A HPV isothermal nucleic acid detection kit based on Argonaute protein and its application method

Abstract

The present invention discloses an HPV isothermal nucleic acid detection kit based on Argonaute protein and an application method thereof, comprising a LAMP amplification reagent and a TbAgo detection reagent. The present invention creatively introduces TbAgo which is compatible with the LAMP reaction temperature and has high catalytic activity, and combines it with the LAMP amplification technology to construct a novel isothermal nucleic acid detection kit for detecting HPV16 and HPV18. The method is simple to operate, and the sensitivity and accuracy are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular relates to an HPV isothermal nucleic acid detection kit based on Argonaute protein and an application method thereof. Background Art

[0002] Cervical cancer is a malignant tumor that occurs in the lower cervix of the female uterus. Its incidence rate ranks second among female malignant tumors in my country, second only to breast cancer. Persistent infection with high-risk human papillomavirus (HPV) is the main factor causing cervical cancer, especially HPV16 and HPV18, which cause about 70% of cases. Therefore, establishing a rapid, sensitive and accurate HPV16 and HPV18 detection method is of great significance for large-scale screening and early diagnosis of cervical cancer.

[0003] Real-time fluorescence quantitative PCR (RT-PCR) is the most widely used HPV detection method at home and abroad. However, since it requires a professional thermal cycler to control the precise temperature changes and professional technicians to operate, it cannot meet the application scenarios of some resource shortages, such as grassroots hospitals, food safety monitoring sites, customs quarantine, etc.

[0004] Isothermal nucleic acid amplification technology can detect nucleic acid molecules at a constant temperature. Compared with PCR technology, it is more suitable for application scenarios with scarce resources, such as loop-mediated isothermal amplification (LAMP) technology. This technology designs 4 specific primers for 6 regions of the target nucleic acid molecule, and uses the characteristics of double-stranded DNA being in a dynamic equilibrium of denaturation and renaturation at 65°C and Bst DNA polymerase with high strand displacement activity to exponentially amplify nucleic acid molecules. Due to the large variety of primers in the system and their high concentration, nonspecific amplification is prone to occur, resulting in false positives.

[0005] In summary, although RT-PCR can provide accurate test results, it cannot be promoted in areas with resource shortages. Although LAMP technology has low requirements for professional equipment and personnel, it still lacks accuracy. Therefore, it is urgent to develop a simple, fast and accurate HPV16 and HPV18 detection method. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0007] In the present invention, the Argonaute protein is an Argonaute protein active under mesophilic conditions, preferably, TbAgo from Thermus brockianus, and the amino acid sequence of TbAgo is as shown in SEQ ID NO:90.

[0008] As one aspect of the present invention, the present invention provides an HPV isothermal nucleic acid detection kit based on Argonaute protein, which includes a LAMP amplification system and a TbAgo detection system;

[0009] Wherein, the LAMP amplification system comprises Bst DNA polymerase, HPV16 primer set, HPV18 primer set, dNTPs and buffer;

[0010] The HPV16 primer set is selected from SEQ ID NO: 1-30, preferably, SEQ ID NO: 1-6.

[0011] The HPV18 primer set is selected from SEQ ID NO: 31-67, preferably, SEQ ID NO: 31-36.

[0012] The TbAgo detection system comprises TbAgo protein, gDNA16 designed for HPV16 and its signal reporter molecule (reporter16), gDNA18 designed for HPV18 and its signal reporter molecule (reporter18), and a buffer.

[0013] The gDNA16 designed for HPV16 is selected from SEQ ID NO: 68-75, preferably, SEQ ID NO: 69; reporter16 is selected from SEQ ID NO: 76-77, preferably, SEQ ID NO: 76.

[0014] The gDNA18 designed for HPV18 is selected from SEQ ID NO: 78-87, preferably, SEQ ID NO: 85; reporter18 is selected from SEQ ID NO: 88-89, preferably, SEQ ID NO: 89.

[0015] The signal reporter molecule is a single-stranded DNA molecule carrying both a fluorescent group and a quenching group, wherein the fluorescent group includes one or more of FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, and Texas Red-X; the quenching group includes one or more of BHQ, TAMRA, DABCYL, and DDQ; the fluorescent group and the quenching group are independently located at the 5' end and the 3' end of the signal reporter molecule.

[0016] The signal reporter molecule can be detected by an enzyme marker or a colloidal gold test strip after being cleaved by the TbAgo protein.

[0017] The buffer comprises a divalent metal ion, wherein the divalent metal ion is Mn 2+ Mg 2+ or Co 2+ .

[0018] The present invention also provides a centrifugal chip suitable for a novel constant temperature nucleic acid detection method based on Argonaute protein. Specifically, the chip comprises a reaction chamber, a serpentine channel, a buffer chamber, and a waste liquid chamber. The centrifugal chip is composed of the first, second, and third layers from top to bottom, wherein the second layer is made of 2 mm thick PMMA material, engraved with a reaction chamber, a serpentine channel, a buffer chamber, and a waste liquid chamber; the first layer is a single-sided pressure-sensitive film, engraved with air holes and sample loading holes; the third layer is a single-sided pressure-sensitive film, which plays a sealing role. The first, second, and third layers are aligned in sequence and pressurized to form a centrifugal chip.

[0019] The centrifugal chip has the following characteristics: the designed sample loading holes are respectively used for LAMP amplification system, TbAgo cutting system for HPV16, TbAgo cutting system for HPV18, and ddH2O loading, wherein ddH2O is used to seal the reaction chamber, so that there is no gas exchange between the inside of the chip and the outside during the amplification reaction, thereby avoiding the formation of aerosol pollution; the air holes are respectively located at both ends of the chamber, thereby ensuring that the fluid can be centrifuged smoothly; reaction chamber 1 performs LAMP amplification; reaction chambers 2 and 3 perform TbAgo cutting respectively; during centrifugation, the serpentine channel slows down the speed at which the LAMP amplification product moves radially outward, thereby ensuring that the amplification product can fill reaction chambers 2 and 3 in sequence, and avoids it skipping reaction chamber 2 and directly entering reaction chamber 3 or the waste liquid chamber.

[0020] The centrifugal chip needs to be used in conjunction with a centrifugal temperature control device.

[0021] The centrifugal temperature control device is composed of four modules, namely a reaction chamber, a motor system, a temperature control system and a general control system.

[0022] Beneficial effects of the invention: In order to achieve the goal of rapid, accurate and sensitive detection of HPV in resource-scarce areas, the present invention creatively introduces TbAgo, which is compatible with the LAMP reaction temperature and has high catalytic activity, and combines it with the LAMP amplification technology to construct a new constant temperature nucleic acid detection kit for detecting HPV16 and HPV18. The method is simple to operate, and the sensitivity and accuracy are greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0024] Figure 1 This is a schematic diagram of the Argonaute protein-based isothermal nucleic acid detection method provided by the present invention.

[0025] Figure 2 The screening results of LAMP amplification primers for HPV16 and HPV18 provided in the embodiments of the present invention.

[0026] Figure 3 This is the amplification effect of the HPV16-5, HPV16-9, HPV18-5, and HPV18-9 primer sets provided in the embodiments of the present invention.

[0027] Figure 4 The screening results of the TbAgo detection system for HPV16 and HPV18 provided in the embodiments of the present invention.

[0028] Figure 5 The detection sensitivity characterization results of HPV16 and HPV18 provided in the embodiments of the present invention.

[0029] Figure 6 This is the orthogonality characterization result of the dual detection system targeting HPV16 and HPV18 provided in the embodiment of the present invention.

[0030] Figure 7 The test results of HPV16 and HPV18 clinical samples provided in the embodiments of the present invention.

[0031] Figure 8 A schematic diagram of a centrifugal temperature control device provided in an embodiment of the present invention.

[0032] Fig. 9 Schematic diagram of a centrifugal chip provided in an embodiment of the present invention.

[0033] Fig.10 The test results of HPV16 and HPV18 clinical samples in the centrifugal chip provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0034] The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0036] Example 1: Screening of LAMP amplification primers for HPV16 and HPV18

[0037] Figure 1 Schematic diagram of the novel isothermal nucleic acid detection method based on Argonaute protein provided by the present invention. The method first exponentially amplifies the target gene to be detected by LAMP technology, and then cuts the amplicon by the programmable nuclease function of TbAgo, thereby achieving secondary amplification and signal output. In order to achieve high-sensitivity detection of HPV16 and HPV18, the corresponding LAMP amplification primers were first screened. Specifically, 6 sets of primers as shown in SEQ ID NO: 1-30 were designed for HPV16, and 7 sets of primers as shown in SEQ ID NO: 31-67 were designed for HPV18. The above sequences are shown in Table 1.

[0038] Table 1

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] According to Bst2.0 The reaction system was prepared according to the DNA polymerase manual, i.e., 1×IsothermalAmplification Buffer, 6mM MgSO4, 1.4mM dNTPs, 0.2μM F3 / B3, 1.6μM FIP / BIP, 0.4μM LF / LB (acyclic primer for some reactions), and 1μL Bst2.0 DNA polymerase. 2μL of plasmid containing the HPV16 target gene was added to the amplification system for HPV16, and 2μL of plasmid containing the HPV18 target gene was added to the amplification system for HPV18. The plasmid concentration was 100fM, and ddH2O was added to 25μL. The reaction was carried out at 65℃ in a metal bath for 40min, and the amplification results were verified by 2% agarose gel electrophoresis.

[0045] The results are as follows Figure 2 As shown in Figure 1, HPV16-4, HPV16-5, HPV16-9, HPV16-11, HPV16-678, HPV18-1, HPV18-5, HPV18-7, HPV18-9, and HPV18-68 were successfully amplified. HPV16-5, HPV16-9, HPV18-5, and HPV18-9 with better amplification efficiency were selected for detailed characterization, that is, amplified for 20, 30, and 40 minutes respectively. The results are shown in Figure 1. Figure 3 As shown, the amplification rates of the HPV16-5 and HPV18-9 primer groups were relatively consistent, with obvious step-like products appearing at 20 minutes; the amplification rates of the HPV16-9 and HPV18-5 primer groups were relatively consistent, with obvious step-like products appearing at 30 minutes, and the amplification rates were slightly slower than those of the HPV16-5 and HPV18-9 primer groups.

[0046] Example 2: Screening of TbAgo detection systems for HPV16 and HPV18

[0047] According to the results of Example 1, the amplified products of the HPV16-5, HPV16-9, HPV18-5, and HPV18-9 primer sets were selected to design the corresponding TbAgo detection system. Specifically, five gDNAs were designed for the single-stranded loop region of the amplified product of the HPV16-5 primer set: 16-5-g1, 16-5-g2, 16-5-g3, 16-5-g4, 16-5-g5, and one reporter: 16-5-T; three gDNAs were designed for the single-stranded loop region of the amplified product of the HPV16-9 primer set: 16-9-g1, 16-9-g2, 16-9-g3, and one reporter: 16-9-T; Five gDNAs were designed for the single-stranded loop region of the amplified product of the 8-5 primer set: 18-5-g1, 18-5-g2, 18-5-g3, 18-5-g4, 18-5-g5, and one reporter: 18-5-T; five gDNAs were designed for the single-stranded loop region of the amplified product of the HPV18-9 primer set: 18-9-g1, 18-9-g2, 18-9-g3, 18-9-g4, 18-9-g5, and one reporter: 18-9-T. The above sequences are shown in Table 2.

[0048] Table 2

[0049]

[0050]

[0051] The TbAgo reaction system is as follows: 1×lsothermal Amplification Buffer, 6mM MgSO4, 1000nM TbAgO, 500nM gDNA, 500nM reporter, 10μL LAMP amplification product, ddH2O to 20μL. The reaction was carried out at 65℃ for 30min, and the reaction solution was cooled to room temperature. 20μL was taken and placed in a microplate reader to measure the fluorescence value (FAM-modified reporter excitation wavelength 494nm, emission wavelength 518nm; ROX-modified reporter excitation wavelength 575nm, emission wavelength 602nm).

[0052] The results are as follows Figure 4The fluorescence signals generated by HPV16-5-g2 and HPV16-9-g2 in the HPV16 detection system were the strongest, and those generated by HPV18-5-g5 and HPV18-9-g3 in the HPV18 detection system were the strongest. The four groups of reaction solutions were subjected to denaturing polyacrylamide gel electrophoresis, and the cleavage products of 15nt, 13nt, 14nt, and 14nt were detected respectively. The sizes were consistent with the theory, indicating that the fluorescence signals were indeed generated by two rounds of directional cleavage of TbAgo.

[0053] Combined with the LAMP primer screening results in Example 1, HPV16-5-g2 and 16-5-T were selected as the optimal TbAgo detection system for HPV16, and HPV18-9-g3 and 18-9-T were selected as the optimal TbAgo detection system for HPV18.

[0054] Example 3: Sensitivity of the Argonaute protein-based isothermal nucleic acid detection method for HPV16 and HPV18

[0055] Taking HPV16 as an example, the specific steps are introduced.

[0056] First, follow Bst2.0 The LAMP amplification system was prepared according to the DNA polymerase instructions. That is, 1×lsothermal Amplification Buffer, 6mM MgSO4, 1.4mM dNTPs, 0.2μM F3_5_16 / B3_5_16, 1.6μM FIP_5_16 / BIP_5_16, 0.4μM LF_5_16 / LB_5_16, 1μL Bst2.0 DNA polymerase, 2μL plasmid containing the HPV16 target gene (concentrations were 10 0 , 10 1 , 10 2 , 10 3 , 10 5 and 10 7 aM), add ddH2O to 15 μL, and place the reaction solution at the bottom of a 1.5 mL EP tube.

[0057] Next, prepare the TbAgo cleavage system: 1×lsothermal Amplification Buffer, 6mM MgSO4, 1000nM TbAgo, 100nM 16-5-q2, 400nM 16-5-T, and add ddH2O to make up to 15μL. Place the reaction solution in the lid of a 1.5mL EP tube.

[0058] The mixture was reacted at 65°C for 15 min in a metal bath, centrifuged at 4000 rpm for 10 s, and then reacted at 65°C for another 15 min in a metal bath. After the reaction solution was cooled to room temperature, 20 μL of the reaction solution was taken and placed in an ELISA instrument to measure the fluorescence value (excitation wavelength 494 nm, emission wavelength 518 nm).

[0059] When detecting HPV18, the amplification primers were 0.2 μM F3918 / B3918, 1.6 μM FIP_9_18 / BIP_9_18, 0.4 μM LF_9_18 / LB_9_18; the TbAgo system was 1000 nM TbAgo, 800 nM 18-9-g3, 800 nM 18-9-T. The rest of the operations were the same as above.

[0060] The results are as follows Figure 5 As shown, the HPV16 and HPV18 detection systems provided by the present invention can detect target nucleic acids as low as 1aM, and have the advantage of high sensitivity.

[0061] Example 4: Orthogonality of the dual detection system targeting HPV16 and HPV18 based on Argonaute protein

[0062] First, prepare the dual LAMP system of HPV16 and HPV18: 1×lsothermal AmplificationBuffer, 6mM MgSo4, 1.4mM dNTPs, 1×HPV18 LAMP primer (0.2μM F3_9_18 / B3_9_18, 1.6μM FI_P_918 / BIP_9_18, 0.4μM LF_9_18 / LB_9_18), 0.375×HPV16 LAMP primer (i.e., 0.075μM F3_5_16 / B3_5_16, 0.6μM FIP_5_16 / BIP_5_16, 0.15μM LF_5_16 / LB_5_16), 1μL Bst2.0 polymerase, 2 μL of HPV16 and HPV18 plasmids of different concentrations, ddH2O to make up to 15 μL, and place the reaction solution at the bottom of a 1.5 mL EP tube.

[0063] Secondly, prepare a double TbAgo cleavage system: 1×lsothermal Amplification Buffer, 6mMMgSo4, 2μM TbAgo, 100nM 16-5-g2, 400nM 16-5-T, 800nM 18-9-g3, 800nM 18-9-T, and add ddH2O to 15μL. Place the reaction solution in the lid of a 1.5mL EP tube.

[0064] The mixture was reacted at 65°C for 15 min, centrifuged at 4000 rpm for 10 s, and then reacted at 65°C for another 15 min. After the reaction solution was cooled to room temperature, 20 μL of the reaction solution was placed in an ELISA instrument to measure the fluorescence value (FAM: excitation wavelength 494 nm, emission wavelength 518 nm, ROX: excitation wavelength 575 nm, emission wavelength 602 nm).

[0065] The results are as follows Figure 6 As shown, the signals of the HPV16 and HPV18 dual detection system provided by the present invention do not interfere with each other at low concentration (100aM) and high concentration (10pM) of target nucleic acid, and have good orthogonality.

[0066] Example 5: Detection effect of dual detection system based on Argonaute protein targeting HPV16 and HPV18 on clinical samples

[0067] The present invention tested 9 clinical samples, all of which were confirmed by local hospitals using PCR capillary electrophoresis fragment analysis technology to determine the infected HPV subtypes.

[0068] First, nucleic acid was extracted according to the instructions of the viral nucleic acid extraction kit, and then detection was performed according to the operating steps in Example 4, where the plasmid containing HPV16 and HPV18 genes in Example 4 was replaced by the extracted nucleic acid.

[0069] The results are as follows Figure 7 The detection results of the detection system provided by the present invention are consistent with the results of the hospital test. The corresponding fluorescence signals were detected in 5 samples infected with HPV16 and 1 sample infected with HPV18, while the 3 samples infected with HPV51, 52, and 53 did not produce fluorescence signals, indicating that the dual detection system based on Argonaute protein and targeting HPV16 and HPV18 provided by the present invention has good accuracy and practicality.

[0070] Example 6: Detection effect of the detection method provided by the present invention in a centrifugal chip

[0071] In order to improve the convenience of operation and biosafety of the detection process, the present invention designs a centrifugal chip. The chip integrates the LAMP amplification and TbAgo cleavage reaction in the novel isothermal nucleic acid detection method based on Argonaute protein provided by the present invention, and can realize integrated detection without liquid transfer, avoiding the opening operation, thereby improving the convenience of operation and biosafety of the detection process.

[0072] In order to heat the centrifugal chip and control the fluid movement in the chip, the present invention also provides a centrifugal temperature control device, which consists of four modules, namely a reaction chamber, a motor system (speed range is 0-6000rpm), a temperature control system (heating temperature setting range is 25-99℃) and a general control system, such as Figure 8 shown.

[0073] Centrifugal chip Fig. 9 As shown. The centrifugal chip is composed of the 1st, 2nd and 3rd layers from top to bottom. Among them, the 2nd layer is 2mm thick PMMA, engraved with reaction chamber, serpentine channel, buffer chamber and waste liquid chamber. The 1st and 3rd layers are both single-sided pressure-sensitive films. The 1st layer is engraved with air holes and sample loading holes. The 1st, 2nd and 3rd layers are aligned and pressed in sequence to form a centrifugal chip. The width of the channel is 0.4mm, the diameter of the sample loading hole and the air hole is 1mm, the diameter of the reaction chamber 1 is 6mm, and the diameters of the reaction chambers 2, 3 and the waste liquid chamber are 3.6mm. Sample loading holes 1-3 are responsible for the dual LAMP amplification system of HPV16 and HPV18, the TbAgo cutting system of HPV18 and the TbAgo cutting system of HPV16 respectively; 4 and 5 are responsible for adding ddH2O to seal the reaction chamber, so that there is no gas exchange between the inside of the chip and the outside during the amplification reaction, thereby avoiding the formation of aerosol contamination. The air holes are located at both ends of the cavity, so as to ensure that the fluid can be centrifuged smoothly. Reaction chamber 1 performs LAMP amplification, while reaction chambers 2 and 3 perform TbAgo cleavage. During centrifugation, the serpentine channel slows down the speed at which the LAMP amplification product moves radially outward, thereby ensuring that the amplification product can fill reaction chambers 2 and 3 in sequence, and prevents it from skipping reaction chamber 2 and directly entering reaction chamber 3 or the waste liquid chamber.

[0074] In order to verify the practicability of the centrifugal chip provided by the present invention, clinical sample testing was performed. The specific operation is as follows:

[0075] (1) Prepare the TbAgo cleavage system for HPV16 and HPV18 respectively: 1× lsothermal Amplification Buffer, 6 mM MgSO4, 1 μM TbAgo, 100 nM 16-5-g2, 400 nM 16-5-T; 1× lsothermal Amplification Buffer, 6 mM MgSO4, 1 μM TbAgo, 800 nM 18-9-g3, 800 nM 18-9-T, the volume of each was 10 μL;

[0076] (2) Place 10 μL of HPV16 and HPV18 TbAgo cleavage systems in reaction chambers 3 and 2, respectively;

[0077] (3) Preparation of HPV16 and HPV18 dual LAMP system: 1×lsothermal Amplification Buffer, 6mM MgSO4, 1.4mM dNTPs, 1×HPV18 LAMP primers (0.2μM F3_9_18 / B3_9_18, 1.6μM FIP_9_18 / BIP_9_18, 0.4μM LF_9_18 / LB_9_18), 0.375×HPV16 LAMP primers (0.075μM F3_5_16 / B3_5_16, 0.6μM FIP_5_16 / BIP_5_16, 0.15μM LF_5_16 / LB_5_16), 1.2μL Bst2.0 DNA polymerase, 3μL of extracted nucleic acid from clinical samples, and ddH2O to make up to 30μL;

[0078] (4) Place 30 μL of HPV16 and HPV18 dual LAMP system in reaction chamber 1;

[0079] (5) Add 5 μL ddH2O to each well 4 and 5;

[0080] (6) Seal sample loading holes 1-5 with a pressure-sensitive film;

[0081] (7) Set the temperature of the heating module of the centrifugal temperature control device to 76°C, and place the chip in the reaction chamber for 15 minutes;

[0082] (8) Centrifugation: counterclockwise 3000 rpm (15 s), clockwise 1000 rpm (5 s), counterclockwise 1000 rpm (5 s), clockwise 4000 rpm (10 s);

[0083] (9) Continue the reaction for 15 min;

[0084] (10) Take out the chip, place it in a blue light imager, observe and record the results.

[0085] result Fig.10 As shown, 5 samples infected with HPV16 produced obvious yellow-green fluorescence; 1 sample infected with HPV18 produced obvious red fluorescence; and 3 samples infected with HPV51, 52, and 53 produced no fluorescence signal. This shows that the dual detection system of HPV16 and HPV18 in the centrifugal chip has good accuracy and practicality.

[0086] SEQ ID NO:90:

[0087]

[0088]

[0089] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An HPV isothermal nucleic acid detection kit based on Argonaute protein, characterized in that: Includes LAMP amplification reagent and TbAgo detection reagent; Wherein, the LAMP amplification reagent comprises an HPV16 primer set and / or an HPV18 primer set, the sequence of the HPV16 primer set is selected from SEQ ID NOs: 1-30; the sequence of the HPV18 primer set is selected from SEQ ID NOs: 31-67; The TbAgo detection reagent includes TbAgo protein, gDNA designed for HPV16 and its signal reporter molecule reporter16 and / or gDNA designed for HPV18 and its signal reporter molecule reporter18; the sequence of the gDNA designed for HPV16 is selected from SEQ ID NO: 68-75, and the sequence of the signal reporter molecule reporter16 is selected from SEQ ID NO: 76-77; the sequence of the gDNA designed for HPV18 is selected from SEQ ID NO: 78-87, and the sequence of the signal reporter molecule reporter18 is selected from SEQ ID NO: 88-89.

2. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1, characterized in that: The LAMP amplification reagent also includes Bst DNA polymerase, dNTPs and a buffer.

3. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1 or 2, characterized in that: The sequence of the HPV16 primer set is shown in SEQ ID NOs: 1-6; the sequence of the HPV18 primer set is shown in SEQ ID NOs: 31-36.

4. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1 or 2, characterized in that: The TbAgo protein includes a wild type TbAgo protein or a mutant thereof, and the amino acid sequence of the TbAgo protein is more than 70% identical to the sequence shown in SEQ ID NO:

90.

5. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1 or 2, characterized in that: The sequence of the gDNA designed for HPV16 is shown in SEQ ID NO: 69; the sequence of the signal reporter molecule reporter16 is shown in SEQ ID NO: 76; the sequence of the gDNA designed for HPV18 is shown in SEQ ID NO: 85; the sequence of the signal reporter molecule reporter18 is shown in SEQ ID NO: 89; the gDNA designed for HPV16 and the gDNA designed for HPV18 are both single-stranded DNA molecules with 5' end phosphorylation.

6. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1 or 2, characterized in that: The signal reporter molecule is a single-stranded DNA molecule carrying both a fluorescent group and a quenching group; wherein the fluorescent group includes: one or more of FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, and Texas Red-X; the quenching group includes: one or more of BHQ, TAMRA, DABCYL, and DDQ; the fluorescent group and the quenching group are independently located at the 5′ end and the 3′ end of the signal reporter molecule.

7. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1 or 2, characterized in that: The signal reporter molecule can be detected by an enzyme marker or a colloidal gold test strip after being cleaved by the TbAgo protein.

8. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 2, characterized in that: The buffer comprises a divalent metal ion, wherein the divalent metal ion is Mn 2+ Mg 2+ or Co 2+ .

9. The HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1 or 2, characterized in that: It also includes a centrifugal chip, which includes a reaction chamber, a serpentine channel, a buffer chamber, and a waste liquid chamber.

10. The application method of the HPV isothermal nucleic acid detection kit based on Argonaute protein according to claim 1, characterized in that: The reaction conditions for LAMP amplification are amplification at a temperature of 60 to 70° C. for 15 to 40 minutes; the conditions for TbAgo detection are reaction at a temperature of 60 to 70° C. for 15 to 30 minutes.

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