigenin antibody and flows to the C line. The biotin end of the ssDNA reporter probe binds to the streptavidin at the C line, so there may also be weak color at the C line.

[0034] Example 2 The specific steps for HPV18 specific testing are as follows: Step 1: Use the nucleic acid extraction module to perform Si-OH MBs-based elution-free nucleic acid enrichment Viral nucleic acid extraction is a critical step in the entire detection process. The Si-OH MBs method is a simple, low-cost, and rapid method for viral extraction. Si-OH MBs have a high specific surface area and can effectively adsorb nucleic acid molecules, achieving efficient enrichment. Compared to other nucleic acid extraction methods, this method can efficiently enrich nucleic acid molecules in low-concentration samples. The specific steps are as follows: (1) Add 30 μL of Si-OH MBs and virus lysis buffer (Lysis buffer, LB, containing 2.5 mM EDTA-Na2, 20 mM Tris, 4 M GuSCN and 2 M NaCl, pH 6.5) to a 1.5 mL EP tube and mix well.

[0035] (2) Add the sample to be tested into the above EP tube.

[0036] (3) Vortex or invert to mix well and incubate at room temperature for 10 min to ensure that the viral particles are fully lysed.

[0037] (4) Place the EP tube on a magnetic stand and wait for 15 seconds, or use a magnet to assist magnetic aggregation, to allow the Si-OH MBs to migrate and aggregate to the bottom of the EP tube. The solution gradually becomes clear, achieving magnetic separation, and the supernatant is slowly removed by aspiration.

[0038] (5) Add 800–1000 μL of freshly prepared 80% ethanol (aq) to the EP tube to wash away any LB or other impurities remaining on the surface of the Si-OH MBs. Leave for 5 s, repeat step (4) to achieve magnetic separation, and slowly remove the supernatant by aspiration.

[0039] (6) Open the lid and dry the Si-OH MBs at room temperature to ensure that the residual ethanol washing solution is removed; (7) Close the EP tube cap tightly and remove it from the magnetic stand for subsequent detection and analysis.

[0040] Step 2: Use a specific amplification module to perform LAMP reaction: (1) Prepare 10× LAMP primer premix: 16 μM inner primers (FIP / BIP), 4 μM loop primer LB, 2 μM outer primers (F3 / B3), and nuclease-free water.

[0041] (2) Prepare LAMP amplification reaction solution (2×WarmStart LAMP reaction solution (with UDG): 12.5 μL, 10× primer premix: 2.5 μL, nuclease-free water: 5 μL) and mix well to obtain LAMP amplification reaction solution. Seal the tube with an equal volume of mineral oil to eliminate bubbles.

[0042] (3) Add 40 μL of LAMP amplification reaction solution to the EP tube in step 1 (7), and amplify at 65°C for 30 min to obtain the LAMP amplification product.

[0043] Step 3: Use the signal activation module to activate the signal: (1) Mixed system: Add 2 μL of LAMP amplification product obtained in step 2 to a reaction tube containing the following components: 0.5 μL Cas12a enzyme (250 nM), 0.5 μL guide RNA (crRNA, 500 nM), 1 μL ssDNA reporter probe (8 nt, 500 nM), 15 μL Buffer 3.1 (reaction buffer), and 1 μL RNase inhibitor (to prevent RNA degradation).

[0044] Incubate the reaction at 47°C for 20 min to trigger the CRISPR / Cas12a reaction. (2) Signal activation module when the target is present: Target recognition: crRNA specifically binds to the target sequence in the LAMP amplicon, activating the nuclease activity of Cas12a. Trans-cleavage activation: The activated Cas12a enzyme produces nonspecific cleavage activity, randomly cleaving the single-stranded DNA reporter in the system. Signal generation: The cleaved reporter is released. Core principle: LAMP amplification → CRISPR / Cas12a recognizes LAMP amplicon → trans-cleaves ssDNA reporter probe → fluorescence or LFA method detects signal.

[0045] Step 4: Use the detection module to perform typing detection: The sample activated by the signal obtained in step 3 is dropped onto the lateral flow test strip and the test result is positive, the T line is colored, and the C line is weakly colored, such as Figure 3 shown.

[0046] Negative control experiment: The method and steps are the same as above. When the target is not present in the sample to be tested, the trans-cleavage activity of Cas12a is not activated, and the ssDNA reporter probe cannot be non-specifically cleaved. The uncut ssDNA reporter probe, after its digoxigenin-modified end binds to the gold nanoparticles modified with digoxigenin antibodies, flows to line C. At the same time, the biotin end of the ssDNA reporter probe binds to the streptavidin at line C, causing line C to develop color. However, since Cas12a is not activated and there is no cleavage product, the digoxigenin-antibody-modified gold nanoparticle complex cannot be captured at line T, so line T does not develop color or only shows weak color (the color depth is relatively lower than line C).

[0047] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. An HPV home self-test kit based on CRISPR / Cas12 and LAMP technology, characterized in that: include: Nucleic acid extraction module; A specific amplification module, comprising a constant temperature amplification reagent, an HPV16-specific LAMP primer set, whose polynucleotide sequences are shown in SEQ ID NO. 1 to SEQ ID NO. 5; and an HPV18-specific LAMP primer set, whose polynucleotide sequences are shown in SEQ ID NO. 6 to SEQ ID NO. 11; A signal activation module comprising a CRISPR / Cas12a / crRNA complex system, wherein the polynucleotide sequence of a crRNA specific for HPV16 is shown in SEQ ID NO. 12; the polynucleotide sequence of a crRNA specific for HPV18 is shown in SEQ ID NO. 13; and the polynucleotide sequence of an ssDNA reporter probe specifically modified with digoxigenin and biotin at both ends is shown in SEQ ID NO. 14; and The detection module includes a lateral flow test strip, which includes a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad arranged in sequence on a PVC base plate, wherein the conjugation pad is sprayed with gold nanoparticles modified with digoxigenin antibodies, and the nitrocellulose membrane is sequentially coated with a quality control line sprayed with streptavidin and a detection line sprayed with a heterologous secondary antibody.

2. The HPV home self-test kit based on CRISPR / Cas12 and LAMP technology according to claim 1, characterized in that When using a specific module for isothermal amplification, prepare HPV16-specific LAMP primer premix and HPV18-specific LAMP primer premix separately and set aside; The HPV16-specific LAMP primer premix includes 16 μM inner primer FIP with a polynucleotide sequence as shown in SEQ ID NO.3, 16 μM inner primer BIP with a polynucleotide sequence as shown in SEQ ID NO.4, 4 μM loop primer LB with a polynucleotide sequence as shown in SEQ ID NO.5, 2 μM outer primer F3 with a polynucleotide sequence as shown in SEQ ID NO.1, 2 μM outer primer B3 with a polynucleotide sequence as shown in SEQ ID NO.2, and 5 μM nuclease-free water. The HPV18-specific LAMP primer premix includes 16 μM inner primer FIP with a polynucleotide sequence as shown in SEQ ID NO.8, 16 μM inner primer BIP with a polynucleotide sequence as shown in SEQ ID NO.9, 4 μM loop primer LB with a polynucleotide sequence as shown in SEQ ID NO.11, 2 μM outer primer F3 with a polynucleotide sequence as shown in SEQ ID NO.6, 2 μM outer primer B3 with a polynucleotide sequence as shown in SEQ ID NO.7, and nuclease-free water.

3. The HPV home self-test kit based on CRISPR / Cas12 and LAMP technology according to claim 1, characterized in that Also includes: The nucleic acid extraction module includes silanol-modified magnetic beads, virus lysis solution, and a magnetic stand or magnet.

4. The HPV home self-test kit based on CRISPR / Cas12 and LAMP technology according to claim 3, characterized in that The nucleic acid extraction process using the nucleic acid extraction module is as follows: 1) Add 30 μL of Si-OH MBs and viral lysis buffer (LB) to a 1.5 mL EP tube and mix thoroughly. The viral lysis buffer (LB) contains 2.5 mM EDTA-Na2, 20 mM Tris, 4 M GuSCN, and 2 M NaCl, pH 6.

5. 2) Add the sample to be tested into the above EP tube; 3) Vortex or invert to mix thoroughly and incubate at room temperature for 10 minutes to ensure that the virus particles in the sample are fully lysed; 4) Place the EP tube on a magnetic stand and wait for 15 seconds, or place a magnet at the bottom of the EP tube to allow the Si-OH MBs to move and aggregate at the bottom of the EP tube for magnetic separation. Afterwards, discard the supernatant. 5) Add 800-1000 μL of 80% ethanol to the EP tube from step 4) to wash away any residual LB or other impurities on the surface of the Si-OH MBs. Leave for 5 s and repeat step 4) to obtain a no-elution nucleic acid extract. Air-dry the extract at room temperature for later use.

5. The HPV home self-test kit based on CRISPR / Cas12 and LAMP technology according to claim 1, characterized in that The minimum detection limit of the lateral flow strip is 100 nM.

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