Hpv limiting amplification primer combination, kit and application thereof

Abstract

This invention relates to the field of biological detection, and more particularly to primer combinations, reagent kits, and their applications. The invention provides primer combinations including: primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII, primer set VIII, primer set IX, primer set X, primer set XI, primer set XII, primer set XIII, primer set XIV, primer set XV, primer set XVI, primer set XVII, primer set XVIII, primer set XIX, primer set XX, primer set XXI, primer set XXII, and primer set XXIII. This invention also provides a reagent kit for rapid HPV detection. This kit can rapidly detect the above 23 HPV types within 20 minutes. The detection limit of the kit is up to 10. 3 The accuracy and specificity are good, with a per-copy / mL ratio.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to HPV-limited amplification primer combinations, kits, and their applications. Background Technology

[0002] HPV infection is closely related to cervical cancer. Therefore, timely detection and identification of HPV types are crucial for effective disease control and selection of appropriate treatment plans. Currently, the main detection method is qPCR, which achieves large-scale in vitro amplification of specific gene fragments in a short time and analyzes the amplified fragments in real time using a fluorescence acquisition instrument, ultimately identifying specific HPV types. Conventional PCR amplification takes 1-2 hours and requires a certain level of sample purity, while loop-mediated isothermal amplification PCR (LAMP) is gradually being used in various fields due to its advantages of simple operation, rapid detection, high specificity, and low cost.

[0003] LAMP fluorescence amplification detection requires the collection and analysis of fluorescence increments during the amplification process. For low-concentration samples, the amplification time is long, requiring 60 or 70 minutes before an "amplification curve" appears for interpretation, almost the same as conventional PCR amplification, thus failing to demonstrate the advantages of LAMP technology. In contrast, loop-mediated isothermal amplification based on hydrogels allows hydrogel monomers to spontaneously polymerize into a gel at room temperature, accelerating the LAMP reaction. After heating at 65°C for 20 minutes, positive and negative samples can be distinguished. It also offers the advantage of rapid detection for low-concentration samples, and the results can be observed under a fluorescence microscope or UV flashlight, requiring less sophisticated equipment. After heating, the reagents become gel-like, and the amplification products are fixed, preventing aerosol pollution to the environment.

[0004] Existing qPCR fluorescence collection technology requires at least one hour to complete a single HPV subtype test, and has high requirements for laboratory environment and equipment. Different HPV types may require different test kits with varying requirements for sample extraction and experimental conditions. HPV testing is very time-consuming. There is a market demand for a kit that can quickly and easily perform multiple tests to facilitate immediate testing according to testing needs. Summary of the Invention

[0005] In view of this, the present invention provides primer combinations, a reagent kit, and their applications. The present invention provides a reagent kit for rapid detection of HPV. This kit can rapidly complete the detection of the above 23 HPV types within 20 minutes. The detection limit of the kit can reach 10. 3 copies / mL, with good accuracy and specificity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides primer combinations, including: (a1) or (a2) or (a3) ​​as follows:

[0008] (a1) It consists of primer sets I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, and XXIII;

[0009] (a2) Composed of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, or twenty-three primer sets I, II, III, IV, V, VI, VII, VIII, IX, X, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XII, XIII, XVI, XVI, XVII, XVIII, XIX, XX, XXI, XII, and XXIII;

[0010] (a3) includes primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII, primer set VIII, primer set IX, primer set X, primer set XI, primer set XII, primer set XIII, primer set XIV, primer set XV, primer set XVI, primer set XVII, primer set XVIII, primer set XIX, primer set XX, primer set XXI, primer set XXII, and primer set XXIII.

[0011] This invention also provides the application of the above primer combinations in any of the following:

[0012] (I) Identifying different types of human papillomavirus;

[0013] (II) Preparation of a kit for identifying different types of human papillomavirus;

[0014] (III) Detect whether the sample to be tested is human papillomavirus;

[0015] (IV) Preparation of a kit for detecting whether a sample to be tested is human papillomavirus.

[0016] The present invention also provides a detection reagent, comprising: the above-described primer combination.

[0017] In some embodiments of the present invention, the above-mentioned detection reagent further includes: magnesium ions, dNTPs, polymerase, TCEP, and hydrogel.

[0018] In some embodiments of the present invention, the hydrogel in the above-mentioned detection reagent is obtained by crosslinking eight-arm polyethylene glycol acrylate and dithiol polyethylene glycol.

[0019] In some embodiments of the present invention, the molar ratio of the octahedral polyethylene glycol acrylate and the dimercaptopolyethylene glycol in the above-mentioned detection reagent is 1:4.

[0020] In some embodiments of the present invention, the final concentration of the eight-arm polyethylene glycol acrylate in the above-mentioned detection reagent is 1.8 mM to 3 mM.

[0021] In some embodiments of the present invention, the final concentration of the eight-arm polyethylene glycol acrylate in the above-mentioned detection reagent is 1.8 mM, 2 mM, 2.5 mM, 2.75 mM or 3 mM.

[0022] In some embodiments of the present invention, the final concentration of the eight-arm polyethylene glycol acrylate in the above-mentioned detection reagent is 2.5 mM.

[0023] In some embodiments of the present invention, the final concentration of the dithiol polyethylene glycol in the above-mentioned detection reagent is 7.2 mM to 12 mM.

[0024] In some embodiments of the present invention, the final concentration of the dithiol polyethylene glycol in the above-mentioned detection reagent is 7.2 mM, 8 mM, 10 mM, 11 mM or 12 mM.

[0025] In some embodiments of the present invention, the final concentration of the dithiol polyethylene glycol in the above-mentioned detection reagent is 10 mM.

[0026] In some embodiments of the present invention, in the above-mentioned detection reagent, the final concentration of the primer set in the primer combination is 0.1× to 1.2×.

[0027] In some embodiments of the present invention, in the above-mentioned detection reagent, the final concentration of the primer set in the primer combination is 0.1×, 0.3×, 0.6×, 0.9× or 1.2×.

[0028] In some embodiments of the present invention, in the above-mentioned detection reagent, the final concentration of the primer set in the primer combination is 0.6×.

[0029] In some embodiments of the present invention, the final concentration of magnesium ions in the above-mentioned detection reagent is 3.2 mM to 6.4 mM.

[0030] In some embodiments of the present invention, the final concentration of magnesium ions in the above-mentioned detection reagent is 3.2 mM, 4 mM, 4.8 mM, 5.6 mM or 6.4 mM.

[0031] In some embodiments of the present invention, the final concentration of magnesium ions in the above-mentioned detection reagent is 4.8 mM.

[0032] In some embodiments of the present invention, the final concentration of the dNTPs in the above-mentioned detection reagent is 0.8 mM to 1.6 mM.

[0033] In some embodiments of the present invention, the final concentration of the dNTPs in the above-mentioned detection reagent is 0.8 mM, 1 mM, 1.2 mM, 1.4 mM or 1.6 mM.

[0034] In some embodiments of the present invention, the final concentration of the dNTPs in the above-mentioned detection reagent is 1.2 mM.

[0035] In some embodiments of the present invention, the concentration of the polymerase in the above-mentioned detection reagent is 6.4 U to 12.8 U / reaction.

[0036] In some embodiments of the present invention, the concentration of the polymerase in the above-mentioned detection reagent is 6.4 U / reaction, 8 U / reaction, 9.6 U / reaction, 11.2 U / reaction, or 12.8 U / reaction.

[0037] In some embodiments of the present invention, the concentration of the polymerase in the above-mentioned detection reagent is 9.6 U / reaction.

[0038] In some embodiments of the present invention, the polymerase in the above-mentioned detection reagent includes Bst enzyme.

[0039] In some embodiments of the present invention, the above-mentioned detection reagent further includes one or more of the following: lyophilization protectant, nucleic acid dye, reducing agent and buffer solution.

[0040] In some embodiments of the present invention, the lyophilization protectant in the above-mentioned detection reagent includes one or more of pullulan, mannitol, BSA, trehalose, glycine, and cyclodextrin.

[0041] In some embodiments of the present invention, the lyophilization protectant in the above-mentioned detection reagent includes one or more of the following: 2% pullulan, 5% mannitol, 10% BSA, 15% trehalose, 10mM glycine and 2% cyclodextrin.

[0042] In some embodiments of the present invention, the lyophilization protectant in the above-mentioned detection reagent includes: lyophilization protectant A and lyophilization protectant B;

[0043] The freeze-drying protectant A comprises: 1.25% mannitol, 0.4% BSA, 1.5% trehalose, 15mM glycine and 1% cyclodextrin; the freeze-drying protectant B comprises: 2% pullulan.

[0044] In some embodiments of the present invention, the buffer solution in the above-mentioned detection reagent comprises: betaine, KCl, Tris-HCl, and (NH4)2SO4; the molar ratio of betaine, KCl, Tris-HCl, and (NH4)2SO4 is 4-6:5-8:2-5:1; and the pH value of Tris-HCl is 7.5-8.8.

[0045] In some embodiments of the present invention, the reducing agent in the above-mentioned detection reagent includes: 100mM TCEP.

[0046] In some embodiments of the present invention, the detection reagents include: reagent 1 and reagent 2;

[0047] The reagent 1 comprises: the dNTPs, the primer combination, the polymerase, the polyethylene glycol acrylate, the TCEP, and the lyophilization protectant B;

[0048] The reagent 2 includes: the buffer solution, the magnesium ions, the reducing agent, the dithiol polyethylene glycol, and the lyophilization protectant A.

[0049] This invention also provides the application of the above-mentioned detection reagent in any of the following:

[0050] (I) Identifying different types of human papillomavirus; and / or

[0051] (II) Preparation of kits for identifying different types of human papillomavirus; and / or

[0052] (III) Detecting whether the sample to be tested is human papillomavirus; and / or

[0053] (IV) Preparation of a kit for detecting whether a sample to be tested is human papillomavirus.

[0054] The present invention also provides a kit comprising: the above-described detection reagents and acceptable adjuvants, carriers or devices.

[0055] This invention also provides a method for identifying different types of human papillomavirus for non-diagnostic purposes, comprising the following steps:

[0056] S1: Obtain the pretreated sample to be tested;

[0057] S2: Mix the sample to be tested with the above-mentioned detection reagents and / or the above-mentioned kits, incubate, and then detect;

[0058] S3: The identification result is obtained based on the presence or absence of fluorescent amplification spots.

[0059] In some embodiments of the present invention, the pretreatment in S1 of the above method employs one or more of the magnetic bead method and the sample release agent method.

[0060] In some embodiments of the present invention, the sample release method described above includes the step of adding a sample release agent; the amount of the sample release agent added is 5 to 15 μL.

[0061] In some embodiments of the present invention, in the above method S2, the incubation time is 10 to 30 minutes.

[0062] In some embodiments of the present invention, the detection described in method S3 above is to acquire images using a fluorescence microscope and / or to acquire images using a mobile phone under ultraviolet irradiation.

[0063] The beneficial effects of this invention include:

[0064] (1) Samples only need to be coarsely processed: Pretreatment can be direct lysis of the sample (room temperature, 5 minutes), the sample pretreatment time is short, and the sample processing effect is close to that of the magnetic bead method;

[0065] (2) It can detect 23 HPV subtypes in 20 minutes. The instrument requirements are lower than those of ordinary PCR. Only a heating device and a fluorescence microscope containing blue light wavelength are needed to complete the test, or a heating device, an ultraviolet flashlight and a mobile phone are needed.

[0066] (3) The method of the present invention has the characteristics of high sensitivity and high specificity. Attached Figure Description

[0067] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0068] Figure 1 Example diagram of fluorescent amplification spots in Example 3; wherein: the left shows a schematic diagram of amplification spots containing green fluorescent spots; the right shows a schematic diagram of interpretation of amplification spots without green fluorescent spots;

[0069] Figure 2 The optimal hydrogel concentration is shown in the diagram.

[0070] Figure 3 Comparison of freeze-dried morphologies;

[0071] Figure 4 The fluorescence spectrum is shown after lyophilization and reconstitution.

[0072] Figure 5 The graph shows the freeze-drying stability test results;

[0073] Figure 6 The diagram shows the interpretation of different detection devices. Detailed Implementation

[0074] This invention discloses primer combinations, reagent kits, and their applications.

[0075] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0076] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0077] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0078] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0079] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0080] In Examples 1 to 13 of this invention, all raw materials and reagents used can be purchased from the market.

[0081] The present invention will be further illustrated below with reference to the embodiments:

[0082] Example 1: Design and Screening of Primer Combinations for HPV Hydrogel-Restricted Amplification

[0083] The LAMP primer sequences for 23 HPV subtypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66, and subtypes 6, 11, 43, 81, and 42) are shown below:

[0084] Table 1. LAMP primer design for 123 different HPV types

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] To verify the amplification efficiency and sensitivity of LAMP primers for 23 HPV types, hydrogel validation was performed using 23 HPV type samples after qPCR determination, with concentrations of 1E6 copies / mL, 1E5 copies / mL, 1E4 copies / mL, 1000 copies / mL, and 500 copies / mL, respectively.

[0094] Table 2. Detection sensitivity results for different HPV types.

[0095]

[0096]

[0097] Conclusion: Using LAMP primer pairs for the detection of different HPV types, the primer pairs listed in Table 1, while ensuring sensitivity and specificity, can accurately detect HPV types at a sample concentration of 1000 copies / mL. The optimal primer sequences are shown below:

[0098] Table 3 Optimal primer sequences for different HPV types

[0099]

[0100]

[0101]

[0102]

[0103] Example 2: Preparation of hydrogel-limited amplification reagent for HPV detection

[0104] The dNTPs and primers used in the examples were obtained from Shanghai Sangon Biotech Co., Ltd.; the Bst polymerase in the LAMP reaction mixture was purchased from Xinhai Gene, the eight-arm PEG acrylate was purchased from Shanghai Tuoyang, and the thiol-PEG-thiol was purchased from Xi'an Kaixin. To address the limitations of existing technologies, this invention provides a kit for rapid detection of 23 HPV subtypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66, 6, 11, 81, 42, 43).

[0105] Table 4. Reagent Kit Components

[0106]

[0107]

[0108]

[0109] The kit contains reagent components A and B. Reagent component A contains dNTPs, primer MIX, Bst enzyme, octagonal polyethylene glycol acrylate, TCEP, lyophilization protectant A, and water. The final concentrations of dNTPs, primer MIX, Bst enzyme, octagonal polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000), and TCEP in the reaction mixture are 1.2 mM, 0.6 × 10⁻⁶, 9.6 U / reaction, 2.5 mM, and 0.33 mM, respectively.

[0110] Component B contains Bst Buffer, magnesium ions, TCEP, dithioglycol, SYBR Green, lyophilization protectant B, and water. The reaction system contains Bst Buffer at a final concentration of 1×, magnesium ions at a final concentration of 4.8 mM, SYBR Green at a final concentration of 1×, TCEP at a final concentration of 1.33 mM, and dithioglycol (SH-PEG-SH, Mw = 4000) at a concentration of 10 mM.

[0111] Table 5 Configuration of Single Reaction Systems

[0112]

[0113] The primer mixture contains FIP, BIP, LF, LB, F3, and B3; FIP, LF, and F3 are mixed in a molar ratio of 8:4:1, with the concentrations of primer BIP, LB, and B3 being the same as FIP, LB, and F3, respectively. In use, the six primers are prepared into a mixture in different proportions. Different primer sequences can be used to detect different HPV types as needed. Specific configuration ratios are shown in Table 6.

[0114] Table 6.10× Primer Mix Configuration

[0115] Component Name Dosage (μL) F3 (50μM) 1 B3 (50μM) 1 FIP (50μM) 8 BIP (50μM) 8 LF (50μM) 4 LB (50μM) 4 DEPC water Add to 30μL

[0116] 10×Bst Buffer is composed of betaine, KCl, Tris-HCl, and (NH4)2SO4 in a molar ratio of 4–6:5–8:2–5:1, and the pH of Tris-HCl is 7.5–8.8.

[0117] 50×SYBR Green dye was obtained by diluting commercially available 10000×SYBR Green I dye by 200 times.

[0118] 0.5 mg / μL of octahedral polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000) and 0.5 mg / μL of dithiol polyethylene glycol (SH-PEG-SH, Mw = 4000) were obtained by dissolving solid powder in DEPC water.

[0119] Freeze-drying protectant A: 1.25% mannitol, 0.4% BSA, 1.5% trehalose, 15mM glycine, 1% cyclodextrin.

[0120] Lyophilization protectant B: 2% pullulan

[0121] Component A was lyophilized at the bottom of a cryopreservation tube, and Component B, a general-purpose component, was lyophilized in a light-protected vial.

[0122] Component B of this invention contains no bioactive ingredients. Besides functioning as a gel network, the thiol-polyethylene glycol-thiol group also acts as a shaping agent during the freeze-drying process. TCEP in component B effectively reduces and stabilizes the thiol (-SH) groups, preventing their oxidation into disulfide bonds (-SS-). Pullulan in freeze-drying protectant B forms a stable protective film during freeze-drying, preventing further contact between oxygen and other oxidants and the thiol-polyethylene glycol-thiol group, thereby reducing oxidation and degradation.

[0123] In some embodiments of the present invention, the freeze-drying protectant A is suitable for both components A and B to be added in an amount of 3 μL.

[0124] Example 3: Use of hydrogel-limited amplification reagent for HPV detection

[0125] (1) Sample processing

[0126] Remove the sample and vortex to mix (frozen samples should be fully thawed at room temperature before use). Take 600–1000 μL of the sample to be tested and centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant and add 300–500 μL of sample processing solution to the precipitate. Vortex to mix until there is no obvious precipitate. Let stand at room temperature for 5 minutes before use for detection.

[0127] (2) Reagent preparation

[0128] Remove each component from the kit and allow it to stand at room temperature until well mixed. Calculate the required number of reactions (n) based on the number of samples to be tested (n), where "2" refers to the number of negative and positive controls. Add 7 mL of nuclease-free water to component B (350T / vial) and mix thoroughly.

[0129] (3) Adding samples

[0130] Prepare n+2 centrifuge tubes. Add 480 μL of component B to each centrifuge tube, and then add 240 μL each of the test sample, negative control, and positive control. Mix and centrifuge for a few seconds. Take 30 μL of the mixture and add it to the reactor containing the lyophilized component A. Mix and centrifuge, and cover with an anti-evaporation membrane.

[0131] (4) Incubation

[0132] The reactor after adding the sample was placed on a heater and incubated at 65°C for 20 minutes.

[0133] (5) Result detection

[0134] Remove the reactor and observe the results under a blue light electron microscope, then select a suitable field of view for storage. Alternatively, take photos with a mobile phone under LED ultraviolet light and then observe the results.

[0135] Result interpretation (see Table 7 for example results).

[0136] Quality control

[0137] Quality control products Quality control requirements HPV negative control product Under blue light excitation in a fluorescence microscope, no green fluorescent spots were observed at any of the wells. HPV positive control product Under blue light excitation in a fluorescence microscope, all wells showed green fluorescent spots.

[0138] (6) Result determination

[0139] Negative determination: Under blue light excitation under a fluorescence microscope, if there is a green fluorescent spot in the housekeeping gene well and no green fluorescent spot in other wells, the sample does not contain HPV virus or the HPV DNA concentration is less than the detection sensitivity or is another HPV type.

[0140] Positive result determination: Under blue light excitation in a fluorescence microscope, if a green fluorescent spot is found in the well containing the housekeeping gene, and a green fluorescent spot is found in any other well, then the result is considered positive for the corresponding genotype. If only one fluorescent spot is found, it is recommended to extract and retest once. If the repeated result still shows a fluorescent spot, the result is considered positive; otherwise, the result is considered negative.

[0141] If the housekeeping gene shows no fluorescent spot, it is recommended to extract DNA again for testing. If the housekeeping gene shows a green fluorescent spot in the repeat test, the repeat test result is acceptable; if the housekeeping gene still shows no green fluorescent spot in the repeat test, but the HPV type test is positive, then it is considered a positive result; if the housekeeping gene still shows no green fluorescent spot in the repeat test and the HPV type test is negative, then the sample is unacceptable.

[0142] Table 7 Examples of fluorescent amplification spots

[0143] Interpretation method Result Interpretation Result diagram Contains green fluorescent amplification spots Positive like Figure 1 As shown on the left No green fluorescent amplification spots Negative like Figure 1 As shown on the right

[0144] Example 4: Optimization of magnesium ion concentration

[0145] Magnesium ions are an essential cofactor for Bst DNA polymerase, crucial for its activity and stability. Both excessively high and low magnesium ion concentrations can affect the efficiency and specificity of the LAMP reaction. Excessively high magnesium ion concentrations may lead to nonspecific amplification, while excessively low concentrations may result in poor reaction initiation or low amplification efficiency. Therefore, optimizing the magnesium ion concentration is key to ensuring efficient amplification and reducing background noise.

[0146] To address the impact of magnesium ion concentration within the system on amplified clinically tested samples and negative samples, the final magnesium ion concentrations were set to 3.2 mM, 4 mM, 4.8 mM, 5.6 mM, and 6.4 mM, with other component concentrations referring to Example 2.

[0147] Table 8 Optimization of Magnesium Ion Concentration

[0148]

[0149]

[0150] Conclusion: Under the premise of ensuring the accuracy of the test results, the final concentration of magnesium ions at 4.8 mM is the best. Too low a concentration will cause the detection limit sample to be unstable, while too high a concentration will cause false positive results.

[0151] Example 4: Optimization of dNTP Concentration

[0152] dNTPs provide essential nucleotide triphosphates for DNA polymerases and are fundamental to DNA synthesis. The concentration of dNTPs must be sufficient to support large-scale DNA synthesis without depleting resources, but also not so high as to inhibit enzyme activity. Inappropriate dNTP concentrations can lead to reduced amplification efficiency or the production of erroneous amplification products. By optimizing dNTP concentrations, the specificity of amplification and the yield of products can be improved.

[0153] To address the impact of dNTP concentrations within the system on amplified clinically detectable samples and negative samples, the final dNTP concentrations were set to 0.8 mM, 1 mM, 1.2 mM, 1.4 mM, and 1.6 mM, with other component concentrations referring to Example 2.

[0154] Table 9 Optimization of dNTP Concentration

[0155]

[0156]

[0157] Conclusion: The optimal concentration of dNTPs is 1.2 mM. Too few dNTPs will lead to reduced amplification efficiency and nonspecific amplification, while too many dNTPs will inhibit the PCR reaction and cause false negative results.

[0158] Example 5: Bst enzyme concentration optimization experiment

[0159] Bst DNA polymerase is the specific enzyme used in LAMP reactions, enabling rapid DNA amplification under isothermal conditions. The enzyme concentration is crucial to the reaction rate and efficiency. Insufficient enzyme concentration may lead to slow or incomplete reactions, while excessive enzyme may increase non-specific amplification, affecting result interpretation. Optimizing the concentration of Bst DNA polymerase ensures efficient DNA synthesis while reducing the risk of erroneous amplification.

[0160] To address the impact of Bst enzyme concentration on the amplification of clinically tested samples and negative samples, the Bst enzyme concentration was set to 6.4 U, 8 U, 9.6 U, 11.2 U, and 12.8 U per reaction, with other component concentrations referring to Example 2.

[0161] Table 10 Bst enzyme concentration optimization experiment

[0162]

[0163]

[0164] Conclusion: The optimal concentration of Bst enzyme is 8 U to 11.2 U per reaction, with the optimal concentration being 9.6 U per reaction. Too low a concentration of Bst enzyme will cause false negatives, while too high a concentration of Bst enzyme will cause false positives.

[0165] Example 6 Primer Concentration Optimization

[0166] The amount of primers in the amplification system affects the amplification effect. Too low a primer amount reduces the amplification product, potentially leading to false negatives; too high a primer concentration promotes non-specific binding and primer dimer formation, resulting in false positives. This invention determines the optimal primer amount by adjusting the ratio of different primer concentrations in the reaction system. The primer concentrations were adjusted to 0.1×, 0.3×, 0.6×, 0.9×, and 1.2×, with other component concentrations referring to Example 2.

[0167] Table 11 Primer Concentration Optimization

[0168]

[0169]

[0170] The detection results are shown in Table 11. At a primer concentration of 1.2×, nonspecificity may occur, and the higher the primer concentration, the more severe the nonspecificity. False negative results were observed at concentrations of 0.1× and 0.3×, while the results were better at primer concentrations between 0.3 and 0.9×. The 0.6× primer concentration showed the best specificity and sensitivity, and was therefore selected as the optimal primer concentration.

[0171] Example 7: Optimization of Hydrogel Concentration

[0172] PEG hydrogels are a type of water-insoluble gel material formed by the crosslinking of hydrophilic polymers. After crosslinking, the polymer aqueous solution forms a hydrogel with a porous structure. By adjusting the molecular weight parameters of the polymer, the pore size and the physicochemical properties of the gel can be controlled.

[0173] The concentration of the eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000) was adjusted to: 1.8 mM, 2 mM, 2.5 mM, 2.75 mM, and 3 mM. The final concentration of HS-PEG-SH was four times that of the eight-arm polyethylene glycol acrylate. The specific adjustment range is shown in the table below. For the concentrations of other components, refer to Example 2.

[0174] Table 12 Optimization of hydrogel concentration

[0175]

[0176]

[0177] The test results are as follows Figure 2As shown, the amplification point gradually decreased with increasing concentrations of 8Arm-PEG-AC-10K and HS-PEG-SH-4k. Concentration 1 showed significant diffusion, while concentration 5 had a smaller amplification point. Considering all factors, concentration 3 (8Arm-PEG-AC-10K 2.5mM, HS-PEG-SH-4k 10mM) was deemed the optimal PEG concentration. The experimental template consisted of 5+E4 copies / mL of samples of each type.

[0178] Example 8: Sample Loading Optimization Experiment

[0179] The amount of DNA loaded into the amplification system has a certain impact on the detection sensitivity and accuracy. Too low a loading amount will lead to insufficient amplification template and reduced sensitivity, while too high a loading amount may contain inhibitors that affect the amplification results. The loading amounts are set to 5 μL, 10 μL, and 15 μL for the sample released by the sample release agent.

[0180] The sample release agent was a mixed solution of 120 mM sodium hydroxide, 0.9% trehalose, 0.2% Gemini surfactant, 0.8% proline, 40 mM sodium carbonate, and 2% 1-butyl-3-methylimidazolium bromide. Gemini surfactant and 1-butyl-3-methylimidazolium bromide synergistically release viral DNA through alkaline lysis with sodium hydroxide, while proline and trehalose improve the preservation stability of the DNA. Sodium carbonate enhances the stability of the alkaline lysis environment.

[0181] Table 13 Optimization Test of Release Agent Sample Amount

[0182]

[0183]

[0184] Conclusion: A sample loading volume of 10 μL resulted in the highest sensitivity without affecting detection accuracy; therefore, the optimal loading volume was 10 μL. A loading volume of 15 μL caused inhibition in some samples.

[0185] Example 9: Optimization of Lyophilization Protectant

[0186] This embodiment uses HPV16 for testing. Different formulations of lyophilization protectant A were added to component A, and different formulations of lyophilization protectant B were added to component B. The components were then placed in a freeze dryer for freeze drying, and the freeze-dried morphology and post-freeze testing effects of the different lyophilization protectants were observed.

[0187] Table 14 Freeze-drying Procedure

[0188]

[0189] Table 15 Formulation of Lyophilization Protectant

[0190] Lyophilization protectant A1 Lyophilization protectant A1 Lyophilization protectant A3 Lyophilization protectant B1 Lyophilization protectant B2 Lyophilization protectant B3 1.25% Mannitol 1.25% Mannitol 1.25% Mannitol 2% pullulanose 1.25% Mannitol 2% pullulanose 0.4% BSA 0.4% BSA 0.4% BSA 0.4% BSA 0.4% BSA 1.5% Trehalose 1.5% Trehalose 1.5% Trehalose 1.5% Trehalose 15mM glycine 15mM glycine 1% Cyclodextrin

[0191] like Figure 3 As shown, freeze-drying protectant B2 exhibits bottom shrinkage, while other freeze-drying protectants show good results. Freeze-drying protectant B1 is partially insoluble or dissolves slowly, while other freeze-drying protectants show good solubility.

[0192] like Figure 4 As shown, after reconstitution of lyophilization protectant B3 with lyophilization protectants A1 / A2 / A3, their amplification efficiency was tested. According to... Figure 4 The fluorescence results after lyophilization and reconstitution showed that lyophilization protectant A3 and lyophilization protectant B3 had the highest amplification efficiency, and this combination was selected as the best protectant.

[0193] Lyophilization protectants A3 and B3 were selected, and components A / B were lyophilized. After being stored at room temperature for different times, the storage stability of the lyophilized reagents was tested, and the results are as follows. Figure 5 As shown.

[0194] Depend on Figure 5 The results showed that the lyophilized reagent could still stably detect HPV16 samples after being stored at room temperature for 15 months.

[0195] Example 10: Optimal reaction time for monitoring amplification points

[0196] The primer sequences corresponding to the 23 HPV types are shown in Table 3. After the primers were mixed, they were prepared into hydrogel systems and fluorescent Lamp systems, respectively.

[0197] Table 16

[0198]

[0199]

[0200] The fluorescence detection system used 10 μL of component A and 10 μL of component B. 10 μL of each component was added separately. 5 copies / mL, 10 3 10 μL of templates for HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66, 6, 11, 81, 42, and 43 (copies / mL) were used as a control, with negative nucleic acid as a control. The hydrogel system is described in Example 3. The detection speed of the two methods for positive samples was compared. The fluorescence PCR instrument program was set to 65℃, 30s, 1 cycle; 65℃, 30s (fluorescence acquisition), 80 cycles. The time for one cycle was 1 minute. The hydrogel heating and fixation detection time was 10 min, 20 min, and 30 min. Blue light excitation was used for microscope to observe the presence of fluorescent spots. The results are shown in Table 17.

[0201] Table 17 Time taken to detect positive samples of different HPV types

[0202]

[0203]

[0204] Conclusion: By comparing hydrogel and fluorescent PCR, positive amplification points appeared in samples of different concentrations within 20 minutes. Due to the need for fluorescence acquisition by the instrument, fluorescent PCR required high-concentration samples to show amplification curves after 17 minutes, and low-concentration samples required even longer. This indicates that hydrogel can complete the detection process within 20 minutes, which has the advantage of rapid detection.

[0205] Example 11 Cross-specificity test

[0206] To verify the accuracy of this kit, and to ensure there was no cross-reactivity between the primers, DNA samples from different HPV types were cross-tested to check for false positives. All HPV type concentrations were 10. 7 After preparing the hydrogel (copies / mL), it was heated in a 65℃ constant temperature heater for 20 minutes and observed using a fluorescence microscope. The results are shown in Table 18.

[0207] Table 18 Cross-specificity test

[0208]

[0209]

[0210]

[0211]

[0212] Conclusion: There was no cross-infection among the primers for different HPV types, the specificity was good, and the positive and negative typing was accurate.

[0213] Example 12 Sample Pretreatment Comparison

[0214] The hydrogel detection method of this invention exhibits high resistance to sample interference and can stably detect samples prepared using various methods. This embodiment uses clinical HPV16 borderline positive samples to investigate the effects of two treatment methods—magnetic bead method and sample release agent—on the sample preparation of hydrogel LAMP.

[0215] (1) Sample preparation: Ten borderline positive cervical swab samples of HPV16 were collected from clinical practice and labeled as TQ1-10.

[0216] (2) Nucleic acid extraction: The nucleic acid extraction process was strictly carried out in accordance with the instructions for each reagent. The final nucleic acid elution / dissolution volume was set to 500 μL for all extraction methods.

[0217] (3) Detection: The extracted nucleic acid was detected using HPV16 fluorescent PCR detection reagent, molecular restriction amplification reagent and LAMP ordinary reagent respectively.

[0218] 1. Sample processing using magnetic beads:

[0219] Remove the sample and vortex to mix (frozen samples should be fully thawed at room temperature before use). Take 1000 μL of the sample to be tested and centrifuge at 12000 rpm for 5 min. Remove 800 μL of supernatant. Extract the remaining 200 μL using the magnetic bead method, with an elution volume of 500 μL.

[0220] 2. Sample processing of the release agent:

[0221] Remove the sample and vortex to mix (frozen samples should be fully thawed at room temperature before use). Take 1000 μL of the sample to be tested into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 5 min, carefully remove the supernatant, add 500 μL of sample release agent, vortex to mix until there is no obvious precipitation, place at room temperature for 5 min, centrifuge at 7000 rpm for 30 s, and use the supernatant for detection.

[0222] The test results are as follows:

[0223] Table 19 Comparison of Sample Preprocessing

[0224]

[0225]

[0226] As shown in the table above, the sample release agent performs worse than the magnetic bead method in fluorescence PCR detection, and there is a risk of detection failure for low-copy samples. In hydrogel-based limited amplification, there is no significant difference between the sample release agent and the magnetic bead method. The hydrogel method has a higher tolerance for impurities in the pre-treated sample (sample release agent) than fluorescence quantitative PCR. Furthermore, the sample release agent is simpler to handle, takes less time, and can complete detection within 20 minutes when used with the hydrogel system.

[0227] Example 13 Interpretation of Different Detection Devices

[0228] The presence of fluorescent spots in the hydrogel amplification system can be observed using a fluorescence microscope or by taking a photo with a mobile phone under ultraviolet light. This invention uses both methods for detection and interpretation, and compares the consistency of the two interpretation methods. The test samples are clinical samples that have undergone fluorescence qPCR assays, and the positive sample concentration is 10. 5copies / mL, 3000 copies / mL, negative sample concentration is 10 5 copies / mL.

[0229] like Figure 6 As shown, the readings obtained by moving the mobile phone under fluorescence microscopy and ultraviolet irradiation are consistent, indicating that the hydrogel system has high applicability to different reaction containers and reading methods. Sample test results can be interpreted when conditions are met: light transmission, transparency, and a clear visual range.

[0230] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer combination, characterized in that, include: The following are options (a1), (a2), or (a3): (a1) It consists of primer sets I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, and XXIII; (a2) Composed of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, or twenty-three primer sets I, II, III, IV, V, VI, VII, VIII, IX, X, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XII, XIII, XVI, XVI, XVII, XVIII, XIX, XX, XXI, XII, and XXIII; (a3) Includes primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII, primer set VIII, primer set IX, primer set X, primer set XI, primer set XII, primer set XIII, primer set XIV, primer set XV, primer set XVI, primer set XVII, primer set XVIII, primer set XIX, primer set XX, primer set XXI, primer set XXII, and primer set XXIII; The primer set I includes: Primer set I-I and / or primer set I-II; The primer set I-I consists of primer I-I-F3, primer I-I-B3, primer I-I-FIP, primer I-I-BIP, primer I-I-LF, and primer I-I-LB; The primer set I-II consists of primer I-II-F3, primer I-II-B3, primer I-II-FIP, primer I-II-BIP, primer I-II-LF, and primer I-II-LB; Primer I-I-F3 and primer I-II-F3 respectively have: (b1) Nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:7; (b2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b1); (b3) A sequence that has at least 80% homology with the nucleotide sequence shown in (b1); (b4) Complementary sequences to sequences shown in (b1), (b2) or (b3); Primer I-I-B3 and primer I-II-B3 respectively have: (b5) Nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:8; (b6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b5); (b7) A sequence that has at least 80% homology with the nucleotide sequence shown in (b5); (b8), complementary sequences to sequences shown in (b5), (b6) or (b7); Primer I-I-FIP and primer I-II-FIP respectively have: (b9) Nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:9; (b10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b9); (b11) A sequence that has at least 80% homology with the nucleotide sequence shown in (b9); (b12), complementary sequences to sequences shown in (b9), (b10) or (b11); The primers I-I-BIP and I-II-BIP respectively have: (b13) Nucleotide sequences as shown in SEQ ID NO:4 and SEQ ID NO:10; (b14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b13); (b15) A sequence that has at least 80% homology with the nucleotide sequence shown in (b13); (b16), complementary sequences to sequences shown in (b13), (b14) or (b15); Primer I-I-LF and primer I-II-LF each have: (b17) Nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:11; (b18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b17); (b19) A sequence that has at least 80% homology with the nucleotide sequence shown in (b17); (b20), complementary sequences to sequences shown in (b17), (b18) or (b19); Primer I-I-LB and primer I-II-LB each have: (b21) Nucleotide sequences as shown in SEQ ID NO:6 and SEQ ID NO:12; (b22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b21); (b23) A sequence that has at least 80% homology with the nucleotide sequence shown in (b21); (b24) Complementary sequences to sequences shown in (b21), (b22) or (b23); The primer set II includes: Primer set II-I and / or primer set II-II; The primer set II-I consists of primer II-I-F3, primer II-I-B3, primer II-I-FIP, primer II-I-BIP, primer II-I-LB, and primer II-I-LF; The primer set II-II consists of primer II-II-F3, primer II-II-B3, primer II-II-FIP, primer II-II-BIP, primer II-II-LF, and primer II-II-LB; Primer II-I-F3 and primer II-II-F3 respectively have: (c1) Nucleotide sequences as shown in SEQ ID NO:13 and SEQ ID NO:19; (c2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c1); (c3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c1); (c4) Complementary sequences to sequences shown in (c1), (c2) or (c3); Primer II-I-B3 and primer II-II-B3 respectively have: (c5) Nucleotide sequences as shown in SEQ ID NO:14 and SEQ ID NO:20; (c6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c5); (c7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c5); (c8), complementary sequences to sequences such as (c5), (c6) or (c7); Primer II-I-FIP and primer II-II-FIP respectively have: (c9) Nucleotide sequences as shown in SEQ ID NO:15 and SEQ ID NO:21; (c10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to have the nucleotide sequence shown in (c9); (c11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c9); (c12), complementary sequences to sequences shown in (c9), (c10) or (c11); Primer II-I-BIP and primer II-II-BIP respectively have: (c13) Nucleotide sequences as shown in SEQ ID NO:16 and SEQ ID NO:22; (c14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c13); (c15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c13); (c16), complementary sequences to sequences shown in (c13), (c14) or (c15); Primer II-I-LF and primer II-II-LF each have: (c17) Nucleotide sequences as shown in SEQ ID NO:18 and SEQ ID NO:23; (c18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c17); (c19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c17); (c20), complementary sequences to sequences such as (c17), (c18) or (c19); Primer II-I-LB and primer II-II-LB each have: (c21) Nucleotide sequences as shown in SEQ ID NO:17 and SEQ ID NO:24; (c22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c21); (c23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c21); (c24), complementary sequences to sequences shown in (c21), (c22) or (c23); The primer set III includes: Primer set III-I and / or primer set III-II; The primer set III-I consists of primer III-I-F3, primer III-I-B3, primer III-I-FIP, primer III-I-BIP, primer III-I-LF, and primer III-I-LB; The primer set III-II consists of primer III-II-F3, primer III-II-B3, primer III-II-FIP, primer III-II-BIP, primer III-II-LF, and primer III-II-LB; Primer III-I-F3 and primer III-II-F3 respectively have: (d1) Nucleotide sequences as shown in SEQ ID NO:25 and SEQ ID NO:31; (d2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d1); (d3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d1); (d4) Complementary sequences to sequences shown in (d1), (d2) or (d3); Primer III-I-B3 and primer III-II-B3 respectively have: (d5) Nucleotide sequences as shown in SEQ ID NO:26 and SEQ ID NO:32; (d6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d5); (d7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d5); (d8), complementary sequences to sequences such as (d5), (d6) or (d7); The primers III-I-FIP and III-II-FIP respectively have: (d9) Nucleotide sequences as shown in SEQ ID NO:27 and SEQ ID NO:33; (d10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d9); (d11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d9); (d12), complementary sequences to sequences such as (d9), (d10) or (d11); The primers III-I-BIP and III-II-BIP respectively have: (d13) Nucleotide sequences as shown in SEQ ID NO:28 and SEQ ID NO:34; (d14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d13); (d15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d13); (d16), complementary sequences to sequences shown in (d13), (d14) or (d15); Primer III-I-LF and primer III-II-LF each have: (d17) Nucleotide sequences as shown in SEQ ID NO:29 and SEQ ID NO:35; (d18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d17); (d19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d17); (d20), complementary sequences to sequences such as (d17), (d18) or (d19); The primers III-I-LB and III-II-LB respectively have: (d21) Nucleotide sequences as shown in SEQ ID NO:30 and SEQ ID NO:36; (d22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d21); (d23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d21); (d24), complementary sequences to sequences shown in (d21), (d22) or (d23); The primer set IV includes: Primer set IV-I and / or primer set IV-II; The primer set IV-I consists of primer IV-I-F3, primer IV-I-B3, primer IV-I-FIP, primer IV-I-BIP, primer IV-I-LF, and primer IV-I-LB; The primer set IV-II consists of primers IV-II-F3, IV-II-B3, IV-II-FIP, IV-II-BIP, IV-II-LF, and IV-II-LB; Primer IV-I-F3 and primer IV-II-F3 respectively have: (e1) Nucleotide sequences as shown in SEQ ID NO:37 and SEQ ID NO:43; (e2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e1); (e3) A sequence that has at least 80% homology with the nucleotide sequence shown in (e1); (e4), complementary sequences to sequences such as (e1), (e2) or (e3); Primer IV-I-B3 and primer IV-II-B3 respectively have: (e5) Nucleotide sequences such as SEQ ID NO:38 and SEQ ID NO:44; (e6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e5); (e7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e5); (e8), complementary sequences to sequences such as (e5), (e6) or (e7); The primers IV-I-FIP and IV-II-FIP respectively have: (e9), nucleotide sequences as shown in SEQ ID NO:39 and SEQ ID NO:45; (e10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e9); (e11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e9); (e12), complementary sequences to sequences such as (e9), (e10) or (e11); The primers IV-I-BIP and IV-II-BIP respectively have: (e13) Nucleotide sequences as shown in SEQ ID NO:40 and SEQ ID NO:46; (e14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e13); (e15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e13); (e16), complementary sequences to sequences such as (e13), (e14) or (e15); Primer IV-I-LF and primer IV-II-LF each have: (e17), nucleotide sequences as shown in SEQ ID NO:41 and SEQ ID NO:47; (e18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e17); (e19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e17); Complementary sequences to sequences such as (e20), (e17), (e18), or (e19); The primers IV-I-LB and IV-II-LB have: (e21) Nucleotide sequences as shown in SEQ ID NO:42 and SEQ ID NO:48; (e22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e21); (e23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e21); (e24), complementary sequences to sequences shown in (e21), (e22) or (e23); The primer set V includes: Primer set V-I and / or primer set V-II; The primer set V-I consists of primer V-I-F3, primer V-I-B3, primer V-I-FIP, primer V-I-BIP, primer V-I-LF, and primer V-I-LB; The primer set V-II consists of primer V-II-F3, primer V-II-B3, primer V-II-FIP, primer V-II-BIP, and primer V-II-LB; The primers V-I-F3 and V-II-F3 respectively have: (f1) Nucleotide sequences as shown in SEQ ID NO:49 and SEQ ID NO:55; (f2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f1); (f3) A sequence that has at least 80% homology with the nucleotide sequence shown in (f1); (f4), complementary sequences to sequences shown in (f1), (f2) or (f3); Primer V-I-B3 and primer V-II-B3 respectively have: (f5) Nucleotide sequences as shown in SEQ ID NO:50 and SEQ ID NO:56; (f6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f5); (f7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f5); (f8), complementary sequences to sequences such as (f5), (f6) or (f7); The primers V-I-FIP and V-II-FIP respectively have: (f9) Nucleotide sequences as shown in SEQ ID NO:51 and SEQ ID NO:57; (f10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f9); (f11) and sequences that have at least 80% homology with the nucleotide sequences shown in (f9); (f12), complementary sequences to sequences shown in (f9), (f10) or (f11); The primers V-I-BIP and V-II-BIP have: (f13) Nucleotide sequences as shown in SEQ ID NO:52 and SEQ ID NO:58; (f14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f13); (f15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f13); (f16), complementary sequences to sequences shown in (f13), (f14) or (f15); The primer V-I-LF has the following characteristics: (f17) The nucleotide sequence shown in SEQ ID NO:53; (f18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f17); (f19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f17); (f20), complementary sequences to sequences such as (f17), (f18) or (f19); The primers V-I-LB and V-II-LB have: (f21) Nucleotide sequences as shown in SEQ ID NO:54 and SEQ ID NO:59; (f22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f21); (f23) A sequence that has at least 80% homology with the nucleotide sequence shown in (f21); (f24), complementary sequences to sequences shown in (f21), (f22) or (f23); The primer set VI includes: Primer set VI-Ⅰ and / or primer set VI-Ⅱ; The primer set VI-I consists of primer VI-I-F3, primer VI-I-B3, primer VI-I-FIP, primer VI-I-BIP, and primer VI-I-LB; The primer set VI-II consists of primer VI-II-F3, primer VI-II-B3, primer VI-II-FIP, primer VI-II-BIP, primer VI-II-LF, and primer VI-II-LB; Primer VI-Ⅰ-F3 and primer VI-Ⅱ-F3 respectively have: (g1) Nucleotide sequences as shown in SEQ ID NO:60 and SEQ ID NO:65; (g2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g1); (g3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g1); (g4), complementary sequences to sequences such as (g1), (g2) or (g3); Primer VI-Ⅰ-B3 and primer VI-Ⅱ-B3 respectively have: (g5) Nucleotide sequences as shown in SEQ ID NO:61 and SEQ ID NO:66; (g6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g5); (g7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g5); (g8), complementary sequences to sequences such as (g5), (g6) or (g7); The primers VI-Ⅰ-FIP and VI-Ⅱ-FIP respectively have: (g9) Nucleotide sequences as shown in SEQ ID NO:62 and SEQ ID NO:67; (g10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g9); (g11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g9); (g12), complementary sequences to sequences such as (g9), (g10) or (g11); The primers VI-Ⅰ-BIP and VI-Ⅱ-BIP respectively have: (g13), nucleotide sequences as shown in SEQ ID NO:63 and SEQ ID NO:68; (g14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g13); (g15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g13); (g16), complementary sequences to sequences shown in (g13), (g14) or (g15); The primer VI-II-LF has the following characteristics: (g17) The nucleotide sequence shown in SEQ ID NO:69; (g18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g17); (g19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g17); Complementary sequences to sequences such as (g20), (g17), (g18), or (g19); The primers VI-Ⅰ-LB and VI-Ⅱ-LB have: (g21) Nucleotide sequences as shown in SEQ ID NO:64 and SEQ ID NO:70; (g22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g21); (g23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g21); (g24), complementary sequences to sequences such as (g21), (g22) or (g23); The primer set VII includes: Primer set VII-Ⅰ and / or primer set VII-Ⅱ; The primer set VII-I consists of primer VII-I-F3, primer VII-I-B3, primer VII-I-FIP, primer VII-I-BIP, primer VII-I-LF, and primer VII-I-LB; The primer set VII-II consists of primer VII-II-F3, primer VII-II-B3, primer VII-II-FIP, primer VII-II-BIP, primer VII-II-LF, and primer VII-II-LB; Primer VII-Ⅰ-F3 and primer VII-Ⅱ-F3 respectively have: (h1) Nucleotide sequences as shown in SEQ ID NO:71 and SEQ ID NO:77; (h2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h1); (h3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h1); (h4), complementary sequences to sequences shown in (h1), (h2) or (h3); Primer VII-Ⅰ-B3 and primer VII-Ⅱ-B3 respectively have: (h5), nucleotide sequences as shown in SEQ ID NO:72 and SEQ ID NO:78; (h6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h5); (h7) and sequences that have at least 80% homology with the nucleotide sequences shown in (h5); (h8), complementary sequences to sequences such as (h5), (h6) or (h7); The primers VII-I-FIP and VII-II-FIP have: (h9), nucleotide sequences as shown in SEQ ID NO:73 and SEQ ID NO:79; (h10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h9); (h11) and sequences that have at least 80% homology with the nucleotide sequences shown in (h9); (h12), complementary sequences to sequences such as (h9), (h10) or (h11); The primers VII-Ⅰ-BIP and VII-Ⅱ-BIP have: (h13), nucleotide sequences as shown in SEQ ID NO:74 and SEQ ID NO:80; (h14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h13); (h15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h13); (h16), complementary sequences to sequences shown in (h13), (h14) or (h15); The primers VII-Ⅰ-LF and VII-Ⅱ-LF have: (h17), nucleotide sequences as shown in SEQ ID NO:75 and SEQ ID NO:81; (h18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h17); (h19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h17); Complementary sequences to sequences such as (h20), (h17), (h18), or (h19); The primers VII-Ⅰ-LB and VII-Ⅱ-LB have: (h21), nucleotide sequences as shown in SEQ ID NO:76 and SEQ ID NO:82; (h22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h21); (h23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h21); (h24), complementary sequences to sequences shown in (h21), (h22) or (h23); Primer set VIII includes: Primer sets VIII-I and / or primer sets VIII-II; The primer set VIII-I consists of primer VIII-I-F3, primer VIII-I-B3, primer VIII-I-FIP, primer VIII-I-BIP, primer VIII-I-LF, and primer VIII-I-LB; The primer set VIII-II consists of primer VIII-II-F3, primer VIII-II-B3, primer VIII-II-FIP, primer VIII-II-BIP, primer VIII-II-LF, and primer VIII-II-LB; Primer VIII-Ⅰ-F3 and primer VIII-Ⅱ-F3 respectively have: (i1) Nucleotide sequences as shown in SEQ ID NO:83 and SEQ ID NO:89; (i2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i1); (i3) A sequence that has at least 80% homology with the nucleotide sequence shown in (i1); (i4), complementary sequences to sequences as shown in (i1), (i2) or (i3); Primer VIII-Ⅰ-B3 and primer VIII-Ⅱ-B3 respectively have: (i5) Nucleotide sequences as shown in SEQ ID NO:84 and SEQ ID NO:90; (i6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i5); (i7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (i5); (i8), complementary sequences to sequences such as (i5), (i6) or (i7); The primers VIII-I-FIP and VIII-II-FIP have: (i9), nucleotide sequences as shown in SEQ ID NO:85 and SEQ ID NO:91; (i10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i9); (i11) and sequences that have at least 80% homology with the nucleotide sequences shown in (i9); (i12), complementary sequences to sequences such as (i9), (i10) or (i11); The primers VIII-Ⅰ-BIP and VIII-Ⅱ-BIP have: (i13) Nucleotide sequences as shown in SEQ ID NO:86 and SEQ ID NO:92; (i14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i13); (i15) A sequence that has at least 80% homology with the nucleotide sequence shown in (i13); (i16), complementary sequences of sequences such as (i13), (i14) or (i15); The primers VIII-Ⅰ-LF and VIII-Ⅱ-LF have: (i17), nucleotide sequences as shown in SEQ ID NO:87 and SEQ ID NO:93; (i18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i17); (i19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (i17); (i20), complementary sequences of sequences such as (i17), (i18) or (i19); The primers VIII-Ⅰ-LB and VIII-Ⅱ-LB have: (i21) Nucleotide sequences as shown in SEQ ID NO:88 and SEQ ID NO:94; (i22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i21); (i23) A sequence that has at least 80% homology with the nucleotide sequence shown in (i21); (i24), complementary sequences to sequences such as (i21), (i22) or (i23); The primer set IX includes: Primer set IX-Ⅰ and / or primer set IX-Ⅱ; The primer set IX-I consists of primer IX-I-F3, primer IX-I-B3, primer IX-I-FIP, primer IX-I-BIP, primer IX-I-LF, and primer IX-I-LB; The primer set IX-II consists of primer IX-II-F3, primer IX-II-B3, primer IX-II-FIP, primer IX-II-BIP, primer IX-II-LF, and primer IX-II-LB; The primers IX-Ⅰ-F3 and IX-Ⅱ-F3 respectively have: (j1) Nucleotide sequences as shown in SEQ ID NO:95 and SEQ ID NO:101; (j2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (j1); (j3) A sequence that has at least 80% homology with the nucleotide sequence shown in (j1); (j4), complementary sequences to sequences such as (j1), (j2) or (j3); The primers IX-Ⅰ-B3 and IX-Ⅱ-B3 respectively have: (j5) Nucleotide sequences such as SEQ ID NO:96 and SEQ ID NO:102; (j6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (j5); (j7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (j5); (j8), complementary sequences to sequences such as (j5), (j6) or (j7); The primers IX-Ⅰ-FIP and IX-Ⅱ-FIP respectively have: (j9) Nucleotide sequences as shown in SEQ ID NO:97 and SEQ ID NO:103; (j10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (j9); (j11) and sequences that have at least 80% homology with the nucleotide sequences shown in (j9); (j12), complementary sequences to sequences such as (j9), (j10) or (j11); The primers IX-Ⅰ-BIP and IX-Ⅱ-BIP have: (j13) Nucleotide sequences as shown in SEQ ID NO:98 and SEQ ID NO:104; (j14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (j13); (j15) A sequence that has at least 80% homology with the nucleotide sequence shown in (j13); (j16), complementary sequences to sequences such as (j13), (j14) or (j15); The primers IX-Ⅰ-LF and IX-Ⅱ-LF respectively have: (j17) Nucleotide sequences as shown in SEQ ID NO:99 and SEQ ID NO:105; (j18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (j17); (j19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (j17); Complementary sequences to sequences such as (j20), (j17), (j18), or (j19); The primers IX-Ⅰ-LB and IX-Ⅱ-LB respectively have: (j21) Nucleotide sequences as shown in SEQ ID NO:100 and SEQ ID NO:106; (j22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (j21); (j23) A sequence that has at least 80% homology with the nucleotide sequence shown in (j21); (j24), complementary sequences to sequences such as (j21), (j22) or (j23); The primer set X includes: Primer set X-I and / or primer set X-II; The primer set X-I consists of primer X-I-F3, primer X-I-B3, primer X-I-FIP, primer X-I-BIP, primer X-I-LF, and primer X-I-LB; The primer set X-II consists of primer X-II-F3, primer X-II-B3, primer X-II-FIP, primer X-II-BIP, primer X-II-LF, and primer X-II-LB; The primers X-Ⅰ-F3 and X-Ⅱ-F3 respectively have: (k1) Nucleotide sequences as shown in SEQ ID NO:107 and SEQ ID NO:113; (k2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (k1); (k3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (k1); (k4), complementary sequences to sequences such as (k1), (k2) or (k3); The primers X-Ⅰ-B3 and X-Ⅱ-B3 respectively have: (k5) Nucleotide sequences as shown in SEQ ID NO:108 and SEQ ID NO:114; (k6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (k5); (k7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (k5); (k8), complementary sequences to sequences such as (k5), (k6) or (k7); The primers X-I-FIP and X-II-FIP respectively have: (k9), nucleotide sequences such as those shown in SEQ ID NO:109 and SEQ ID NO:115; (k10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (k9); (k11) and (k9) are sequences that have at least 80% homology with the nucleotide sequences shown. (k12), complementary sequences to sequences such as (k9), (k10) or (k11); The primers X-I-BIP and X-II-BIP respectively have: (k13), nucleotide sequences as shown in SEQ ID NO:110 and SEQ ID NO:116; (k14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (k13); (k15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (k13); (k16), complementary sequences to sequences such as (k13), (k14) or (k15); The primers X-I-LF and X-II-LF respectively have: (k17), nucleotide sequences as shown in SEQ ID NO:111 and SEQ ID NO:117; (k18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (k17); (k19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (k17); Complementary sequences to sequences such as (k20), (k17), (k18), or (k19); The primers X-Ⅰ-LB and X-Ⅱ-LB have: (k21), nucleotide sequences as shown in SEQ ID NO:112 and SEQ ID NO:118; (k22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (k21); (k23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (k21); (k24), complementary sequences to sequences such as (k21), (k22) or (k23); The primer set XI includes: Primer set XI-Ⅰ and / or primer set XI-Ⅱ; The primer set XI-Ⅰ consists of primer XI-Ⅰ-F3, primer XI-Ⅰ-B3, primer XI-Ⅰ-FIP, primer XI-Ⅰ-BIP, and primer XI-Ⅰ-LB; The primer set XI-II consists of primer XI-II-F3, primer XI-II-B3, primer XI-II-FIP, primer XI-II-BIP, and primer XI-II-LF; The primers XI-Ⅰ-F3 and XI-Ⅱ-F3 respectively have: (l1) Nucleotide sequences as shown in SEQ ID NO:119 and SEQ ID NO:124; (l2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (l1); (l3) A sequence that has at least 80% homology with the nucleotide sequence shown in (l1); (l4) Complementary sequences to sequences shown in (l1), (l2) or (l3); The primers XI-Ⅰ-B3 and XI-Ⅱ-B3 respectively have: (l5) Nucleotide sequences as shown in SEQ ID NO:120 and SEQ ID NO:125; (l6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (l5); (l7) A sequence that has at least 80% homology with the nucleotide sequence shown in (l5); (l8), complementary sequences to sequences such as (l5), (l6) or (l7); The primers XI-Ⅰ-FIP and XI-Ⅱ-FIP respectively have: (l9) Nucleotide sequences as shown in SEQ ID NO:121 and SEQ ID NO:126; (l10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (l9); (l11) and sequences that have at least 80% homology with the nucleotide sequences shown in (l9); (l12), complementary sequences to sequences shown in (l9), (l10) or (l11); The primers XI-Ⅰ-BIP and XI-Ⅱ-BIP respectively have: (l13) Nucleotide sequences as shown in SEQ ID NO:122 and SEQ ID NO:127; (l14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (l13); (l15) A sequence that has at least 80% homology with the nucleotide sequence shown in (l13); (l16), complementary sequences to sequences shown in (l13), (l14) or (l15); The primer XI-Ⅱ-LF has the following characteristics: (l17) The nucleotide sequence shown in SEQ ID NO:128; (l18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (l17); (l19) A sequence that has at least 80% homology with the nucleotide sequence shown in (l17); (l20), complementary sequences to sequences such as (l17), (l18) or (l19); The primer XI-Ⅰ-LB has the following characteristics: (l21) The nucleotide sequence shown in SEQ ID NO:123; (l22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (l21); (l23) A sequence that has at least 80% homology with the nucleotide sequence shown in (l21); (l24), complementary sequences to sequences shown in (l21), (l22) or (l23); The primer set XII includes: Primer set XII-Ⅰ and / or primer set XII-Ⅱ; The primer set XII-I consists of primer XII-I-F3, primer XII-I-B3, primer XII-I-FIP, primer XII-I-BIP, primer XII-I-LF, and primer XII-I-LB; The primer set XII-II consists of primer XII-II-F3, primer XII-II-B3, primer XII-II-FIP, primer XII-II-BIP, and primer XII-II-LF; The primers XII-Ⅰ-F3 and XII-Ⅱ-F3 respectively have: (m1), nucleotide sequences as shown in SEQ ID NO:129 and SEQ ID NO:135; (m2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (m1); (m3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (m1); (m4), complementary sequences to sequences such as (m1), (m2) or (m3); The primers XII-Ⅰ-B3 and XII-Ⅱ-B3 respectively have: (m5), nucleotide sequences as shown in SEQ ID NO:130 and SEQ ID NO:136; (m6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (m5); (m7) and (m5) are sequences that have at least 80% homology with the nucleotide sequences shown. (m8), complementary sequences to sequences such as (m5), (m6) or (m7); The primers XII-Ⅰ-FIP and XII-Ⅱ-FIP respectively have: (m9), nucleotide sequences as shown in SEQ ID NO:131 and SEQ ID NO:137; (m10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (m9); (m11) and (m9) are sequences that have at least 80% homology with the nucleotide sequences shown. (m12), complementary sequences to sequences such as (m9), (m10) or (m11); The primers XII-Ⅰ-BIP and XII-Ⅱ-BIP respectively have: (m13), nucleotide sequences as shown in SEQ ID NO:132 and SEQ ID NO:138; (m14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (m13); (m15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (m13); (m16), complementary sequences to sequences such as (m13), (m14) or (m15); The primers XII-Ⅰ-LF and XII-Ⅱ-LF respectively have: (m17), nucleotide sequences as shown in SEQ ID NO:133 and SEQ ID NO:139; (m18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (m17); (m19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (m17); Complementary sequences to sequences such as (m20), (m17), (m18), or (m19); The primer XII-Ⅰ-LB has the following characteristics: (m21), as shown in SEQ ID NO:134; (m22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (m21); (m23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (m21); (m24), complementary sequences to sequences such as (m21), (m22) or (m23); The primer set XIII includes: Primer set XIII-I and / or primer set XIII-II; The primer set XIII-I consists of primer XIII-I-F3, primer XIII-I-B3, primer XIII-I-FIP, primer XIII-I-BIP, primer XIII-I-LF, and primer XIII-I-LB; The primer set XIII-II consists of primer XIII-II-F3, primer XIII-II-B3, primer XIII-II-FIP, primer XIII-II-BIP, primer XIII-II-LB and primer XIII-II-LF; The primers XIII-Ⅰ-F3 and XIII-Ⅱ-F3 respectively have: (n1) Nucleotide sequences as shown in SEQ ID NO:140 and SEQ ID NO:146; (n2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (n1); (n3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (n1); (n4), complementary sequences to sequences such as (n1), (n2) or (n3); The primers XIII-Ⅰ-B3 and XIII-Ⅱ-B3 respectively have: (n5) Nucleotide sequences as shown in SEQ ID NO:141 and SEQ ID NO:147; (n6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (n5); (n7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (n5); (n8), complementary sequences to sequences such as (n5), (n6) or (n7); The primers XIII-Ⅰ-FIP and XIII-Ⅱ-FIP respectively have: (n9) Nucleotide sequences as shown in SEQ ID NO:142 and SEQ ID NO:148; (n10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (n9); (n11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (n9); (n12), complementary sequences to sequences such as (n9), (n10) or (n11); The primers XIII-Ⅰ-BIP and XIII-Ⅱ-BIP respectively have: (n13) Nucleotide sequences as shown in SEQ ID NO:143 and SEQ ID NO:149; (n14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (n13); (n15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (n13); (n16), complementary sequences to sequences such as (n13), (n14) or (n15); The primers XIII-Ⅰ-LF and XIII-Ⅱ-LF respectively have: (n17) Nucleotide sequences as shown in SEQ ID NO:144 and SEQ ID NO:150; (n18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (n17); (n19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (n17); (n20), complementary sequences to sequences such as (n17), (n18) or (n19); The primers XIII-Ⅰ-LB and XIII-Ⅱ-LB respectively have: (n21) Nucleotide sequences as shown in SEQ ID NO:145 and SEQ ID NO:151; (n22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (n21); (n23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (n21); (n24), complementary sequences to sequences such as (n21), (n22) or (n23); The primer set XIV includes: Primer set XIV-Ⅰ and / or primer set XIV-Ⅱ; The primer set XIV-I consists of primer XIV-I-F3, primer XIV-I-B3, primer XIV-I-FIP, primer XIV-I-BIP, primer XIV-I-LF, and primer XIV-I-LB; The primer set XIV-II consists of primer XIV-II-F3, primer XIV-II-B3, primer XIV-II-FIP, primer XIV-II-BIP, primer XIV-II-LF, and primer XIV-II-LB; The primers XIV-Ⅰ-F3 and XIV-Ⅱ-F3 respectively have: (o1) Nucleotide sequences as shown in SEQ ID NO:152 and SEQ ID NO:158; (o2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (o1); (o3) A sequence that has at least 80% homology with the nucleotide sequence shown in (o1); (o4), complementary sequences to sequences such as (o1), (o2) or (o3); The primers XIV-Ⅰ-B3 and XIV-Ⅱ-B3 respectively have: (o5) Nucleotide sequences as shown in SEQ ID NO:153 and SEQ ID NO:159; (o6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (o5); (o7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (o5); (o8), complementary sequences to sequences such as (o5), (o6) or (o7); The primers XIV-Ⅰ-FIP and XIV-Ⅱ-FIP respectively have: (o9) Nucleotide sequences as shown in SEQ ID NO:154 and SEQ ID NO:160; (o10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (o9); (o11) and sequences that have at least 80% homology with the nucleotide sequences shown in (o9); (o12), complementary sequences to sequences such as (o9), (o10) or (o11); The primers XIV-Ⅰ-BIP and XIV-Ⅱ-BIP respectively have: (o13) Nucleotide sequences as shown in SEQ ID NO:155 and SEQ ID NO:161; (o14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (o13); (o15) A sequence that has at least 80% homology with the nucleotide sequence shown in (o13); (o16), complementary sequences to sequences shown in (o13), (o14) or (o15); The primers XIV-Ⅰ-LF and XIV-Ⅱ-LF each have: (o17), nucleotide sequences as shown in SEQ ID NO:156 and SEQ ID NO:162; (o18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (o17); (o19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (o17); (o20), complementary sequences to sequences such as (o17), (o18) or (o19); The primers XIV-Ⅰ-LB and XIV-Ⅱ-LB each have: (o21) Nucleotide sequences as shown in SEQ ID NO:157 and SEQ ID NO:163; (o22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (o21); (o23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (o21); (o24), complementary sequences to sequences shown in (o21), (o22) or (o23); The primer set XV includes: Primer set XV-Ⅰ and / or primer set XV-Ⅱ; The primer set XV-I consists of primer XV-I-F3, primer XV-I-B3, primer XV-I-FIP, primer XV-I-BIP, primer XV-I-LF, and primer XV-I-LB; The primer set XV-II consists of primer XV-II-F3, primer XV-II-B3, primer XV-II-FIP, primer XV-II-BIP, primer XV-II-LF, and primer XV-II-LB; The primers XV-Ⅰ-F3 and XV-Ⅱ-F3 respectively have: (p1) Nucleotide sequences as shown in SEQ ID NO:164 and SEQ ID NO:170; (p2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (p1); (p3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (p1); (p4), complementary sequences to sequences shown in (p1), (p2) or (p3); The primers XV-Ⅰ-B3 and XV-Ⅱ-B3 respectively have: (p5) Nucleotide sequences as shown in SEQ ID NO:165 and SEQ ID NO:171; (p6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (p5); (p7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (p5); (p8), complementary sequences to sequences shown in (p5), (p6) or (p7); The primers XV-Ⅰ-FIP and XV-Ⅱ-FIP respectively have: (p9) Nucleotide sequences as shown in SEQ ID NO:166 and SEQ ID NO:172; (p10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (p9); (p11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (p9); (p12), complementary sequences to sequences shown in (p9), (p10) or (p11); The primers XV-Ⅰ-BIP and XV-Ⅱ-BIP respectively have: (p13) Nucleotide sequences as shown in SEQ ID NO:167 and SEQ ID NO:173; (p14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (p13); (p15) A sequence that has at least 80% homology with the nucleotide sequence shown in (p13); (p16), complementary sequences to sequences shown in (p13), (p14) or (p15); The primers XV-Ⅰ-LF and XV-Ⅱ-LF respectively have: (p17) Nucleotide sequences as shown in SEQ ID NO:168 and SEQ ID NO:174; (p18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (p17); (p19) A sequence that has at least 80% homology with the nucleotide sequence shown in (p17); (p20), complementary sequences to sequences shown in (p17), (p18) or (p19); The primers XV-Ⅰ-LB and XV-Ⅱ-LB respectively have: (p21) Nucleotide sequences as shown in SEQ ID NO:169 and SEQ ID NO:175; (p22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (p21); (p23) A sequence that has at least 80% homology with the nucleotide sequence shown in (p21); (p24), complementary sequences to sequences shown in (p21), (p22) or (p23); The primer set XVI includes: Primer set XVI-Ⅰ and / or primer set XVI-Ⅱ; The primer set XVI-I consists of primer XVI-I-F3, primer XVI-I-B3, primer XVI-I-FIP, primer XVI-I-BIP, primer XVI-I-LF, and primer XVI-I-LB; The primer set XVI-II consists of primer XVI-II-F3, primer XVI-II-B3, primer XVI-II-FIP, primer XVI-II-BIP, primer XVI-II-LF, and primer XVI-II-LB; The primers XVI-Ⅰ-F3 and XVI-Ⅱ-F3 respectively have: (q1) Nucleotide sequences as shown in SEQ ID NO:176 and SEQ ID NO:182; (q2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (q1); (q3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (q1); (q4), complementary sequences to sequences such as (q1), (q2) or (q3); The primers XVI-Ⅰ-B3 and XVI-Ⅱ-B3 respectively have: (q5) Nucleotide sequences as shown in SEQ ID NO:177 and SEQ ID NO:183; (q6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (q5); (q7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (q5); (q8), complementary sequences to sequences such as (q5), (q6) or (q7); The primers XVI-Ⅰ-FIP and XVI-Ⅱ-FIP respectively have: (q9) Nucleotide sequences as shown in SEQ ID NO:178 and SEQ ID NO:184; (q10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (q9); (q11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (q9); (q12), complementary sequences to sequences such as (q9), (q10) or (q11); The primers XVI-Ⅰ-BIP and XVI-Ⅱ-BIP respectively have: (q13) Nucleotide sequences as shown in SEQ ID NO:179 and SEQ ID NO:185; (q14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (q13); (q15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (q13); (q16), complementary sequences to sequences such as (q13), (q14) or (q15); The primers XVI-Ⅰ-LF and XVI-Ⅱ-LF respectively have: (q17) Nucleotide sequences as shown in SEQ ID NO:180 and SEQ ID NO:186; (q18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (q17); (q19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (q17); Complementary sequences to sequences such as (q20), (q17), (q18), or (q19); The primers XVI-Ⅰ-LB and XVI-Ⅱ-LB respectively have: (q21) Nucleotide sequences as shown in SEQ ID NO:181 and SEQ ID NO:187; (q22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (q21); (q23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (q21); (q24), complementary sequences to sequences such as (q21), (q22) or (q23); The primer set XVII includes: Primer set XVII-Ⅰ and / or primer set XVII-Ⅱ; The primer set XVII-I consists of primer XVII-I-F3, primer XVII-I-B3, primer XVII-I-FIP, primer XVII-I-BIP, primer XVII-I-LF, and primer XVII-I-LB; The primer set XVII-II consists of primer XVII-II-F3, primer XVII-II-B3, primer XVII-II-FIP, primer XVII-II-BIP, primer XVII-II-LF, and primer XVII-II-LB; The primers XVII-Ⅰ-F3 and XVII-Ⅱ-F3 respectively have: (r1) Nucleotide sequences as shown in SEQ ID NO:188 and SEQ ID NO:194; (r2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (r1); (r3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (r1); (r4), complementary sequences to sequences such as (r1), (r2) or (r3); The primers XVII-Ⅰ-B3 and XVII-Ⅱ-B3 respectively have: (r5) Nucleotide sequences as shown in SEQ ID NO:189 and SEQ ID NO:195; (r6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (r5); (r7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (r5); (r8), complementary sequences to sequences such as (r5), (r6) or (r7); The primers XVII-Ⅰ-FIP and XVII-Ⅱ-FIP respectively have: (r9) Nucleotide sequences as shown in SEQ ID NO:190 and SEQ ID NO:196; (r10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (r9); (r11) is a sequence that has at least 80% homology with the nucleotide sequence shown in (r9); (r12), complementary sequences to sequences such as (r9), (r10) or (r11); The primers XVII-Ⅰ-BIP and XVII-Ⅱ-BIP respectively have: (r13) Nucleotide sequences as shown in SEQ ID NO:191 and SEQ ID NO:197; (r14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (r13); (r15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (r13); (r16), complementary sequences to sequences such as (r13), (r14) or (r15); The primers XVII-Ⅰ-LF and XVII-Ⅱ-LF each have: (r17) Nucleotide sequences as shown in SEQ ID NO:192 and SEQ ID NO:198; (r18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (r17); (r19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (r17); Complementary sequences to sequences such as (r20), (r17), (r18), or (r19); The primers XVII-Ⅰ-LB and XVII-Ⅱ-LB respectively have: (r21) Nucleotide sequences as shown in SEQ ID NO:193 and SEQ ID NO:199; (r22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (r21); (r23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (r21); (r24), complementary sequences to sequences such as (r21), (r22) or (r23); The primer set XVIII includes: Primer set XVIII-Ⅰ and / or primer set XVIII-Ⅱ; The primer set XVIII-I consists of primer XVIII-I-F3, primer XVIII-I-B3, primer XVIII-I-FIP, primer XVIII-I-BIP, primer XVIII-I-LF, and primer XVIII-I-LB; The primer set XVIII-II consists of primer XVIII-II-F3, primer XVIII-II-B3, primer XVIII-II-FIP, primer XVIII-II-BIP, primer XVIII-II-LF, and primer XVIII-II-LB; The primers XVIII-Ⅰ-F3 and XVIII-Ⅱ-F3 respectively have: (s1) Nucleotide sequences as shown in SEQ ID NO:200 and SEQ ID NO:206; (s2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (s1); (s3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (s1); (s4), complementary sequences to sequences such as (s1), (s2) or (s3); The primers XVIII-Ⅰ-B3 and XVIII-Ⅱ-B3 respectively have: (s5) Nucleotide sequences as shown in SEQ ID NO:201 and SEQ ID NO:207; (s6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (s5); (s7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (s5); (s8), complementary sequences to sequences such as (s5), (s6) or (s7); The primers XVIII-Ⅰ-FIP and XVIII-Ⅱ-FIP respectively have: (s9), nucleotide sequences as shown in SEQ ID NO:202 and SEQ ID NO:208; (s10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (s9); (s11) and (s9) are sequences that have at least 80% homology with the nucleotide sequences shown. (s12), complementary sequences to sequences such as (s9), (s10) or (s11); The primers XVIII-Ⅰ-BIP and XVIII-Ⅱ-BIP respectively have: (s13) Nucleotide sequences as shown in SEQ ID NO:203 and SEQ ID NO:209; (s14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (s13); (s15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (s13); (s16), complementary sequences to sequences such as (s13), (s14) or (s15); The primers XVIII-Ⅰ-LF and XVIII-Ⅱ-LF respectively have: (s17) Nucleotide sequences as shown in SEQ ID NO:204 and SEQ ID NO:210; (s18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (s17); (s19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (s17); Complementary sequences to sequences such as (s20), (s17), (s18), or (s19); The primers XVIII-Ⅰ-LB and XVIII-Ⅱ-LB respectively have: (s21) Nucleotide sequences as shown in SEQ ID NO:205 and SEQ ID NO:211; (s22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (s21); (s23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (s21); (s24), complementary sequences to sequences such as (s21), (s22) or (s23); The primer set XIX includes: Primer set XIX-Ⅰ and / or primer set XIX-Ⅱ; The primer set XIX-Ⅰ consists of primer XIX-Ⅰ-F3, primer XIX-Ⅰ-B3, primer XIX-Ⅰ-FIP, primer XIX-Ⅰ-BIP, primer XIX-Ⅰ-LF, and primer XIX-Ⅰ-LB; The primer set XIX-II consists of primer XIX-II-F3, primer XIX-II-B3, primer XIX-II-FIP, primer XIX-II-BIP, primer XIX-II-LF, and primer XIX-II-LB; The primers XIX-Ⅰ-F3 and XIX-Ⅱ-F3 respectively have: (t1), nucleotide sequences as shown in SEQ ID NO:212 and SEQ ID NO:218; (t2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (t1); (t3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (t1); (t4), complementary sequences to sequences such as (t1), (t2) or (t3); The primers XIX-Ⅰ-B3 and XIX-Ⅱ-B3 respectively have: (t5), nucleotide sequences as shown in SEQ ID NO:213 and SEQ ID NO:219; (t6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (t5); (t7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (t5); (t8), complementary sequences to sequences such as (t5), (t6) or (t7); The primers XIX-Ⅰ-FIP and XIX-Ⅱ-FIP respectively have: (t9), nucleotide sequences as shown in SEQ ID NO:214 and SEQ ID NO:220; (t10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (t9); (t11) and (t9) are sequences that have at least 80% homology with the nucleotide sequences shown. (t12), complementary sequences to sequences such as (t9), (t10) or (t11); The primers XIX-Ⅰ-BIP and XIX-Ⅱ-BIP respectively have: (t13) Nucleotide sequences as shown in SEQ ID NO:215 and SEQ ID NO:221; (t14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (t13); (t15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (t13); (t16), complementary sequences to sequences such as (t13), (t14) or (t15); The primers XIX-Ⅰ-LF and XIX-Ⅱ-LF each have: (t17), nucleotide sequences as shown in SEQ ID NO:216 and SEQ ID NO:222; (t18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (t17); (t19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (t17); (t20), complementary sequences to sequences such as (t17), (t18) or (t19); The primers XIX-Ⅰ-LB and XIX-Ⅱ-LB respectively have: (t21) Nucleotide sequences as shown in SEQ ID NO:217 and SEQ ID NO:223; (t22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (t21); (t23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (t21); (t24), complementary sequences to sequences such as (t21), (t22) or (t23); The primer set XX includes: Primer set XX-Ⅰ and / or primer set XX-Ⅱ; The primer set XX-I consists of primer XX-I-F3, primer XX-I-B3, primer XX-I-FIP, primer XX-I-BIP, primer XX-I-LF, and primer XX-I-LB; The primer set XX-II consists of primer XX-II-F3, primer XX-II-B3, primer XX-II-FIP, primer XX-II-BIP, and primer XX-II-LB; The primers XX-Ⅰ-F3 and XX-Ⅱ-F3 respectively have: (u1), nucleotide sequences as shown in SEQ ID NO:224 and SEQ ID NO:230; (u2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (u1); (u3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (u1); (u4), complementary sequences to sequences such as (u1), (u2) or (u3); The primers XX-Ⅰ-B3 and XX-Ⅱ-B3 respectively have: (u5), nucleotide sequences such as those shown in SEQ ID NO:225 and SEQ ID NO:231; (u6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (u5); (u7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (u5); (u8), complementary sequences to sequences such as (u5), (u6) or (u7); The primers XX-Ⅰ-FIP and XX-Ⅱ-FIP respectively have: (u9), nucleotide sequences such as those shown in SEQ ID NO:226 and SEQ ID NO:232; (u10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (u9); (u11) and (u9) are sequences that have at least 80% homology with the nucleotide sequences shown. (u12), complementary sequences to sequences such as (u9), (u10) or (u11); The primers XX-Ⅰ-BIP and XX-Ⅱ-BIP respectively have: (u13), nucleotide sequences as shown in SEQ ID NO:227 and SEQ ID NO:233; (u14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (u13); (u15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (u13); (u16), complementary sequences to sequences such as (u13), (u14) or (u15); The primer XX-Ⅰ-LF has the following characteristics: (u17), nucleotide sequence as shown in SEQ ID NO:228; (u18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (u17); (u19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (u17); (u20), complementary sequences to sequences such as (u17), (u18) or (u19); The primers XX-Ⅰ-LB and XX-Ⅱ-LB respectively have: (u21), nucleotide sequences as shown in SEQ ID NO:229 and SEQ ID NO:234; (u22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (u21); (u23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (u21); (u24), complementary sequences to sequences such as (u21), (u22) or (u23); The primer set XXI includes: Primer set XXI-Ⅰ and / or primer set XXI-Ⅱ; The primer set XXI-Ⅰ consists of primers XXI-Ⅰ-F3, XXI-Ⅰ-B3, XXI-Ⅰ-FIP, XXI-Ⅰ-BIP, XXI-Ⅰ-LF, and XXI-Ⅰ-LB; The primer set XXI-II consists of primers XXI-II-F3, XXI-II-B3, XXI-II-FIP, XXI-II-BIP, XXI-II-LF, and XXI-II-LB. The primers XXI-Ⅰ-F3 and XXI-Ⅱ-F3 respectively have: (v1), nucleotide sequences as shown in SEQ ID NO:235 and SEQ ID NO:241; (v2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (v1); (v3) A sequence that has at least 80% homology with the nucleotide sequence shown in (v1); (v4), complementary sequences to sequences such as (v1), (v2) or (v3); The primers XXI-Ⅰ-B3 and XXI-Ⅱ-B3 respectively have: (v5), nucleotide sequences as shown in SEQ ID NO:236 and SEQ ID NO:242; (v6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (v5); (v7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (v5); (v8), complementary sequences to sequences such as (v5), (v6) or (v7); The primers XXI-Ⅰ-FIP and XXI-Ⅱ-FIP respectively have: (v9), nucleotide sequences as shown in SEQ ID NO:237 and SEQ ID NO:243; (v10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (v9); (v11) and (v9) are sequences that have at least 80% homology with the nucleotide sequences shown. (v12), complementary sequences to sequences such as (v9), (v10) or (v11); The primers XXI-Ⅰ-BIP and XXI-Ⅱ-BIP respectively have: (v13), nucleotide sequences as shown in SEQ ID NO:238 and SEQ ID NO:244; (v14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (v13); (v15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (v13); (v16), complementary sequences to sequences shown in (v13), (v14) or (v15); The primers XXI-Ⅰ-LF and XXI-Ⅱ-LF respectively have: (v17), nucleotide sequences as shown in SEQ ID NO:239 and SEQ ID NO:245; (v18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (v17); (v19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (v17); Complementary sequences to sequences such as (v20), (v17), (v18), or (v19); The primers XXI-Ⅰ-LB and XXI-Ⅱ-LB respectively have: (v21), nucleotide sequences as shown in SEQ ID NO:240 and SEQ ID NO:246; (v22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (v21); (v23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (v21); (v24), complementary sequences to sequences such as (v21), (v22) or (v23); The primer set XXII includes: Primer set XXII-Ⅰ and / or primer set XXII-Ⅱ; The primer set XXII-I consists of primers XXII-I-F3, XXII-I-B3, XXII-I-FIP, XXII-I-BIP, XXII-I-LF, and XXII-I-LB; The primer set XXII-II consists of primers XXII-II-F3, XXII-II-B3, XXII-II-FIP, XXII-II-BIP, XXII-II-LB, and XXII-II-LF. The primers XXII-Ⅰ-F3 and XXII-Ⅱ-F3 respectively have: (w1) Nucleotide sequences as shown in SEQ ID NO:247 and SEQ ID NO:253; (w2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (w1); (w3) is a sequence that has at least 80% homology with the nucleotide sequence shown in (w1); (w4), complementary sequences to sequences as shown in (w1), (w2) or (w3); The primers XXII-Ⅰ-B3 and XXII-Ⅱ-B3 respectively have: (w5) Nucleotide sequences as shown in SEQ ID NO:248 and SEQ ID NO:254; (w6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (w5); (w7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (w5); (w8), complementary sequences to sequences such as (w5), (w6) or (w7); The primers XXII-Ⅰ-FIP and XXII-Ⅱ-FIP respectively have: (w9), nucleotide sequences as shown in SEQ ID NO:249 and SEQ ID NO:255; (w10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (w9); (w11) and (w9) are sequences that have at least 80% homology with the nucleotide sequences shown. (w12), complementary sequences to sequences such as (w9), (w10) or (w11); The primers XXII-Ⅰ-BIP and XXII-Ⅱ-BIP respectively have: (w13), nucleotide sequences as shown in SEQ ID NO:250 and SEQ ID NO:256; (w14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (w13); (w15) is a sequence that has at least 80% homology with the nucleotide sequence shown in (w13); (w16), complementary sequences to sequences such as (w13), (w14) or (w15); The primers XXII-Ⅰ-LF and XXII-Ⅱ-LF respectively have: (w17), nucleotide sequences as shown in SEQ ID NO:251 and SEQ ID NO:258; (w18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (w17); (w19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (w17); Complementary sequences to sequences such as (w20), (w17), (w18), or (w19); The primers XXII-Ⅰ-LB and XXII-Ⅱ-LB respectively have: (w21), nucleotide sequences as shown in SEQ ID NO:252 and SEQ ID NO:257; (w22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (w21); (w23) is a sequence that has at least 80% homology with the nucleotide sequence shown in (w21); (w24), complementary sequences to sequences such as (w21), (w22) or (w23); The primer set XXIII includes: Primer set XXIII-Ⅰ and / or primer set XXIII-Ⅱ; The primer set XXIII consists of primers XXIII-I-F3, XXIII-I-B3, XXIII-I-BIP, XXIII-I-FIP, XXIII-I-LB, and XXIII-I-LF; The primer set XXIII-II consists of primer XXIII-II-F3, primer XXIII-II-B3, primer XXIII-II-FIP, primer XXIII-II-BIP, primer XXIII-II-LF, and primer XXIII-II-LB; The primers XXIII-Ⅰ-F3 and XXIII-Ⅱ-F3 respectively have: (x1) Nucleotide sequences as shown in SEQ ID NO:259 and SEQ ID NO:265; (x2) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (x1); (x3) A sequence that has at least 80% homology with the nucleotide sequence shown in (x1); (x4), complementary sequences to sequences such as (x1), (x2) or (x3); The primers XXIII-Ⅰ-B3 and XXIII-Ⅱ-B3 respectively have: (x5) Nucleotide sequences as shown in SEQ ID NO:260 and SEQ ID NO:266; (x6) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (x5); (x7) is a sequence that has at least 80% homology with the nucleotide sequence shown in (x5); (x8), complementary sequences to sequences such as (x5), (x6) or (x7); The primers XXIII-Ⅰ-FIP and XXIII-Ⅱ-FIP respectively have: (x9) Nucleotide sequences as shown in SEQ ID NO:262 and SEQ ID NO:267; (x10) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (x9); (x11) and sequences that have at least 80% homology with the nucleotide sequences shown in (x9); (x12), complementary sequences to sequences such as (x9), (x10) or (x11); The primers XXIII-Ⅰ-BIP and XXIII-Ⅱ-BIP respectively have: (x13) Nucleotide sequences as shown in SEQ ID NO:261 and SEQ ID NO:268; (x14) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (x13); (x15) A sequence that has at least 80% homology with the nucleotide sequence shown in (x13); (x16), complementary sequences to sequences shown in (x13), (x14) or (x15); The primers XXIII-Ⅰ-LF and XXIII-Ⅱ-LF respectively have: (x17) Nucleotide sequences as shown in SEQ ID NO:264 and SEQ ID NO:269; (x18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (x17); (x19) is a sequence that has at least 80% homology with the nucleotide sequence shown in (x17); (x20), complementary sequences to sequences such as (x17), (x18) or (x19); The primers XXIII-Ⅰ-LB and XXIII-Ⅱ-LB respectively have: (x21) Nucleotide sequences as shown in SEQ ID NO:263 and SEQ ID NO:270; (x22) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (x21); (x23) A sequence that has at least 80% homology with the nucleotide sequence shown in (x21); (x24), complementary sequences to sequences such as (x21), (x22) or (x23).

2. The application of the primer combination as described in claim 1 in any of the following: (I) Identifying different types of human papillomavirus; and / or (II) Preparation of kits for identifying different types of human papillomavirus; and / or (III) Detecting whether the sample to be tested is human papillomavirus; and / or (IV) Preparation of a kit for detecting whether a sample to be tested is human papillomavirus.

3. A detection reagent, characterized in that, include: The primer combination as described in claim 1.

4. The detection reagent as described in claim 3, characterized in that, Also includes: Magnesium ions, dNTPs, polymerase, TCEP, and hydrogel.

5. The detection reagent as described in claim 4, characterized in that, The hydrogel was obtained by crosslinking eight-arm polyethylene glycol acrylate and dithiol polyethylene glycol; the molar ratio of the eight-arm polyethylene glycol acrylate to the dithiol polyethylene glycol was 1:

4.

6. The detection reagent as described in claim 4 or 5, characterized in that, The final concentration of the primer set in the primer combination is 0.1× to 1.2×; the final concentration of the magnesium ions is 3.2 mM to 6.4 mM.

7. The detection reagent according to any one of claims 4 to 6, characterized in that, The final concentration of the dNTPs is 0.8 mM to 1.6 mM; the concentration of the polymerase is 6.4 U to 12.8 U / reaction.

8. The detection reagent according to any one of claims 3 to 7, characterized in that, Also includes: One or more of the following: lyophilization protectant, nucleic acid dye, reducing agent, and buffer.

9. The detection reagent as described in claim 8, characterized in that, The freeze-drying protectant includes one or more of pullulan, mannitol, BSA, trehalose, glycine, and cyclodextrin.

10. A method for identifying different types of human papillomavirus (HPV) for non-diagnostic purposes, characterized in that, Includes the following steps: S1: Obtain the pretreated sample to be tested; S2: Mix the sample to be tested with the detection reagent as described in any one of claims 3 to 9, incubate, and then detect; S3: The identification result is obtained based on the presence or absence of fluorescent amplification spots.

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