A method for detecting norovirus based on ultrafast reverse transcription-free exponential amplification reaction (RTF-EXPAR)

Abstract

The application discloses a method for detecting norovirus based on super-fast reverse transcription-free exponential amplification reaction (RTF-EXPAR), belongs to the technical field of norovirus detection, and relates to application of the super-fast reverse transcription-free exponential amplification reaction for detecting norovirus, and comprises norovirus RNA fragments and binder DNA (Binder DNA). The detection steps comprise sample providing, reaction system construction, isothermal amplification reaction and product detection. The application provides a primer reagent combination for detecting norovirus based on RTF-EXPAR, solves the technical problem that there is no technology for detecting norovirus in the prior art, realizes rapid detection from a sample to a result and guarantees effectiveness, has high specificity and low detection limit in the aspect of detection, needs a short time for amplification reaction, and the reaction result can be observed by naked eyes. The detection method provided by the application can skip the step of "reverse transcription of RNA into cDNA", directly utilizes DNA-RNA heterologous double-strand cutting to start the reaction, simplifies a process and shortens time.

Description

Technical Field

[0001] This invention relates to the field of norovirus detection technology, specifically to a method for detecting norovirus based on ultra-rapid reverse transcription exponential amplification reaction (RTF-EXPAR). Background Technology

[0002] Norovirus (NoV) is one of the leading pathogens causing acute nonbacterial gastroenteritis in humans. It is characterized by extremely low infectious doses, diverse transmission routes, and widespread susceptibility, playing a dominant role in diarrhea outbreaks globally. Belonging to the Caliciviridae family, it is a non-enveloped, single-stranded, positive-sense RNA virus with a relatively simple genome structure but a rapid mutation rate. It exhibits strong resistance to the environment, being acid- and heat-resistant and insensitive to common disinfectants such as alcohol. Based on the homology of the VP1 gene sequence encoding the major capsid protein, norovirus can be divided into several genotypes. GI and GII are the two main genotypes that infect humans, while other genotypes primarily infect animals and rarely humans. Among the many genotypes, GI.1 was the first prototype strain discovered and identified, providing important reference for basic research on norovirus. GII.4, on the other hand, has been the most widespread and prevalent genotype globally in recent decades, causing the most outbreaks. It is characterized by strong transmissibility, high antigenic variation, and high susceptibility in the population. It is listed by the World Health Organization as one of the foodborne viruses requiring close monitoring.

[0003] Norovirus is highly contagious; it can infect humans even at very low viral loads, and its transmission efficiency is significantly higher than other enteroviruses. The main sources of infection are patients in the acute phase, asymptomatic carriers, and those shedding the virus during the recovery period. Even after clinical symptoms subside, viral shedding can continue for weeks, further increasing the risk of transmission. The primary transmission route is the fecal-oral route, but it can also be transmitted through aerosols produced by vomiting over short distances, and indirectly through contaminated water, food, everyday items, and environmental surfaces. Consuming raw or undercooked shellfish often leads to outbreaks. The general population is susceptible to norovirus, and long-lasting cross-immunity is difficult to establish after infection, leading to repeated infections. The disease exhibits a clear seasonality, with winter and spring being peak seasons. It easily triggers outbreaks and even epidemics in densely populated and relatively enclosed spaces. Furthermore, it can survive in the environment for extended periods, posing a continuous threat to the safety and control of food processing, catering services, and port quarantine.

[0004] While existing norovirus detection technologies are systematic, their limitations in sensitivity, versatility, timeliness, and practicality hinder effective control. From a technical perspective, the core challenge lies in the fact that extremely low infectious doses of the virus become the primary source of error. Among detection technologies, real-time quantitative PCR (RT-qPCR) boasts excellent sensitivity and specificity, but it heavily relies on specialized laboratories, sophisticated instruments, and skilled operators, with testing times reaching 2-4 hours, failing to meet the immediate decision-making needs of epidemic sites. Rapid antigen detection methods such as immunochromatographic test strips typically have 1-3 orders of magnitude lower sensitivity than RT-qPCR, easily resulting in missed detections in early infection stages or at low viral loads, and their antibodies are susceptible to rapid viral mutations, leading to detection escape. While emerging CRISPR-Cas technology has made progress in speed and ease of operation, its resistance to interference in complex real-world samples, stability, and commercial maturity remain insufficient.

[0005] Furthermore, norovirus has low viral load and long survival time in environmental and food samples, which further increases the difficulty of accurate quantification and efficient screening. In addition, the industry standardization system is not yet perfect, and the results of different methods are not comparable enough, all of which restrict the promotion and application of norovirus detection towards accuracy, speed and large scale.

[0006] In summary, existing norovirus detection technologies fall short in terms of genotyping specificity, detection speed, ease of operation, and field applicability, failing to effectively meet the practical needs of aquatic product safety supervision, port quarantine, grassroots epidemic monitoring, and rapid tracing of sudden outbreaks. To accurately control foodborne transmission and outbreaks of norovirus, strengthen the virus monitoring system for key foods such as shellfish, and improve on-site real-time and routine grassroots testing capabilities, there is an urgent need to develop a new virus detection technology that integrates ultra-fast, independent reverse transcription-free, highly specific, and easy-to-operate features. This technology would provide crucial technical support for the early detection and treatment of norovirus and the protection of public health safety. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting norovirus based on ultra-rapid reverse transcription exponential amplification reaction (RTF-EXPAR), addressing the problems of complex operation and long processing time in existing norovirus type I (NoV. GI) and type II (GI) detection techniques mentioned in the background. This invention innovatively develops an ultra-rapid detection technology based on RTF-EXPAR. It breaks through by employing a one-step isothermal amplification system (50℃) and completely avoids the reverse transcription step required by traditional RT-PCR, achieving rapid detection from sample to result while ensuring validity.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR), comprising primer nucleotide sequences such as norovirus RNA fragments, binder DNA, trigger X, and template DNA (Trigger X'-X'), and the method for detecting norovirus includes the following steps: S1. First, sample provision is required: Provide the sample to be tested. The sample can be from the environment, food or clinical excrement. For environmental or food samples, routine pretreatment is required to enrich and extract viral RNA.

[0009] S2. Next, the reaction system needs to be constructed: the RTF-EXPAR reaction system needs to be constructed. This system is the core of the invention, and its key lies in containing a specially designed combination of primers and functional proteins. Specifically, the system comprises: norovirus target RNA specific binder DNA (Binder DNA): this DNA molecule is designed with a region completely complementary to the target RNA sequence and a specific sequence recognized by a nicking enzyme; template DNA (Template X'-X'): ​​this template comprises two parts, one is a replication region consistent with the trigger X sequence, and the other is a template region complementary to the trigger X; strand displacement DNA polymerase (e.g., Bst 2.0 DNA polymerase): used to extend the primer and displace the downstream DNA strand during polymerization; nicking enzyme (e.g., Nt.BstNBI): capable of cleaving only one strand after recognizing a specific sequence on double-stranded DNA, creating a nick to provide an extension starting point for the polymerase; dNTPs (including dATP, dCTP, dGTP, dTTP): serving as raw materials for the amplification reaction; reaction buffer, providing a suitable pH and ionic environment; and also includes single-stranded DNA binding protein (SSB) and bovine serum albumin (BSA) to stabilize the reaction system and improve amplification efficiency.

[0010] S3. Then, an isothermal amplification reaction is required: Add the RNA solution of the sample to be tested prepared in step S1 to the RTF-EXPAR reaction system in step S2, mix well, and place it in a constant temperature device (such as a water bath or a constant temperature metal bath) to carry out the isothermal amplification reaction for a certain period of time at a constant temperature.

[0011] The reaction principle is briefly described as follows: When the target norovirus RNA is present in the system, the binder DNA hybridizes with it to form a DNA-RNA heteroduplex. Under the action of nicking enzymes and polymerases, the binder DNA is recognized and nicked, releasing a short single-stranded DNA segment, namely Trigger X, through a strand displacement reaction. Trigger X then binds to an excess of Template X'-X', initiating the exponential amplification cycle of EXPAR, which generates a large amount of newly synthesized labeled DNA in a short time.

[0012] S4. Finally, product detection is required: The amplification products from step S3 are detected, and the results can be interpreted using one of the following two methods based on the signal type: Fluorescence method: Fluorescently labeled bases (such as fluorescein-labeled dATP) are added to the dNTPs in the reaction system. The amplification products will be incorporated into the fluorescent groups. The growth curve of the fluorescence signal is recorded using a portable fluorescence detector. A typical "S"-shaped amplification curve indicates a positive result.

[0013] Nucleic acid test strip method (LFD): Antigen molecules (such as FITC or Digoxin) are pre-labeled on the template DNA of the reaction system. After amplification, the product is diluted with pure water, and a certain amount is added to the sample pad of the nucleic acid test strip. Through chromatography, the results can be observed with the naked eye within a certain period of time to determine whether the control line and the test line (T line) are colored, thereby judging negative and positive results.

[0014] Preferably, the binder DNA includes a sequence region that specifically hybridizes with norovirus target RNA and a sequence region that can be recognized and cleaved by a nicking enzyme, and the template DNA (Template X'-X') includes a sequence region complementary to the trigger X released from the binder DNA and its own complementary strand template region.

[0015] Preferably, the reaction system further includes single-stranded DNA binding protein (SSB) and bovine serum albumin (BSA), the constant temperature is 45℃ to 55℃, preferably 50℃, and the isothermal amplification reaction time is 20 to 40 minutes, preferably 30 minutes.

[0016] Preferably, the amplification product is diluted 15-25 times with pure water, and 60-100 μL is added to the sample application area of ​​the test strip. The color development result is observed within 15 minutes. The judgment criteria are: only the C line is colored, which is negative; and the simultaneous color development of the C line and the T line is positive.

[0017] Preferably, the norovirus is type GI or type GII norovirus, and the applications include: monitoring norovirus in environmental water sources, screening norovirus during food processing, or quarantine of norovirus in import and export goods at ports.

[0018] Preferably, the application primer reagent composition is used in any of the following.

[0019] A1. Application in the preparation of products for the detection or auxiliary detection of norovirus.

[0020] A2. Application in the preparation of products for testing whether samples contain norovirus.

[0021] A3. Application in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by norovirus.

[0022] A4. Application in the preparation of products for screening diseases caused by norovirus.

[0023] Preferably, the detection process uses a triple nucleic acid test strip (lateral flow dipstick, LFD), which is an endpoint detection technology for visually observing amplification products. It uses a chromatographic double antibody sandwich method to rapidly detect nucleic acid amplification products and forms a "fluorescein antibody label" at the detection line position.

[0024] Preferably, the norovirus reagent combination comprises: Part A: 1.50 μL water, 2.50 μL 10-fold isothermal amplification buffer, 3.75 μL BSA solution, 2.00 μL LBst 2.0 DNA polymerase (1.6 U / μL), 0.75 μL Nt.BstNBI (10 U / μL).

[0025] Part B: 6.30 μL water, 5.00 μL 10-fold isothermal amplification buffer, 1.00 μL Template X'-X' (1 μM), 2.40 μL MgSO4 (100 mM), 1.00 μL dNTPs (10 nM, dATP, dCTP, dGTP, dTTP), 0.5 μL dsGreen (1:5 dilution in DMSO, from 100-fold to 20-fold), then 0.30 μL SSB solution.

[0026] Part C: 1 μL BstNI (10 U / μL), 2 μL Binder DNA X (1 μL), then take 3 μL viral RNA (1.2 nmol) or a negative sample. Incubate in a PCR instrument at 50°C for 5 min; Addition steps: Add part B (16.5 μL) to the PCR tube, then add part C (6 μL), then add part a (10.5 μL), and then seal the tube.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The detection method provided by this invention can skip the "RNA reverse transcription to cDNA" step and directly use DNA-RNA heteroduplex cleavage to initiate the reaction, simplifying the process and shortening the time.

[0028] Compared with existing norovirus detection technologies, the detection method provided by this invention is simple and rapid to operate, requiring no expensive thermal cycling detection equipment. The method completes the test in just 30 minutes, and the results are visible on the test strip, observable with the naked eye. It is suitable for emergency and on-site testing needs due to its short testing time. Relying on the exponential amplification of EXPAR, the detection sensitivity is extremely high, capable of capturing trace amounts of viral RNA. This makes it suitable for the rapid and accurate detection of RNA viruses like norovirus, and has potential application value in norovirus screening and diagnosis. It also provides insights for the simplification and upgrading of nucleic acid detection technology. Attached Figure Description

[0029] Figure 1 Primer sequence design table for detecting norovirus type I (NoV.GⅠ) and type II (GⅡ) using the RTF-EXPAR system of this invention; Figure 2 The effectiveness of the RTF-EXPAR system of this invention in detecting norovirus type I (NoV. GI) and type II (GII) as a best primer selection and blank control is demonstrated. Figure 3 The diagram shows the specificity of the RTF-EXPAR system of this invention in detecting norovirus type I (NoV. GI) and type II (GII). Figure 4 The graph shows the sensitivity of the RTF-EXPAR system of the present invention in detecting different concentrations of Template X'-X' for norovirus type I (NoV. GI) and type II (GII); Figure 5 The image shows the effect of the RTF-EXPAR system of the present invention in detecting different concentrations of viral RNA of norovirus type I (NoV.GⅠ) and type II (GⅡ). Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 are within the scope of protection of the present invention.

[0031] Example 1: Please see Figure 1-5 This embodiment provides primer sequence design and detection method for detecting norovirus type I (NoV.GI) and type II (GII) using the RTF-EXPAR system, including primer design for norovirus type I (NoV.GI) and type II (GII) viral RNA fragments, three different binder DNAs, trigger X, three different template DNAs (TemplateX'-X'), and specifically fluorescently labeled dATP bases.

[0032] The method for detecting norovirus using the RTF-EXPAR system includes the following steps: First, prepare three solutions, totaling 33 μL: A, B, and C. Mix the three solutions in the given reagent order. Before use, place the frozen norovirus RNA sample on ice to thaw slowly. Once thawed, immediately remove the required amount of norovirus RNA. Then, refreeze the remaining sample at -80 °C. Thaw other biological reagents slowly on ice.

[0033] Part A consists of: 1.50 μL water, 2.50 μL 10-fold isothermal amplification buffer, 3.75 μL BSA solution, 2.00 μL Bst 2.0 DNA polymerase (1.6 U / μL), and 0.75 μL Nt.BstNBI (10 U / μL).

[0034] Part B: 6.30 μL water, 5.00 μL 10-fold isothermal amplification buffer, 1.00 μL Template X'-X' (1 μM), 2.40 μL MgSO4 (100 mM), 1.00 μL dNTPs (10 nM, dATP, dCTP, dGTP, dTTP), 0.5 μL dsGreen (1:5 diluted in DMSO, from 100-fold to 20-fold), then 0.30 μL SSB solution; Part C: 1.00 μL BstNI (10 U / μL), 2.00 μL Binder DNA X (1 μL), then take 3.00 μL viral RNA (1.2 nmol) or a negative sample, and incubate in a PCR instrument at 50°C for 5 min.

[0035] Addition steps: Add part B (16.5 μL) to the PCR tube, then add part C (6 μL), and finally add part a (10.5 μL). Seal the tube and mix well.

[0036] Amplification steps: Amplify at a constant temperature of 50℃ for 30 minutes in a PCR instrument.

[0037] Detection procedure: Dilute the amplification product 20 times with pure water, then add 80 μL to the sample application area of ​​the test strip and observe the color development within 15 minutes; the criteria for judging the detection result are as follows: When the control line (C line) of the test strip develops color but the test line does not, the result is considered negative. When both the control line (C line) and the test line (T line) of the test strip show color simultaneously, the result is considered positive.

[0038] Example 2: This example demonstrates the impact of different Binder DNA X and Template X'-X' sequences on the detection results.

[0039] Using the reaction system and detection method described in Example 1, primers for norovirus type I (NoV. GI) and type II (GII) were used as templates, including Binder DNA X1, Template X'-X'1, Binder DNA X2, Template X'-X'2, and Binder DNA X3, Template X'-X'3, with water as a negative control. The mixture was incubated at 50°C for 30 minutes for detection. Results are as follows: Figure 2As shown, the Norovirus 1 (NoV. GI) test strips contain, in order: Binder DNA X1, Template X'-X'1; Binder DNA X2, Template X'-X'2; Binder DNA X3, Template X'-X'3, and a negative control. No T line indicates a negative result. This shows that the optimal primers for the Norovirus 1 detection system are the first primer pair, and the absence of a T line in the negative control indicates that the system itself does not exhibit an amplification reaction. Similarly, the Norovirus 2 (NoV. GI) test strips contain, in order: Binder DNA X1, Template X'-X'1; Binder DNA X2, Template X'-X'2; Binder DNA X3, Template X'-X'3, and a negative control. No T line indicates a negative result. This shows that the optimal primers for the Norovirus 2 detection system are the first primer pair, and the absence of a result in the negative control indicates that the system itself does not exhibit an amplification reaction.

[0040] Example 3: This embodiment demonstrates the effectiveness and specificity of the RTF-EXPAR-based norovirus detection system in detecting different RNA viruses.

[0041] Using the reaction system and detection method described in Example 1, in Part C, norovirus RNA and two other RNA viruses were used as detection targets for incubation reactions. Figure 3 As shown, the results are as follows: Norovirus GⅠ RNA and other RNA viruses, in order; Norovirus GⅡ RNA and other RNA viruses, in order.

[0042] according to Figure 3 As shown, only norovirus RNA yielded a positive result, while the other viruses yielded negative results. This demonstrates the specific recognition step of this detection system—that norovirus RNA can only bind to the designed Binder DNA X when the target virus is present in the sample. The detection method and primer reagent set established in this invention only show a positive result when norovirus RNA is used as a template, ensuring the accuracy of the detection and avoiding non-specific reactions.

[0043] Example 4: This embodiment demonstrates the effect of different concentrations of Template X'-X' on the detection results of the RTF-EXPAR detection system.

[0044] Using the reaction system and detection method described in Example 1, templates X'-X' of norovirus type I B were added to the system in the following gradient volumes: 2 μl, 1 μl, 0.9 μl, 0.8 μl, 0.7 μl, and 0.6 μl, respectively. Water was added to each volume to make up the total volume of 33 μL. An isothermal amplification reaction was then performed, followed by detection experiments. Similarly, templates X'-X' of norovirus type II B were added to the system in the following gradient volumes: 3 μl, 2 μl, 1 μl, 0.9 μl, 0.8 μl, and 0.7 μl, respectively. Water was added to each volume to make up the total volume of 33 μL. An isothermal amplification reaction was then performed, followed by detection experiments. The results are as follows Figure 4 The results are shown below. For norovirus type I, the Template X'-X' gradient volumes are 2 μl, 1 μl, 0.9 μl, 0.8 μl, 0.7 μl, and 0.6 μl, respectively. A Template X'-X' volume of 0.7 μl still indicates a positive result. For norovirus type II, the Template X'-X' gradient volumes are 3 μl, 2 μl, 1 μl, 0.9 μl, 0.8 μl, and 0.7 μl, respectively. A Template X'-X' volume of 0.7 μl in the detection system also indicates a positive result.

[0045] according to Figure 4 As shown, this detection system can still bind and initiate amplification detection even when the content of Template X'-X' is very low.

[0046] Example 5: This embodiment demonstrates the effect of different concentrations of norovirus RNA on the detection results in the RTF-EXPAR detection system.

[0047] Using the reaction system and detection method described in Example 1, RNA from part C (1.2 nmol) of the norovirus type I detection system was added to the system at its original concentration and at dilutions of 2, 10, 50, 100, 200, and 300 times, with each dilution of norovirus type I RNA serving as a template. RTF-EXPAR amplification was then performed, and detection limit experiments were conducted. The intensity of the T line on each test strip was then compared. Similarly, RNA from part C (1.2 nmol) of the norovirus type II detection system was added to the system at its original concentration and at dilutions of 10, 50, 100, 200, 300, and 500 times, with each dilution of norovirus type II RNA serving as a template. RTF-EXPAR amplification was then performed, and detection limit experiments were conducted. The intensity of the T line on each test strip was then compared. The detection results are as follows: Figure 5The results shown are, in order, the detection results of norovirus type I RNA at the original concentration and diluted 2, 10, 50, 100, 200, and 300 times; and the detection results of norovirus type II RNA at the original concentration and diluted 10, 50, 100, 200, 300, and 500 times. according to Figure 5 As a result, the norovirus RNA load remained positive even at extremely low levels. Therefore, the detection method established in this invention demonstrates that this test can still detect viruses at extremely low concentrations.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting norovirus based on ultra-rapid reverse transcription exponential amplification reaction (RTF-EXPAR), characterized in that, The primer nucleotide sequences, including norovirus RNA fragments, binder DNA, trigger X, and template DNA (Trigger X'-X'), are used to detect norovirus. The method includes the following steps: S1. First, sample provision is required: provide the sample to be tested. The sample can be from the environment, food or clinical excrement. For environmental or food samples, routine pretreatment is required to enrich and extract viral RNA. S2. Next, the reaction system needs to be constructed: the RTF-EXPAR reaction system needs to be constructed. This system is the core of the invention, and its key lies in containing a specially designed combination of primers and functional proteins. Specifically, the system comprises: norovirus target RNA specific binder DNA (Binder DNA): this DNA molecule is designed with a region completely complementary to the target RNA sequence and a specific sequence recognized by a nicking enzyme; template DNA (Template X'-X'): ​​this template comprises two parts, one is a replication region consistent with the trigger X sequence, and the other is a template region complementary to the trigger X; strand displacement DNA polymerase (e.g., Bst 2.0 DNA polymerase): used to extend the primer and displace the downstream DNA strand during polymerization; nicking enzyme (e.g., Nt.BstNBI): capable of cleaving only one strand after recognizing a specific sequence on double-stranded DNA, creating a nick to provide an extension starting point for the polymerase; dNTPs (including dATP, dCTP, dGTP, dTTP): serving as raw materials for the amplification reaction; reaction buffer, providing a suitable pH and ionic environment; and also includes single-stranded DNA binding protein (SSB) and bovine serum albumin (BSA) to stabilize the reaction system and improve amplification efficiency; S3. Then, an isothermal amplification reaction needs to be performed: Add the RNA solution of the sample to be tested prepared in step S1 to the RTF-EXPAR reaction system in step S2, mix well, and place it in a constant temperature device (such as a water bath or a constant temperature metal bath) to carry out the isothermal amplification reaction at a constant temperature for a certain period of time. The reaction principle is briefly described as follows: When the target norovirus RNA is present in the system, the Binder DNA hybridizes with it to form a DNA-RNA heteroduplex. Under the action of nicking enzymes and polymerases, the Binder DNA is recognized and nicked, and then a short single-stranded DNA, namely Trigger X, is released through a strand displacement reaction. Trigger X then binds to an excess of TemplateX'-X', initiating the exponential amplification cycle of EXPAR, which can generate a large amount of newly synthesized labeled DNA in a short time. S4. Finally, product detection is required: The amplification products from step S3 are detected, and the results can be interpreted using one of the following two methods based on the signal type: Fluorescence method: Fluorescently labeled bases (such as fluorescein-labeled dATP) are added to the dNTPs in the reaction system. The amplification products will be incorporated with fluorescent groups. The growth curve of the fluorescence signal is recorded using a portable fluorescence detector. A typical "S"-shaped amplification curve indicates a positive result. Nucleic acid test strip method (LFD): Antigen molecules (such as FITC or Digoxin) are pre-labeled on the template DNA of the reaction system. After amplification, the product is diluted with pure water, and a certain amount is added to the sample pad of the nucleic acid test strip. Through chromatography, the results can be observed with the naked eye within a certain period of time to determine whether the control line and the test line (T line) are colored, thereby judging negative and positive results.

2. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The binder DNA contains a sequence region that specifically hybridizes with norovirus target RNA, and a sequence region that can be recognized and cleaved by nicking enzymes. The template DNA (Template X'-X') contains a sequence region complementary to the trigger X released from the binder DNA, and its own complementary strand template region.

3. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The reaction system also includes single-stranded DNA binding protein (SSB) and bovine serum albumin (BSA), the constant temperature is 45℃~55℃, preferably 50℃; the isothermal amplification reaction time is 20~40 minutes, preferably 30 minutes.

4. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The amplification product is diluted 15-25 times with pure water, and 60-100 μL is added to the sample application area of ​​the test strip. The color development result is observed within 15 minutes. The judgment criteria are: only the C line is colored, which is negative; the simultaneous color development of the C line and the T line is positive.

5. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The norovirus is type GI or type GII norovirus, and the applications include: monitoring norovirus in environmental water sources, screening norovirus during food processing, or quarantine of norovirus in import and export goods at ports.

6. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The application of the primer reagent composition is carried out in any of the following applications: A1. Application in the preparation of products for the detection or auxiliary detection of norovirus; A2. Application in the preparation of products for testing whether samples contain norovirus; A3. Application in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by norovirus; A4. Application in the preparation of products for screening diseases caused by norovirus.

7. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The detection process uses a triple nucleic acid test strip (lateral flow dipstick, LFD), which is an endpoint detection technology for visually observing amplification products. It uses a chromatographic double antibody sandwich method to rapidly detect nucleic acid amplification products, forming a "fluorescein antibody label" at the detection line position.

8. The method for detecting norovirus based on ultra-fast reverse transcription exponential amplification reaction (RTF-EXPAR) according to claim 1, characterized in that: The norovirus reagent combination includes: Part A: 1.50 μL water, 2.50 μL 10-fold isothermal amplification buffer, 3.75 μL BSA solution, 2.00 μL Bst2.0 DNA polymerase (1.6 U / μL), 0.75 μL Nt.BstNBI (10 U / μL); Part B: 6.30 μL water, 5.00 μL 10-fold isothermal amplification buffer, 1.00 μL Template X'-X' (1 μM), 2.40 μL MgSO4 (100 mM), 1.00 μL dNTPs (10 nM, dATP, dCTP, dGTP, dTTP), 0.5 μL dsGreen (1:5 diluted in DMSO, from 100-fold to 20-fold), then 0.30 μL SSB solution; Part C: 1 μL BstNI (10 U / μL), 2 μL Binder DNA X (1 μL), then take 3 μL viral RNA (1.2 nmol) or a negative sample. Incubate in a PCR instrument at 50°C for 5 min; Addition steps: Add part B (16.5 μL) to the PCR tube, then add part C (6 μL), then add part a (10.5 μL), and then seal the tube.

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