Primer probe composition for detecting GI genotype and / or GII genotype norovirus and application

Abstract

The invention discloses a primer probe composition for detecting GI genotype and / or GII genotype norovirus and application of the primer probe composition. The primer probe composition comprises a first primer pair and a first probe aiming at the GI genotype norovirus, and a second primer pair and a second probe aiming at the GI genotype norovirus, wherein the first primer pair comprises a first primer of which the sequence is as shown in SEQ ID NO.1 and a second primer of which the sequence is as shown in SEQ ID NO.2, and the sequence of the first probe is as shown in SEQ ID NO.3; the second primer pair and the second probe aim at GII genotype norovirus; wherein the second primer pair comprises a third primer with a sequence as shown in SEQ ID NO.4 and a fourth primer with a sequence as shown in SEQ ID NO.5, and the sequence of the second probe is as shown in SEQ ID NO.6. The kit provided by the invention can specifically detect GI and GII genotype noroviruses, is simple to operate, and can realize on-site rapid distinguishing and detection of different genotypes of noroviruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus molecular detection, and specifically relates to a primer-probe combination and application thereof for detecting GI genotype and / or GII genotype Norovirus. Background Art

[0002] Norovirus, also known as Norwalk virus, is the prototype representative strain of the Norovirus genus in the human Caliciviridae family. Norovirus has many common characteristics: a diameter of about 26 to 35 nm, no envelope, a rough surface, a spherical shape, icosahedral symmetry, and a single-stranded positive-strand RNA genome.

[0003] Norovirus is genetically subdivided into six established genomes, denoted GI to GVI, with more than 30 different genotypes. Among them, GI genotype and GII genotype Norovirus are the most common strains associated with the epidemiology of acute gastroenteritis cases. Norovirus can easily contaminate food and beverages because the virus is very small in diameter and ingestion of less than 100 will make people sick. Worldwide, norovirus causes more than 50% of non-bacterial diarrhea. Vomiting and diarrhea are the most common symptoms of infection with norovirus. Some infected people also have fever, and severe cases will have symptoms such as dehydration. However, norovirus cannot grow and reproduce on artificial culture media. Therefore, it is crucial to establish an effective and accurate method for detecting norovirus.

[0004] Clinical diagnosis of norovirus is mainly based on epidemiological data, clinical manifestations and routine laboratory test results. These diagnostic methods are not only cumbersome to operate, but can only provide a preliminary diagnosis. To truly achieve a definitive diagnosis, a secondary test is required. Summary of the invention

[0005] The main purpose of the present invention is to provide a primer-probe combination and application for detecting GI genotype and / or GII genotype Norovirus, so as to overcome the shortcomings of the prior art that the GI and GII genotypes cannot be effectively distinguished and on-site visual screening cannot be achieved.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0007] The embodiment of the present invention provides a primer-probe combination for detecting GI genotype and / or GII genotype Norovirus, comprising:

[0008] A first primer pair and a first probe for GI genotype Norovirus; wherein the first primer pair comprises a first primer having a sequence as shown in SEQ ID NO.1 and a second primer having a sequence as shown in SEQ ID NO.2, and the sequence of the first probe is shown in SEQ ID NO.3;

[0009] A second primer pair and a second probe for GII genotype Norovirus; wherein the second primer pair includes a third primer whose sequence is shown in SEQ ID NO.4 and a fourth primer whose sequence is shown in SEQ ID NO.5, and the sequence of the second probe is shown in SEQ ID NO.6.

[0010] The embodiment of the present invention also provides a lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus, comprising a sample pad, a gold label pad, an NC membrane, a water absorbent pad and a bottom plate, wherein the NC membrane is provided with a detection line T1, a detection line T2 and a quality control line C;

[0011] Wherein, the detection line T1 is fixed with digoxin antibody; the detection line T2 is fixed with streptavidin; and the quality control line C is fixed with anti-mouse secondary antibody.

[0012] The embodiments of the present invention also provide the use of the aforementioned primer-probe combination for detecting GI genotype and / or GII genotype Norovirus or the lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus in the preparation of a product for visually detecting GI genotype and / or GII genotype Norovirus.

[0013] An embodiment of the present invention also provides a kit for visually detecting GI genotype and / or GII genotype Norovirus, which includes: the aforementioned primer-probe combination for detecting GI genotype and / or GII genotype Norovirus, and a lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention designs corresponding specific primers and probes for the GI genotype and GII genotype Norovirus gene regions, which can detect GI and GII genotype Noroviruses, and has no cross-reaction with other viruses that have homology with the Norovirus nucleic acid sequence and are likely to cause the same or similar clinical symptoms, such as Rabbit hemorrhagic disease virus, Sapporo virus, Swine vesicular exanthem virus, etc., and the detection effect has good specificity;

[0016] (2) The present invention combines PCR technology with lateral flow technology, which can exponentially amplify the target gene fragment, making the detection effect ultra-sensitive and the detection result intuitive and easy to judge; the present invention designs primer pairs and probes for GI and GII genotypes respectively, realizes the specific amplification and visual identification of Norovirus GI and GII, and achieves the purpose of on-site screening of Norovirus GI and GII genotypes; the detection results of the present invention are relatively reliable, and the interference of complex samples on the experimental results can be eliminated. The detection results are not affected by PCR inhibitors, and the false negative or false positive of PCR caused by sample complexity can be avoided; BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 It is a schematic diagram of a process for visually detecting GI genotype and / or GII genotype Norovirus in a typical embodiment of the present invention;

[0019] Figure 2 This is a result diagram of visual detection of GI and GII genotypes using the designed GI and GII genotype primer and probe combination in Example 4 of the present invention;

[0020] Figure 3 This is a normalized quantitative analysis result diagram of the grayscale value of the T line detected on the visualized lateral flow test strip in Example 5 of the present invention. DETAILED DESCRIPTION

[0021] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Specifically, as one aspect of the technical solution of the present invention, a primer-probe composition for detecting GI genotype and / or GII genotype Norovirus comprises:

[0023] A first primer pair and a first probe for GI genotype Norovirus; wherein the first primer pair comprises a first primer having a sequence as shown in SEQ ID NO.1 and a second primer having a sequence as shown in SEQ ID NO.2, and the sequence of the first probe is shown in SEQ ID NO.3;

[0024] A second primer pair and a second probe for GII genotype Norovirus; wherein the second primer pair includes a third primer whose sequence is shown in SEQ ID NO.4 and a fourth primer whose sequence is shown in SEQ ID NO.5, and the sequence of the second probe is shown in SEQ ID NO.6.

[0025] In some preferred embodiments, the sequences in the primer-probe combination are shown in Table 1:

[0026] Table 1 Sequences in the primer-probe combination of the present invention

[0027]

[0028]

[0029] Note: Y, R, and W are composite bases, R = A or G, W = A or T, and Y = C or T;

[0030] In some preferred embodiments, the 5' end of the first probe is modified with a fluorescein isothiocyanate group, and the 3' end of the first probe is modified with a biotin group.

[0031] In some preferred embodiments, the 5' end of the second probe is modified with a digoxigenin group, and the 3' end of the first probe is modified with a fluorescein isothiocyanate group.

[0032] In some preferred embodiments, the 5' end and 3' end of the primer of the primer probe composition are not modified.

[0033] Another aspect of the embodiments of the present invention further provides a lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus, comprising a sample pad, a gold label pad, an NC membrane, an absorbent pad and a bottom plate, wherein the NC membrane is provided with a detection line T1, a detection line T2 and a quality control line C;

[0034] Wherein, the detection line T1 is fixed with digoxin antibody; the detection line T2 is fixed with streptavidin; and the quality control line C is fixed with anti-mouse secondary antibody.

[0035] The lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus in the present invention is different from the traditional molecular test strip in identifying and detecting the amplified product. The present invention causes hydrolysis of the functionalized probe during the amplification process for different GI and GII genotypes, and does not cause the appearance of a signal on the detection T line. At the same time, based on the detection principle of probe recognition, false positive results caused by primer dimers and nonspecific amplification during the PCR amplification process are effectively avoided.

[0036] Another aspect of an embodiment of the present invention also provides the use of the aforementioned primer-probe composition for detecting GI genotype and / or GII genotype Norovirus or the aforementioned lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus in the preparation of a product for visually detecting GI genotype and / or GII genotype Norovirus.

[0037] In some preferred embodiments, the method for detecting GI genotype and / or GII genotype Norovirus using the product comprises:

[0038] Extracting RNA from the sample to be tested;

[0039] The RNA is amplified by PCR using the aforementioned primer-probe combination for detecting GI genotype and / or GII genotype Norovirus, and the amplified product is then analyzed using a lateral flow test strip to detect the detection result, thereby achieving visual detection of the sample to be tested.

[0040] In some preferred embodiments, the method further comprises: first reverse transcribing the extracted RNA to obtain cDNA; preferably, AMV reverse transcriptase is used for the reverse transcription.

[0041] In some preferred embodiments, the working concentration of the primers in the primer-probe composition is 5-20 μM.

[0042] In some preferred embodiments, the working concentration of the probe in the primer-probe composition is 0.05-0.2 μM.

[0043] In some preferred embodiments, the reaction procedure for PCR amplification includes: pre-denaturation at 95°C for 3-5 min, denaturation at 94°C for 20-30 s, annealing at 55°C for 20-30 s, extension at 72°C for 20-30 s, and extension at 72°C for 5-8 min; preferably, the number of cycles of the denaturation, annealing and extension process is 35-40.

[0044] In some preferred embodiments, when amplification is performed using a negative control sample, the detection zone of the lateral flow test strip exhibits a visible signal.

[0045] In some preferred embodiments, when a positive control sample is used for amplification, the probe is hydrolyzed during the amplification process, and the detection area of ​​the lateral flow test strip will not show a visible signal, thereby avoiding false positive results caused by dimers and nonspecific amplification during the amplification process.

[0046] In some more specific embodiments, the method of using the product to detect GI genotype and / or GII genotype Norovirus comprises:

[0047] (1) Preparation of gold-antibody conjugates: Add 1 mL of AuNPs (gold nanoparticles with a particle size of 20 to 50 nm) solution and 15 μL of K2CO3 solution (0.1 M) to a 1.5 mL clean centrifuge tube, mix well, then add 10 μL of FITC antibody solution (1 mg / mL), and couple at room temperature for 1 h; then add 100 μL of 10% HSA and block at room temperature for 30 min; centrifuge at 13,000 rpm for 10 min, remove the supernatant, resuspend the precipitate with 100 μL of resuspension solution (0.01 M Tris-HCl, 5% sucrose, 1% trehalose, 1.5% BSA, pH 8.0), and store at 4°C for later use.

[0048] (2) Preparation of lateral flow test strips: The size of the test strips was 3 mm × 60 mm. The test strips consisted of five modules: a sample pad, a gold label pad, an NC membrane, a water-absorbing pad, and a bottom plate. The specific preparation method of each module was as follows: the sample pad was cut and soaked in a sample pad treatment solution (0.05 M Tris-HCl, 0.15 M NaCl, 0.25% Tris-100, pH 8.0) for 2 h, dried at 37°C and then used for later use; the gold label pad was cut and soaked in a gold label pad treatment solution (10 mM PB, 5% sucrose, 1% trehalose, 0.3% Tris-20, 0.25% PEG 20000, pH 8.0) 7.4) soak for 2h, dry at 37℃, add 4μL of the conjugate described in step (1) on the dried gold label pad, dry at 37℃ and store for later use; the NC membrane is used to fix two detection (T) lines and quality control (C) lines, 0.5mg / mL digoxin antibody is fixed on the T1 line, 1.5mg / mL streptavidin (SAV) is fixed on the T2 line, and 0.5mg / mL mouse secondary antibody is fixed on the C line. The spray speed of the membrane sprayer is set to 0.5μL / cm. After spraying, the NC membrane is dried in a 37℃ oven and stored for later use; the absorbent pad is used to collect the aqueous phase and provide driving force for the reaction solution to flow along the test strip; the bottom plate is used as a base to assemble the above four modules, and the four modules are connected in sequence on the bottom plate, overlapping each other by about 2mm.

[0049] (3) PCR amplification: The total amplification volume was 25 μL, including 1 μL cDNA template, 0.5 μL 10 μM Norovirus F / R primer, 12.5 μL Taq-HS Probe qPCR Premix, 1.5 μL 0.01 μM probe, and 6.5 μL double distilled water. The amplification program was set as pre-denaturation at 93–97°C for 4–10 min, denaturation at 93–97°C for 20–30 s, annealing at 56–60°C for 20–30 s, extension at 70–75°C for 20–30 s, and extension at 70–75°C for another 4–10 min, for 35–40 cycles.

[0050] (4) Test samples corresponding to the GI and GII genotype Noroviruses: Add GI genotype Norovirus cDNA and primer pairs and probes to the amplification system, anneal at 57°C, and cycle number 35. After amplification, 20 μL of amplified product and 30 μL of 1×PBS are vortexed and mixed, and then dropped onto the pre-assembled lateral flow test strip sample pad. After waiting for 5 minutes, the test results can be obtained based on the test line and quality control line of the test strip. The detection steps for the GII genotype Norovirus are the same as those for the GI genotype Norovirus.

[0051] (5) Detection of actual samples: Pre-treat actual samples of unknown GI and GII genotype Norovirus to obtain single-stranded positive-strand RNA, add AMV reverse transcriptase to obtain target template cDNA, and obtain the detection results according to the specific steps in (1), (2), (3) and (4).

[0052] Specifically, a visual detection method is adopted, in which a probe with a double label (FITC label and Biotin affinity label) is used. When the amplified product containing the probe flows to the lateral flow test strip, it encounters the test strip area that can identify the affinity label (usually a line, i.e., the "detection line", with streptavidin) and is enriched to show a visual signal; wherein, in the amplified product of the negative control sample, the probe structure is intact, and when the amplified product flows to the lateral flow test strip, the test strip area that can identify the affinity label shows a visual signal; in the amplified product of the positive control sample, the probe will be consumed, and when the amplified product flows to the lateral flow test strip, the test strip area that can identify the affinity label will not show a visual signal.

[0053] The detection mechanism of the present invention is mainly as follows: when GI genotype and GII genotype Norovirus are present, the viral RNA is first reverse transcribed to obtain cDNA, and then amplified by PCR, so that the dual-labeled probe is cut off, and the amplification product without the dual-labeled probe flows to the lateral flow test strip and encounters the test strip area that can identify the affinity label (two lines, namely "detection line 1" and "detection line 2", respectively carrying digoxin antibody and streptavidin), and will not be enriched. At this time, the detection line will not show a visual signal, thereby realizing the detection of GI genotype and GII genotype Norovirus.

[0054] Another aspect of an embodiment of the present invention also provides a kit for visually detecting GI genotype and / or GII genotype Norovirus, which includes: the aforementioned primer-probe combination for detecting GI genotype and / or GII genotype Norovirus, and the aforementioned lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus.

[0055] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0056] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0057] Example 1 Design of primer probe

[0058] This example provides a primer-probe combination that can achieve specific amplification and differentiation of the GI and GII genotypes of Norovirus, overcoming the defects of the traditional primer-probe system that cannot effectively differentiate the GI and GII genotypes and the technical bottleneck that the traditional amplification product-based identification is prone to cause false positive detection results.

[0059] (1) Using the cDNA obtained by reverse transcription of the single-stranded RNA of the GI genotype Norovirus as the target gene, specific primers and probes suitable for polymerase chain reaction and lateral flow chromatography technology were designed. The sequences of the primers and probes are as follows:

[0060] Target gene upstream primer sequence GI-F: 5'-CGCTGGATGCGNTTCCAT-3'

[0061] Target gene downstream primer sequence GI-R: 5′-CCTTAGACGCCATCATCATTTAC-3′

[0062] Target gene probe sequence GI-P: 5′-FITC-TGGACAGGAGAYCGCRATCT-Biotin-3′

[0063] (2) Using the cDNA obtained by reverse transcription of single-stranded RNA of GII genotype Norovirus as the target gene, specific primers and probes suitable for polymerase chain reaction and lateral flow technology were designed. The sequences of the primers and probes are as follows:

[0064] Upstream primer sequence of target gene GII-F: 5′-ATGTTCAGRTGGATGAGRTTCTCWGA-3′

[0065] Target gene downstream primer sequence GII-R: 5′-TCGACGCCATCTTCATTCACA-3′

[0066] Target gene probe sequence GII-P: 5'-Dig-AGCACGTGGGAGGGCGATCG-FITC-3'

[0067] Example 2 Preparation of Lateral Flow Test Strips

[0068] (1) Preparation of gold-antibody conjugate: Add 1 mL of AuNPs solution and 15 μL of 0.1 M K2CO3 solution to a 1.5 mL clean centrifuge tube, mix well, then add 10 μL of FITC antibody solution (1 mg / mL), and couple at room temperature for 1 h; then add 100 μL of 10% HSA and block at room temperature for 30 min; centrifuge at 13,000 rpm for 10 min, remove the supernatant, resuspend the precipitate with 100 μL of resuspension solution (0.01 M Tris-HCl, 5% sucrose, 1% trehalose, 1.5% BSA, pH 8.0), and store for later use.

[0069] (2) Preparation of lateral flow test strips: The size of the test strips was 3 mm × 60 mm. The specific preparation method was as follows: the sample pad was cut and soaked in a sample pad treatment solution (0.05 M Tris-HCl, 0.15 M NaCl, 0.25% Tris-100, pH 8.0) for 2 h, dried at 37°C and set aside; the gold label pad was cut and soaked in a gold label pad treatment solution (10 mM PB, 5% sucrose, 1% trehalose, 0.3% Tris-20, 0.25% PEG 20000, pH 7.4) for 2 h, dried at 37°C, 4 μL of the conjugate described in step (1) was added to the dry gold label pad, dried at 37°C and stored for later use; the NC membrane was used to fix the detection (T1 and T2) lines and the quality control (C) line, and 0.5 me / mL of digoxin antibody (Digoxin) was fixed on the T1 line using a film sprayer. antibody), 1.5 mg / mL streptavidin (SAV) was fixed on the T2 line, and 0.5 mg / mL goat-anti-mouse secondary antibody (Goat-anti-mouseantibody) was fixed on the C line. The spray rate was set to 0.5 μL / cm. After spraying, the NC membrane was stored in a 37°C oven for drying and stored for future use; the absorbent pad was used to collect the aqueous phase and provide a driving force for the reaction solution to flow along the test strip; the bottom plate was used as a base to assemble the above four modules, and the four modules were connected in sequence on the bottom plate, overlapping each other by about 2 mm.

[0070] Example 3 Preparation of the template to be detected

[0071] To obtain viral genomic RNA from human feces, it is recommended to use a commercial kit to extract the viral genome, and then use AMV reverse transcriptase to obtain the first-strand cDNA, and the obtained cDNA is used as the target template for amplification.

[0072] Example 4 Primer-probe combination detection method for GI genotype and GII genotype Norovirus (test strip method)

[0073] This embodiment provides a detection kit prepared using the primer-probe combination provided in Embodiment 1, wherein the kit also includes a primer pair and probe for GI genotype Norovirus, a primer pair and probe for GII genotype gene, a negative control, a positive control, double distilled water, and an enzyme premix.

[0074] Among them, the enzyme premix includes Taq-HSDNA polymerase, glycerol and BSA, deoxyribonucleoside triphosphate, tris(hydroxymethylaminomethane) (Tris) and magnesium ions, all from Yugong Technology Co., Ltd.

[0075] The recommended reaction system is shown in Table 2 (25 μL).

[0076] Table 2 Reaction system

[0077] Components Dosage Taq-HS Probe qPCR Premix 12.5μL GI-F (10 μM) 0.5μL GI-R (10 μM) 0.5μL GI-P (0.01 μM) 1.5μL GII-F (10 μM) 0.5μL GII-R (10μM) 0.5μL GII-P (0.01 μM) 1.5μL Double distilled water 6.5μL template 1μL

[0078] The above system was used for reaction. The templates added were the norovirus positive control and the negative control was a solution without the target gene (adding double distilled water). The reaction was carried out according to the specific amplification reaction procedure: 95°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min; the results were as follows: Figure 2 As shown, it can be seen that the designed primer-probe combination can specifically amplify the Norovirus GI and GII genotypes, respectively, causing the hydrolysis of the nucleic acid probe in the amplification system, thereby not causing the appearance of the T line on the lateral flow test strip, indicating a positive result.

[0079] It can be seen that the kit of the present invention has good specificity and helps to improve the accuracy and efficiency of current norovirus detection.

[0080] Example 5

[0081] Based on the detection scheme of Example 4, a mixed sample containing viruses with similar clinical symptoms (rabbit hemorrhagic disease virus, Sapporo-like virus, swine vesicular exanthema virus) and target norovirus GI or GII and a separate comparison sample (rabbit hemorrhagic disease virus, Sapporo-like virus, swine vesicular exanthema virus) were further amplified and visualized, and the gray value of the detection T line on the visualized lateral flow test strip was normalized and quantitatively analyzed. The results are as follows: Figure 3 As shown, it can be seen that compared with the model intensity of the T line of the blank control sample group, based on the designed primer and probe combination specific for Norovirus GI and GII, only the target genotypes GI and GII of Norovirus can be identified and visualized; at the same time, in the presence of non-target viral genes, there will be no interference with the identification of target genes GI and GII; in addition, for genes of non-target viruses that exist alone, the primer and probe combination designed by the present invention for Norovirus GI and GII genotypes will not produce amplification and visual detection responses for other viral genes, and the signal on the lateral flow detection T line will not be weakened, indicating that the primer and probe combination designed by the present invention for Norovirus GI and GII genotypes has good specificity. In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0082] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting GI genotype and / or GII genotype Norovirus, characterized in that: include: A first primer pair and a first probe for GI genotype Norovirus; wherein the first primer pair comprises a first primer having a sequence as shown in SEQ ID NO.1 and a second primer having a sequence as shown in SEQ ID NO.2, and the sequence of the first probe is shown in SEQ ID NO.3; A second primer pair and a second probe for GII genotype Norovirus; wherein the second primer pair includes a third primer whose sequence is shown in SEQ ID NO.4 and a fourth primer whose sequence is shown in SEQ ID NO.5, and the sequence of the second probe is shown in SEQ ID NO.

6.

2. The primer-probe composition according to claim 1, characterized in that: The 5′ end of the first probe is modified with a fluorescein isothiocyanate group, and the 3′ end of the first probe is modified with a biotin group; And / or, the 5′ end of the second probe is modified with a digoxigenin group, and the 3′ end of the first probe is modified with a fluorescein isothiocyanate group.

3. A lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus, comprising a sample pad, a gold label pad, an NC membrane, a water absorbent pad and a bottom plate, characterized in that: The NC membrane is provided with a detection line T1, a detection line T2 and a quality control line C; Wherein, the detection line T1 is fixed with digoxin antibody; the detection line T2 is fixed with streptavidin; and the quality control line C is fixed with anti-mouse secondary antibody.

4. Use of the primer-probe combination for detecting GI genotype and / or GII genotype Norovirus according to any one of claims 1-2 or the lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus according to claim 3 in the preparation of a product for visually detecting GI genotype and / or GII genotype Norovirus.

5. The use according to claim 4, characterized in that The method for using the product to detect GI genotype and / or GII genotype Norovirus includes: Extracting RNA from the sample to be tested; The RNA is amplified by PCR using the primer-probe combination for detecting GI genotype and / or GII genotype Norovirus according to claim 1 or 2, and the amplified product is then analyzed and detected using a lateral flow test strip, thereby achieving visual detection of the sample to be tested based on the detection result of the lateral flow test strip.

6. The use according to claim 4, characterized in that The method further comprises: firstly reverse transcribing the extracted RNA to obtain cDNA; preferably, AMV reverse transcriptase is used for the reverse transcription.

7. The use according to claim 4, characterized in that: The working concentration of the primers in the primer-probe combination is 5-20 μM; And / or, the working concentration of the probe in the primer-probe composition is 0.05-0.2 μM.

8. The use according to claim 4, characterized in that The reaction procedure for PCR amplification includes: pre-denaturation at 95°C for 3-5 min, denaturation at 94°C for 20-30 s, annealing at 55°C for 20-30 s, extension at 72°C for 20-30 s, and extension at 72°C for 5-8 min; preferably, the number of cycles of the denaturation, annealing and extension process is 35-40.

9. The use according to claim 4, characterized in that: When amplification was performed using a negative control sample, the detection area of ​​the lateral flow test strip showed a visible signal; And / or, when a positive control sample is used for amplification, the probe is hydrolyzed during the amplification process, and the detection area of ​​the lateral flow test strip will not show a visible signal, thereby avoiding false positive results caused by dimers and nonspecific amplification during the amplification process.

10. A kit for visually detecting GI genotype and / or GII genotype Norovirus, characterized in that include: The primer-probe combination for detecting GI genotype and / or GII genotype Norovirus according to claim 1 or 2, and the lateral flow test strip for detecting GI genotype and / or GII genotype Norovirus according to claim 3.

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