One-pot rapid nucleic acid detection system based on SfaTnpB combined isothermal amplification technology and application of one-pot rapid nucleic acid detection system

Abstract

The invention relates to the technical field of nucleic acid on-site rapid visual detection, in particular to a one-pot rapid nucleic acid detection system based on an SfaTnpB combined isothermal amplification technology and application of the one-pot rapid nucleic acid detection system. According to the invention, an RAA isothermal amplification technology and an LAMP isothermal amplification technology are respectively combined with SfaTnpB nuclease, and finally, an RAA-SfaTnpB one-pot method system and an LAMP-SfaTnpB one-pot method system are successfully established. The one-pot system established by the invention has the advantages of high sensitivity, high specificity, fast reaction, strong accuracy and no need of complex equipment, the detection result can be visually interpreted by naked eyes, the system has high convenience and practicability, the adaptability and practicability of the system are further improved, and the rapid detection requirements in various scenes can be flexibly met. The system has a wide application prospect in the fields of rapid pathogen detection, genotype detection, early diagnosis of infectious diseases and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid on-site visualization detection of nucleic acids, in particular to a one-pot rapid nucleic acid detection system based on SfaTnpB combined isothermal amplification technology and its application. Background Art

[0002] Traditional nucleic acid detection methods (such as qPCR) usually rely on complex nucleic acid extraction processes, thermal cycle amplification, and electrophoresis analysis. They not only take a long time (about 2 - 4 hours), but also require professional laboratory equipment and technical personnel to operate, and there is a potential risk of cross-contamination. Although recent isothermal amplification technologies (such as LAMP and RPA) have made certain progress in simplifying operations, these current methods still face many challenges: on the one hand, the multi-step, multi-tube operation process increases the possibility of sample contamination; on the other hand, the high sensitivity of the isothermal amplification reaction itself also brings the problem of increased false positive rate.

[0003] SfaTnpB is a novel TnpB-like nuclease, derived from the SfaTnpB transposon family, with a small protein size of only 369 amino acids. This enzyme can recognize the conserved TAM sequence 5’-NTAC in vitro, showing excellent trans-cleavage activity and extremely high target recognition specificity, and having a wide reaction temperature adaptability (16℃ - 55℃). It is worth mentioning that after the engineering optimization of ωRNA, the trans-cleavage activity of SfaTnpB can be significantly enhanced, showing excellent potential for in vitro nucleic acid detection.

[0004] Although there have been studies attempting to integrate the target amplification and reporter cleavage steps (such as a "one-pot" reaction system), most strategies still have limitations such as low sensitivity, low specificity, unstable visualization results, or the need to add additional chromogenic reagents. Therefore, there is an urgent need to develop a new one-pot nucleic acid detection method with high sensitivity, high specificity, fast reaction, high accuracy, no need for complex instruments, and the detection results can be directly judged by the naked eye to better meet the actual application needs of on-site rapid screening in grass-roots medical care, public health emergencies, livestock pathogen prevention and control, etc. Summary of the Invention

[0005] The purpose of the present invention is to provide a one-pot rapid nucleic acid detection system based on SfaTnpB combined isothermal amplification technology and its application to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above purpose, the present invention provides the following solution:

[0007] The present invention provides a one-pot rapid nucleic acid detection system based on SfaTnpB combined isothermal amplification technology, and the one-pot rapid nucleic acid detection system includes an SfaTnpB premix and an isothermal amplification premix;

[0008] The SfaTnpB premix includes SfaTnpB protein, ωRNA and ssDNA-reporter; the Scaffold sequence of the ωRNA is as shown in SEQ ID NO.16; the TAM sequence of the SfaTnpB protein is as shown in ATAC or TTAC; the nucleotide sequence of the ssDNA-reporter is as shown in SEQ ID NO.11;

[0009] The isothermal amplification premix includes primers and an isothermal amplification reaction premix;

[0010] The isothermal amplification techniques include RPA, RAA, LAMP, NASBA, RCA, MCDA, SDA or CPA.

[0011] The molar ratio of the SfaTnpB protein to the ωRNA is 1:5 to 2:1.

[0012] Preferably, when the isothermal amplification technique is RAA, the isothermal amplification reaction premix includes buffer V, magnesium acetate solution and DEPC water; when the isothermal amplification technique is LAMP, the isothermal amplification reaction premix includes Bst, dNTP, Isothermal buffer, MgSO4 and DEPC water.

[0013] Preferably, the molar ratio of the SfaTnpB protein to the ωRNA is 1:3.

[0014] More preferably, the SfaTnpB premix further includes Buffer; the Buffer includes NEBuffer TM r2.1, NEBuffer TM 3, NEBuffer™ r3.1, NEBuffer TM 4, rCutSmart™ Buffer or ThermoFastDigest buffer.

[0015] More preferably, the Buffer is optimally Thermo FastDigest buffer.

[0016] The present invention provides the application of the above one-pot rapid nucleic acid detection system in the preparation of products for detecting viruses.

[0017] The present invention provides a product for detecting viruses, and the product includes the above one-pot rapid nucleic acid detection system.

[0018] Preferably, the virus includes one or more of human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 35, human papillomavirus type 39, human papillomavirus type 45, human papillomavirus type 51, human papillomavirus type 52, human papillomavirus type 56, human papillomavirus type 58, human papillomavirus type 59, human papillomavirus type 66, human papillomavirus type 68, and African swine fever virus.

[0019] Preferably, when the isothermal amplification technology is RAA, the ωRNA sequence targeting human papillomavirus type 16 is as shown in SEQ ID NO.13 or SEQ ID NO.61; the ωRNA sequence targeting human papillomavirus type 18 is as shown in SEQ ID NO.15 or SEQ ID NO.62; the ωRNA sequence targeting human papillomavirus type 31 is as shown in SEQ ID NO.63; the ωRNA sequence targeting human papillomavirus type 33 is as shown in SEQ ID NO.64; the ωRNA sequence targeting human papillomavirus type 35 is as shown in SEQ ID NO.66; the ωRNA sequence targeting human papillomavirus type 39 is as shown in SEQ ID NO.66; the ωRNA sequence targeting human papillomavirus type 45 is as shown in SEQ ID NO.67; the ωRNA sequence targeting human papillomavirus type 51 is as shown in SEQ ID NO.68; the ωRNA sequence targeting human papillomavirus type 52 is as shown in SEQ ID NO.69; the ωRNA sequence targeting human papillomavirus type 56 is as shown in SEQ ID NO.70; the ωRNA sequence targeting human papillomavirus type 58 is as shown in SEQ ID NO.71; the ωRNA sequence targeting human papillomavirus type 59 is as shown in SEQ ID NO.72; the ωRNA sequence targeting human papillomavirus type 66 is as shown in SEQ ID NO.73; the ωRNA sequence targeting human papillomavirus type 68 is as shown in SEQ ID NO.74;

[0020] When the isothermal amplification technology is LAMP, the ωRNA sequence targeting human papillomavirus type 16 is as shown in SEQ ID NO.37; the ωRNA sequence targeting human papillomavirus type 18 is as shown in SEQ ID NO.38; the ωRNA sequence targeting African swine fever virus is as shown in SEQ ID NO.50.

[0021] Preferably, when the isothermal amplification technology is RAA, the primers include primer pairs for amplifying human papillomavirus type 16, primer pairs for amplifying human papillomavirus type 18, or universal primer pairs for human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 35, human papillomavirus type 39, human papillomavirus type 45, human papillomavirus type 51, human papillomavirus type 52, human papillomavirus type 56, human papillomavirus type 58, human papillomavirus type 59, human papillomavirus type 66, and human papillomavirus type 68; the primer pair for amplifying human papillomavirus type 16 includes an upstream primer shown in SEQ ID NO. 17 and a downstream primer shown in SEQ ID NO. 18; the primer pair for amplifying human papillomavirus type 18 includes an upstream primer shown in SEQ ID NO. 19 and a downstream primer shown in SEQ ID NO. 20; the universal primer pair includes an upstream primer shown in SEQ ID NO. 75 and a downstream primer shown in SEQ ID NO. 76;

[0022] When the isothermal amplification technology is LAMP, the primers include one or several of the primer pairs for amplifying human papillomavirus type 16, the primer pairs for amplifying human papillomavirus type 18, and the primer pairs for amplifying African swine fever virus; the primer pair for amplifying human papillomavirus type 16 includes LAMP-HPV16-F3 shown in SEQ ID NO. 39, LAMP-HPV16-B3 shown in SEQ ID NO. 40, LAMP-HPV16-FIP shown in SEQ ID NO. 41, LAMP-HPV16-BIP shown in SEQ ID NO. 42, and LAMP-HPV16-LB shown in SEQ ID NO. 43; the primer pair for amplifying human papillomavirus type 18 includes LAMP-HPV18-F3 shown in SEQ ID NO. 44, LAMP-HPV18-B3 shown in SEQ ID NO. 45, LAMP-HPV18-FIP shown in SEQ ID NO. 46, LAMP-HPV18-BIP shown in SEQ ID NO. 47, and LAMP-HPV18-LB shown in SEQ ID NO. 48; the primer pair for amplifying African swine fever virus includes p72-F3 shown in SEQ ID NO. 51, p72-B3 shown in SEQ ID NO. 52, p72-FIP shown in SEQ ID NO. 53, p72-BIP shown in SEQ ID NO. 54, and p72-LB shown in SEQ ID NO. 55.

[0023] Preferably, the test samples of the product include one or more of blood, cells, cervical smear, cervical liquid-based cells, vaginal swab serum, saliva, body fluid, plasma, urine, bronchoalveolar lavage fluid, cerebrospinal fluid, gastric juice, tonsils, lymph nodes, and feces.

[0024] Preferably, when the isothermal amplification technology is RAA, based on 50 μL, the isothermal amplification premix includes one dry powder reaction tube, 25 μL buffer V, 2 μL upstream primer, 2 μL downstream primer, 5 μL magnesium acetate solution, and the balance of DEPC water;

[0025] When the isothermal amplification technology is LAMP, based on 35 μL, the isothermal amplification premix includes 1.2 μL primer mixture, 1 μL Bst, 3.5 μL dNTP, 2.5 μL Isothermal buffer, 1.5 μL MgSO4, and the balance of DEPC water.

[0026] More preferably, the product includes a reagent, a kit, or a chip.

[0027] As an additional aspect, the present invention provides the application of the above one-pot rapid nucleic acid detection system or the above product in the on-site rapid detection of plants, animals, humans, and microorganisms.

[0028] As an additional aspect, the present invention provides a detection method for the above product.

[0029] When the isothermal amplification technology is RAA, the detection method includes the step of simultaneously adding the isothermal amplification premix and the SfaTnpB premix to the bottom of the reaction tube, mixing well, and placing it at 37°C to 42°C for reaction for 30 to 50 min;

[0030] When the isothermal amplification technology is LAMP, the detection method includes the step of simultaneously adding the isothermal amplification premix and the SfaTnpB premix to the bottom of the reaction tube, mixing well, and placing it at 46°C to 50°C for reaction for 30 to 50 min.

[0031] More preferably, when the isothermal amplification technology is RAA, the optimal reaction temperature is 37°C, and the optimal reaction time is 40 min;

[0032] When the isothermal amplification technology is LAMP, the optimal reaction temperature is 48°C, and the optimal reaction time is 40 min.

[0033] The present invention discloses the following technical effects:

[0034] The present invention combines the RAA and LAMP isothermal amplification technologies with the SfaTnpB nuclease respectively, and systematically optimizes the entire detection system. Finally, the RAA-SfaTnpB one-pot system and the LAMP-SfaTnpB one-pot system are successfully established. The present invention first discovers that SfaTnpB can be efficiently combined with the RAA or LAMP isothermal amplification technology to achieve rapid one-pot detection of nucleic acids. For different systems, corresponding detection systems are established. The one-pot detection system disclosed in the present invention can simultaneously complete multi-target amplification and ssDNA reporter molecule cleavage in a single tube, that is, the entire detection process only requires one sample addition. At the same time, it has the advantages of high sensitivity, high specificity, high accuracy, fast reaction, simple operation, and no need for complex equipment, significantly improving the detection efficiency. Moreover, the detection results can be directly judged by the naked eye, suitable for on-site rapid detection, with high convenience and practicality, further improving the adaptability and practicality of the system, and can flexibly meet the rapid detection needs in various scenarios. Compared with the traditional "amplification-detection" two-step method, this method effectively avoids problems such as aerosol contamination and cumbersome operation caused by the transfer of amplification products. The present invention shows broad application prospects in the fields of rapid pathogen detection, genotype detection, and early diagnosis of infectious diseases. At the same time, the present invention also has the following advantages:

[0035] 1. The present invention provides a one-pot rapid detection system based on the combination of SfaTnpB and RAA isothermal amplification technology. The whole reaction is carried out in one pot, and the detection process can be completed within 30 - 50 minutes at 37°C - 42°C.

[0036] 2. The present invention provides a one-pot rapid detection system based on the combination of SfaTnpB and LAMP isothermal amplification technology. The whole reaction is carried out in one pot, and the detection process can be completed within 30 - 50 minutes.

[0037] 3. The present invention provides two one-pot detection systems based on SfaTnpB nuclease, which are adapted to different reaction temperatures, and can improve the universality of different pathogen targets to be detected to a greater extent. Description of the Drawings

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0039] Figure 1 It is the working flow chart of the RAA-SfaTnpB one-pot nucleic acid detection system;

[0040] Figure 2 Figure showing the comparison of fluorescence intensities between the experimental group and the control group when the SfaTnpB protein and ωRNA are at different molar ratios;

[0041] Figure 3 Figure for the comparative analysis of dynamic fluorescence signals of the one-pot nucleic acid detection system based on SfaTnpB and LbCas12a for classical and non-classical TAM / PAM targets; where, "+" represents the addition of dsDNA (template plasmid); "-" represents no addition of dsDNA, i.e., the negative control group;

[0042] Figure 4 Figure for the evaluation of the detection sensitivity of HPV16-L1 plasmid (A) and HPV18-L1 plasmid (B) by the one-pot nucleic acid detection system based on RAA-SfaTnpB; where, the control is the negative control group;

[0043] Figure 5 Figure for the evaluation of the detection performance of HPV16 and HPV18 clinical samples by the one-pot nucleic acid detection system based on RAA-SfaTnpB; where, PC: Positive Control, positive control group (HPV16-L1 and HPV18-L1 plasmids diluted as templates); NC: Negative Control, negative control group;

[0044] Figure 6 Flowchart of the one-pot nucleic acid detection system of LAMP-SfaTnpB;

[0045] Figure 7 Figure for the comparison of the fluorescence kinetic performance of the one-pot nucleic acid detection systems of LAMP-SfaTnpB and LAMP-AapCas12b; where, "+" represents the addition of dsDNA (template plasmid); "-" represents no addition of dsDNA, i.e., the negative control group;

[0046] Figure 8 Figure for the evaluation of the detection sensitivity of HPV16-L1 plasmid (A) and HPV18-L1 plasmid (B) by the one-pot nucleic acid detection system based on LAMP-SfaTnpB; where, the control is the negative control group;

[0047] Figure 9 Figure for the evaluation of the detection performance of HPV16 and HPV18 clinical samples by the one-pot nucleic acid detection system based on LAMP-SfaTnpB; where, PC: Positive Control, positive control (HPV16-L1 and HPV18-L1 plasmids diluted as templates); NC: Negative Control, negative control group;

[0048] Figure 10Results graph of qPCR detection of HPV16 and HPV18 clinical samples; among which, A is the qPCR detection of HPV16 clinical samples; B is the qPCR detection of HPV18 clinical samples;

[0049] Figure 11 Graph for evaluating the detection sensitivity of pMD18T-ASFV-p72 plasmid based on the LAMP-SfaTnpB one-pot nucleic acid detection system; among which, the control is the negative control group;

[0050] Figure 12 Graph for evaluating the detection performance of ASFV clinical samples based on the LAMP-SfaTnpB one-pot nucleic acid detection system; among which, PC: Positive Control, positive control (the diluted pMD18T-ASFV-P72 plasmid is used as the template); NC: Negative Control, negative control group;

[0051] Figure 13 Results graph of qPCR detection of ASFV clinical samples;

[0052] Figure 14 Graph for the mismatch tolerance test results of the SfaTnpB protein for different base pairing positions between the spacer and the target; among which, M: mismatch; Ctrl: Control, negative control group;

[0053] Figure 15 Graph for the identification and analysis of specific ωRNAs corresponding to three high-risk HPV subtypes, namely HPV35 (A), HPV16 (B), and HPV18 (C);

[0054] Figure 16 Graph for the identification and analysis of specific ωRNAs corresponding to three high-risk HPV subtypes, namely HPV33 (A), HPV45 (B), and HPV52 (C);

[0055] Figure 17 Graph for the identification and analysis of specific ωRNAs corresponding to three high-risk HPV subtypes, namely HPV31 (A), HPV39 (B), and HPV51 (C);

[0056] Figure 18 Graph for the identification and analysis of specific ωRNAs corresponding to three high-risk HPV subtypes, namely HPV56 (A), HPV59 (B), and HPV68 (C);

[0057] Figure 19 Identification and analysis of specific ωRNAs corresponding to two high-risk HPV subtypes, namely HPV58 (A) and HPV66 (B);

[0058] Figure 20Detection result diagram of a one-tube method for clinical samples of different subtypes of HPV; among them, Ctrl: Control, negative control group. Detailed implementation manners

[0059] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0060] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0061] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0062] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention's specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention's specification are obvious to those skilled in the art. The present invention's specification and examples are merely exemplary.

[0063] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0064] Term description

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0066] SfaTnpB nuclease: It is a novel TnpB nuclease derived from the SfaTnpB transposon family, with a protein size of only 369 amino acids, and its reaction temperature range is 16°C to 55°C.

[0067] ωRNA (RNA-guided nuclease activator): ωRNA is a small molecule RNA related to TnpB, usually encoded by the host genome. It binds to the TnpB protein to form a complex for recognizing and targeting specific DNA sequences. This RNA-guided mechanism is similar to crRNA in the CRISPR-Cas system.

[0068] TAM (TnpB-recognized sequence, TnpB-Associated Motif): TAM is the specific DNA sequence recognized and bound by the TnpB nuclease. These sequences are usually located near the target gene and are used to guide the precise cleavage or editing of the TnpB nuclease in the genome.

[0069] Trans cleavage: It refers to that after the TnpB nuclease recognizes and binds to the target DNA, it can not only cleave the DNA sequence paired with ωRNA, but also cleave adjacent or other DNA molecules.

[0070] Materials, reagents, etc. used in the examples can be obtained from commercial channels without special instructions.

[0071] Among them, the sample sources: Human papillomavirus type 16 (HPV16) and human papillomavirus type 18 (HPV18) in the examples were provided by the Affiliated Obstetrics and Gynecology Hospital of Nankai University. Human papillomavirus type 31 (HPV31), human papillomavirus type 33 (HPV33), human papillomavirus type 35 (HPV35), human papillomavirus type 39 (HPV39), human papillomavirus type 45 (HPV45), human papillomavirus type 51 (HPV51), human papillomavirus type 52 (HPV52), human papillomavirus type 56 (HPV56), human papillomavirus type 58 (HPV58), human papillomavirus type 59 (HPV59), human papillomavirus type 66 (HPV66) and human papillomavirus type 68 (HPV68) were provided by Peking University Shenzhen Hospital. African swine fever virus (ASFV) was provided by Hunan Shengce Biotechnology Co., Ltd.

[0072] Reagents: The RAA nucleic acid amplification kit (basic) was purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd., product number: B00000A-48; HiScribe ® T7 High Yield RNA Synthesis Kit was purchased from NEB, product number: E2040S; Bst 3.0 DNA Polymerase was purchased from NEB, product number: M0374S; dNTP Mix was purchased from NEB, product number: N0447S; 10× FastDigest buffer was purchased from Thermo Scientific, product number: B64; EnGen ®LbaCas12a (Cpf1) was purchased from NEB, catalog number: M0653T; AapCas12b was purchased from Tolo Biotech Co., Ltd., catalog number: 32118.

[0073] It should be understood that these examples are only for illustrating the present invention and not for limiting the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, they are generally in accordance with conventional conditions or the conditions recommended by the manufacturer.

[0074] Example 1 Development of a one-pot nucleic acid detection system based on RAA-SfaTnpB

[0075] The working flowchart of the one-pot nucleic acid detection system based on RAA-SfaTnpB is as Figure 1

[0076] In this embodiment, the SfaTnpB protein (amino acid sequence shown in SEQ ID NO.2) was successfully expressed and purified through a prokaryotic expression system. Meanwhile, the ωRNA corresponding to SfaTnpB and the crRNA corresponding to LbCas12a were prepared by in vitro transcription technology. Based on the RAA isothermal amplification technology, a one-pot detection system of RAA-SfaTnpB was established, and the effects of the classical TAM sequence ("TTAC") and the non-classical TAM sequence ("ATAC") on the detection efficiency of this system were systematically compared. The specific workflow is shown in Figure 1 . To evaluate the performance advantages of this system, a control detection system of RAA-LbCas12a was established in parallel for comparative analysis by referring to the published one-pot detection method of RPA-CRISPR / Cas12a in the literature "A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses".

[0077] The target dsDNA selected in this embodiment is a partial fragment of the sheep GAPDH gene. When the TAM of SfaTnpB is TTAC, its target sequence (GAPDH-T1) is: 5’-CAAGTTCCACGGCACAGTCAAGGCAGAGAAC GGGAAGCTCGTCATCAATGGAAAG GCCATCACCATCTTCCAGGA GTAAGTGTGGGAGATGGAACAGGAAGAACTTGGCCTTGGAGGAGGCACTGGGACGGGGTGATCACCTGCGGTCTGTGGTACCGTGTCCCTGAGCTTTGTCCTGTCTTTCTCATAGGCGAGATCC-3’ (SEQ ID NO.3). The reverse complementary sequence marked in bold is the TAM, and the reverse complementary sequence of the underlined sequence is the targeting sequence; the corresponding ωRNA sequence is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUG AGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA UCCUGGAAGAUGGUGAU GGC -3’ (the underlined part is the targeting sequence, SEQ ID NO.4).

[0078] When the TAM of SfaTnpB is ATAC, its target sequence (GAPDH-T2) is: 5’-CAAGTTCCAC GGCACAGTCAAGGCAGAGAACGGGAAGCTCGTCATCAATGGAAAG GCCATCACCATCTTCCAGGA GTATGTGTGGGAGATGGAACAGGAAGAACTTGGCCTTGGAGGAGGCACTGGGACGGGGTGATCACCTGCGGTCTGTGGTACCGTGTCCCTGAGCTTTGTCCTGTCTTTCTCATAGGCGAGATCC-3’ (SEQ ID NO.5). The reverse complementary sequence marked in bold is the TAM, and the reverse complementary sequence of the underlined sequence is the targeting sequence; the corresponding ωRNA sequence is shown in SEQ ID NO.4.

[0079] When the PAM of LbCas12a is TTTC, its target sequence (GAPDH-T3) is: 5’-CAAGTTCCACGGCAC AGTCAAGGCAGAGAACGGGAAGCTCGTCATCAATGGAAAG GCCATCACCATCTTCCAGGA GAAAGTGTGGGAGATGGAACAGGAAGAACTTGGCCTTGGAGGAGGCACTGGGACGGGGTGATCACCTGCGGTCTGTGGTACCGTGTCCCTGAGCTTTGTCCTGTCTTTCTCATAGGCGAGATCC-3’ (SEQ ID NO.6). The reverse complementary sequence marked in bold is the PAM, and the reverse complementary sequence of the underlined sequence is the targeting sequence. The corresponding crRNA sequence is 5’-AAUUUCUACUGUUGUAGAU UCCUGGAAGAUGGUGAUGGC -3’ (SEQ ID NO.7), (the underlined part is the targeting sequence).

[0080] When the PAM of LbCas12a is GTTC, its target sequence (GAPDH-T4) is: 5’-CAAGTTCCACGGCA CAGTCAAGGCAGAGAACGGGAAGCTCGTCATCAATGGAAAG GCCATCACCATCTTCCAGGAGAACGTGTGGGAGATGGAACAGGAAGAACTTGGCCTTGGAGGAGGCACTGGGACGGGGTGATCACCTGCGGTCTGTGGTACCGTGTCCCTGAGCTTTGTCCTGTCTTTCTCATAGGCGAGATCC-3’ (SEQ ID NO.8). The reverse complementary sequence marked in bold is the PAM, and the reverse complementary sequence of the sequence where the underline is located is the targeting sequence. The corresponding crRNA sequence is shown in SEQ ID NO.7.

[0081] The target sequences of the two different TAM / PAMs targeted by SfaTnpB and LbCas12a above were amplified by overlap PCR and purified, and then cloned into the pMD18T vector respectively to obtain plasmids pMD18T-GAPDH-T1, pMD18T-GAPDH-T2, pMD18T-GAPDH-T3 and pMD18T-GAPDH-T4 as templates. To ensure that the targeting sequences are the same, except for the TAM / PAM sequences, the spacer sequence of SfaTnpB is the same as the spacer sequence of LbCas12a. RAA-GAPDH-F: CAAGTTCCACGGCACAGTCAAGGCAGAGAACG (SEQ ID NO.9) and RAA-GAPDH-R: GGATCTCGCCTATGAGAAAGACAGGACAAAGCTC (SEQ ID NO.10) were used as RAA amplification primers.

[0082] Reaction system of the RAA-SfaTnpB one-pot method: The RAA premix was 25 μL buffer V (from the RAA nucleic acid amplification kit), 2 μL 10 μM forward primer F, 2 μL 10 μM reverse primer R, one dry powder reaction tube and 5 μL magnesium acetate solution, supplemented with DEPC water to 50 μL. Add 250 nM SfaTnpB protein, 700 nM ωRNA, 250 nM ssDNA-reporter (5’-ROX / GTATCCAGTGCG / BHQ2-3’, SEQ ID NO.11) and 1 μL template plasmid (pMD18T-GAPDH-T1 or pMD18T-GAPDH-T2) to 19.3 μL of the RAA premix. The final system is 25 μL, and the molar ratio of SfaTnpB protein to ωRNA in this system is 1:3.

[0083] In this example, the molar ratio of SfaTnpB protein to ωRNA was optimized, and the results are as Figure 2As shown in the figure. The results showed that in the experimental groups containing the target, when the molar ratios of SfaTnpB protein to ωRNA were 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5 respectively, the fluorescence intensities of all experimental groups were significantly higher than those of the control group without the target. In particular, when the molar ratio of SfaTnpB protein to ωRNA was 1:3, the detection system exhibited the strongest fluorescence signal.

[0084] Reaction system of the RAA-LbCas12a one-pot method: First, prepare the CRISPR-Cas12a premix, which is a mixture of 1 μL crRNA (1.2 μM), 1 μL LbCas12a (1 μM), 2 μL 10X NEB Buffer 2.1, and 0.3 μL 100 μM ssDNA-reporter (SEQ ID NO.11). Then configure the RAA premix (25 μL Buffer V, 2 μL 10 μM forward primer F, 2 μL 10 μM reverse primer R, one dry powder reaction tube, and 5 μL magnesium acetate solution, made up to 50 μL with DEPC water). Take 19.3 μL of the RAA premix and 4.3 μL of the LbCas12a premix respectively, and add 1 μL of the template plasmid (pMD18T-GAPDH-T3 or pMD18T-GAPDH-T4). The final volume of the system is 25 μL.

[0085] Among them, no template plasmid was added to all negative control groups. The reaction tubes were placed in a 37 °C metal bath and reacted for 0 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min respectively. After each reaction time ended, it was inactivated at 80 °C for 5 min. The reaction solution was transferred to an enzyme-labeled plate, and each well was diluted with 80 μL of DEPC water. The fluorescence values of each reaction at 561 - 601 nm were read by an enzyme-labeled instrument. The results are as Figure 3 shown. The RAA-SfaTnpB one-pot method system demonstrated better detection performance, with higher fluorescence signal intensity and faster reaction. Further analysis of the influence of the TAM sequence found that when using the non-classical TAM ("ATAC"), its fluorescence rising curve was significantly better than that of the classical TAM ("TTAC"), indicating that the "ATAC" TAM was more suitable for the RAA-SfaTnpB one-pot detection system.

[0086] Example 2 High-sensitivity detection of clinical samples by the RAA-SfaTnpB one-pot nucleic acid detection system

[0087] In this example, to evaluate the detection sensitivity of the RAA-SfaTnpB one-pot nucleic acid detection system, different copy numbers (10 4~10 -1 pUC57-HPV16-L1 plasmid and pUC57-HPV18-L1 plasmid standards at (

[0088] In this example, the pUC57-HPV16-L1 plasmid selected was synthesized by GenScript and cloned into the pUC57 backbone. The HPV16-L1 fragment sequence it contains is: 5’-CTGTCCCAGTATCTAAGGTTGTAAGCACGGATG AATATGTTGCACGCACAAACATATATTATCATGCAGGAACATCCAGACTACTTGCAGTTGGACATCCCTATTTTCCTATTAAAAAACCTAACAATAACAAAATATTAGTTCCTAAAGTATCAGGATTACAATACAGGGTATTTAGAATACATTTACCTGACCCCAATAAGTTTGGTTTTCCTGACACCTCATTTTATAATCCAGATACACAGCGGCTGGTTTGGGCCTGTGTAGGTGTTGAGGTAGGTCGTGGTCAGCCATTAGGTGTGGGCATTAGTGGCCATCCTTTATTAAATAAATTGGATGACACTGAAAATGCTAGTGCTTATGCAGCAAATGC AGGTGTGGATAATAGAGAATGTATATCTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCAGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATTCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCTATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGCGACAGCTTA-3’ (SEQ ID NO.12); The reverse complementary sequence marked in bold is TAM, and the reverse complementary sequence of the underlined sequence is the targeting sequence. The corresponding ωRNA sequence is 5’-AUCAGCAUAGGGAACUAUAU GUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA AUUCUCUAUUAUCCACACCU -3’ (SEQ ID NO.13, the underlined part is the targeting sequence).

[0089] The pUC57-HPV18-L1 plasmid was synthesized by GenScript and cloned into the pUC57 backbone. The HPV18-L1 fragment sequence it contains is: 5’-GCTAAAGGCACTGCTTGTAAATCGCGTCCTTTATCACAGGGCGATTGCCC CCCTTTAGAACTTAAAAACACAGTTTTGGAAGATGGTGATATGGTAGATACTGGATATGGTGCCATGGACTTTAGTACATTGCAAGATACTAAATGTGAGGTACCATTGGATATTTGTCAGTCTATTTGTAAATATCCTGATTATTTACAAATGTCTGCAGATCCTTATGGGGATTCCATGTTTTTTTGCTTACGGCGTGAGCAGCTTTTTGCTAGGCATTTTTGGAATAGGGCAGGTACTATGGGTGACACTGTGCCTCAATC CTTATATATTAAAGGCACAGGTATGCGTGCTTCACCTGGCAGCTGTGTGTATTCTCCCTCTCCAAGTGGCTCTATTGTTACCTCTGACTCCCAGTTGTTTAATAAACCATATTGGTTACATAAGGCACAGGGTCATAACAATGGTATCTGCTGGCATAATCAATTATTTGTTACTGTGGTAGATACCACTCGTAGTACCAATTTAACAATATGTGCTTCTACACAGTCTCCTGTACCTGGGCAATATGATGCTACCA-3’ (SEQ ID NO.14); The reverse complementary sequence marked in bold is TAM, and the reverse complementary sequence of the underlined sequence is the targeting sequence. The corresponding ωRNA sequence is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CUGUGCCUUUAAUAUAUAAG -3’ (SEQ ID NO.15, the underlined part is the targeting sequence), the Scaffold sequence of ωRNA is shown in SEQ ID NO.16, specifically: AUCAGCAUAGGGAACUAUAUGUU CUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA.

[0090] The sensitivity of the RAA-SfaTnpB one-pot system for detecting HPV16 / 18-L1 plasmids was determined. Gradient-diluted pUC57-HPV16-L1 plasmids (primers: RAA-HPV16-F / R) and pUC57-HPV18-L1 plasmids (primers: RAA-HPV18-F / R) were used as templates, respectively. The HPV16 primer sequences were 5'-AATCCAGATACACAGCGGCT GGTTTGGGCCTGT-3' (RAA-HPV16-F, SEQ ID NO.17) and 5'-TTTGCCCCAGTGTTCCCC TATAGGTGGTTT-3' (RAA-HPV16-R, SEQID NO.18), and the HPV18 detection primer sequences were 5'-GCAAGATACTAAATGTGAGGTACCATTGGAT-3' (RAA-HPV18-F, SEQ ID NO.19) and 5'-AGTCAGAGGTAACAATAGAGCCACTTGGAGA-3 (RAA-HPV18-R, SEQ IDNO.20). The one-pot detection system was as follows: The RAA premix included 25 μL of buffer V, 2 μL of 10 μM forward primer F, 2 μL of 10 μM reverse primer R, one dry powder reaction tube, and 5 μL of magnesium acetate solution, and was made up to 50 μL with DEPC water. 250 nM of SfaTnpB protein (the SfaTnpB protein obtained in Example 1, the same in subsequent experiments), 700 nM of ωRNA, 250 nM of ssDNA-reporter (SEQ ID NO.11), and 1 μL of template plasmid (pUC57-HPV16-L1 or pUC57-HPV18-L1) were added to 19.3 μL of the RAA premix, and the final volume of the system was 25 μL. The template plasmid was not added to the negative control group. The reaction tubes were placed in a 37°C metal bath for 40 min. After the reaction, they were inactivated at 80°C for 5 min. The reaction solution was transferred to an enzyme-labeled plate, and each well was diluted with 80 μL of DEPC water. The fluorescence values of each reaction at 561 - 601 nm were read using an enzyme-labeled instrument.

[0091] The results are shown in Figure 4 Figure [X] (not provided in the original, so 'Figure [X]' is used as a placeholder). The detection limit of the RAA-SfaTnpB one-pot detection system for pUC57-HPV16-L1 plasmids was up to 4.1 copies / μL (copies / μL), and for pUC57-HPV18-L1 plasmids it was up to 3.8 copies / μL (copies / μL), achieving single-copy detection. This further demonstrated that the RAA-SfaTnpB one-pot detection system had high-sensitivity detection performance.

[0092] Example 3: Efficient and accurate detection of HPV16 / 18 clinical samples using the RAA-SfaTnpB one-pot system

[0093] In this example, for 35 clinical samples infected with HPV16 / HPV18, DNA solutions of the 35 samples were obtained using the DNA crude extraction method, and the RAA-SfaTnpB one-pot system was used to detect the infection status of the clinical samples. To verify the reliability of the detection results, the same batch of samples was rechecked by qPCR detection at the same time.

[0094] The DNA crude extraction process was as follows: 80 μL of QuickExtract DNA Extraction Solution (Lucigen) was mixed with 80 μL of the sample preservation solution. The reaction program of the PCR instrument was: 65°C for 10 min; 95°C for 5 min. The solution after the reaction was used as the template for the RAA-SfaTnpB one-pot detection. The detection of the infection with HPV16 and HPV18 was carried out separately. The RAA-SfaTnpB one-pot detection system was as follows: The RAA premix included 25 μL of buffer V, 2 μL of 10 μM forward primer F, 2 μL of 10 μM reverse primer R, a dry powder reaction tube, and 5 μL of magnesium acetate solution, and was made up to 50 μL with DEPC water. 250 nM of SfaTnpB protein, 700 nM of ωRNA (SEQ ID NO.13 or SEQ ID NO.15), 250 nM of ssDNA-reporter (SEQ ID NO.11), and 3 μL of the template solution were added to 19.3 μL of the RAA premix, and the final system was 25 μL. Among them, PC was the positive control group, that is, the template was the plasmid (pUC57-HPV16-L1 or pUC57-HPV18-L1) with 3 10 copies / μL, and NC was the negative control group, that is, no template was added. The reaction tubes were placed in a 37°C metal bath and reacted for 40 min. After the reaction, it was inactivated at 80°C for 5 min. The reaction solution was transferred to an enzyme-labeled plate, and 80 μL of DEPC water was added to each well for dilution. The fluorescence value of each reaction at 561 - 601 nm was read by an enzyme-labeled instrument.

[0095] Referring to the reported detection system, qPCR targets the E6 gene of HPV16 and HPV18, and the corresponding qPCR detection primers are: HPV16-qPCR-F: 5'-AATGTTTCAGGACCCACAGG-3 (SEQ ID NO.21)' and HPV16-qPCR-R: 5'-GTTGCTTGCAGT ACACACATTC-3' (SEQ ID NO.22), HPV18-qPCR-F: 5'-ACCCTACAAGCTACCTGATCT-3' (SEQ ID NO.23) and HPV18-qPCR-R: 5'-ACCTCTGTAAGTTCCAATACTGTC-3' (SEQ ID NO.24). The TaqMan probes used are: HPV16-E6-probe: 5’-FAM / ACCACAGTTAT GCACAGAGCTGCA / BHQ1-3’ (SEQ ID NO.25) and HPV18-E6-probe: 5’-FAM / ACGGAACTG AACACTTCACTGCAAGA / BHQ1-3’ (SEQ ID NO.26). The qPCR system includes 1 μL of 10 μM forward primer F, 1 μL of 10 μM reverse primer R, 0.4 μL of 10 μM probe of 2× enzyme reaction solution (Luna ® Universal qPCR MasterMix, NEB), and 3 μL of template, made up to 20 μL with DEPC water. The reaction conditions of qPCR are: 95°C, 3 min; (95°C, 10 s, 60°C, 30 s) × 40 cycles.

[0096] The results are as Figure 5 shown. Samples No. 3 (S3), No. 7 (S7), No. 13 (S13), No. 14 (S14), No. 20 (S20), No. 23 (S23), No. 28 (S28) and No. 30 (S30) are HPV16-infected samples; samples No. 17 (S17), No. 18 (S18), No. 19 (S19), No. 21 (S21), No. 22 (S22), No. 24 (S24) and No. 25 (S25) are HPV18-infected samples; the detection results are consistent with Figure 10 those in (samples with Ct value ≤ 35 are determined as positive infection samples) in the qPCR detection results, proving that the RAA-SfaTnpB one-pot system can achieve accurate detection of HPV16 / HPV18 clinical samples.

[0097] Example 4 Development of a LAMP-SfaTnpB one-pot nucleic acid detection system

[0098] The working process of the LAMP-SfaTnpB one-pot nucleic acid detection system is as follows Figure 6 shown. In this example, the SfaTnpB protein (amino acid sequence shown in SEQ ID NO.2) was successfully expressed and purified through a prokaryotic expression system. Meanwhile, in vitro transcription technology was used to prepare the ωRNA corresponding to SfaTnpB and the sgRNA corresponding to AapCas12b. Based on the LAMP isothermal amplification technology, a LAMP-SfaTnpB one-pot detection system was established. The specific working process is shown in Figure 5 . And referring to the published LAMP-CRISPR / Cas12b one-pot detection method, a LAMP-AapCas12b control detection system was established in parallel for comparative analysis.

[0099] In this example, the selected target dsDNA is a partial fragment of the sheep GAPDH gene. When the TAM of SfaTnpB is TTAC, its target sequence (GAPDH-T5) is: 5’-GTATGATTCCACCCATGGCAAGTTCCACGGCAC AGTCAAGGCAGAGAACGGGAAGCTCGTCATCAATGGAAAG GCCATCACCATCTTCCAGGA GTAAGTGTGGGAGATGGAACAGGAAGAACTTGGCCTTGGAGGAGGCACTGGGACGGGGTGATCACCTGCGGTCTGTGGTACCGTGTCCCTGAGCTTTGTCCTGTCTTTCTCATAGGCGAGATCCTGC-3’ (SEQ ID NO.27). The reverse complementary sequence marked in bold is the TAM, and the reverse complementary sequence of the underlined sequence is the targeting sequence. The corresponding ωRNA sequence is shown in SEQ ID NO.4; when the AapCas12b PAM is TTTC, its target sequence (GAPDH-T6) is: 5’-GTATGATTCCACCCATGGCAAGT TCCACGGCACAGTCAAGGCAGAGAACGGGAAGCTCGTCATCAATGGAAAG GCCATCACCATCTTCCAGGAGAAAGTGTGGGAGATGGAACAGGAAGAACTTGGCCTTGGAGGAGGCACTGGGACGGGGTGATCACCTGCGGTCTGTGGTACCGTGTCCCTGAGCTTTGTCCTGTCTTTCTCATAGGCGAGATCCTGC-3’ (SEQ ID NO.28). The reverse complementary sequence marked in bold is PAM, and the reverse complementary sequence of the sequence underlined is the targeting sequence. The corresponding sgRNA sequence is 5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCAC UCCU GGAAGAUGGUGAUGGC -3’ (SEQ ID NO.29) (underlined is the targeting sequence).

[0100] The target sequences of the two different TAM / PAMs targeted by the above SfaTnpB and AapCas12b were amplified by overlap PCR and purified, and then cloned into the pMD18T vector respectively to obtain the plasmids pMD18T-GAPDH-T5 and pMD18T-GAPDH-T6 as templates. To ensure the consistency of the targeting sequences, except for the TAM / PAM sequences, the SfaTnpB spacer sequence is the same as the AapCas12b spacer sequence. Using GAPDH-F3: GTATGATTCCACCCATGGCA (SEQ ID NO.30), GAPDH-B3: GCAGGATCTCGCCTATGAGA (SEQ ID NO.31), GAPDH-FIP: CCTGGAAGATGGTGATGGCCTTAGTTCCACGGCACAGTCA (SEQ ID NO.32), GAPDH-BIP: AGGAAGAACTTGGCCTTGGAGGGGGACACGGTACCACAGA (SEQ ID NO.33), GAPDH-LF: CGAGCTTCCCGTTCTCTGC (SEQ ID NO.34) and GAPDH-LB: TGGGACGGGGTGATCACCTG (SEQ ID NO.35) as the LAMP amplification primers.

[0101] Reaction system of the LAMP-SfaTnpB one-pot system: 1.2 μL (1.6 μM GAPDH-FIP / GAPDH-BIP, 0.2 μM GAPDH-F3 / GAPDH-B3, and 0.4 μM GAPDH-LF / GAPDH-LB) primer mixture, 1 μL Bst 3.0 DNA Polymerase, 3.5 μL dNTP, 2.5 μL Isothermal buffer, 1.5 μL MgSO4, 250 nM SfaTnpB protein, 700 nM ωRNA, 3 μL 10×FastDigest Buffer, 250 nM ssDNA-reporter (SEQ ID NO.11), and 1 μL template plasmid (pMD18T-GAPDH-T5). The final volume of the system is 35 μL. Template DNA is not added to the control group. The reaction is set at 4,8 °C in a metal bath for 0 min, 15 min, 30 min, 45 min, and 60 min, and then inactivated at 80 °C for 5 min.

[0102] Reaction system of the LAMP-AapCas12b one-pot system: The total volume of the LAMP-AapCas12b one-pot system is 25 μL, which contains 2.5 μL Isothermal buffer, 6 mM MgSO4, 1.4 mM dNTP, 0.2 μM GAPDH-F3 / GAPDH-B3 primers, 1.6 μM GAPDH-FIP / GAPDH-BIP primers, 100 nM AapCas12b, 100 nM sgRNA, 250 nM ssDNA-reporter (SEQ ID NO.11), 8 U Bst 3.0 DNA Polymerase, and 1 μL template plasmid (pMD18T-GAPDH-T6).

[0103] Among them, template plasmids are not added to all negative control groups. The reaction is set at 60 °C in a metal bath for 0 min, 15 min, 30 min, 45 min, and 60 min, and then inactivated at 80 °C for 5 min. After the reaction, the reaction solution is transferred to an ELISA plate, and 80 μL of DEPC water is added to each well for dilution. The fluorescence value of each reaction at 561 - 601 nm is read by an ELISA reader.

[0104] The results are as Figure 7As shown, the LAMP-SfaTnpB one-pot system exhibits better detection performance than the LAMP-AapCas12b one-pot system, with higher fluorescence signal intensity and faster reaction, indicating that a one-pot detection system for SfaTnpB based on LAMP amplification has been successfully established.

[0105] Example 5 High-sensitivity detection achieved by the LAMP-SfaTnpB one-pot nucleic acid detection system

[0106] In this example, to evaluate the detection sensitivity of the LAMP-SfaTnpB one-pot nucleic acid detection system, standard products of pUC57-HPV16-L1 plasmid and pUC57-HPV18-L1 plasmid with different copy numbers (10 4 ~10 -1 copies / μL) were prepared by gradient dilution method as detection templates. By systematically analyzing the limit of detection (LOD) of templates with different concentrations, the sensitivity performance of this nucleic acid detection system was comprehensively evaluated.

[0107] The pUC57-HPV16-L1 plasmid selected in this example was synthesized by GenScript and cloned into the pUC57 backbone. The HPV16-L1 fragment sequence it contains is: 5’-TGGTTGATACTGGCTTTGGTGCTATGGACTTTACTA CATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCTATTTGCAAATATCCAGATTATATTAAAATGGTGTCAGAACCATATGGCGACAGCTTATTTTTTTATTTACGAAGGGAACAAATGTTTGTTAGACATTTATTTAATAGGGCTGGTACTGTTGGTGAAAATGTACCAGACGATTTATACATTAAAGGCTCTGGGTCTACTGCAAATTTAGCCAGTTCAAATTATTTTCCTACACCTAGTGGTTCTATGGTTAC CTCTGATGCCCAAATATTCAATAAACCTTATTGGTTACAACGAGCACAGGGCCACAATAATGGCATTTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACGTTATGACATACATACATTCTATGAATTCCACTATTTTGGAGGACTGGAATTTTGGTCTACAACCTCCCCCAGGAGGCACACTAGAAGATACTTATAGGTTTGTAACATCCCAGGCAATTGCTTGTCAAAAACATACACCTCCAGCACCTAAAGAAGATCCCCTTAAAAAATACACTTTTTGGGAAGTAAATTTAAAGGAAAAGTTTTCTGCAGACCTAGATCAGTTTCCTTTAGGAC-3’ (SEQ ID NO.36); The bold-marked sequence is TAM, and the underlined sequence is the targeting sequence. The corresponding ωRNA sequence is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CUCUGAUGCCCAAAUAUUCA -3’ (SEQ ID NO.37, the underlined part is the targeting sequence).

[0108] The pUC57-HPV18-L1 plasmid was synthesized by GenScript and cloned into the pUC57 backbone. The nucleotide sequence of the HPV18-L1 fragment it contains is shown in SEQ ID NO.14; The bold-marked sequence is TAM, and the underlined sequence is the targeting sequence. The corresponding ωRNA sequence is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGG UAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA GGCGUGAGCAGCUUU UUGCU -3’ (SEQ ID NO.38, the underlined part is the targeting sequence);

[0109] The sensitivity of the HPV16 / 18-L1 plasmid was detected using the LAMP-SfaTnpB one-pot system, and serially diluted HPV16-L1 plasmid (primers: LAMP-HPV16-F3 / B3 / FIP / BIP / LB) and HPV18-L1 plasmid (primers: LAMP-HPV18-F3 / B3 / FIP / BIP / LB) were used as templates, respectively. The HPV16 primer sequences were LAMP-HPV16-F3: 5'-AGACGATTTATACATTAAAGGCT-3' (SEQ ID NO.39), LAMP-HPV16-B3: 5'-CATATTTGTACTGCGTGTAGT-3' (SEQ ID NO.40), LAMP-HPV16-FIP: 5'-AGGTAACCATAGAACCACTAGGTGTTGGGTCTACTGCAAATTTAGC-3' (SEQ ID NO.41), LAMP-HPV16-BIP: 5'-TAAACCTTATTGGTTACAACGAGCACAACAACAGTAACAAATAGTTGG-3' (SEQ ID NO.42), and LAMP-HPV16-LB: 5'-CAGGGCCACAATAATGGCATTT-3' (SEQ ID NO.43); the HPV18 primer sequences were LAMP-HPV18-F3: 5'-ACAAATG TCTGCAGATCCTT-3' (SEQ ID NO.44), LAMP-HPV18-B3: 5'-GGTAACAATAGAGCCACTTG-3' (SEQ ID NO.45), LAMP-HPV18-FIP: 5'-GCCCTATTCCAAAAATGCCTAGCGGGATTCCATGTTTTTTTGCTT-3' (SEQ ID NO.46), LAMP-HPV18-BIP: 5'-GGTGACACTGTGCCTC AATCGGGAGAATACACACAGCT-3' (SEQ ID NO.47), and LAMP-HPV18-LB: 5'-GGTATGCGTGCTTCACCTGG-3' (SEQ IDNO.48).

[0110] LAMP-SfaTnpB one-pot detection system: 1.2 μL (1.6 μM FIP / BIP, 0.2 μM F3 / B3, and 0.4 μM LB) primer mixture, 1 μL Bst 3.0 DNA Polymerase, 3.5 μL dNTP, 2.5 μL Isothermal buffer, 1.5 μL MgSO4, 250 nM SfaTnpB protein, 700 nM ωRNA, 3 μL 10× FastDigest Buffer, 250 nM ssDNA-reporter (SEQ ID NO.11), and 1 μL template plasmid (pUC57-HPV16-L1 or pUC57-HPV18-L1), with a final volume of 35 μL.

[0111] The negative control group did not add the template plasmid. The reaction tubes were placed in a 48°C metal bath for 40 min. After the reaction, they were inactivated at 80°C for 5 min. The reaction solution was transferred to an ELISA plate, and each well was diluted with 80 μL of DEPC water. The fluorescence values of each reaction at 561 - 601 nm were read using an ELISA reader.

[0112] The results are as Figure 8 shown. The detection limit of the LAMP-SfaTnpB one-pot detection system for the pUC57-HPV16-L1 plasmid was up to 4.12 copies / μL, and for the pUC57-HPV18-L1 plasmid was 3.8 copies / μL. This further demonstrated that the LAMP-SfaTnpB one-pot detection system had high-sensitivity detection performance.

[0113] Example 6 High-efficiency and accurate detection of HPV16 / HPV18 clinical samples using the LAMP-SfaTnpB one-pot system

[0114] In this example, for 35 clinical samples infected with HPV16 / HPV18, DNA solutions of the 35 samples were obtained using the DNA crude extraction method, and the LAMP-SfaTnpB one-pot system was further used to detect the infection status of the clinical samples. To verify the reliability of the detection results, the same batch of samples was rechecked by qPCR detection.

[0115] The process of crude DNA extraction is as follows: 80 μL of QuickExtract DNA Extraction Solution (Lucigen) is mixed with 80 μL of sample preservation solution, and the mixture is reacted in a PCR instrument at 65 °C for 10 min first, and then at 95 °C for 5 min. The solution after the reaction is used as the template for the LAMP-SfaTnpB one-pot method. Detection is carried out separately for the cases of HPV16 and HPV18 infection. The LAMP-SfaTnpB one-pot detection system: 1.2 μL (1.6 μM FIP / BIP, 0.2 μM F3 / B3, and 0.4 μM LB) primer mixture (the specific sequences of the primers are shown in Example 5, and the primers for amplifying HPV16 and HPV18 are used separately), 1 μL of Bst3.0 DNA Polymerase, 3.5 μL of dNTP, 2.5 μL of Isothermal buffer, 1.5 μL of MgSO4, 250 nM of SfaTnpB protein, 700 nM of ωRNA, 3 μL of 10×FastDigest Buffer, 250 nM of ssDNA-reporter (SEQ ID NO.11), and 3 μL of template solution. The final system is 35 μL. Among them, PC is the positive control group, that is, the template is a plasmid of 10 3 copies / μL, and NC is the negative control group, that is, no template is added. The reaction tubes are placed in a 48 °C metal bath and reacted for 50 min. After the reaction, it is inactivated at 80 °C for 5 min. The reaction solution is transferred to an enzyme-labeled plate, and 80 μL of DEPC water is added to each well for dilution. The fluorescence values of each reaction at 561 - 601 nm are read by an enzyme-labeled instrument. Referring to the reported detection system, qPCR targets the E6 genes of HPV16 and HPV18, and the corresponding qPCR detection primers are: HPV16-qPCR-F (SEQ ID NO.21), HPV16-qPCR-R (SEQ ID NO.22), HPV18-qPCR-F (SEQ ID NO.23), HPV18-qPCR-R (SEQ IDNO.24). The TaqMan probes used are: HPV16-E6-probe (SEQ ID NO.25) and HPV18-E6-probe (SEQ ID NO.26). The qPCR system includes 1 μL of 10 μM F, 1 μL of 10 μM R, 2× enzyme reaction solution (Luna ®Universal qPCR Master Mix, NEB) 0.4 μL of 10 μM probe and 3 μL of template, made up to 20 μL with DEPC water. The reaction conditions for qPCR were: 95°C for 3 min; (95°C for 10 s, 60°C for 30 s) × 40 cycles. The results were as Figure 9 shown. Samples No. 3 (S3), No. 7 (S7), No. 13 (S13), No. 14 (S14), No. 20 (S20), No. 23 (S23), No. 28 (S28) and No. 30 (S30) were HPV16-infected samples; Samples No. 17 (S17), No. 18 (S18), No. 19 (S19), No. 21 (S21), No. 22 (S22), No. 24 (S24) and No. 25 (S25) were HPV18-infected samples; The test results were consistent with Figure 5 those of the RAA-SfaTnpB one-pot system in Figure 10 and the qPCR test results in

[0116] Example 7: The LAMP-SfaTnpB one-pot nucleic acid detection system achieves highly sensitive and accurate detection of African swine fever virus

[0117] In this example, the detection performance of the one-pot nucleic acid detection system based on SfaTnpB for African swine fever virus (ASFV) was systematically evaluated. First, pMD18T-ASFV-p72 plasmid standards with different copy numbers (3.5×10 5 ~3.5×10 -1 copies / μL) were prepared by serial dilution for sensitivity testing; Subsequently, 22 clinical samples were tested and verified by qPCR method to comprehensively evaluate the rapidity, accuracy and robustness of the detection system.

[0118] The p72 fragment sequence contained in the pMD18T-ASFV-p72 plasmid selected in this example was: 5’-CAGCACAGCTGAACCGTTCTGAAGAAGAAGAAAGTTAATAGCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCT CC CGTGGCTTCAAAGCAAAGGTAATCATCATCGCACCCGGATCATCGGGGGTTTTAATCGCATTGCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGAAATTTATCGATAAGATTGATACCATGAGCAGTTACGGAAATGTTTTTAATAATAGGTAATGTGATCGGATACGTAACGGGGCTAATATCAGATATAGATGAACATGCGTCTGGAAGAGCTGTATCTCTATCCTGAAAGCTTATCTCTGCGTGGTGAGTGGGCTGCATAATGGCGTTAACAACATGTCCGAACTTGTGCCAATCTCGGTGTTGATGAGGATTTTGATCGGAGATGTTCCAGGTAGGTTTTAATCCTATAAACATATATTCAATGGGCCATTTAAGAGCAGACATTAGTTTTTCATCGTGGTGGTTATTGTTGGTGTGGGTCAC-3’ (SEQ ID NO.49); The reverse complementary sequence marked in bold is TAM, and the reverse complementary sequence where the underline is located is the targeting sequence. The corresponding ωRNA sequence is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGG GUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CUUUGCUUUGAAGCCACGGG- 3' (SEQ ID NO.50, the underlined part is the targeting sequence); the LAMP primer sequences used are p72-F3: 5'-CCCAACTAATATAAAATTCTCTTGC-3' (SEQ ID NO.51), p72-B3: 5'-GCTCATGGTATCAATCTTATCGA-3' (SEQ ID NO.52), p72-FIP: 5'-TGATGATTACCTTTGCTTTGAAGCCGGATACGTTAATATGACCACTG-3' (SEQ ID NO.53), p72-BIP: 5'-CCGGATCATCGGGGGTTTTAATTTCCATCAAAGTTCTGCA-3' (SEQ ID NO.54) and p72-LB: 5'-CGCATTGCCTCCGTAGTGG-3' (SEQ ID NO.55). The process of crude DNA extraction from clinical samples is as follows: Mix 80 μL of QuickExtract DNA Extraction Solution (Lucigen) with 80 μL of sample preservation solution, and the reaction program in the PCR instrument is: 65°C, 10 min; 95°C, 5 min, and the solution after the reaction is used as the reaction template. LAMP-SfaTnpB one-pot detection system: 1.2 μL (1.6 μM p72-FIP / p72-BIP, 0.2 μM p72-F3 / p72-B3 and 0.4 μM p72-LB) primer mixture, 1 μL of Bst 3.0 DNA Polymerase, 3.5 μL of dNTP, 2.5 μL of Isothermal buffer, 1.5 μL of MgSO4, 250 nM of SfaTnpB protein, 700 nM of ωRNA, 3 μL of 10×FastDigest Buffer, 250 nM of ssDNA-reporter (SEQ ID NO.11) and 1 μL of template plasmid (pMD18T-ASFV-p72) or 3 μL of sample solution, and the final system is 35 μL.

[0119] Among them, no template DNA was added to the negative control group. The reaction solution was transferred to an enzyme-labeled plate, and 80 μL of DEPC water was added to each well for dilution. The fluorescence values of each reaction at 561 - 601 nm were read by an enzyme-labeled instrument. The detection process of qPCR referred to the ASFV diagnostic method published by OIE, and the primer sequences were qPCR-p72-F: 5'-CTGCTCATGGTAT CAATCTTATCGA-3' (SEQ ID NO.56) and qPCR-p72-R: 5'-GATACCACAAGATCAGCCGTA-3' (SEQ ID NO.57).

[0120] As Figure 11 shown, the detection limit of the LAMP-SfaTnpB one-pot method for ASFV can reach 3.17 copies / μL, indicating that this system has excellent detection sensitivity and can meet the requirements of various application scenarios. In the clinical sample verification, the detection results of this system for 22 samples showed 12 positive and 10 negative ( Figure 12 ), and all detection results were completely consistent with the qPCR identification results ( Figure 13 , samples with Ct value ≤ 35 were determined as positive infection samples). These data fully confirmed that the LAMP-SfaTnpB one-pot method system has excellent detection performance, including high sensitivity, high accuracy and good stability, providing a reliable basis for its application in clinical diagnosis and field detection.

[0121] Example 8 Screening of Specific ωRNAs Corresponding to 14 High-Risk HPV Subtypes

[0122] In this example, ωRNAs for specific genotyping were designed and screened for 14 high-risk HPV subtypes. First, the specificity of SfaTnpB for single-base discrimination was evaluated through in vitro trans-cleavage experiments. Base-by-base mismatch tests were performed on the spacers at three different targets, and it was found that when the mismatched base between the spacer and the target sequence (TS) was in the 6 - 12 nt interval, the recognition efficiency of SfaTnpB was significantly reduced. Based on this, by comparing the L1 gene sequences of 14 high-risk HPV subtypes, highly specific ωRNAs were designed and screened for each subtype for HPV genotyping detection.

[0123] In this embodiment, the three targets for SfaTnpB mismatch tolerance are ASFV-p72, CSFV-E2, and DNMT1. The corresponding wild-type spacer sequences are 5'-CTTTGCTTTGAAGCCACGGG-3' (SEQ ID NO.58), 5'-CACTTCCGTTCTCAGAGTTG-3' (SEQ ID NO.59), and 5'-ATCTGCAAAATCGCTTTGGC-3' (SEQ ID NO.60).

[0124] In this embodiment, the GenBank numbers of the 14 high-risk HPV subtypes selected are as follows: for HPV16: LC368980.1; for HPV18: KC470210.1; for HPV31: OP712005.1; for HPV33: HQ537701.1; for HPV35: MT217174.1; for HPV39: OP971074.1; for HPV45: EF202156.1; for HPV51: OR997935.1; for HPV52: LC270053.1; for HPV56: LR862043.1; for HPV58: LR861973.1; for HPV59: ON365489.1; for HPV66: EF177185.1; for HPV68: KC470280.1.

[0125] The ωRNA sequences corresponding to the 14 high-risk HPV subtypes are as follows. The ωRNA sequence targeting HPV16 is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CAAUGUUGCAGUAAAUCCAG -3’ (SEQ ID NO.61, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV18 is 5’-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUAC CCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA AUAACUGUGUCUGCU UAUAA-3' (SEQ ID NO.62, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV31 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAU GAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA UACAAGGACUACCUUUACCC -3' (SEQ ID NO.63, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV33 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUA AUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA UUGGAUGUAAGCCUCCAACA -3' (SEQ ID NO.64, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV35 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGG GUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CUGGUUAGCAUUACAAGGUG -3' (SEQ ID NO.65, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV39 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAG GGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA AUUAUUGGGCUUGCAUGCCU -3' (SEQ IDNO.66, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV45 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA AGCUGUUAUUACGCAGGAUG -3' (SEQ ID NO.67, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV51 is 5'-AUCAGCAUAGGGAACU AUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA AAGUUCCAGGGGGGGGCAGU-3' (SEQ ID NO.68, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV52 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CAGGUU UACCAGCAUAUUUG -3' (SEQ ID NO.69, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV56 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUAC CCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA AATGTTCACCCATAGCGGGA -3' (SEQ ID NO.70, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV58 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGAGAUA CCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CCCAAUGCUCACCAG UGGGA -3' (SEQ ID NO.71, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV59 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGAGGGGUAAUGA GAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA ACGUGAUAAUGUAUCUGUGG -3' (SEQ ID NO.72, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV66 is 5'-AUCAGCAUAGGGAACUAUAUGUUCUGCCCUAUGA GGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAA CUGCUAAAUUAGAGACCUCA -3' (SEQ ID NO.73, the underlined part is the targeting sequence); the ωRNA sequence targeting HPV68 is 5'-AUCAGCAUAGGGAACUAUAUG UUCUGCCCUAUGAGGGGUAAUGAGAUACCCUAAUCUUGAGGAUCACUCAAGAAUCCCACCCGUAAUUUAGGGUGGGAGUCUCAATGTCCTTAGGATTTCTGTTA - 3' (SEQ ID NO.74, the underlined part is the targeting sequence); The reaction system is 20 μL, including 2 μL of 10×CutSmart Buffer, 250 nM of SfaTnpB protein, 700 nM of ωRNA (one of SEQ ID NOs. 61 - SEQ ID NO.74), 200 ng of each different HPV subtype target, and 0.2 μL of 100 μM ssDNA - reporter.

[0126] Among them, the negative control group does not add the target. Set the kinetic reaction program in the microplate reader, with a temperature of 37°C, read the fluorescence signal every 2 minutes and shake, and the wavelength range is 561 - 601 nm.

[0127] The results of the SfaTnpB single - base mismatch tolerance analysis ( Figure 14 ) show that when a base mismatch occurs between the spacer and the target sequence (TS) in the 6 - 12 nt region, the catalytic activity of SfaTnpB is significantly reduced, and no obvious fluorescence signal is generated. This feature enables the mismatch sites in this region to effectively distinguish different genotypes. Based on this property, the present invention has successfully designed 14 ωRNAs for specific genotyping of high - risk HPV subtypes through systematic screening ( Figures 15 - 19 ). The verification experiment shows that all ωRNAs can specifically recognize their corresponding HPV subtypes, and no cross - reaction is detected among different subtypes, confirming that this detection system has a high - specificity genotyping ability.

[0128] Example 9: The RAA - SfaTnpB one - pot system enables efficient and accurate detection of clinical samples of different high - risk HPV subtypes

[0129] In this example, for 28 clinical samples infected with different high - risk HPV subtypes, DNA solutions of 28 samples were obtained by DNA crude extraction method, and the RAA - SfaTnpB one - pot system was used to detect the infection status of clinical samples.

[0130] The process of crude DNA extraction is as follows: 80 μL of QuickExtract DNA Extraction Solution (Lucigen) is mixed with 80 μL of sample preservation solution. The reaction program of the PCR instrument is: 65°C for 10 min; 95°C for 5 min. The solution after the reaction is used as the template for the RAA-SfaTnpB one-pot detection. The RAA-SfaTnpB one-pot detection system is as follows: The RAA premix includes 25 μL of buffer V, 2 μL of 10 μM forward primer F, 2 μL of 10 μM reverse primer R, one dry powder reaction tube, and 5 μL of magnesium acetate solution, and is made up to 50 μL with DEPC water. The sequence of the forward primer F is: 5’-GGNCAGCCWTTAGGTGTTGGYMTWAGTGGTCATCC-3’ (SEQ ID NO.75, where N represents A / T / C / G; W represents A / T; Y represents C / T; M represents A / C); the sequence of the reverse primer R is: 5’-TCCATRGCSCCASATCCTGTRTCHACCATRTCMCC-3’ (SEQ ID NO.76, where R represents A / G; S represents C / G; H represents A / T / C; M represents A / C); 250 nM of SfaTnpB protein, 700 nM of ωRNA (one of SEQ ID NO.61 - SEQ ID NO.74), 250 nM of ssDNA-reporter (SEQ IDNO.11), and 3 μL of template solution are added to 19.3 μL of the RAA premix, and the final system is 25 μL.

[0131] Among them, NC is the negative control group, that is, no template is added. The reaction tubes are placed in a 37°C metal bath for 40 min. After the reaction, they are inactivated at 80°C for 5 min. After the reaction, they are placed under a blue light instrument for taking pictures.

[0132] The results are as Figure 20As shown, sample 1 (S1) is an HPV51 infected person, sample 2 (S2) is a person not infected with 14 high-risk types of HPV, sample 3 (S3) is a co-infected person with HPV33 and HPV51, sample 4 (S4) is a co-infected person with HPV66 and HPV52, sample 5 (S5) is a co-infected person with HPV35 and HPV66, sample 6 (S6) is an HPV16 infected person, sample 7 (S7) is an HPV33 infected person, sample 8 (S8) is a person not infected with 14 high-risk types of HPV, sample 9 (S9) is a co-infected person with HPV39 and HPV52, sample 10 (S10) is a co-infected person with HPV58 and HPV39, sample 11 (S11) is a person not infected with 14 high-risk types of HPV, sample 12 (S12) is an HPV39 infected person, sample 13 (S13) is an HPV52 infected person, sample 14 (S14) is an HPV59 infected person, sample 15 (S15) is an HPV18 infected person, sample 16 (S16) is an HPV68 infected person, sample 17 (S17) is an HPV33 infected person, sample 18 (S18) is a person not infected with 14 high-risk types of HPV, sample 19 (S19) is an HPV35 infected person, sample 20 (S20) is an HPV58 infected person, sample 21 (S21) is a person not infected with 14 high-risk types of HPV, sample 22 (S22) is an HPV18 infected person, sample 23 (S23) is an HPV31 infected person, sample 24 (S24) is a person not infected with 14 high-risk types of HPV, sample 25 (S25) is an HPV52 infected person, sample 26 (S26) is an HPV58 infected person, sample 27 (S27) is an HPV51 infected person, sample 28 (S28) is a co-infected person with HPV56 and HPV66.

[0133] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A one-pot rapid nucleic acid detection system based on SfaTnpB combined with isothermal amplification technology, characterized in that: The one-pot rapid nucleic acid detection system includes a SfaTnpB premix and an isothermal amplification premix; The SfaTnpB premix includes SfaTnpB protein, ωRNA and ssDNA-reporter; the scaffold sequence of the ωRNA is shown in SEQ ID NO.16; the TAM sequence of the SfaTnpB protein is shown in ATAC or TTAC; the nucleotide sequence of the ssDNA-reporter is shown in SEQ ID NO.11; The isothermal amplification premix comprises primers and an isothermal amplification reaction premix; The isothermal amplification techniques include RPA, RAA, LAMP, NASBA, RCA, MCDA, SDA or CPA.

2. The one-pot rapid nucleic acid detection system according to claim 1, characterized in that: The molar ratio of the SfaTnpB protein to the ωRNA is 1:5 to 2:

1.

3. The one-pot rapid nucleic acid detection system according to claim 2, characterized in that: The molar ratio of the SfaTnpB protein to the ωRNA is 1:

3.

4. The one-pot rapid nucleic acid detection system according to any one of claims 1 to 3, characterized in that: When the isothermal amplification technique is RAA, the isothermal amplification reaction premix includes buffer V, magnesium acetate solution and DEPC water; when the isothermal amplification technique is LAMP, the isothermal amplification reaction premix includes Bst, dNTP, ISothermal buffer, MgSO4 and DEPC water.

5. Use of the one-pot rapid nucleic acid detection system according to any one of claims 1 to 4 in the preparation of products for detecting viruses.

6. A product for detecting viruses, characterized in that: The product includes the one-pot rapid nucleic acid detection system described in any one of claims 1 to 4.

7. The product according to claim 6, characterized in that The viruses include one or more of human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 35, human papillomavirus type 39, human papillomavirus type 45, human papillomavirus type 51, human papillomavirus type 52, human papillomavirus type 56, human papillomavirus type 58, human papillomavirus type 59, human papillomavirus type 66, human papillomavirus type 68 and African swine fever virus.

8. The product according to claim 6 or 7, characterized in that When the isothermal amplification technique is RAA, the ωRNA sequence targeting the human papillomavirus type 16 is shown in SEQ ID NO.13 or SEQ ID NO.61; the ωRNA sequence targeting the human papillomavirus type 18 is shown in SEQ ID NO.15 or SEQ ID NO.62; the ωRNA sequence targeting the human papillomavirus type 31 is shown in SEQ ID NO.63; the ωRNA sequence targeting the human papillomavirus type 33 is shown in SEQ ID NO.64; the ωRNA sequence targeting the human papillomavirus type 35 is shown in SEQ ID NO.66; the ωRNA sequence targeting the human papillomavirus type 39 is shown in SEQ ID NO.66; the ωRNA sequence targeting the human papillomavirus type 45 is shown in SEQ ID NO.67; the ωRNA sequence targeting the human papillomavirus type 51 is shown in SEQ ID NO.68; the ωRNA sequence targeting the human papillomavirus type 52 is shown in SEQ ID NO. The ωRNA sequence targeting human papillomavirus 56 is shown in SEQ ID NO.69; the ωRNA sequence targeting human papillomavirus 56 is shown in SEQ ID NO.70; the ωRNA sequence targeting human papillomavirus 58 is shown in SEQ ID NO.71; the ωRNA sequence targeting human papillomavirus 59 is shown in SEQ ID NO.72; the ωRNA sequence targeting human papillomavirus 66 is shown in SEQ ID NO.73; and the ωRNA sequence targeting human papillomavirus 68 is shown in SEQ ID NO.

74. When the isothermal amplification technology is LAMP, the ωRNA sequence targeting human papillomavirus type 16 is shown as SEQ ID NO.37; the ωRNA sequence targeting human papillomavirus type 18 is shown as SEQ ID NO.38; and the ωRNA sequence targeting African swine fever virus is shown as SEQ ID NO.

50.

9. The product according to claim 6 or 7, characterized in that When the isothermal amplification technique is RAA, the primers include a primer pair for amplifying human papillomavirus type 16, a primer pair for amplifying human papillomavirus type 18, or a universal primer pair for human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 35, human papillomavirus type 39, human papillomavirus type 45, human papillomavirus type 51, human papillomavirus type 52, human papillomavirus type 56, human papillomavirus type 58, human papillomavirus type 59, human papillomavirus type 66, and human papillomavirus type 68; the primer pair for amplifying human papillomavirus type 16 includes an upstream primer as shown in SEQ ID NO. 17 and a downstream primer as shown in SEQ ID NO. 18; the primer pair for amplifying human papillomavirus type 18 includes a primer pair as shown in SEQ ID NO. The upstream primer shown in SEQ ID NO.19 and the downstream primer shown in SEQ ID NO.20; the universal primer pair includes the upstream primer shown in SEQ ID NO.75 and the downstream primer shown in SEQ ID NO.76; When the isothermal amplification technique is LAMP, the primers include one or more pairs of primer pairs for amplifying human papillomavirus type 16, a primer pair for amplifying human papillomavirus type 18, and a primer pair for amplifying African swine fever virus; the primer pair for amplifying human papillomavirus type 16 includes LAMP-HPV16-F3 as shown in SEQ ID NO.39, LAMP-HPV16-B3 as shown in SEQ ID NO.40, LAMP-HPV16-FIP as shown in SEQ ID NO.41, LAMP-HPV16-BIP as shown in SEQ ID NO.42, and LAMP-HPV16-LB as shown in SEQ ID NO.43; the primer pair for amplifying human papillomavirus type 18 includes LAMP-HPV18-F3 as shown in SEQ ID NO.44, LAMP-HPV18-B3 as shown in SEQ ID NO.45, LAMP-HPV18-FIP as shown in SEQ ID NO.46, LAMP-HPV18-LB as shown in SEQ ID NO.

47. LAMP-HPV18-BIP shown in NO.47 and LAMP-HPV18-LB shown in SEQ ID NO.48; the primer pair for amplifying African swine fever virus includes p72-F3 shown in SEQ ID NO.51, p72-B3 shown in SEQ ID NO.52, p72-FIP shown in SEQ ID NO.53, p72-BIP shown in SEQ ID NO.54 and p72-LB shown in SEQ ID NO.

55.

10. The product according to any one of claims 6 to 9, characterized in that: The samples to be tested for the product include one or more of blood, cells, cervical scrapings, cervical fluid-based cells, vaginal swab serum, saliva, body fluids, plasma, urine, bronchial lavage fluid, cerebrospinal fluid, gastric juice, tonsils, lymph nodes and feces.

11. The product according to any one of claims 6 to 9, characterized in that: When the isothermal amplification technique is RAA, the isothermal amplification premix includes, based on 50 μL, a dry powder reaction tube, 25 μL of buffer V, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of magnesium acetate solution, and the balance of DEPC water; When the isothermal amplification technique is LAMP, based on 35 μL, the isothermal amplification premix includes 1.2 μL primer mixture, 1 μL Bst, 3.5 μL dNTP, 2.5 μL ISothermal buffer, 1.5 μL MgSO4 and the balance DEPC water.

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