Hantavirus detection system and detection method based on isothermal amplification and crisper / cas12 signal amplification

Abstract

This invention discloses a Hantan virus detection system and method based on isothermal amplification and CRISPR / Cas12 signal amplification, belonging to the field of pathogenic microorganism and drug resistance gene detection technology. Multiple sets of starting primers and amplification primers targeting different conserved regions of the Hantan virus genome are designed to form multi-target parallel amplification. The 3' phosphorylated sequence of the starting primer is cleaved by BstNI restriction enzyme to activate extension, and cascade isothermal amplification is achieved by combining Nt.BstNBI and a large fragment of Bst DNA polymerase. The Cas12 protein and crRNA recognize the activation sequence in the amplification product, triggering the signal output of the fluorescent reporter probe. This detection method does not require a thermal cycler, has a detection limit of 10 copies / μL, exhibits high tolerance to viral mutations and RNA degradation, and can be optionally equipped with an integrated sample processing module for rapid field detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a Hantan virus detection system and method based on isothermal amplification and CRISPR / Cas12 signal amplification. Background Technology

[0002] Orthohantaviruses are a class of pathogens primarily carried by rodents, particularly rats. According to the latest classification by the International Committee on Taxonomy of Viruses (ICTV), these viruses belong to the class Bunyaviricetes, order Elliovirales, family Hantaviridae, and genus Orthohantavirus. This genus contains approximately 70 species, of which about 20 can cause serious illness in humans, clinically manifesting primarily as hemorrhagic fever with renal syndrome (HFRS, also known as epidemic hemorrhagic fever in my country) and Hantavirus pulmonary syndrome (HPS).

[0003] HFRS is classified as a Class B notifiable infectious disease in my country. Orthohantavirus is an enveloped, single-stranded, negative-strand RNA virus. The viral particles are spherical or oval, with an average diameter of approximately 120 nanometers. Its genome consists of three gene segments: large (L), medium (M), and small (S), which encode RNA-dependent RNA polymerase, envelope glycoproteins (Gn and Gc, formerly known as G1 and G2), and nucleocapsid proteins, respectively. Among the various serotypes causing HFRS, Hantan virus (HTNV), Seohantavirus (SEOV), Pumala virus (PUUV), and Dobraval-Belgrade virus (DOBV) are the main pathogens. HTNV and Seohantavirus are the predominantly circulating serotypes in my country.

[0004] Currently, detection methods for Hantan virus (HTNV) mainly include serological testing (such as ELISA) and nucleic acid testing (such as RT-PCR). However, existing technologies still have the following shortcomings: 1) Strong dependence on testing equipment, making them unsuitable for rapid on-site testing. Traditional nucleic acid testing methods, such as RT-PCR, rely on sophisticated thermal cycling instruments for temperature control. These instruments are large, costly, and inconvenient to carry, making them difficult to promote and apply in primary healthcare institutions, field monitoring stations, or mobile testing scenarios. 2) Single-site primer design easily leads to false negative results. Hantan virus is an RNA virus with a high rate of genetic mutation. Existing detection methods mostly use primers or probes targeting a single conserved region of the viral genome. Once the target site mutates, the primer may not bind effectively, resulting in false negative results and reducing the reliability and universality of the detection. To overcome the above technical problems, this invention is based on the HTNV 76-118 standard strain sequence. This patent selects five primer pairs from the conserved viral sequence, resulting in a wider recognition range and better compatibility with random mutations in the viral sequence and differences between strains. In addition, this patent uses nucleic acid isothermal amplification technology, which does not rely on thermal cycling and is more in line with the requirements of rapid on-site testing.

[0005] In conclusion, developing a technical solution capable of detecting HTNV in human, rodent, and environmental samples with high sensitivity and robustness is of great significance for improving my country's early diagnostic capabilities for HFRS, on-site monitoring levels, and public health emergency response efficiency. Summary of the Invention

[0006] To address the technical problems of existing Hantan virus detection methods, which rely on large-scale thermal cycling equipment, are prone to false negatives due to viral mutations, and have insufficient sensitivity, this invention aims to provide a Hantan virus detection system and method based on isothermal amplification and CRISPR / Cas12 signal amplification, as well as primer combinations, kits, and applications for a rapid HFRS detection method that does not rely on complex equipment, has a multi-target fault-tolerant mechanism, and strong signal amplification capabilities.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a method for detecting Hantan virus based on isothermal amplification and CRISPR / Cas signal amplification, comprising the following steps: The sample to be tested was mixed with the starting primer, and the cleavage enzyme was added for incubation. The blocking modification at the 3′ end of the starting primer was removed to obtain the reaction system. Add amplification primers, chain displacement polymerase and nickase to the above reaction system, and carry out isothermal amplification reaction to obtain amplification products; The amplification product is mixed with the Cas protein-guide RNA complex and the signal reporter probe and incubated. The signal generated after the signal reporter probe is cleaved is detected to determine whether Hantan virus is present in the sample to be tested.

[0008] The 3' end of the starting primer has a blocking modification, and it does not have extension activity until the blocking modification is removed. The amplification primer contains a region complementary to a portion of the sequence of the starting primer and an activation sequence.

[0009] Preferably, there are at least two sets of starting primers, each set of starting primers targeting different conserved regions of the Hantan virus genome; and at least two sets of amplification primers, each set of amplification primers corresponding to a set of starting primers.

[0010] More preferably, there are five sets of starting primers and five sets of amplification primers. These five sets of starting and amplification primers target five different conserved regions of the Hantan virus genome, forming a multi-target parallel amplification mechanism. By using five sets of primers targeting different conserved regions of the Hantan virus genome to form a multi-target parallel amplification mechanism, even if a mutation occurs at a certain site of the virus or that region is lost due to RNA degradation, the remaining primers can still initiate amplification normally, thereby effectively avoiding false negative results and significantly improving the universality and robustness of the detection system against viral mutations and sample degradation.

[0011] Preferably, the blocking modification is a phosphorylation modification; the cleaving enzyme is a restriction endonuclease, more preferably a BstNI endonuclease.

[0012] Preferably, the nicking enzyme is a nicking endonuclease, more preferably Nt.BstNBI endonuclease; the strand displacement polymerase is a large fragment of Bst DNA polymerase.

[0013] Preferably, the isothermal amplification reaction temperature is 52℃~62℃, and the amplification time is 30 min~90 min.

[0014] Preferably, the isothermal amplification reaction temperature is 58°C and the amplification time is 1 hour; the fluorescence detection is performed at room temperature and the incubation time is 10 minutes.

[0015] Preferably, when the sample to be tested is rodent tissue, environmental swab or fecal sample, an integrated sample processing module is used to lyse and release nucleic acids before the amplification step, and the resulting lysate is directly added to the amplification reaction system as a template.

[0016] The lysis buffer contains proteinase K, detergent, and nucleic acid stabilizer. The lysis conditions are heating at 65°C for 10-15 minutes, enabling rapid processing of various sample types such as tissues, swabs, and feces.

[0017] Secondly, the present invention provides a Hantan virus detection system based on isothermal amplification and CRISPR / Cas12 signal amplification, comprising: The starting primer has a blocking modification at its 3′ end, and the starting primer does not have extension activity before the blocking modification is removed; Amplification primers comprising a region complementary to a portion of the sequence of the starting primer and an activation sequence; A cleavage enzyme is used to remove the blocking modification at the 3′ end of the starting primer; Chain displacement polymerase and nickase are used to amplify nucleic acids under isothermal conditions to produce amplification products containing the activated sequence. Cas protein and its guide RNA are used to specifically recognize the activation sequence in the amplification product and cleave the signal reporter probe; The signal reporting probe generates a detectable signal after being cut.

[0018] Preferably, the starting primers are in at least two sets, each set of starting primers targeting different conserved regions of the Hantan virus genome; the amplification primers are in at least two sets, each set of amplification primers corresponding to each set of starting primers.

[0019] Preferably, the blocking modification is phosphorylation modification; the cleavage enzyme is BstNI endonuclease; the cleavage enzyme is Nt.BstNBI endonuclease; the strand displacement polymerase is a large fragment of Bst DNA polymerase; the Cas protein is Cas12 protein; and the signal reporter probe is an oligonucleotide probe labeled with a fluorescent group and a quencher group.

[0020] Preferably, the system further includes an integrated sample processing module, which includes sample processing tubes pre-loaded with lysis buffer and nucleic acid stabilizer for on-site sample lysis and nucleic acid release.

[0021] Thirdly, this invention provides a Hantan virus detection kit based on isothermal amplification and CRISPR / Cas12 signal amplification, comprising: The starting primer has a blocking modification at its 3′ end; Amplification primers comprising a region complementary to a portion of the sequence of the starting primer and an activation sequence; A cleavage enzyme is used to remove the blocking modification at the 3′ end of the starting primer; Chain displacement polymerase and nickase are used to amplify nucleic acids under isothermal conditions; Cas protein and its guide RNA are used to specifically recognize activation sequences in amplification products and cleave signal reporter probes; The signal reporting probe generates a detectable signal after being cut.

[0022] Preferably, there are at least two sets of starting primers, each set of starting primers targeting different conserved regions of the Hantan virus genome; and at least two sets of amplification primers, each set of amplification primers corresponding to a set of starting primers.

[0023] Preferably, the blocking modification is phosphorylation modification; the cleavage enzyme is BstNI endonuclease; the cleavage enzyme is Nt.BstNBI endonuclease; the strand displacement polymerase is a large fragment of Bst DNA polymerase; the Cas protein is Cas12 protein; and the signal reporter probe is an oligonucleotide probe labeled with a fluorescent group and a quencher group.

[0024] Preferably, each component is packaged independently in lyophilized or solution form.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The Hantan virus detection method based on the system provided by this invention involves a four-step process: primer activation, isothermal amplification, Cas12 fluorescence detection, and UV / fluorescence observation. The entire process can be completed within 1.5 hours, requiring no thermal cycling equipment. It is simple to operate and provides intuitive results (presence or absence of fluorescence), making it suitable for rapid screening in scenarios such as grassroots disease control, field monitoring, and customs ports. It achieves miniaturization and portability of the detection equipment, making it suitable for rapid on-site detection. Based on isothermal amplification technology combined with fluorescence signal capture, the entire reaction process does not rely on large equipment such as thermal cycling instruments; only a simple fluorescence colorimetric light source is needed to complete the entire detection process. This characteristic makes this invention highly integrated and portable, particularly suitable for field, grassroots, or mobile on-site operating environments. It significantly improves detection sensitivity, enabling reliable identification of low-abundance targets.

[0026] The system provided by this invention achieves isothermal amplification without the need for thermal cycling instruments through the synergistic cooperation of multiple primer sets, BstNI activation, Nt.BstNBI and Bst DNA polymerase cascade amplification, Cas12-crRNA recognition, and fluorescent reporter probes. Combined with CRISPR / Cas12 signal cascade amplification, the detection sensitivity is significantly improved. At the same time, the design of multiple primer sets provides a foundation for subsequent anti-mutation and anti-degradation, giving the detection system the comprehensive advantages of portability, high sensitivity, and high robustness.

[0027] This invention introduces a CRISPR / Cas12 system as a signal amplification medium. Through the incidental cleavage activity of the Cas12 protein, target recognition events can be efficiently converted into amplified fluorescent signals, thereby further cascading amplification of the detection signal based on existing isothermal amplification, effectively improving the detection sensitivity of Hantan virus. This avoids the risk of false negatives caused by viral mutations and enhances the universality and robustness of the detection.

[0028] The Hantan virus detection kit provided by this invention integrates the starting primer, amplification primer, cutting enzyme, strand displacement polymerase, nicking enzyme, Cas protein-guide RNA complex, and signal reporter probe into a single detection product. Users only need to add samples and incubate at a constant temperature to complete the detection, eliminating the need for thermal cyclers and professional operators, thus greatly reducing the barrier to entry. Each component of the kit can be packaged independently in lyophilized or solution form, facilitating transportation and long-term storage. Combining multi-target primer design and cascade signal amplification mechanism, the kit has the comprehensive advantages of high sensitivity, high robustness, and extremely simple operation, and can be widely used in rapid on-site detection scenarios such as clinical laboratories, disease control centers, field monitoring stations, and customs ports, showing good prospects for industrialization and commercialization. Attached Figure Description

[0029] Figure 1 The schematic diagram of the reaction flow of the Hantan virus detection system and method of this invention is as follows: A is the starting primer (P1-P5) which binds complementary to viral RNA and releases the phosphorylated 3' tail under the action of BstNI endonuclease, activating it into primers with extension activity; B is the activated primer set which binds to the amplification primer set (A1-A5) respectively, and extends under the action of Bst DNA Polymerase Large Fragment. Subsequently, Nt.BstNBI endonuclease causes the double strand to form a single-strand gap at a specific position. This gap can then undergo a strand displacement reaction under the action of Bst DNA Polymerase Large Fragment to form a new strand and initiate isothermal cycling; C is all the nucleic acid fragments generated by isothermal cycling, some of which can be used as primers to participate in isothermal amplification, and the other part can become the recognition sequence of CAS12; D is the CAS12 recognition of the isothermal amplification product sequence and generates a fluorescent signal by trans-cleavage activity cleaving the fluorescent probe (FAM-TTTTTTTT-BHQ).

[0030] Figure 2 The graph shows the detection results of the detection method of the present invention applied to the HTNV standard strain. In the graph, A is a bar graph of fluorescence intensity measured using a multifunctional microplate reader (BioTek-Synergy LX) (excitation wavelength 485 nm, emission wavelength 528 nm); B is a graph of the UV naked eye test results of the sensitivity of the detection method of the present invention; and C is a real-time fluorescence quantitative test curve of the sensitivity of the detection method of the present invention. Figure 3 The graph shows the verification results of the compatibility of the detection method of the present invention with viral sequence mutations and the specificity of real clinical samples. In this graph, A is a bar chart of mutation compatibility test results; B is the ELISA reader quantitative results of the specificity verification of real samples; and C is the real-time fluorescence amplification curve of each sample (Roche-cobas z 480). Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0032] I. Material Sources and Reagent Preparation Virus strains and samples: Hantan virus (HTNV) 76-118 standard strain (preserved in the laboratory sample bank). Clinical serum samples, rodent tissues, environmental swabs, etc., were legally sourced and ethically approved.

[0033] Main reagents: BstNI endonuclease, Nt.BstNBI endonuclease, and Bst DNA polymerase (large fragment) were purchased from Beyotime Biotechnology (Shanghai); LbaCas12a protein was purchased from EZassay (Shenzhen); dNTPs, RNase inhibitors, and recombinant albumin were purchased from Beyotime Biotechnology (Shanghai). The fluorescent reporter probe FAM-TTTTTTTT-BHQ was synthesized by EZassay (Shenzhen); primers and crRNA were synthesized by Sangon Biotech (Shanghai) and purified by PAGE.

[0034] Buffer preparation: Buffer A (starting mixture): 100 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 100 µg / mL Recombinant Albumin, diluted to volume with deionized water, sterile filtered, aliquoted and stored at -20℃.

[0035] Buffer B (amplification mixture): 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100. After sterile filtration, aliquot and store at -20℃. Before use, add 1 IU / µL Nt.BstNBI and 1 IU / µL Bst DNA polymerase (large fragment).

[0036] Fluorescence detection buffer system: 100 mM Tris-HCl (pH 7.5), 500 mM NaCl, 100 mM MgCl2, 1 mg / mL Recombinant Albumin, supplemented with 120 nM Reporter (FAM-TTTTTTTT-BHQ), 50 nM crRNA, and 50 nM LbaCas12a before use.

[0037] II. Primer and crRNA Sequence Design Five sets of start primers were designed targeting the conserved regions of the L, M, and S segments in the genome of HTNV 76-118 strain (GenBank accession numbers: M14626, M14627, M14628). All primers have a phosphorylation (-p) modification at their 3' ends. The sequences are as follows:

[0038] Amplification primers (each set contains a sequence complementary to the corresponding start primer and a downstream universal extension region): Each set of amplification primers contains a template sequence corresponding to the start primer and a downstream universal extension region, as follows:

[0039] The crRNA sequence (designed to target the common activation sequence in HTNV amplification products) is: 5'-UAAUUUCUACUAAGUGUAGAUAUCAACUACUAUACUCUAA-3' (e.g., SEQ ID NO.11).

[0040] The present invention will now be described in further detail with reference to the accompanying drawings. The specific steps are as follows: The detection process of this invention is described in [reference]. Figure 1 The four stages shown are operated as follows: Step 1: Initial primer activation stage ( Figure 1 (A) Take 5 μL of the nucleic acid sample (RNA) to be tested, add 45 μL of buffer A, so that the final concentration of the starting primers (P1-P5) is 10 nM each.

[0041] Preheat at 58°C for 2 minutes, add 0.5 IU BstNI restriction enzyme, and continue incubation at 58°C for 15 minutes.

[0042] like Figure 1 As shown in Figure A, after the initiating primer binds complementary to the viral RNA, the BstNI endonuclease removes the phosphorylated short sequence at its 3′ end, releasing the 3′-OH terminus with elongation activity, thus completing primer activation.

[0043] Step 2: Cascaded isothermal amplification stage ( Figure 1 (B) Add five sets of amplification primers (A1 to A5, 100 nM each) to the above reaction solution and mix well.

[0044] Add buffer B (containing Nt.BstNBI and Bst DNA polymerase fragments) to make the volume ratio of buffer B to the product mixture from step 1 4:1 (i.e., add 40 μL of buffer B to a total volume of 50 μL).

[0045] Incubate at a constant temperature of 58℃ for 1 hour.

[0046] like Figure 1 As shown in Figure B, the activated initiating primer binds to the amplification primer and extends under the action of the large fragment of Bst DNA polymerase; subsequently, the Nt.BstNBI endonuclease generates a single-strand gap at a specific position on the double strand, and the polymerase restarts strand displacement synthesis from the gap, forming a cascade cycle to achieve exponential nucleic acid amplification.

[0047] Step 3: CRISPR / Cas12 recognition and fluorescence signal generation ( Figure 1 C and Figure 1 (D) Take 5 μL of the amplification product from step 2 and add it to 45 μL of fluorescence detection buffer (containing the fluorescent reporter probe FAM-TTTTTTTT-BHQ, crRNA, and LbaCas12a protein).

[0048] Incubate at room temperature (approximately 25°C) in the dark for 10–20 minutes.

[0049] like Figure 1 As shown in Figure C, the activation sequence in the isothermal amplification product is specifically recognized by the Cas12-crRNA complex; as Figure 1 As shown in Figure D, the activated Cas12 protein exhibits trans-cleavage activity, non-specifically cleaving the fluorescent reporter probe, separating the FAM fluorescent group from the BHQ quencher group, and generating a detectable green fluorescent signal.

[0050] Step 4: Result Interpretation Qualitative observation: Irradiate the reaction tube (corresponding wavelength 302 nm or 365 nm) with an ultraviolet lamp. Figure 2 (A) Positive samples showed obvious green fluorescence.

[0051] Quantitative detection: Fluorescence intensity was measured using a multi-functional microplate reader (excitation 485 nm / emission 528 nm) or a quantitative real-time PCR instrument (corresponding to...). Figure 2 China A and Figure 2 (C). Positive criterion: Fluorescence intensity is significantly higher than the negative control (without template control) by more than 3 times. III. Specific Implementation Examples Example 1: Detection of HTNV standard strains Step 1: Isothermal amplification (initial activation phase) RNA from HTNV strains 76-118 (obtained via in vitro transcription and quantified to 10⁻⁶) was collected. 4 Add 5 μL of the starting primer set (10 mM each, 5 primers in total), 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, and 100 µg / ml Recombinant Albumin.

[0053] Preheat the mixture at 58°C for 2 minutes, then add 0.5 IU of BstNI restriction enzyme and continue incubation at 58°C for 15 minutes.

[0054] Add 100 mM of each of the five sets of amplification primers to the above reaction solution and mix well.

[0055] Add buffer B consisting of: 1 IU Nt.BstNBI, 1 IU Bst DNA Polymerase (LargeFragment), 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Triton X-100 to a total volume of 50 μL. The volume ratio of buffer B to the product mixture from step 1 is 4:1 (i.e., add 40 μL of buffer B). Incubate at 58°C for 1 hour to complete isothermal amplification.

[0056] Step 2: CRISPR / Cas12 fluorescence detection Take 5 μL of the amplification product from step 1 and add it to 45 μL of fluorescence detection buffer (100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl2, 1 mg / mL Recombinant Albumin, 120 nM Reporter (FAM-TTTTTTTT-BHQ), 50 nM crRNA, 50 nM LbaCas12a). Incubate at room temperature (approximately 25°C) in the dark for 10 minutes.

[0057] Observe the fluorescence color using a UV lamp (wavelength 302 nm or 365 nm); or measure the fluorescence intensity using a multi-functional microplate reader (BioTek-Synergy LX) (excitation wavelength 485 nm, emission wavelength 528 nm). Result interpretation: If obvious green fluorescence appears in the reaction tube (under UV light), and quantitative detection is performed using a multi-functional microplate reader, and the fluorescence intensity of the positive tube is significantly higher than that of the negative control (without template control), with a difference of more than 3 times, it is judged as HTNV positive. Figure 2 (A). This demonstrates that the system can effectively detect HTNV standard strains, and the results offer the dual advantages of being both qualitatively intuitive and quantitatively accurate.

[0058] Example 2: Sensitivity Detection Take the in vitro transcribed HTNV RNA and dilute it 10-fold serially with TE buffer to 10. 6 10 5 10 4 10 3 10 2 10 1 10 0 copies / μL. Detection was performed according to the method in Example 1, with a template-free negative control included. Fluorescence color was observed using a UV lamp (wavelength 302 nm or 365 nm); or fluorescence intensity was measured using a quantitative real-time PCR instrument (e.g., Roche-cobas z 480) (excitation wavelength 485 nm, emission wavelength 528 nm).

[0059] Result determination: Under UV light observation with the naked eye, the minimum resolution threshold between the positive and negative tubes is approximately 10. 3 copies / μL, that is, when the concentration is ≥10 3 At copies / μL, the presence or absence of green fluorescence can be reliably distinguished by the naked eye. Real-time fluorescence monitoring was performed using a Roche-cobas z 480 real-time PCR instrument. 1 Even at 10 copies / μL, the positive amplification curve and the negative control can still be clearly distinguished, with a detection limit of 10 copies / μL. This is comparable to conventional RT-PCR (detection limit approximately 10 copies / μL). 3 Compared to (copies / μL), the sensitivity of this invention is improved by about 100 times.

[0060] Results: When the HTNV RNA concentration was 10... 1 At a detection limit of 10 copies / μL, the fluorescence signal was still significantly higher than that of the negative control (p<0.05), and the detection limit reached 10 copies / μL. This is significantly higher than that of conventional RT-PCR (detection limit approximately 10 copies / μL). 3 Compared to (copies / μL), the sensitivity is improved by about 100 times.

[0061] Example 3: Mutation Compatibility Test Two mutant RNA fragments were synthesized: HTNV M1 fragment with a single-base mutation (G→A at position 93), the mutation site being located within the primer P1 region; and HTNV M2 fragment with multiple-base mutations (GA at position 93; CT at position 203; TC at position 303; CG at position 616), the mutation sites being located within the P1-P4 regions respectively. The fragment concentration was 10. 4 copies / μL. Detection was performed using the five primer sets of this invention and the corresponding amplification primers.

[0062] The results are as follows Figure 3 As shown in Figure A, all three reaction tubes, HTNV-WT, HTNV M1, and HTNV M2, produced strong positive fluorescence signals with no significant difference in signal intensity. This result demonstrates that even if the viral genome mutates at individual primer target sites, the multi-target parallel amplification mechanism (five sets of primers) employed in this invention can still effectively initiate amplification and trigger Cas12 detection, thereby robustly avoiding false negatives caused by single-site mutations.

[0063] Example 4: Specificity Validation of Real Clinical Samples We collected several serum samples from individuals infected with HTNV, influenza virus (H1N1), hepatitis B virus (HBV), and healthy individuals.

[0064] Serum samples from individuals infected with HTNV, influenza virus (H1N1), and hepatitis B virus (HBV) were collected separately. HTNV was detected using the method described in this patent. Fluorescence intensity (excitation wavelength 485 nm, emission wavelength 528 nm) was measured using a multi-functional microplate reader (BioTek-Synergy LX) and a quantitative PCR instrument (Roche-cobas z 480). Results: In the multi-functional microplate reader test, the reaction wells of the HTNV patient samples showed significant green fluorescence, which was more than 3 times higher than that of the control samples. Figure 3 (B) In the amplification curves recorded by the real-time PCR instrument, only the reaction wells of HTNV patient samples showed typical S-shaped positive amplification curves and were judged as positive by the instrument. Other pathogen and healthy human samples all showed negative straight lines. Figure 3 (C)

[0065] The above results indicate that the method of the present invention has high clinical specificity for HTNV, is not affected by other common pathogens (H1N1, HBV), and is suitable for accurate identification of clinical serum samples.

[0066] IV. Test Kit This invention can be assembled into a detection kit, comprising: Starting primer mixture (P1~P5, SEQ ID NO.1~5); Amplification primer mixture (A1~A5, SEQ ID NO.6~10); BstNI endonuclease, Nt.BstNBI endonuclease, Bst DNA polymerase (large fragment); LbaCas12a protein, crRNA (SEQ ID NO.11), and fluorescent reporter probe (FAM-TTTTTTTT-BHQ); Buffer A, Buffer B, and fluorescence detection buffer system; An optional integrated sample processing module (including lysis tubes, lysis buffer, and nucleic acid stabilizer) can be added. Positive control (HTNV RNA standard) and negative control (nuclease-free water).

[0067] The kit can be stored at -20°C and has a shelf life of 12 months.

[0068] The detection system and method provided by this invention do not rely on large-scale thermal cycling equipment, are easy to operate, have a short detection time (approximately 1.5 hours in total), high sensitivity (up to 10 copies / μL), and high robustness against viral mutations and RNA degradation. They can be formulated into reagent kits, suitable for rapid on-site testing scenarios such as clinical laboratories, disease control centers, field monitoring stations, and customs ports, and have promising prospects for industrial applications.

[0069] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for detecting Hantan virus based on isothermal amplification and CRISPR / Cas12 signal amplification, characterized in that, Includes the following steps: The sample to be tested was mixed with the starting primer, and the cleavage enzyme was added for incubation. The blocking modification at the 3' end of the starting primer was removed to obtain the reaction system. Add amplification primers, chain displacement polymerase and nicking enzyme to the above reaction system, and perform isothermal amplification reaction to obtain amplification products; The amplification product is mixed with the Cas protein-guide RNA complex and the signal reporter probe and incubated. The signal generated after the signal reporter probe is cleaved is detected to determine whether Hantan virus is present in the sample to be tested.

2. The method for detecting Hantan virus according to claim 1, characterized in that, The starting primers consist of at least two sets, each targeting a different conserved region of the Hantan virus genome; the amplification primers consist of at least two sets, each corresponding to a set of starting primers.

3. The method for detecting Hantan virus according to claim 1, characterized in that, The blocking modification is a phosphorylation modification; the cleaving enzyme is a restriction endonuclease.

4. The method for detecting Hantan virus according to claim 1, characterized in that, The nicking enzyme is a nicking restriction endonuclease; the strand displacement polymerase is a large fragment of Bst DNA polymerase.

5. The method for detecting Hantan virus according to claim 1, characterized in that, The isothermal amplification reaction temperature is 52℃~62℃, and the amplification time is 30 min~90 min.

6. The method for detecting Hantan virus according to claim 1, characterized in that, When the sample to be tested is rodent tissue, environmental swab, or fecal sample, an integrated sample processing module is used to lyse and release nucleic acids before the amplification step, and the resulting lysate is directly added to the amplification reaction system as a template.

7. The Hantan virus detection system based on isothermal amplification and CRISPR / Cas12 signal amplification as described in any one of claims 1 to 6, characterized in that, include: The starting primer has a blocking modification at its 3′ end, and the starting primer does not have extension activity before the blocking modification is removed; Amplification primers comprising a region complementary to a portion of the sequence of the starting primer and an activation sequence; A cleavage enzyme is used to remove the blocking modification at the 3′ end of the starting primer; Chain displacement polymerase and nickase are used to amplify nucleic acids under isothermal conditions to produce amplification products containing the activated sequence. Cas protein and its guide RNA are used to specifically recognize the activation sequence in the amplification product and cleave the signal reporter probe; The signal reporting probe generates a detectable signal after being cut.

8. The Hantan virus detection system according to claim 7, characterized in that, The starting primers consist of at least two sets, each targeting a different conserved region of the Hantan virus genome; the amplification primers consist of at least two sets, each corresponding to a set of starting primers.

9. The Hantan virus detection system according to claim 7, characterized in that, The blocking modification is phosphorylation modification; the cleavage enzyme is BstNI endonuclease; the cleavage enzyme is Nt.BstNBI endonuclease; the strand displacement polymerase is a large fragment of BstDNA polymerase; the Cas protein is Cas12 protein; and the signal reporter probe is an oligonucleotide probe labeled with a fluorescent group and a quencher group.

10. A Hantan virus detection kit based on isothermal amplification and CRISPR / Cas12 signal amplification, characterized in that, include: The starting primer has a blocking modification at its 3' end; Amplification primers comprising a region complementary to a portion of the sequence of the starting primer and an activation sequence; A cleavage enzyme is used to remove the blocking modification at the 3' end of the starting primer; Chain displacement polymerase and nickase are used to amplify nucleic acids under isothermal conditions; Cas protein and its guide RNA are used to specifically recognize activation sequences in amplification products and cleave signal reporter probes; The signal reporting probe generates a detectable signal after being cut.

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