Microbial limitation amplification detection technique for the reproductive tract

Abstract

The present application relates to the field of biological detection, and particularly relates to a variety of reproductive tract microorganism detection technology based on hydrogel limiting position. The present application provides primer combinations, including: the primer group I, the primer group II, the primer group III, the primer group IV, the primer group V, the primer group VI, the primer group VII and the primer group VIII. The LAMP isothermal amplification reagent of the present application can detect eight kinds of reproductive tract microorganisms at one time within 20 minutes. The requirement degree of the instrument is lower than that of common PCR, and only a constant temperature heating device and a fluorescence microscope containing blue light wavelength, or a constant temperature heating device, an ultraviolet light source and a mobile phone can complete the detection. Due to the adsorption impurity characteristics of the hydrogel, the sample after preliminary lysis can be detected, the sample pretreatment time is greatly shortened, the operation is reduced, and one-step rapid detection of the sample is realized.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a detection technology for various reproductive tract microorganisms based on locomotor amplification. Background Technology

[0002] The most common diseases in women are reproductive tract infections, among which bacterial, fungal, trichomoniasis, mycoplasma, and chlamydia infections are more prevalent. Some of these infections are highly contagious, capable of parasitizing the host's cells and causing changes in bodily functions, even leading to cellular abnormalities in severe cases. Therefore, timely detection and identification of pathogens are crucial for effective disease control and the selection of appropriate treatment plans. Compared to traditional detection technologies centered on pathogen isolation and culture, new detection technologies go beyond simply culturing and isolating pathogens, significantly shortening the identification cycle while offering accuracy, speed, and high throughput.

[0003] Currently, the main detection method is still PCR detection, which can achieve large-scale in vitro amplification of specific gene fragments in a short time, and analyze the amplified fragments in real time using a fluorescence acquisition instrument, ultimately achieving the identification of specific pathogens. Since ordinary PCR amplification takes 1-2 hours and has certain requirements for sample purity, LAMP isothermal amplification PCR, with its advantages of simple operation, rapid detection, high specificity, and low cost, is gradually being used in various fields.

[0004] Compared to fluorescence amplification detection on qPCR instruments, LAMP requires the collection and analysis of fluorescence increments during the amplification process. Furthermore, 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 can distinguish between positive and negative samples after heating at 65°C for 20 minutes. It also offers the advantage of rapid detection for low-concentration samples, and the results can be observed under a fluorescence microscope or a UV flashlight, requiring less sophisticated equipment. After heating, the reagents become gel-like, fixing the amplification products and preventing aerosol pollution. Thiol-containing polymers spontaneously polymerize into gels at room temperature without initiation, thus not affecting the LAMP reaction system. A hydrogel system combining eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw=20000) and dithiol polyethylene glycol (HS-PEG-SH, Mw=4000) monomers was selected.

[0005] Existing qPCR fluorescence acquisition technology requires at least one hour to complete a single reproductive tract microbial examination and has high requirements for laboratory environment and equipment. Different pathogens may require different test kits with varying requirements for sample extraction and experimental conditions. The detection of multiple reproductive tract microorganisms is very time-consuming. There is a market demand for a kit that can quickly and easily perform multiple tests to facilitate immediate detection according to testing needs. Summary of the Invention

[0006] In view of this, the present invention provides a rapid detection technology for multiple reproductive tract microorganisms based on hydrogel-limited amplification. The LAMP isothermal amplification reagent of the present invention can detect eight reproductive tract microorganisms simultaneously within 20 minutes. The instrument requirements are lower than those of conventional PCR; only a heating device and a fluorescence microscope containing blue light wavelengths or ultraviolet light illumination, along with a mobile phone, are needed to complete the detection. Furthermore, due to the impurity adsorption properties of hydrogels, samples after preliminary lysis can be used for detection, greatly shortening sample pretreatment time, reducing operations, and achieving rapid one-step sample detection.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides primer combinations, including: (a1) or (a2) or (a3) ​​as follows:

[0009] (a1) It consists of primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII and primer set VIII;

[0010] (a2) It consists of any one, two, three, four, five, six or seven of the primer sets I, II, III, IV, V, VI, VII and VIII;

[0011] (a3) includes primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII and primer set VIII;

[0012] The primer set I includes one or more of primer set I-I, primer set I-II, and primer set I-III;

[0013] 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;

[0014] The primer set consists of primer I-II-F3, primer I-II-B3, primer I-II-FIP, primer I-II-BIP, and primer I-II-LF;

[0015] The primer set consists of primers I-III-F3, I-III-B3, I-III-FIP, I-III-BIP, and I-III-LF;

[0016] The primer set II includes one or more of primer set II-I, primer set II-II, and primer set II-III;

[0017] 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-LF, and primer II-I-LB;

[0018] The primer set II-II consists of primer II-II-F3, primer II-II-B3, primer II-II-FIP, primer II-II-BIP, and primer II-II-LF;

[0019] The primer set II-III consists of primer II-III-F3, primer II-III-B3, primer II-III-FIP, primer II-III-BIP, and primer II-III-LF;

[0020] The primer set III includes one or more of primer set III-I, primer set III-II, and primer set III-III;

[0021] 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;

[0022] The primer set III-II consists of primer III-II-F3, primer III-II-B3, primer III-II-FIP, primer III-II-BIP, and primer III-II-LF;

[0023] The primer set III-III consists of primers III-III-F3, III-III-B3, III-III-FIP, III-III-BIP, III-III-LF, and III-III-LB.

[0024] The primer set IV includes one or more of primer set IV-I, primer set IV-II, and primer set IV-III;

[0025] The primer set IV-I consists of primer IV-I-F3, primer IV-I-B3, primer IV-I-FIP, primer IV-I-BIP, and primer IV-I-LF;

[0026] 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;

[0027] The primer set IV-III consists of primer IV-III-F3, primer IV-III-B3, primer IV-III-FIP, primer IV-III-BIP, and primer IV-III-LF;

[0028] The primer set V includes one or more of primer set V-I, primer set V-II, and primer set V-III;

[0029] 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;

[0030] The primer set V-II consists of primer V-II-F3, primer V-II-B3, primer V-II-FIP, primer V-II-BIP, primer V-II-LF, and primer V-II-LB;

[0031] The primer set V-III consists of primer V-III-F3, primer V-III-B3, primer V-III-FIP, primer V-III-BIP, primer V-III-LF, and primer V-III-LB;

[0032] The primer set VI includes one or more of primer set VI-Ⅰ, primer set VI-Ⅱ, and primer set VI-Ⅲ;

[0033] 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-LF;

[0034] The primer set VI-II consists of primer VI-II-F3, primer VI-II-B3, primer VI-II-FIP, primer VI-II-BIP, and primer VI-II-LF;

[0035] The primer set VI-III consists of primer VI-III-F3, primer VI-III-B3, primer VI-III-FIP, primer VI-III-BIP, primer VI-III-LF and primer V-III-LB;

[0036] The primer set VII includes one or more of primer set VII-I, primer set VII-II, and primer set VII-III;

[0037] 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;

[0038] 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;

[0039] The primer set VII-III consists of primer VII-III-F3, primer VII-III-B3, primer VII-III-FIP, primer VII-III-BIP, primer VII-III-LF, and primer VII-III-LB;

[0040] The primer set VIII includes one or more of primer sets VIII-I, VIII-II, and VIII-III;

[0041] 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;

[0042] 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;

[0043] The primer set VIII-III consists of primer VIII-III-F3, primer VIII-III-B3, primer VIII-III-FIP, primer VIII-III-BIP, primer VIII-III-LF, and primer VIII-III-LB.

[0044] In some embodiments of the present invention, in the above primer combination, the molar ratio 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 in primer group I-I is: (1~2):(1~2):(4~8):(4~8):(2~4):(2~4);

[0045] The molar ratio of primers I-II-F3, I-II-B3, I-II-FIP, I-II-BIP and I-II-LF in primer set I-II is: (1-2): (1-2): (4-8): (4-8): (2-4);

[0046] The molar ratio of primers I-III-F3, I-III-B3, I-III-FIP, I-III-BIP, I-III-LF, and I-III-LB in primer sets I-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0047] The molar ratio of primers II-I-F3, II-I-B3, II-I-FIP, II-I-BIP, II-I-LF, and II-I-LB in primer set II-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0048] The molar ratio of primers II-II-F3, II-II-B3, II-II-FIP, II-II-BIP and II-II-LF in primer set II-II is (1-2):(1-2):(4-8):(4-8):(2-4);

[0049] The molar ratio of primers II-III-F3, II-III-B3, II-III-FIP, II-III-BIP and II-III-LF in primer set II-III is: (1-2): (1-2): (4-8): (4-8): (2-4);

[0050] The molar ratio of primers III-I-F3, III-I-B3, III-I-FIP, III-I-BIP, III-I-LF, and III-I-LB in primer set III-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0051] The molar ratio of primers III-II-F3, III-II-B3, III-II-FIP, III-II-BIP and III-II-LF in primer set III-II is (1-2):(1-2):(4-8):(4-8):(2-4);

[0052] The molar ratio of primers III-III-F3, III-III-B3, III-III-FIP, III-III-BIP, III-III-LF, and III-III-LB in primer set III-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0053] The molar ratio of primers IV-I-F3, IV-I-B3, IV-I-FIP, IV-I-BIP and IV-I-LF in primer set IV-I is (1-2):(1-2):(4-8):(4-8):(2-4);

[0054] The molar ratio of primers IV-II-F3, IV-II-B3, IV-II-FIP, IV-II-BIP, IV-II-LF, and IV-II-LB in primer set IV-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0055] The molar ratio of primers IV-III-F3, IV-III-B3, IV-III-FIP, IV-III-BIP and IV-III-LF in primer set IV-III is (1-2):(1-2):(4-8):(4-8):(2-4);

[0056] The molar ratio of primers V-I-F3, V-I-B3, V-I-FIP, V-I-BIP, V-I-LF, and V-I-LB in primer set V-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0057] The molar ratio of primers V-II-F3, V-II-B3, V-II-FIP, V-II-BIP, V-II-LF, and V-II-LB in primer set V-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0058] The molar ratio of primers V-III-F3, V-III-B3, V-III-FIP, V-III-BIP, V-III-LF, and V-III-LB in primer set V-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0059] The molar ratio of primers VI-I-F3, VI-I-B3, VI-I-FIP, VI-I-BIP and VI-I-LF in primer set VI-I is (1-2): (1-2): (4-8): (4-8): (2-4);

[0060] The molar ratio of primers VI-II-F3, VI-II-B3, VI-II-FIP, VI-II-BIP and VI-II-LF in primer set VI-II is (1-2): (1-2): (4-8): (4-8): (2-4);

[0061] The molar ratio of primers VI-III-F3, VI-III-B3, VI-III-FIP, VI-III-BIP, VI-III-LF, and VI-III-LB in primer set VI-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0062] The molar ratio of primers VII-I-F3, VII-I-B3, VII-I-FIP, VII-I-BIP, VII-I-LF, and VII-I-LB in primer set VII-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0063] The molar ratio of primers VII-II-F3, VII-II-B3, VII-II-FIP, VII-II-BIP, VII-II-LF, and VII-II-LB in primer set VII-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0064] The molar ratio of primers VII-III-F3, VII-III-B3, VII-III-FIP, VII-III-BIP, VII-III-LF, and VII-III-LB in primer set VII-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0065] The molar ratio of primers VIII-I-F3, VIII-I-B3, VIII-I-FIP, VIII-I-BIP, VIII-I-LF, and VIII-I-LB in primer set VIII-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0066] The molar ratio of primers VIII-II-F3, VIII-II-B3, VIII-II-FIP, VIII-II-BIP, VIII-II-LF, and VIII-II-LB in primer set VIII-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4);

[0067] The molar ratio of primers VIII-Ⅲ-F3, VIII-Ⅲ-B3, VIII-Ⅲ-FIP, VIII-Ⅲ-BIP, VIII-Ⅲ-LF and VIII-Ⅲ-LB in primer set VIII-Ⅲ is (1~2):(1~2):(4~8):(4~8):(2~4):(2~4).

[0068] This invention also provides the application of the above primer combinations in any of the following:

[0069] (I) Identify one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis;

[0070] (II) Prepare a kit for identifying one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis.

[0071] (III) Detect whether the sample to be tested contains one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis;

[0072] (IV) Prepare a kit to detect whether the sample to be tested contains one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis.

[0073] The present invention also provides a kit comprising the above-described primer combination.

[0074] In some embodiments of the present invention, the kit further includes dNTPs, polymerase, magnesium ions, and hydrogel.

[0075] In some embodiments of the present invention, the final concentration of the dNTP in the above-mentioned kit is 0.6 to 1.4 mM.

[0076] In some embodiments of the present invention, the final concentration of the dNTP in the above-mentioned kit is 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM or 1.4 mM.

[0077] In some embodiments of the present invention, the final concentration of the dNTP in the above-mentioned kit is 1.0 mM.

[0078] In some embodiments of the present invention, the polymerase in the above-mentioned kit includes Bst enzyme, and the final concentration of Bst enzyme is 6.4 to 12.8 U / reaction.

[0079] In some embodiments of the present invention, the polymerase in the above-mentioned kit includes Bst enzyme, and the final concentration of Bst enzyme is 8-11.2 U / reaction.

[0080] In some embodiments of the present invention, the final concentration of the Bst enzyme in the above-mentioned kit is 6.4 U / reaction, 8 U / reaction, 9.6 U / reaction, 11.2 U / reaction, or 12.8 U / reaction.

[0081] In some embodiments of the present invention, the final concentration of the Bst enzyme in the above-mentioned kit is 8U, 9.6U / reaction, or 11.2U / reaction.

[0082] In some embodiments of the present invention, the final concentration of the Bst enzyme in the above-described kit is 9.6 U / reaction.

[0083] In some embodiments of the present invention, the final concentration of magnesium ions in the above-mentioned kit is 2 to 6 mM.

[0084] In some embodiments of the present invention, the final concentration of magnesium ions in the above-mentioned kit is 2mM, 3mM, 4mM, 5mM or 6mM.

[0085] In some embodiments of the present invention, the final concentration of magnesium ions in the above-mentioned kit is 4 mM.

[0086] In some embodiments of the present invention, the hydrogel in the above-mentioned kit is obtained by crosslinking eight-arm polyethylene glycol acrylate and dithiol polyethylene glycol.

[0087] In some embodiments of the present invention, the molar ratio of the octahedral polyethylene glycol acrylate to the dimercaptopolyethylene glycol in the above-mentioned kit is 1:4.

[0088] In some embodiments of the present invention, the final concentration of the eight-arm polyethylene glycol acrylate in the above-mentioned kit is 2.5 mM.

[0089] In some embodiments of the present invention, the final concentration of the dithiol polyethylene glycol in the above-mentioned kit is 10 mM.

[0090] In some embodiments of the present invention, the kit further includes: a lyophilization protectant, a reducing agent, a nucleic acid dye, and a buffer solution.

[0091] In some embodiments of the present invention, the lyophilization protectant in the above-mentioned kit comprises: lyophilization protectant A and lyophilization protectant B; lyophilization protectant A comprises: 12.5 g / L mannitol, 4 g / L BSA, 15 g / L trehalose, 15 mM glycine, and 10 g / L cyclodextrin; lyophilization protectant B comprises: 20 g / L pullulan.

[0092] This invention also provides a method for detecting, for non-diagnostic purposes, whether a sample contains Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis, comprising the following steps:

[0093] S1: Obtain the nucleic acid from the pretreated test sample;

[0094] S2: Using the nucleic acid extracted in S1 as a template, loop-mediated isothermal amplification was performed using the primers in the above primer combinations to obtain the amplification results;

[0095] S3: The identification result is obtained based on the number of fluorescent amplification spots.

[0096] In some embodiments of the present invention, the pretreatment in the above method includes: a release agent method or a magnetic bead method.

[0097] In some embodiments of the present invention, the amplification time in the above method is 20 to 30 minutes.

[0098] In some embodiments of the present invention, the identification results are obtained by fluorescence microscopy imaging or by taking photos with a mobile phone under ultraviolet irradiation.

[0099] In some embodiments of the present invention, in the above method, if the primer set I is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is candidate to contain Candida albicans;

[0100] If primer set II is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Chlamydia trachomatis;

[0101] If the primer set III is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Neisseria gonorrhoeae;

[0102] If the primer set IV is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Ureaplasma urealyticum;

[0103] If the primer set V is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Mycoplasma hominis;

[0104] If primer set VI is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Mycoplasma genitalium;

[0105] If primer set VII is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain vaginal Gardner;

[0106] Using primer set VIII, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate for containing Trichomonas vaginalis.

[0107] In some embodiments of the present invention, the sample volume added during amplification in the above method is 5 to 15 μL.

[0108] In some embodiments of the present invention, the sample volume added during amplification in the above method is 5 μL, 10 μL or 15 μL.

[0109] In some embodiments of the present invention, the sample volume added during amplification in the above method is 10 μL.

[0110] The beneficial effects of this invention include:

[0111] (1) This invention provides a hydrogel-based detection technology for amplifying and detecting multiple reproductive tract microorganisms. The pathogen detection reagents include: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, Trichomonas vaginalis LAMP primer-probe combination, amplification reagents, and a hydrogel (the main components of which are dithiol polyethylene glycol and octa-arm-PEG-acrylate). After incubation for 20 minutes, the wells are observed under a fluorescence microscope. Wells with green fluorescent amplification spots are positive for the corresponding pathogens. The detection limit of the kit can reach 10. 3 The accuracy and specificity are good, with a per-copy / mL ratio.

[0112] (2) In the method provided by the present invention, the sample only needs to be coarsely processed: the preliminary lysis can be the 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;

[0113] (3) The method provided by this invention can detect 8 kinds of reproductive tract microorganisms in 20 minutes at a time. The instrument requirements are lower than those of ordinary PCR. Only a fluorescence microscope or ultraviolet flashlight with blue light wavelength, a mobile phone, and a heating device are needed to complete the detection.

[0114] (4) The method of the present invention has the characteristics of high sensitivity and high specificity. Attached Figure Description

[0115] 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.

[0116] Figure 1 Graph showing the detection results of different concentrations of Chlamydia trachomatis;

[0117] Figure 2 The image shows the test results of positive samples for eight pathogens.

[0118] Figure 3 The graph shows the detection results of different dNTP concentrations;

[0119] Figure 4 The graph shows the detection results for different Bst enzyme concentrations;

[0120] Figure 5 The figure shows the comparison results of different hydrogel concentrations;

[0121] Figure 6 Showing the amplification time of different Candida albicans samples;

[0122] Figure 7 The image shows a LAMP fluorescent PCR amplification pattern.

[0123] Figure 8 The graph shows the cross-specificity detection results of primers for 8 pathogens;

[0124] Figure 9 The results of detection using different sample pretreatment methods are shown in the figure.

[0125] Figure 10 The image shows a comparison between microscopic observation and ultraviolet light irradiation results;

[0126] Figure 11 The graph shows the test results of different freeze-drying protectants. Detailed Implementation

[0127] This invention discloses a rapid detection technology for various reproductive tract microorganisms based on hydrogel-restricted loop-mediated isothermal amplification.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] In Examples 1 to 13 of this invention, all raw materials and reagents used can be purchased from the market.

[0134] The present invention will be further illustrated below with reference to the embodiments:

[0135] Example 1: Primer combination for teratogenic amplification of reproductive tract infection pathogens

[0136] The LAMP primer sequences for Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis are shown below:

[0137] Table 1 Primer Design for Pathogens of Reproductive Tract Infections

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] To verify the amplification efficiency and sensitivity of LAMP primers for eight pathogens, qPCR was used to perform limit amplification on samples of the eight pathogens after they were identified, with concentrations of 1E6 copies / mL, 1E5 copies / mL, 1E4 copies / mL, 1000 copies / mL, and 500 copies / mL, respectively. Figure 1 The image shows the detection results of different concentrations of Chlamydia trachomatis.

[0146] Table 2 Detection sensitivity of different pathogens

[0147]

[0148]

[0149] Conclusion: Using LAMP primers, the detection of different pathogens was accurate at a sample concentration of 1000 copies / mL while maintaining both sensitivity and specificity. Detection was unstable below 1000 copies / mL, indicating that the designed primers had a sensitivity of 1000 copies / mL. The optimal primer sequences are shown below:

[0150] Table 3 Optimal primer sequences for different pathogens

[0151]

[0152]

[0153] This kit detects 1×10⁻⁶ cells / day. 3 A diagram illustrating eight pathogens at concentrations of copies / mL, including Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis. Figure 2 As shown.

[0154] Example 2: Preparation and Use of Reagents for Hydrogel-Limited Amplification for Detection of Pathogens in Reproductive Tract Infections

[0155] 1. Preparation of hydrogel-limited amplification reagent

[0156] 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 polyethylene glycol acrylate was purchased from Shanghai Tuoyang, and the thiol-PEG-thiol was purchased from Xi'an Kaixin.

[0157] To address the limitations of existing technologies, this invention provides a kit for the rapid detection of common pathogens such as Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis.

[0158] The kit contains reagent components A and B, both in lyophilized powder form. Reagent component A contains dNTPs, primers, Bst enzyme, octagonal polyethylene glycol acrylate, TCEP, lyophilization protectant A, and water. The final concentrations of dNTPs, primers, Bst enzyme, octagonal polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000), octagonal polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000), and TCEP are 1mM (total concentration of component B). The amount of lyophilization protectant A added is 3μL.

[0159] Component B contains Bst Buffer, magnesium ions, TCEP, dithiol polyethylene glycol, and lyophilization protectant B. The final concentration of Bst Buffer in the reaction system is 1×, the final concentration of magnesium ions is 4mM, the final concentration of SYBR Green is 1×, the final concentration of TCEP is 1mM, the concentration of dithiol polyethylene glycol (HS-PEG-SH, Mw=4000) is 10mM (total concentration of component A), and the amount of lyophilization protectant B added is 3μL. The specific configuration is shown in Table 4.

[0160] Table 4 Reaction System Configuration

[0161]

[0162]

[0163] 10×BstBuffer 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 value of Tris-HCl is 7.5–8.8.

[0164] 100×SYBR Green dye is obtained by diluting commercially available 10000×SYBR Green I dye by 100 times.

[0165] 0.5 mg / μL of octahedral polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000) and 0.5 mg / μL of dithiol polyethylene glycol (HS-PEG-SH, Mw = 4000) were obtained by dissolving solid powder in DEPC water.

[0166] 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 F3 being the same as those of FIP, LB, and B3. In use, the six primers are prepared into a mixture in different proportions. Some primer pairs contain only one loop primer, with the other replaced by water. Different primer sequences can be used to detect different pathogens. Specific configuration ratios are shown in Table 5.

[0167] Table 510× Primer Mixture Preparation

[0168] 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

[0169] Lyophilization protectant A: 12.5 g / L mannitol, 4 g / L BSA, 15 g / L trehalose, 15 mM glycine, 10 g / L cyclodextrin.

[0170] Lyophilization protectant B: 20 ​​g / L pullulan polysaccharide.

[0171] 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.

[0172] Component B of this invention contains no bioactive ingredients. Besides forming a gel network, the thiol-polyethylene glycol-thiol group also acts as a plasticizer 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.

[0173] In some embodiments of the present invention, the freeze-drying protectants A and B are respectively applicable to components A and B, and the amount added is 3 μL.

[0174] 2. Use of hydrogel-limited amplification for the detection of pathogens causing reproductive tract infections

[0175] 1) Sample processing

[0176] Remove the sample and vortex to mix (frozen samples should be fully thawed at room temperature before use). Take 500–1000 μL of the sample to be tested and centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant and add 100–300 μL of sample release agent to the precipitate. Vortex to mix until there is no obvious precipitate. Let stand at room temperature for 5 minutes. The supernatant is used for detection.

[0177] 2) Reagent preparation

[0178] Take out components A and B from the kit in Table 3. Reconstitute at a rate of 10 μL per reaction. Add 10 μL of reconstituted component A to the bottom of each reaction tube and 10 μL of component B to the cap.

[0179] 3) Sample addition

[0180] Add 10 μL of the sample to be tested to each of the prepared reaction tubes. Mix and centrifuge for a few seconds, then add the mixture to the reactor and cover with an anti-evaporation membrane.

[0181] 4) Incubation

[0182] The reactor after adding the sample was placed on a heater and incubated at 65°C for 20 minutes.

[0183] 5) Result detection

[0184] Remove the reactor and observe the results under a fluorescence microscope at blue light wavelength. Select a suitable field of view for preservation. Alternatively, take photos using a mobile phone under ultraviolet light.

[0185] 6) Result Interpretation

[0186] Negative: No green fluorescent amplification spots.

[0187] Positive: Contains multiple green fluorescent amplification spots (≥1).

[0188] Example 3: Optimization of magnesium ion concentration

[0189] Magnesium ions are an essential cofactor for Bst nucleic acid 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.

[0190] 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 2mM, 3mM, 4mM, 5mM, and 6mM, with other component concentrations referring to Example 2.

[0191] Table 6. Detection results for different magnesium ion concentrations

[0192]

[0193]

[0194] Conclusion: Under the premise of ensuring the accuracy of the test results, the final concentration of magnesium ions at 4 mM is the best. Too low a concentration will cause the detection limit sample to be unstable, and too high a concentration will cause false positive results.

[0195] Example 4: Optimization of dNTP Concentration

[0196] dNTPs provide essential nucleotide triphosphates for nucleic acid polymerases and are fundamental to nucleic acid synthesis. The concentration of dNTPs must be sufficient to support large-scale nucleic acid synthesis without depleting resources, but also not so high as to inhibit enzyme activity. Inappropriate dNTP concentrations may lead to reduced amplification efficiency or the production of erroneous amplification products. By optimizing the dNTP concentration, the specificity of amplification and the yield of products can be improved.

[0197] To investigate the impact of dNTP concentration within the system on samples amplified to the clinical detection limit and negative samples, the final dNTP concentrations were set to 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, and 1.4 mM. Figure 3 (As shown in Example 2), the concentrations of other components are as described in Example 2.

[0198] Table 7. Detection results of different dNTP concentrations

[0199]

[0200]

[0201] Conclusion: The optimal concentration of dNTPs is 1.0 mM. Too few dNTPs will lead to nonspecific amplification and false positive results, while too many dNTPs will inhibit the PCR reaction and result in false negative results.

[0202] Example 5: Bst enzyme concentration optimization experiment

[0203] Bst nucleic acid polymerase is the specific enzyme used in LAMP reactions, enabling rapid nucleic acid 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 nucleic acid polymerase ensures efficient nucleic acid synthesis while reducing the risk of erroneous amplification.

[0204] To investigate the effect of Bst enzyme concentration on the amplification of clinically detectable and negative samples, the Bst enzyme concentrations were set at 6.4 U, 8 U, 9.6 U, 11.2 U, and 12.8 U per reaction. Figure 4(As shown in Example 2), the concentrations of other components are as described in Example 2.

[0205] Table 8. Detection results for different Bst concentrations

[0206]

[0207] 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.

[0208] Example 6 Primer Concentration Optimization

[0209] 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 test samples were clinical samples that had undergone fluorescence qPCR assays, with a positive sample concentration of 10... 3 The primer concentrations were adjusted to 0.2×, 0.5×, and 0.8×, with other component concentrations following the guidelines in Example 2. The results are shown in Table 9.

[0210] Table 9. Detection results at different primer concentrations

[0211]

[0212] The test results show that samples with too low a primer concentration will have false negative results, while samples with too high a primer concentration will have false positive results. The optimal primer concentration is determined to be 0.5×.

[0213] Example 7: Optimization of Hydrogels at Different Concentrations

[0214] Eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000) and dithiol polyethylene glycol (HS-PEG-SH, Mw = 4000) combine under specific conditions to form a porous hydrogel. Different concentrations of the reaction system result in different pore sizes, thus affecting the size of the fluorescent amplification spots. This invention optimizes the optimal concentration ratio of eight-arm polyethylene glycol acrylate and dithiol polyethylene glycol on the hydrogel reaction system. The test samples were clinical samples assessed by fluorescent qPCR, with a positive sample concentration of 10. 5copies / mL. The concentration of the eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000) was adjusted to 2 mM, 2.5 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.

[0215] The results are as follows Figure 5 As shown in Table 10.

[0216] Table 10 Optimization of Hydrogel Concentration

[0217] Concentration 1 Concentration 2 Concentration 3 8Arm-PEG-AC-20K 2mM 2.5mM 3mM HS-PEG-SH-4k 8mM 10mM 12mM

[0218] The test results show that lower concentrations result in larger hydrogel pores and larger fluorescent amplification spots; higher concentrations result in smaller pores and smaller fluorescent amplification spots. However, this has no impact on result interpretation. Concentration 2 produces the most uniform spots, making it suitable for instrument interpretation, thus determining concentration 2 as the optimal reaction concentration.

[0219] Example 8: Sample Dosage Optimization Experiment

[0220] The amount of nucleic acid added to the amplification system has a certain impact on the detection sensitivity and accuracy. Too low a amount of added sample will lead to insufficient amplification template and reduced sensitivity, while too high a amount of template may contain inhibitors, affecting the amplification results. The sample addition amounts are set to 5 μL, 10 μL, and 15 μL for the sample release agent.

[0221] Table 11 Detection results at different sample dosages

[0222]

[0223] Conclusion: The highest sensitivity was achieved with a sample volume of 10 μL, without affecting detection accuracy; therefore, the optimal sample volume is 10 μL. In some samples, inhibition occurred with a sample volume of 15 μL.

[0224] Example 9: Optimal reaction time for monitoring amplification points

[0225] The primer sequences corresponding to the eight pathogens are shown in Table 1. After mixing the primers, they were configured into a limited amplification system and a fluorescence system (see Table 2).

[0226] Component A and component B were added in volumes of 10 μL each. 10 μL of nucleic acid templates from clinical samples extracted using magnetic beads or treated with release agents were added, respectively, for Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis. The template concentration was 10 μL for each sample. 6 copies / mL, 10 5 copies / mL, 103 The samples were counted at 100 copies / mL, with negative nucleic acid as a control. The detection speed of the two methods for positive samples was compared. The fluorescent LAMP PCR program was set to 65℃, 30s, 1 cycle; 65℃, 30s (fluorescence acquisition), 80 cycles. One cycle took approximately 1 minute. The time for limit amplification, heat fixation, and detection was 10min, 20min, and 30min. Blue light excitation was used to observe the presence of fluorescent spots under a microscope. The results are as follows: Figure 6 , Figure 7 As shown in Table 12.

[0227] Table 12 Time taken to detect positive samples for different pathogens

[0228]

[0229]

[0230] Conclusion: By comparing the limiting amplification with 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 17 cycles (17 minutes) for high-concentration samples to show amplification curves, and low-concentration samples required even longer. This indicates that limiting amplification can complete the detection within 20 minutes during the sample detection process, and has the advantage of rapid detection.

[0231] Example 10 Cross-specificity test

[0232] To verify the accuracy of this kit, and to ensure no cross-reactivity between primers, nucleic acids from different pathogen samples were cross-tested to check for false positives. The pathogen concentration was 10⁻⁶. 7 After preparing the hydrogel (copies / mL), it was placed in a 65℃ constant temperature device and heated for 20 minutes, then observed using a fluorescence microscope. The results are as follows: Figure 8 As shown.

[0233] Conclusion: There was no cross-infection among the primers for each pathogen, the specificity was good, and the positive and negative typing was accurate.

[0234] Example 11 Sample Pretreatment

[0235] The sample release agent was a mixed solution of 200 mM sodium hydroxide, 0.9% trehalose, 0.20 g / L Gemini surfactant, 0.8% proline, 40 mM sodium carbonate, and 20 g / L 1-butyl-3-methylimidazolium bromide ([BMIM]Br). 1-Butyl-3-methylimidazolium bromide exhibits good lytic activity against fungal cell walls, particularly in disrupting the lipid bilayer structure of the cell membrane. Proline and trehalose can improve the preservation stability of nucleic acids. Sodium carbonate can improve the stability of alkaline lysis environments. Clinical borderline positive samples (1000 copies / mL) and negative samples were treated using both the release agent method and the magnetic bead method (detailed steps refer to the instruction manual), and the detection results of the two methods were compared. All clinical samples were subjected to fluorescence qPCR detection and determination. The detection results are shown below. Figure 9 As shown in Table 13:

[0236] Table 13 Detection results of different pathogen pretreatment methods

[0237]

[0238] Conclusion: For samples with detection limits, there is no significant difference between the magnetic bead method and the release agent method; both can accurately distinguish sample types. The release agent method is simpler to operate and takes less time. When combined with a limit amplification system, detection can be completed within 30 minutes.

[0239] Example 12 Interpretation of Different Detection Devices

[0240] Based on the presence of fluorescent spots in the limited amplification system, observation can be achieved through fluorescence microscopy imaging and mobile phone photography under ultraviolet light. This invention uses both methods for detection and interpretation, comparing the consistency of the two interpretation methods. The test samples are clinical samples that have undergone fluorescence qPCR assays. The results are as follows: Figure 10 As shown.

[0241] Conclusion: The results of the fluorescence microscope and the ultraviolet flashlight irradiation were consistent, indicating that the limited amplification system is highly applicable to different reaction containers and interpretation methods. When the detection device is transparent, has good light transmittance, and a clear visual range, the sample detection results can be interpreted.

[0242] Example 13 Optimization of Lyophilization Protectant

[0243] This embodiment uses Candida albicans for testing. 3 μL of different formulations of lyophilization protectant A were added to each sample A, and the samples were placed in a lyophilizer for lyophilization. The lyophilized morphology was observed. Similarly, 3 μL of different formulations of lyophilization protectant B were added to each sample B, and the lyophilized morphology of the different protectants was observed. The lyophilization procedure is shown in Table 14.

[0244] Table 14 Freeze-drying Procedure

[0245]

[0246] Table 15 Formulation of Lyophilization Protectant

[0247] Lyophilization protectant A1 Lyophilization protectant A1 Lyophilization protectant A3 Lyophilization protectant B1 Lyophilization protectant B2 Lyophilization protectant B3 12.5g / L mannitol 12.5g / L mannitol 12.5g / L mannitol 20g / L pullulan 12.5g / L mannitol 20g / L pullulan 4g / LBSA 4g / LBSA 4g / LBSA 4g / LBSA 4g / LBSA 15g / L Trehalose 15g / L Trehalose 15g / L Trehalose 15g / L Trehalose 15mM glycine 15mM glycine 10g / L cyclodextrin

[0248] like Figure 11 As shown, freeze-drying protectants A1, A2, and A3 all showed good freeze-drying properties; freeze-drying protectants B1 and B2 were loose and could not be shaped after freeze-drying, while freeze-drying protectant B3 showed the best effect. It is recommended to use freeze-drying protectant B3 in combination with A1, A2, and A3 for testing.

[0249] like Figure 11 As shown, after reconstitution of lyophilization protectant B3 with lyophilization protectants A1 / A2 / A3, their amplification efficiency was tested. According to... Figure 11 The fluorescence results after lyophilization and reconstitution show that the amplification sites formed after lyophilization protectant B3 was reconstituted with lyophilization protectants A1 and A2 exhibited diffusion and could not be formed; the amplification sites formed after reconstitution with lyophilization protectant A3 were normal and had the highest amplification efficiency. Therefore, this combination was selected as the best protectant.

[0250] Lyophilization protectants A3 and B3 were selected, and components A / B were lyophilized and stored at room temperature for different times. The storage stability of the lyophilized reagents was tested, and the results are shown in Table 16.

[0251] As shown in Table 16, the lyophilized reagent can still stably detect Candida albicans samples after being stored at room temperature for 15 months, using a sample with 1000 copies / mL as the detection limit.

[0252] Table 16 Results of the storage stability of lyophilized reagents

[0253] Pathogens 0 months 3 months 6 months 9 months 12 months 15 months Candida albicans Positive Positive Positive Positive Positive Positive Chlamydia trachomatis Positive Positive Positive Positive Positive Positive Neisseria gonorrhoeae Positive Positive Positive Positive Positive Positive Ureaplasma urealyticum Positive Positive Positive Positive Positive Positive Mycoplasma hominis Positive Positive Positive Positive Positive Positive Mycoplasma genitalium Positive Positive Positive Positive Positive Positive Vaginal Gardner Positive Positive Positive Positive Positive Positive Trichomonas vaginalis Positive Positive Positive Positive Positive Positive

Claims

1. A primer combination, characterized in that, include: The following are options (a1), (a2), or (a3): (a1) It consists of primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII and primer set VIII; (a2) It consists of any one, two, three, four, five, six or seven of the primer sets I, II, III, IV, V, VI, VII and VIII; (a3) includes primer set I, primer set II, primer set III, primer set IV, primer set V, primer set VI, primer set VII and primer set VIII; The primer set I includes one or more of primer set I-I, primer set I-II, and primer set I-III; 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 I-I-F3 has the following characteristics: (b1) The nucleotide sequence shown in SEQ ID NO:1; (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); The primer I-I-B3 has the following characteristics: (b5) The nucleotide sequence shown in SEQ ID NO:2; (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); The primer I-I-FIP has the following characteristics: (b9) The nucleotide sequence shown in SEQ ID NO:3; (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 primer I-I-BIP has the following characteristics: (b13) The nucleotide sequence shown in SEQ ID NO:4; (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); The primer I-I-LF has the following characteristics: (b17) The nucleotide sequence shown in SEQ ID NO:5; (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); The primer I-I-LB has the following characteristics: (b21) The nucleotide sequence shown in SEQ ID NO:6; (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 I-II consists of primer I-II-F3, primer I-II-B3, primer I-II-FIP, primer I-II-BIP, and primer I-II-LF; The primer I-II-F3 has the following characteristics: (b25) The nucleotide sequence shown in SEQ ID NO:7; (b26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b25); (b27) A sequence that has at least 80% homology with the nucleotide sequence shown in (b25); (b28), complementary sequences to sequences shown in (b25), (b26) or (b27); The primer I-II-B3 has the following characteristics: (b29) The nucleotide sequence shown in SEQ ID NO:8; (b30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b29); (b31) A sequence that has at least 80% homology with the nucleotide sequence shown in (b29); (b32), complementary sequences to sequences shown in (b29), (b30) or (b31); The primer I-II-FIP has the following characteristics: (b33) The nucleotide sequence shown in SEQ ID NO:9; (b34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b33); (b35) A sequence that has at least 80% homology with the nucleotide sequence shown in (b33); (b36), complementary sequences to sequences shown in (b33), (b34) or (b35); The primer I-II-BIP has the following characteristics: (b37) The nucleotide sequence shown in SEQ ID NO:10; (b38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b37); (b39) A sequence that has at least 80% homology with the nucleotide sequence shown in (b37); (b40), complementary sequences to sequences shown in (b37), (b38) or (b39); The primer I-II-LF has the following characteristics: (b41) The nucleotide sequence shown in SEQ ID NO:11; (b42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b41); (b43) A sequence that has at least 80% homology with the nucleotide sequence shown in (b41); (b44), complementary sequences to sequences shown in (b41), (b42) or (b43); The primer set I-III consists of primer I-III-F3, primer I-III-B3, primer I-III-FIP, primer I-III-BIP, primer I-III-LF, and primer I-I-LB; The primer I-III-F3 has the following characteristics: (b45) The nucleotide sequence shown in SEQ ID NO:12; (b46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b45); (b47) A sequence that has at least 80% homology with the nucleotide sequence shown in (b45); (b48), complementary sequences to sequences shown in (b45), (b46) or (b47); The primer I-III-B3 has the following characteristics: (b49) The nucleotide sequence shown in SEQ ID NO:13; (b50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b49); (b51) A sequence that has at least 80% homology with the nucleotide sequence shown in (b49); (b52), complementary sequences to sequences shown in (b49), (b50) or (b51); The primer I-III-FIP has the following characteristics: (b53) The nucleotide sequence shown in SEQ ID NO:14; (b54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b53); (b55) A sequence that has at least 80% homology with the nucleotide sequence shown in (b53); (b56), complementary sequences to sequences shown in (b53), (b54) or (b55); The primer I-III-BIP has the following characteristics: (b57) The nucleotide sequence shown in SEQ ID NO:15; (b58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b57); (b59) A sequence that has at least 80% homology with the nucleotide sequence shown in (b57); (b60), complementary sequences to sequences such as (b57), (b58) or (b59); The primer I-III-LF has the following characteristics: (b61) The nucleotide sequence shown in SEQ ID NO:16; (b62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b61); (b63) A sequence that has at least 80% homology with the nucleotide sequence shown in (b61); (b64), complementary sequences to sequences shown in (b61), (b62) or (b63); The primer I-III-LB has the following characteristics: (b65) The nucleotide sequence shown in SEQ ID NO:17; (b66) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (b65); (b67) A sequence that has at least 80% homology with the nucleotide sequence shown in (b65); (b68), complementary sequences to sequences shown in (b65), (b66) or (b67); The primer set II includes one or more of primer set II-I, primer set II-II, and primer set II-III; 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-LF, and primer II-I-LB; The primer II-I-F3 has the following characteristics: (c1) The nucleotide sequence shown in SEQ ID NO:18; (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); The primer II-I-B3 has the following characteristics: (c5) The nucleotide sequence shown in SEQ ID NO:19; (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); The primer II-I-FIP has the following characteristics: (c9) The nucleotide sequence shown in SEQ ID NO:20; (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); The primer II-I-BIP has the following characteristics: (c13) The nucleotide sequence shown in SEQ ID NO:21; (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); The primer II-I-LF has the following characteristics: (c17) The nucleotide sequence shown in SEQ ID NO:22; (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); The primer II-I-LB has the following characteristics: (c21) The nucleotide sequence shown in SEQ ID NO:23; (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 II-II consists of primer II-II-F3, primer II-II-B3, primer II-II-FIP, primer II-II-BIP, and primer II-II-LF; The primer II-II-F3 has the following characteristics: (c25) The nucleotide sequence shown in SEQ ID NO:24; (c26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c25); (c27) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c25); (c28), complementary sequences to sequences shown in (c25), (c26) or (c27); The primer II-II-B3 has the following characteristics: (c29) The nucleotide sequence shown in SEQ ID NO:25; (c30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c29); (c31) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c29); (c32), complementary sequences to sequences shown in (c29), (c30) or (c31); The primer II-II-FIP has the following characteristics: (c33) The nucleotide sequence shown in SEQ ID NO:26; (c34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c33); (c35) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c33); (c36), complementary sequences to sequences shown in (c33), (c34) or (c35); The primer II-II-BIP has the following characteristics: (c37) The nucleotide sequence shown in SEQ ID NO:27; (c38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c37); (c39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c37); (c40), complementary sequences to sequences such as (c37), (c38) or (c39); The primer II-II-LF has the following characteristics: (c41) The nucleotide sequence shown in SEQ ID NO:28; (c42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c41); (c43) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c41); (c44), complementary sequences to sequences such as (c41), (c42) or (c43); The primer set II-III consists of primer II-III-F3, primer II-III-B3, primer II-III-FIP, primer II-III-BIP, and primer II-III-LF; The primer II-III-F3 has the following characteristics: (c45) The nucleotide sequence shown in SEQ ID NO:29; (c46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c45); (c47) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c45); (c48), complementary sequences to sequences such as (c45), (c46) or (c47); The primer II-III-B3 has the following characteristics: (c49) The nucleotide sequence shown in SEQ ID NO:30; (c50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to have the nucleotide sequence shown in (c49); (c51) and the sequence having at least 80% homology with the nucleotide sequence shown in (c49); (c52), complementary sequences to sequences such as (c49), (c50) or (c51); The primers II-III-FIP have: (c53) The nucleotide sequence shown in SEQ ID NO:31; (c54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c53); (c55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c53); (c56), complementary sequences to sequences such as (c53), (c54) or (c55); The primers II-III-BIP have the following characteristics: (c57) The nucleotide sequence shown in SEQ ID NO:32; (c58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c57); (c59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c57); Complementary sequences to sequences such as (c60), (c57), (c58), or (c59); The primers II-III-LF have: (c61) The nucleotide sequence shown in SEQ ID NO:33; (c62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (c61); (c63) is a sequence that has at least 80% homology with the nucleotide sequence shown in (c61); (c64), complementary sequences to sequences shown in (c61), (c62) or (c63); The primer set III includes one or more of primer set III-I, primer set III-II, and primer set III-III; 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 III-I-F3 has the following characteristics: (d1) The nucleotide sequence shown in SEQ ID NO:34; (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); The primer III-I-B3 has the following characteristics: (d5) Any nucleotide sequence as shown in SEQ ID NO:35; (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 primer III-I-FIP has the following characteristics: (d9) The nucleotide sequence shown in SEQ ID NO:36; (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 primer III-I-BIP has the following characteristics: (d13) The nucleotide sequence shown in SEQ ID NO:37; (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); The primer III-I-LF has the following characteristics: (d17) The nucleotide sequence shown in SEQ ID NO:38; (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 primer III-I-LB has the following characteristics: (d21) The nucleotide sequence shown in SEQ ID NO:39; (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 III-II consists of primer III-II-F3, primer III-II-B3, primer III-II-FIP, primer III-II-BIP, and primer III-II-LF; The primer III-II-F3 has the following characteristics: (d25) The nucleotide sequence shown in SEQ ID NO:40; (d26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d25); (d27) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d25); (d28), complementary sequences to sequences such as (d25), (d26) or (d27); The primer III-II-B3 has the following characteristics: (d29) Any nucleotide sequence as shown in SEQ ID NO:41; (d30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d29); (d31) and (d29) are sequences that have at least 80% homology with the nucleotide sequences shown. (d32), complementary sequences to sequences such as (d29), (d30) or (d31); The primer III-II-FIP has the following characteristics: (d33), nucleotide sequence as shown in SEQ ID NO:42; (d34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d33); (d35) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d33); (d36), complementary sequences to sequences such as (d33), (d34) or (d35); The primer III-II-BIP has the following characteristics: (d37) The nucleotide sequence shown in SEQ ID NO:43; (d38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d37); (d39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d37); (d40), complementary sequences to sequences such as (d37), (d38) or (d39); The primer III-II-LF has the following characteristics: (d41) The nucleotide sequence shown in SEQ ID NO:44; (d42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d41); (d43) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d41); (d44), complementary sequences to sequences such as (d41), (d42) or (d43); The primer set III-III consists of primers III-III-F3, III-III-B3, III-III-FIP, III-III-BIP, III-III-LF, and III-III-LB. The primer III-III-F3 has the following characteristics: (d45) The nucleotide sequence shown in SEQ ID NO:45; (d46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d45); (d47) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d45); (d48), complementary sequences to sequences such as (d45), (d46) or (d47); The primer III-III-B3 has the following characteristics: (d49) Any nucleotide sequence as shown in SEQ ID NO:46; (d50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d49); (d51) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d49); (d52), complementary sequences to sequences such as (d49), (d50) or (d51); The primer III-III-FIP has the following characteristics: (d53) The nucleotide sequence shown in SEQ ID NO:47; (d54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d53); (d55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d53); (d56), complementary sequences to sequences such as (d53), (d54) or (d55); The primer III-III-BIP has the following characteristics: (d57) The nucleotide sequence shown in SEQ ID NO:48; (d58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d57); (d59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d57); Complementary sequences to sequences such as (d60), (d57), (d58), or (d59); The primer III-III-LF has the following characteristics: (d61) The nucleotide sequence shown in SEQ ID NO:49; (d62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d61); (d63) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d61); (d64), complementary sequences to sequences shown in (d61), (d62) or (d63); The primer III-III-LB has the following characteristics: (d65) The nucleotide sequence shown in SEQ ID NO:50; (d66) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (d65); (d67) is a sequence that has at least 80% homology with the nucleotide sequence shown in (d65); (d68), complementary sequences to sequences such as (d65), (d66) or (d67); The primer set IV includes one or more of primer set IV-I, primer set IV-II, and primer set IV-III; The primer set IV-I consists of primer IV-I-F3, primer IV-I-B3, primer IV-I-FIP, primer IV-I-BIP, and primer IV-I-LF; The primer IV-I-F3 has the following characteristics: (e1) The nucleotide sequence shown in SEQ ID NO:51; (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); The primer IV-I-B3 has the following characteristics: (e5) The nucleotide sequence shown in SEQ ID NO:52; (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 have: (e9) The nucleotide sequence shown in SEQ ID NO:53; (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 primer IV-I-BIP has the following characteristics: (e13) The nucleotide sequence shown in SEQ ID NO:54; (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); The primers IV-I-LF have: (e17) The nucleotide sequence shown in SEQ ID NO:55; (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 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; The primer IV-II-F3 has the following characteristics: (e21) The nucleotide sequence shown in SEQ ID NO:56; (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 IV-II-B3 has the following characteristics: (e25) The nucleotide sequence shown in SEQ ID NO:57; (e26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e25); (e27) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e25); (e28), complementary sequences to sequences such as (e25), (e26) or (e27); The primers IV-II-FIP have: (e29) The nucleotide sequence shown in SEQ ID NO:58; (e30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e29); (e31) and sequences that have at least 80% homology with the nucleotide sequences shown in (e29); (e32), complementary sequences to sequences shown in (e29), (e30) or (e31); The primers IV-II-BIP have the following characteristics: (e33) Nucleotide sequences as shown in SEQ ID NO:59; (e34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e33); (e35) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e33); (e36), complementary sequences to sequences such as (e33), (e34) or (e35); The primers IV-II-LF have: (e37) The nucleotide sequence shown in SEQ ID NO:60; (e38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e37); (e39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e37); (e40), complementary sequences to sequences such as (e37), (e38) or (e39); The primers IV-II-LB have: (e41) The nucleotide sequence shown in SEQ ID NO:61; (e42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e41); (e43) A sequence that has at least 80% homology with the nucleotide sequence shown in (e41); (e44), complementary sequences to sequences such as (e41), (e42) or (e43); The primer set IV-III consists of primer IV-III-F3, primer IV-III-B3, primer IV-III-FIP, primer IV-III-BIP, and primer IV-III-LF; The primer IV-III-F3 has the following characteristics: (e45) The nucleotide sequence shown in SEQ ID NO:62; (e46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e45); (e47) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e45); (e48), complementary sequences to sequences such as (e45), (e46) or (e47); The primer IV-III-B3 has the following characteristics: (e49) Nucleotide sequences as shown in SEQ ID NO:63; (e50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e49); (e51) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e49); (e52), complementary sequences to sequences such as (e49), (e50) or (e51); The primers IV-III-FIP have the following characteristics: (e53) The nucleotide sequence shown in SEQ ID NO:64; (e54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e53); (e55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e53); (e56), complementary sequences to sequences such as (e53), (e54) or (e55); The primers IV-III-BIP have the following characteristics: (e57) The nucleotide sequence shown in SEQ ID NO:65; (e58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e57); (e59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e57); (e60), complementary sequences to sequences such as (e57), (e57) or (e58); The primers IV-III-LF have: (e61) The nucleotide sequence shown in SEQ ID NO:66; (e62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (e61); (e63) is a sequence that has at least 80% homology with the nucleotide sequence shown in (e61); (e64), complementary sequences to sequences shown in (e61), (e62) or (e63); The primer set V includes one or more of primer set V-I, primer set V-II, and primer set V-III; 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 V-I-F3 has the following characteristics: (f1) The nucleotide sequence shown in SEQ ID NO:67; (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 such as (f1), (f2) or (f3); The primer V-I-B3 has the following characteristics: (f5) The nucleotide sequence shown in SEQ ID NO:68; (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 primer V-I-FIP has the following characteristics: (f9) The nucleotide sequence shown in SEQ ID NO:69; (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 primer V-I-BIP has the following characteristics: (f13) The nucleotide sequence shown in SEQ ID NO:70; (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:71; (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 primer V-I-LB has the following characteristics: (f21) The nucleotide sequence shown in SEQ ID NO:72; (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 V-II consists of primer V-II-F3, primer V-II-B3, primer V-II-FIP, primer V-II-BIP, primer V-II-LF, and primer V-II-LB; The primer V-II-F3 has the following characteristics: (f25) The nucleotide sequence shown in SEQ ID NO:73; (f26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f25); (f27) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f25); (f28), complementary sequences to sequences shown in (f25), (f26) or (f27); The primer V-II-B3 has the following characteristics: (f29) The nucleotide sequence shown in SEQ ID NO:74; (f30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f29); (f31) and sequences that have at least 80% homology with the nucleotide sequences shown in (f29); (f32), complementary sequences to sequences shown in (f29), (f30) or (f31); The primer V-II-FIP has the following characteristics: (f33) The nucleotide sequence shown in SEQ ID NO:75; (f34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f33); (f35) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f33); (f36), complementary sequences to sequences shown in (f33), (f34) or (f35); The primer V-II-BIP has the following characteristics: (f37) The nucleotide sequence shown in SEQ ID NO:76; (f38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f37); (f39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f37); Complementary sequences to sequences such as (f40), (f37), (f38), or (f39); The primer V-II-LF has the following characteristics: (f41) The nucleotide sequence shown in SEQ ID NO:77; (f42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f41); (f43) A sequence that has at least 80% homology with the nucleotide sequence shown in (f41); (f44), complementary sequences to sequences shown in (f41), (f42) or (f43); The primer V-II-LB has the following characteristics: (f45) The nucleotide sequence shown in SEQ ID NO:78; (f46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f45); (f47) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f45); (f48), complementary sequences to sequences shown in (f45), (f46) or (f47); The primer set V-III consists of primer V-III-F3, primer V-III-B3, primer V-III-FIP, primer V-III-BIP, primer V-III-LF, and primer V-III-LB; The primer V-Ⅲ-F3 has the following characteristics: (f49) The nucleotide sequence shown in SEQ ID NO:79; (f50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f49); (f51) and sequences that have at least 80% homology with the nucleotide sequences shown in (f49); (f52), complementary sequences to sequences shown in (f49), (f50) or (f51); The primer V-Ⅲ-B3 has the following characteristics: (f53) The nucleotide sequence shown in SEQ ID NO:80; (f54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f53); (f55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f53); (f56), complementary sequences to sequences such as (f53), (f54) or (f55); The primer V-Ⅲ-FIP has the following characteristics: (f57) The nucleotide sequence shown in SEQ ID NO:81; (f58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f57); (f59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f57); Complementary sequences to sequences such as (f60), (f57), (f58), or (f59); The primer V-Ⅲ-BIP has the following characteristics: (f61) The nucleotide sequence shown in SEQ ID NO:82; (f62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f61); (f63) A sequence that has at least 80% homology with the nucleotide sequence shown in (f61); (f64), complementary sequences to sequences shown in (f61), (f62) or (f63); The primer V-Ⅲ-LF has the following characteristics: (f65) The nucleotide sequence shown in SEQ ID NO:83; (f66) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f65); (f67) is a sequence that has at least 80% homology with the nucleotide sequence shown in (f65); (f68), complementary sequences to sequences shown in (f65), (f66) or (f67); The primer V-Ⅲ-LB has the following characteristics: (f69) The nucleotide sequence shown in SEQ ID NO:84; (f70) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (f69); (f71) and sequences that have at least 80% homology with the nucleotide sequences shown in (f69); (f72), complementary sequences to sequences shown in (f69), (f70) or (f71); The primer set VI includes one or more of primer set VI-Ⅰ, primer set VI-Ⅱ, and primer set VI-Ⅲ; 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-LF; The primer VI-Ⅰ-F3 has the following characteristics: (g1) The nucleotide sequence shown in SEQ ID NO:85; (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); The primer VI-Ⅰ-B3 has the following characteristics: (g5) The nucleotide sequence shown in SEQ ID NO:86; (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 primer VI-Ⅰ-FIP has the following characteristics: (g9) The nucleotide sequence shown in SEQ ID NO:87; (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 primer VI-Ⅰ-BIP has the following characteristics: (g13) The nucleotide sequence shown in SEQ ID NO:88; (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-Ⅰ-LF has the following characteristics: (g17) The nucleotide sequence shown in SEQ ID NO:89; (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 primer set VI-II consists of primer VI-II-F3, primer VI-II-B3, primer VI-II-FIP, primer VI-II-BIP, and primer VI-II-LF; The primer VI-Ⅱ-F3 has the following characteristics: (g21) The nucleotide sequence shown in SEQ ID NO:90; (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 VI-Ⅱ-B3 has the following characteristics: (g25) The nucleotide sequence shown in SEQ ID NO:91; (g26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g25); (g27) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g25); (g28), complementary sequences to sequences such as (g25), (g26) or (g27); The primer VI-II-FIP has the following characteristics: (g29) The nucleotide sequence shown in SEQ ID NO:92; (g30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g29); (g31) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g29); (g32), complementary sequences to sequences such as (g29), (g30) or (g31); The primer VI-II-BIP has the following characteristics: (g33), nucleotide sequence as shown in SEQ ID NO:93; (g34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g33); (g35) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g33); (g36), complementary sequences to sequences such as (g33), (g34) or (g35); The primer VI-II-LF has the following characteristics: (g37), nucleotide sequence as shown in SEQ ID NO:94; (g38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g37); (g39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g37); Complementary sequences to sequences such as (g40), (g37), (g38), or (g39); The primer set VI-III consists of primer VI-III-F3, primer VI-III-B3, primer VI-III-FIP, primer VI-III-BIP, primer VI-III-LF and primer V-III-LB; The primer VI-Ⅲ-F3 has the following characteristics: (g41) The nucleotide sequence shown in SEQ ID NO:95; (g42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g41); (g43) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g41); (g44), complementary sequences to sequences such as (g41), (g42) or (g43); The primer VI-Ⅲ-B3 has the following characteristics: (g45) The nucleotide sequence shown in SEQ ID NO:96; (g46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g45); (g47) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g45); (g48), complementary sequences to sequences such as (g45), (g46) or (g47); The primer VI-Ⅲ-FIP has the following characteristics: (g49) The nucleotide sequence shown in SEQ ID NO:97; (g50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g49); (g51) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g49); (g52), complementary sequences to sequences such as (g49), (g50) or (g51); The primer VI-Ⅲ-BIP has the following characteristics: (g53), nucleotide sequence as shown in SEQ ID NO:98; (g54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g53); (g55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g53); (g56), complementary sequences to sequences such as (g53), (g54) or (g55); The primer VI-Ⅲ-LF has the following characteristics: (g57) The nucleotide sequence shown in SEQ ID NO:99; (g58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g57); (g59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g57); Complementary sequences to sequences such as (g60), (g57), (g58), or (g59); The primer VI-Ⅲ-LB has the following characteristics: (g60), nucleotide sequence as shown in SEQ ID NO:100; (g61) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (g60); (g62) is a sequence that has at least 80% homology with the nucleotide sequence shown in (g60); (g63), complementary sequences to sequences such as (g60), (g61) or (g62); The primer set VII includes one or more of primer set VII-I, primer set VII-II, and primer set VII-III; 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 VII-Ⅰ-F3 has the following characteristics: (h1) The nucleotide sequence shown in SEQ ID NO:101; (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); The primer VII-Ⅰ-B3 has the following characteristics: (h5) The nucleotide sequence shown in SEQ ID NO:102; (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 primer VII-I-FIP has the following characteristics: (h9) The nucleotide sequence shown in SEQ ID NO:103; (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 primer VII-I-BIP has the following characteristics: (h13) The nucleotide sequence shown in SEQ ID NO:104; (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 primer VII-I-LF has the following characteristics: (h17), nucleotide sequence as shown in SEQ ID NO:105; (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 primer VII-Ⅰ-LB has the following characteristics: (h21) The nucleotide sequence shown in SEQ ID NO:106; (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); 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; The primer VII-II-F3 has the following characteristics: (h25), nucleotide sequences as shown in SEQ ID NO:107; (h26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h25); (h27) and sequences that have at least 80% homology with the nucleotide sequences shown in (h25); (h28), complementary sequences to sequences such as (h25), (h26) or (h27); The primer VII-II-B3 has the following characteristics: (h29), nucleotide sequences as shown in SEQ ID NO:108; (h30) is a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h29); (h31) and sequences that have at least 80% homology with the nucleotide sequences shown in (h29); (h32), complementary sequences to sequences shown in (h29), (h30) or (h31); The primer VII-II-FIP has the following characteristics: (h33), nucleotide sequences as shown in SEQ ID NO:109; (h34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h33); (h35) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h33); (h36), complementary sequences to sequences such as (h33), (h34) or (h35); The primer VII-II-BIP has the following characteristics: (h37), nucleotide sequences as shown in SEQ ID NO:110; (h38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h37); (h39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h37); Complementary sequences to sequences such as (h40), (h37), (h38), or (h39); The primer VII-II-LF has the following characteristics: (h41) The nucleotide sequence shown in SEQ ID NO:111; (h42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h41); (h43) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h41); (h44), complementary sequences to sequences such as (h41), (h42) or (h43); The primer VII-II-LB has the following characteristics: (h45), as shown in SEQ ID NO:112; (h46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h45); (h47) and sequences that have at least 80% homology with the nucleotide sequences shown in (h45); (h48), complementary sequences to sequences such as (h45), (h46) or (h47); The primer set VII-III consists of primer VII-III-F3, primer VII-III-B3, primer VII-III-FIP, primer VII-III-BIP, primer VII-III-LF, and primer VII-III-LB; The primer VII-Ⅲ-F3 has the following characteristics: (h49), nucleotide sequence as shown in SEQ ID NO:113; (h50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h49); (h51) and sequences that have at least 80% homology with the nucleotide sequences shown in (h49); (h52), complementary sequences to sequences such as (h49), (h50) or (h51); The primer VII-Ⅲ-B3 has the following characteristics: (h53), nucleotide sequences as shown in SEQ ID NO:114; (h54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h53); (h55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h53); (h56), complementary sequences to sequences such as (h53), (h54) or (h55); The primer VII-III-FIP has the following characteristics: (h57), nucleotide sequences as shown in SEQ ID NO:115; (h58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h57); (h59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h57); Complementary sequences to sequences such as (h60), (h57), (h58), or (h59); The primer VII-III-BIP has the following characteristics: (h61) The nucleotide sequence shown in SEQ ID NO:116; (h62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h61); (h63) is a sequence that has at least 80% homology with the nucleotide sequence shown in (h61); (h64), complementary sequences to sequences such as (h61), (h62) or (h63); The primer VII-III-LF has the following characteristics: (h65), nucleotide sequence as shown in SEQ ID NO:117; (h66) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (h65); (h67) and sequences that have at least 80% homology with the nucleotide sequences shown in (h65); (h68), complementary sequences to sequences such as (h65), (h66) or (h67); The primer VII-III-LB has the following characteristics: (h69), nucleotide sequences as shown in SEQ ID NO:118; (h70), a nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to have the nucleotide sequence shown in (h69); (h71) and sequences that have at least 80% homology with the nucleotide sequences shown in (h69); (h72), complementary sequences to sequences such as (h69), (h70) or (h71); The primer set VIII includes one or more of primer sets VIII-I, VIII-II, and VIII-III; 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 VIIII-Ⅰ-F3 has the following characteristics: (i1) The nucleotide sequence shown in SEQ ID NO:119; (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); The primer VIII-Ⅰ-B3 has the following characteristics: (i5) The nucleotide sequence shown in SEQ ID NO:120; (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 primer VIII-I-FIP has the following characteristics: (i9) The nucleotide sequence shown in SEQ ID NO:121; (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 primer VIII-I-BIP has the following characteristics: (i13) The nucleotide sequence shown in SEQ ID NO:122; (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 primer VIII-I-LF has the following characteristics: (i17) The nucleotide sequence shown in SEQ ID NO:123; (i18) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i17); (i19) A sequence that has at least 80% homology with the nucleotide sequence shown in (i17); (i20), complementary sequences to sequences such as (i17), (i18) or (i19); The primer VIII-I-LB has the following characteristics: (i21) The nucleotide sequence shown in SEQ ID NO:124; (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 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; The primer VIII-II-F3 has the following characteristics: (i25) The nucleotide sequence shown in SEQ ID NO:125; (i26) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i25); (i27) and sequences that have at least 80% homology with the nucleotide sequences shown in (i25); (i28), complementary sequences to sequences such as (i25), (i26) or (i27); The primer VIII-II-B3 has the following characteristics: (i29) The nucleotide sequence shown in SEQ ID NO:126; (i30) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i29); (i31) and sequences that have at least 80% homology with the nucleotide sequences shown in (i29); (i32), complementary sequences to sequences such as (i29), (i30) or (i31); The primer VIII-II-FIP has the following characteristics: (i33), nucleotide sequence as shown in SEQ ID NO:127; (i34) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i33); (i35) A sequence that has at least 80% homology with the nucleotide sequence shown in (i33); (i36), complementary sequences to sequences such as (i33), (i34) or (i35); The primer VIII-II-BIP has the following characteristics: (i37) The nucleotide sequence shown in SEQ ID NO:128; (i38) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i37); (i39) is a sequence that has at least 80% homology with the nucleotide sequence shown in (i37); (i40), complementary sequences to sequences such as (i37), (i38) or (i39); The primer VIII-II-LF has the following characteristics: (i41) The nucleotide sequence shown in SEQ ID NO:129; (i42) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i41); (i43) A sequence that has at least 80% homology with the nucleotide sequence shown in (i41); (i44), complementary sequences to sequences such as (i41), (i42) or (i43); The primer VIII-II-LB has the following characteristics: (i45) The nucleotide sequence shown in SEQ ID NO:130; (i46) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i45); (i47) A sequence that has at least 80% homology with the nucleotide sequence shown in (i45); (i48), complementary sequences to sequences such as (i45), (i46) or (i47); The primer set VIII-III consists of primer VIII-III-F3, primer VIII-III-B3, primer VIII-III-FIP, primer VIII-III-BIP, primer VIII-III-LF, and primer VIII-III-LB; The primer VIII-Ⅲ-F3 has the following characteristics: (i49) The nucleotide sequence shown in SEQ ID NO:131; (i50) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i49); (i51) and sequences that have at least 80% homology with the nucleotide sequences shown in (i49); (i52), complementary sequences to sequences such as (i49), (i50) or (i51); The primer VIII-Ⅲ-B3 has the following characteristics: (i53) The nucleotide sequence shown in SEQ ID NO:132; (i54) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i53); (i55) is a sequence that has at least 80% homology with the nucleotide sequence shown in (i53); (i56), complementary sequences to sequences such as (i53), (i54) or (i55); The primer VIII-Ⅲ-FIP has the following characteristics: (i57) The nucleotide sequence shown in SEQ ID NO:133; (i58) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i57); (i59) is a sequence that has at least 80% homology with the nucleotide sequence shown in (i57); Complementary sequences to sequences such as (i60), (i57), (i58), or (i59); The primer VIII-Ⅲ-BIP has the following characteristics: (i61) The nucleotide sequence shown in SEQ ID NO:134; (i62) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i61); (i63) A sequence that has at least 80% homology with the nucleotide sequence shown in (i61); (i64), complementary sequences to sequences such as (i61), (i62) or (i63); The primer VIII-Ⅲ-LF has the following characteristics: (i65), as shown in SEQ ID NO:135; (i66) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i65); (i67) A sequence that has at least 80% homology with the nucleotide sequence shown in (i65); (i68), complementary sequences to sequences such as (i65), (i66) or (i67); The primer VIII-Ⅲ-LB has the following characteristics: (i69) The nucleotide sequence shown in SEQ ID NO:136; (i70) A nucleotide sequence obtained by modifying, substituting, deleting and / or adding one or more bases to the nucleotide sequence shown in (i69); (i71) and sequences that have at least 80% homology with the nucleotide sequences shown in (i69); Complementary sequences to sequences such as (i72), (i69), (i70), or (i71).

2. The primer combination as described in claim 1, characterized in that, The molar ratio of primers I-I-F3, I-I-B3, I-I-FIP, I-I-BIP, I-I-LF, and I-I-LB in primer set I-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers I-II-F3, I-II-B3, I-II-FIP, I-II-BIP and I-II-LF in primer set I-II is: (1-2): (1-2): (4-8): (4-8): (2-4); The molar ratio of primers I-III-F3, I-III-B3, I-III-FIP, I-III-BIP, I-III-LF, and I-III-LB in primer sets I-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers II-I-F3, II-I-B3, II-I-FIP, II-I-BIP, II-I-LF, and II-I-LB in primer set II-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers II-II-F3, II-II-B3, II-II-FIP, II-II-BIP and II-II-LF in primer set II-II is (1-2):(1-2):(4-8):(4-8):(2-4); The molar ratio of primers II-III-F3, II-III-B3, II-III-FIP, II-III-BIP and II-III-LF in primer set II-III is: (1-2): (1-2): (4-8): (4-8): (2-4); The molar ratio of primers III-I-F3, III-I-B3, III-I-FIP, III-I-BIP, III-I-LF, and III-I-LB in primer set III-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers III-II-F3, III-II-B3, III-II-FIP, III-II-BIP and III-II-LF in primer set III-II is (1-2):(1-2):(4-8):(4-8):(2-4); The molar ratio of primers III-III-F3, III-III-B3, III-III-FIP, III-III-BIP, III-III-LF, and III-III-LB in primer set III-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers IV-I-F3, IV-I-B3, IV-I-FIP, IV-I-BIP and IV-I-LF in primer set IV-I is (1-2):(1-2):(4-8):(4-8):(2-4); The molar ratio of primers IV-II-F3, IV-II-B3, IV-II-FIP, IV-II-BIP, IV-II-LF, and IV-II-LB in primer set IV-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers IV-III-F3, IV-III-B3, IV-III-FIP, IV-III-BIP and IV-III-LF in primer set IV-III is (1-2):(1-2):(4-8):(4-8):(2-4); The molar ratio of primers V-I-F3, V-I-B3, V-I-FIP, V-I-BIP, V-I-LF, and V-I-LB in primer set V-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers V-II-F3, V-II-B3, V-II-FIP, V-II-BIP, V-II-LF, and V-II-LB in primer set V-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers V-III-F3, V-III-B3, V-III-FIP, V-III-BIP, V-III-LF, and V-III-LB in primer set V-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VI-I-F3, VI-I-B3, VI-I-FIP, VI-I-BIP and VI-I-LF in primer set VI-I is (1-2): (1-2): (4-8): (4-8): (2-4); The molar ratio of primers VI-II-F3, VI-II-B3, VI-II-FIP, VI-II-BIP and VI-II-LF in primer set VI-II is (1-2): (1-2): (4-8): (4-8): (2-4); The molar ratio of primers VI-III-F3, VI-III-B3, VI-III-FIP, VI-III-BIP, VI-III-LF, and VI-III-LB in primer set VI-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VII-I-F3, VII-I-B3, VII-I-FIP, VII-I-BIP, VII-I-LF, and VII-I-LB in primer set VII-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VII-II-F3, VII-II-B3, VII-II-FIP, VII-II-BIP, VII-II-LF, and VII-II-LB in primer set VII-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VII-III-F3, VII-III-B3, VII-III-FIP, VII-III-BIP, VII-III-LF, and VII-III-LB in primer set VII-III is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VIII-I-F3, VIII-I-B3, VIII-I-FIP, VIII-I-BIP, VIII-I-LF, and VIII-I-LB in primer set VIII-I is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VIII-II-F3, VIII-II-B3, VIII-II-FIP, VIII-II-BIP, VIII-II-LF, and VIII-II-LB in primer set VIII-II is: (1-2): (1-2): (4-8): (4-8): (2-4): (2-4); The molar ratio of primers VIII-Ⅲ-F3, VIII-Ⅲ-B3, VIII-Ⅲ-FIP, VIII-Ⅲ-BIP, VIII-Ⅲ-LF and VIII-Ⅲ-LB in primer set VIII-Ⅲ is (1~2):(1~2):(4~8):(4~8):(2~4):(2~4).

3. The application of the primer combination as described in claim 1 or 2 in any of the following: (I) Identify one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis; (II) Prepare a kit for identifying one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis. (III) Detect whether the sample to be tested contains one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis; (IV) Prepare a kit to detect whether the sample to be tested contains one or more of the following: Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and Trichomonas vaginalis.

4. A reagent kit, characterized in that, include: The primer combination as described in claim 1 or 2.

5. The kit according to claim 4, characterized in that, Also includes: dNTPs, polymerase, magnesium ions, and hydrogel.

6. The reagent kit as described in claim 5, characterized in that, The final concentration of the dNTPs is 0.6–1.4 mM; the polymerase includes Bst enzyme, the final concentration of which is 6.4–12.8 U per reaction; and the final concentration of the magnesium ions is 2–6 mM.

7. The kit as described in claim 5 or 6, characterized in that, The hydrogel was obtained by crosslinking eight-arm polyethylene glycol acrylate and dithiol polyethylene glycol in a molar ratio of 1:

4.

8. The kit according to any one of claims 4 to 7, characterized in that, Also includes: Lyophilization protectant, reducing agent, nucleic acid dye and buffer.

9. A method for detecting whether a sample contains Candida albicans, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Gardnerella vaginalis, and / or Trichomonas vaginalis for non-diagnostic purposes, characterized in that, Includes the following steps: S1: Obtain the nucleic acid from the pretreated test sample; S2: Using the nucleic acid extracted in S1 as a template, loop-mediated isothermal amplification is performed using the primers in the primer combinations described in claim 1 or 2 to obtain the amplification results; S3: The identification result is obtained based on the number of fluorescent amplification spots.

10. The method as described in claim 9, characterized in that, If primer set I is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate for containing Candida albicans; If primer set II is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Chlamydia trachomatis; If the primer set III is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Neisseria gonorrhoeae; If the primer set IV is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Ureaplasma urealyticum; If the primer set V is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Mycoplasma hominis; If primer set VI is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain Mycoplasma genitalium; If primer set VII is used, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate to contain vaginal Gardner; Using primer set VIII, specific amplification using the nucleic acid as a template can be achieved, and the sample to be tested contains or is a candidate for containing Trichomonas vaginalis.

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