Primer and probe for synchronously detecting type II norovirus, group A rotavirus and adenovirus and application

Abstract

The invention provides a synchronous detection primer for type II norovirus, group A rotavirus and adenovirus, which comprises specific primer pairs designed for conserved genes of three pathogens of type II norovirus, group A rotavirus and adenovirus, namely an ORF1 / ORF2 primer pair, a VP6 gene primer pair and a Hexon gene primer pair. The invention also provides a detection probe, which comprises an ORF1 / ORF2 capture probe, a VP6 capture probe and a Hexon capture probe. Meanwhile, the invention further provides application of the primer and the probe in preparation of infectious diarrhea related virus detection products, a specific detection kit and a specific detection method. According to the present invention, the multiple detection of three common diarrhea viruses can be achieved, the detection can be completed in a short time, the amplification product can be rapidly and accurately identified, the color development can be performed, the detection sensitivity and the specificity can be improved, the large-scale instrument is not required, and the cost is low.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a primer, probe, and application for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus. Background Technology

[0002] Viral diarrhea is a common disease threatening global public health, with a high incidence rate, especially among infants, the elderly, and immunocompromised populations. Its causes are complex and diverse, typically caused by multiple pathogens such as rotavirus, norovirus, adenovirus, and astrovirus. While the symptoms of diarrhea caused by different viruses are similar, significant differences exist in epidemiological characteristics, disease severity, and control measures. Therefore, timely and accurate identification of the viral pathogen causing diarrhea is crucial for precise treatment and infection control.

[0003] Traditional laboratory diagnostic methods, such as virus isolation and culture, electron microscopy, and immunological testing, are time-consuming (usually 3-7 days), have low sensitivity, are cumbersome to operate, and require highly skilled personnel and experimental conditions. In clinical applications, rapid diagnosis is crucial for the treatment of patients with viral diarrhea, especially infants and the elderly. If some pathogens (such as norovirus) are not identified in time, they may lead to serious consequences such as hospital-acquired infection outbreaks, severe dehydration, electrolyte imbalances, and even death.

[0004] While existing PCR detection technologies have high sensitivity and specificity, they generally require expensive quantitative PCR instruments, strict laboratory zoning, long testing cycles (usually 2-4 hours), and professional operators, making it difficult to widely implement in outpatient and emergency departments, primary healthcare institutions, and resource-limited areas, and thus failing to meet the clinical needs for rapid screening and on-site detection of diarrhea pathogens.

[0005] Therefore, there is an urgent need for a new method that can simultaneously detect multiple diarrhea viruses in a short time, is easy to operate, and is cost-effective. This is of great practical significance for the clinical differential diagnosis of viral diarrhea, infection control in hospitals, and public health emergency response. Summary of the Invention

[0006] The technical problem this invention aims to solve is to provide primers, probes, and applications for the simultaneous detection of type II norovirus, group A rotavirus, and adenovirus. Through specially designed primers and probes, combined with RPA (recombinase polymerase amplification)-lateral flow immunochromatography, it achieves rapid, simultaneous identification and multiplex detection of these three major viral diarrhea pathogens: type II norovirus, group A rotavirus, and adenovirus. Specifically, the primers are designed targeting the conserved gene sequences of the three viruses, while incorporating nucleic acid modifications and iSpC12 spacer structures. This improves amplification specificity and efficiency while effectively preventing primer dimer formation, ensuring accurate and stable amplified products. Furthermore, combining the rapid amplification advantages of RPA technology with the convenient detection characteristics of lateral flow immunochromatography strips significantly shortens detection time. The specific capture probes on the chromatography strips enable rapid and accurate identification and color development of amplified products, significantly improving detection sensitivity and specificity, and reducing the risk of misdiagnosis and missed diagnosis. Moreover, this technology requires no large or complex instruments, has low detection costs, is easy to operate, and is suitable for large-scale screening and primary healthcare settings.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A primer set for simultaneous detection of norovirus type II, rotavirus A, and adenovirus includes specific primer pairs designed for conserved genes of these three pathogens, with the sequences as follows: Type II norovirus ORF1 / ORF2 primer pair: ORF1 / ORF2-F: 5'-FITC-TTTACGTGCCCAGACAAGAGCCAATGTTCA-3'; ORF1 / ORF2-R:5'-TAGATAGATAGATAGA / iSpC12 / GTCACTCGACGCCATCTTCATTCACAAAAC-3'; Group A rotavirus VP6 gene primer pair: VP6-F: 5'-FITC-GAACGGAAATGACTTTCAAACTGGAGGAA-3'; VP6-R: 5'-CATTCTGCTTCCAAGT / iSpC12 / CTCAATCGTAGTTCTGGCGGTCTCAACAT-3'; Adenovirus Hexon gene primer pair: Hexon-F:5'-FITC-GAACTACATTGGCTTTAGGGATAACTTCATCG-3'; Hexon-R:5'-TCGACTGAGAATTGAC / iSpC12 / AACTGTCAACAGCCTGATTCCACATAGAA-3'.

[0008] As one of the preferred embodiments of the present invention, in the downstream primer R of the three primer pairs, the 5' end is a labeled nucleic acid sequence, and the nucleic acid sequence is designed with a C12 backbone structure modification between it and the adjacent unmodified sequence, namely the iSpC12 spacer, to prevent primer dimer formation.

[0009] As one of the preferred embodiments of the present invention, the ORF1 / ORF2 primer pair, the VP6 gene primer pair, and the Hexon gene primer pair are used for the detection of type II norovirus, group A rotavirus, and adenovirus, respectively.

[0010] The application of the above primers in the preparation of a detection product for infectious diarrhea-associated viruses, wherein the infectious diarrhea-associated viruses include type II norovirus, group A rotavirus, and adenovirus.

[0011] A simultaneous detection probe for type II norovirus, group A rotavirus, and adenovirus, comprising an ORF1 / ORF2 capture probe, a VP6 capture probe, and a Hexon capture probe, with the following sequences: ORF1 / ORF2 capture probe: TCTATCTATCTATCTATTTTTT-Biotin; VP6 capture probe: ACTTGGAAGCAGAATGTTTTTT-Biotin; Hexon capture probe: GTCAATTCTCAGTCGATTTTTT-Biotin.

[0012] As one of the preferred embodiments of the present invention, the ORF1 / ORF2 capture probe, VP6 capture probe and Hexon capture probe are detection line probes designed for type II norovirus, group A rotavirus and adenovirus, respectively, so that the amplification product can bind to the capture probe during the test strip chromatography process, thereby making the detection line colorimetric.

[0013] An application of the above-mentioned probe in the preparation of a detection product for infectious diarrhea-related viruses, wherein the infectious diarrhea-related viruses include type II norovirus, group A rotavirus, and adenovirus.

[0014] A kit for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus, comprising: The above primers; The aforementioned probe; Lateral flow immunochromatographic strips; RPA amplification reagents; Detection system for amplified product loading chromatography strips.

[0015] As one of the preferred embodiments of the present invention, the lateral flow immunochromatographic strip is composed of a sample pad, a gold-labeled pad, an NC membrane, an absorbent pad, and a base plate; the sample pad is used to load the sample, the gold-labeled pad is used to store the AuNP-Ab conjugate, the NC membrane is used to fix the detection line and the control line, the absorbent pad is used to collect the aqueous phase and provide driving force for the flow of the reaction solution on the test strip, and the base plate serves as a backing to assemble the sample pad, the gold-labeled pad, the NC membrane, and the absorbent pad into one unit.

[0016] A method for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus for non-diagnostic purposes, using the aforementioned kit, is as follows: (1) Using the RNA of the sample to be tested as a template, the primers and RPA amplification reagents are used to form an RPA amplification system for RPA amplification; (2) Fix the probe to the NC membrane detection line position of the side-flow immunochromatographic strip; (3) The RPA amplification product obtained in step (1) is combined with the loading buffer, and the infection type of type II norovirus, group A rotavirus and adenovirus in the sample is determined by the detection results of the side-flow immunochromatographic strip detection system.

[0017] As one of the preferred embodiments of the present invention, the sample to be tested is a fecal sample.

[0018] As one of the preferred embodiments of the present invention, the RPA amplification system is used to amplify the conserved genes of the three pathogens: type II norovirus, group A rotavirus, and adenovirus. The specific composition is 1.5 μL of each primer, 29.4 μL of A buffer, 2.5 μL of B buffer, 5 μL of RNA template, and 4.1 μL of ddH2O. The amplification conditions are: amplification at a constant temperature of 39℃ for 20 min.

[0019] As one of the preferred embodiments of the present invention, the lateral flow immunochromatographic strip detection system is used to develop the detection line and control line of the lateral flow immunochromatographic strip after the RPA amplification product is combined with the loading buffer. The specific composition is as follows: 0.5 μL of RPA amplification product and 50 μL of loading buffer; wherein, the loading buffer includes 50 mmol / L Tris-HCl, 0.9% sodium chloride, 0.5% Tween-20, 0.05% P300, and pH 8.0.

[0020] The advantages of this invention compared to the prior art are: (1) Rapid and multiplex detection: The present invention uses RPA-lateral flow immunochromatographic strip technology (RPA-LFS) to rapidly detect and accurately genotype three diarrhea-associated viruses (type II norovirus, group A rotavirus, and adenovirus). The entire detection process takes only 30 to 40 minutes, which can meet the needs of rapid detection.

[0021] (2) High sensitivity and specificity: The present invention has a sensitivity of up to 10 for detecting type II norovirus and group A rotavirus. 1 Copies / μL, adenovirus sensitivity up to 10 2 Copies / μL; This method can accurately detect samples with low viral load, which helps reduce false negative and false positive results, and has good detection performance and sensitivity in clinical diagnosis and application.

[0022] (3) Easy to operate and no large instruments required: This invention does not require large and complex instruments and can realize rapid bedside testing in multiple scenarios. Even personnel with minimal training can operate it, which increases its applicability in different medical environments, especially suitable for remote areas with limited resources.

[0023] (4) Low cost: The present invention only costs about RMB 50 to test one sample, and does not require high instrument costs. The price is lower than similar testing products on the market, making it suitable for large-scale screening.

[0024] (5) Easy to promote and mass-produce: This invention does not require a large amount of reagents and materials, can be mass-produced, and does not require special instruments and training. It is economically feasible and can be quickly expanded to large-scale applications. It helps in the early detection of diarrhea-related viruses and is crucial for clinical situations that require rapid response. Attached Figure Description

[0025] Figure 1 This is an electrophoresis diagram verifying the effectiveness of the primers in the validation example (in the figure, lanes 1-7 are RPA amplification results of different viral templates based on the primers of this invention; lane 8 is a negative control without template). Figure 2 This is a schematic diagram of the RPA-lateral flow immunochromatographic strip technique used in the validation example; Figure 3 This is a diagram showing the results of the lateral flow immunochromatographic strips in the validation example (in the diagram, strips 1 to 7 represent positive results for NoV GII, RVA, Ade, NoV GII+RVA, Ade+NoV GII, RVA+Ade, and Ade+RVA+NoV GII, respectively; strip 8 indicates that the analytical template was replaced with water). Figure 4 This is a graph showing the detection limit test results for NoV GII, RVA, and Ade in the validation example; Figure 5 The results of lateral flow immunochromatographic strips for different diarrhea patients in the validation case are shown (in the figure, 1 to 9 correspond to the sample numbers of different positive patients). Figure 6 This is the RPA-LFS cross-reactivity validation result for 11 common pathogens in the validation examples. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the reagents and experimental methods used in the following embodiments and verification examples are all conventional reagents and methods in the art, and will not be described again.

[0027] The primer and probe sequences involved in the following examples are shown in Table 1.

[0028] Table 1 Primer and probe sequences

[0029] Example 1 This embodiment provides a primer for the simultaneous detection of type II norovirus, group A rotavirus, and adenovirus, including a type II norovirus ORF1 / ORF2 gene primer pair, a group A rotavirus VP6 gene primer pair, and an adenovirus Hexon gene primer pair, each designed for conserved genes of the three diarrhea-associated viruses (type II norovirus, group A rotavirus, and adenovirus).

[0030] In the above three primer pairs (type II norovirus ORF1 / ORF2 gene primer pair, group A rotavirus VP6 gene primer pair, and adenovirus Hexon gene primer pair), the upstream primers (ORF1 / ORF2-F, VP6-F, and Hexon-F) are all labeled with FITC antibody at the 5' end for subsequent binding to the AuNP-FITC antibody conjugate on the gold pad; the downstream primers (ORF1 / ORF2-R, VP6-R, and Hexon-R) have nucleic acid sequence markers introduced at the 5' end, and iSpC12 spacers are inserted between the marker sequence and the primer body to prevent primer dimer formation. The specific sequences are shown in Table 1.

[0031] Example 2 This embodiment presents a simultaneous detection probe for type II norovirus, group A rotavirus, and adenovirus, including ORF1 / ORF2 capture probes (CP). NoV GII ), VP6 capture probe (CP) RVA ) and Hexon capture probe (CP) Ade The sequence is shown in Table 1.

[0032] Among them, the ORF1 / ORF2 capture probe, VP6 capture probe and Hexon capture probe are detection line probes designed for type II norovirus, group A rotavirus and adenovirus, respectively, so that the amplification products can bind to the capture probes during the test strip chromatography process, thereby making the detection line color.

[0033] Example 3 This embodiment provides a kit for the simultaneous detection of type II norovirus, group A rotavirus, and adenovirus, comprising: primers (Example 1), probes (Example 2), lateral flow immunochromatographic strips, RPA amplification reagent, and a lateral flow immunochromatographic strip detection system.

[0034] In this embodiment, the lateral flow immunochromatographic strip consists of a sample pad, a gold-labeled pad, an NC membrane, an absorbent pad, and a base plate. The sample pad is used to load the sample, the gold-labeled pad is used to store the AuNP-Ab conjugate, the NC membrane is used to fix the detection line and the control line, the absorbent pad is used to collect the aqueous phase and provide driving force for the flow of the reaction solution on the test strip, and the base plate serves as a liner to assemble the sample pad, gold-labeled pad, NC membrane, and absorbent pad into one unit.

[0035] Primers and RPA amplification reagents constituted the RPA amplification system, used to amplify conserved genes of three diarrheal viruses. The RPA amplification system consisted of: 1.5 μL of each primer, 29.4 μL of buffer A, 2.5 μL of buffer B, 5 μL of RNA template, and 4.1 μL of ddH2O. Buffers A and B were obtained from Anpu Future Biotechnology Co., Ltd., using the RNA Isothermal Rapid Amplification Kit (Basic Type).

[0036] The probe is immobilized on the lateral flow immunochromatographic strip and subsequently hybridizes with the amplification product.

[0037] The lateral flow immunochromatographic strip detection system is used for the hybridization and color development of RPA amplification products with probes on the lateral flow immunochromatographic strip. The system consists of: RPA amplification products and loading buffer. The loading buffer includes 50 mmol / L Tris-HCl, 0.9% sodium chloride, 0.5% Tween-20, 0.05% P300, and pH 8.0.

[0038] Example 4 This embodiment presents a method for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus for non-diagnostic purposes, using the kit from Example 3. The method is as follows: S1. Nucleic acid extraction (nucleic acid extraction kit from Guangzhou Da'an Gene): Add 50 μL of proteinase K to a 1.5 mL sterile centrifuge tube.

[0039] Take a new 1.5 mL sterile centrifuge tube and add 500 μL of physiological saline. Add 0.1 g of solid fecal sample or 0.1 mL of liquid fecal sample to the tube and mix using a vortex mixer. Centrifuge at 12000 g for 10 min at room temperature. The supernatant is the sample to be tested.

[0040] Add 200 μL of fecal suspension to a 1.5 mL centrifuge tube containing 50 μL of proteinase K.

[0041] Add 200 μL of lysis working buffer (i.e. lysis buffer containing carrier RNA), tighten the cap, vortex for 15 seconds to mix thoroughly, centrifuge at high speed for 10 seconds (to prevent air bubbles from forming during incubation), and incubate at 72°C for 10 minutes; the elution buffer can be preheated at 72°C at the same time.

[0042] Add 250 μL of ethanol, tighten the cap, and vortex for 15 seconds.

[0043] Transfer the entire mixture to a centrifuge column and centrifuge at 12000g for 1 min at room temperature. Transfer the centrifuge column to a new collection tube. Add 500 μL of inhibitor removal solution to the centrifuge column, centrifuge at 12000g for 1 min at room temperature, and then transfer the centrifuge column to a new collection tube.

[0044] Add 500 μL of deionized liquid to the centrifuge column, centrifuge at 12000g for 1 min at room temperature, and then transfer the centrifuge column to a new collection tube.

[0045] Add another 500 μL of deionized liquid to the centrifuge column, centrifuge at 12000g for 1 min at room temperature, and then transfer the centrifuge column to a new collection tube.

[0046] Centrifuge the column-collection tube at 14000g for 3 minutes at room temperature to remove residual ethanol.

[0047] Remove the centrifuge column and place it in a new 1.5mL centrifuge tube. Open the cap of the centrifuge tube and incubate at 72°C for 2 minutes (use a dry incubator, do not use a water bath).

[0048] Carefully add 50 μL of preheated elution buffer (72°C) directly above the membrane on the centrifuge column. Tightly cap the tube, let it stand at room temperature for 1 minute, then centrifuge at 14000g for 1 minute. The nucleic acid solution in the centrifuge tube is ready for immediate use. If storage is required, store at -20°C.

[0049] S2, RPA primer and capture probe design: Multiple published sequences of norovirus type II ORF1 / ORF2, rotavirus group A VP6, and adenovirus Hexon were downloaded from the NCBI website. Highly conserved sequences were obtained through multiple sequence alignment using SnapGene software. Based on these conserved sequences, multiple RPA primer pairs targeting these three viruses were designed using Primer Premier 5 software (Premier Biosoft International, CA, USA). The species specificity of the designed primers was verified using the NCBI-BLAST online tool. Sequences modified with C12 spacers and NAT (Nucleic Acid Labeling) for amplification using the optimal primer set were designed (Example 1), along with capture probe sequences (Example 2), and synthesized by a biotechnology company (the upstream primers for the primer pairs were synthesized by Nanjing Qingke Biotechnology Co., Ltd., and the downstream primers and probes for the required primer pairs were synthesized by Shanghai Sangon Biotech Co., Ltd.).

[0050] S3, RPA amplification: Using RNA from the sample to be tested as a template, an RPA amplification system was constructed using primers and RPA amplification reagents to perform RPA amplification.

[0051] Reaction system (NoV GII, RVA, Ade triple reaction system): A buffer 29.4 μL, each primer 1.5 μL, RNA template 5 μL, ddH2O 4.1 μL, B buffer 2.5 μL; Amplification conditions: Amplification at 39℃ for 20 min.

[0052] The obtained mRPA products were characterized by 3.5% agarose gel electrophoresis.

[0053] S4. Preparation of loading buffer: Includes 50 mmol / L Tris-HCl, 0.9% sodium chloride, 0.5% Tween-20, 0.05% P300, pH 8.0.

[0054] S5. Preparation of lateral flow immunochromatographic strips: (1) Preparation of gold nanoparticles (AuNPs) Take a 250mL Erlenmeyer flask, soak it in the prepared aqua regia, rinse it with ultrapure water, and dry it for later use. Add 120 mL of chloroauric acid solution (0.01%, w / v) to the treated conical flask and heat to boiling; While stirring vigorously, quickly add 1.5 mL of trisodium citrate solution (1%, w / v). Continue heating and stirring until the solution color stabilizes, then continue heating for 15 minutes. After cooling to room temperature, add ultrapure water to a final volume of 100 mL and store at 4°C. Chloroauric acid solution and trisodium citrate solution were both purchased from Sinopharm.

[0055] (2) Preparation of AuNPs-FITC antibody conjugate (AuNP-Ab) Add 1 mL of AuNPs solution to a clean centrifuge tube, and add 8 μL of K2CO3 solution (0.1 mol / L) to adjust the pH of the solution; the K2CO3 solution was purchased from Sinopharm.

[0056] Add 2 μL of FITC antibody (1 mg / mL) and mix by inversion at room temperature for 1 h; FITC antibody was purchased from Guangzhou Geman Biotechnology Co., Ltd.

[0057] Add 100 μL of BSA solution (10%, w / v) to block the remaining sites on the surface of AuNPs, vortex mix, and incubate at room temperature for 1 h; BSA solution was purchased from Shanghai Yuanye Biotechnology.

[0058] Centrifuge at 7900 r / min for 10 min, discard the supernatant, and reconstitute the precipitate with 100 μL of BSA solution (10%, w / v); AuNP-Ab conjugates were stored at 4°C for subsequent preparation of gold-labeled pads. (3) The size of the lateral flow immunochromatographic strip (LFS) is 3mm × 60mm, and it consists of five modules. The specific preparation method is as follows: The sample pad is used to load the sample. To improve the absorbency and surface activity of the sample pad and avoid the influence of complex matrices on the detection results, the sample pad is soaked in a sample pad treatment solution (0.05 mol / L Tris-HCl, 0.15 mol / L NaCl, 0.25% Triton-100, pH 8.0) for 2 hours after cutting, and then dried at 30°C for later use. The sample pad was purchased from Shanghai Jinbiao, Tris-HCl and NaCl were purchased from Sinopharm, and Triton-100 was purchased from Aladdin.

[0059] The gold-labeled pads are used to store AuNP-Ab conjugates. To improve the pads' load-bearing capacity and ensure the dispersibility of the AuNP-Ab conjugates, the gold-labeled pads were soaked in a gold-labeled pad treatment solution (10 mmol / L PB, 5% sucrose, 1% trehalose, 0.3% Tween-20, 0.25% PEG 20000, pH 7.4) for 2 hours after cutting, and then dried at 30°C. Then, 6 μL of AuNP-Ab conjugate was added to the dried gold-labeled pads, dried at 30°C, and stored for later use. PB, 5% sucrose, 1% trehalose, and 0.3% Tween-20 were all purchased from Sinopharm, and PEG 20000 was purchased from Aladdin.

[0060] The NC membrane is used to immobilize three T-lines and one C-line. Three biotinylated capture probes are immobilized on the three T-lines, while the T1-line immobilizes the CP capture probe targeting norovirus type II. NoV GII The T2 line is fixed with a capture probe CP targeting serotype A rotavirus. RVA The T3 line is fixed with a capture probe CP targeting adenovirus. Ade The C-line was fixed with goat anti-mouse secondary antibody, which was diluted to 1 mg / mL with 10 mmol / L PB solution before spraying. All biotinylated capture probes needed to be pre-bound with streptavidin: 2.5 μL of biotinylated capture probe (100 μmol / L), 2.5 μL of streptavidin (5 mg / mL), and 15 μL of 1×PBS were added to the PCR tube, and incubated at room temperature for 1 h. The spraying speed of the spraying apparatus was set to 0.5 μL / cm. After spraying, the NC membrane was dried in a 30°C oven and stored. The NC membrane was purchased from Sartorius, the streptavidin from Shanghai Yisheng Biotechnology, and the goat anti-mouse secondary antibody from Jiangsu Dongkang Biotechnology.

[0061] The absorbent pad is used to collect the aqueous phase and provides the driving force for the reaction solution to flow down the test strip. The absorbent pad was purchased from Shanghai Jiening.

[0062] The base plate serves as a lining for assembling the aforementioned four modules. The four modules are connected sequentially on the base plate, overlapping each other by approximately 2mm. The base plate was purchased from Shanghai Jinbiao.

[0063] S6. The amplification product is hybridized with the capture probe on the lateral flow immunochromatographic strip, and then colored: 0.5 μL of RPA amplification product and 50 μL of loading buffer (50 mmol / L Tris-HCl, 0.9% sodium chloride, 0.5% Tween-20, 0.05% P300, pH 8.0) are mixed thoroughly and then dropped onto the LFS sample pad. After reacting for 10 minutes, the color development of the test strip is observed, and the result can be judged visually.

[0064] Verification Example This validation example is used to verify the feasibility of the above method (Example 4), combining clinical sample testing to achieve nucleic acid extraction, RPA amplification, lateral flow immunochromatographic strip detection, and color development, including the following specific steps: I. Nucleic acid extraction: Same as Example 4.

[0065] II. RPA Primer and Capture Probe Design: Multiple published sequences of norovirus type II ORF1 / ORF2, rotavirus group A VP6, and adenovirus Hexon were downloaded from the NCBI website. Multiple sequence alignment was performed using SnapGene software to obtain highly conserved sequences. Based on these conserved sequences, multiple RPA primer pairs targeting these three viruses were designed using Primer Premier 5 software (Premier Biosoft International, CA, USA). The species specificity of the designed primers was verified using the NCBI-BLAST online tool. Sequences modified with C12 spacers and NATs for amplification using the optimal primer set were designed (Example 1), and capture probe sequences were designed (Example 2), and synthesized by a biotechnology company. To verify the feasibility of the RPA primers, standard strains of norovirus type II ORF1 / ORF2, rotavirus group A VP6, and adenovirus were used to perform RPA amplification and electrophoresis verification on the designed primers, with results as follows: Figure 1 As shown, the primers of this invention can effectively amplify the conserved genes of these three pathogens (Norovirus type II ORF1 / ORF2 gene, Group A rotavirus VP6 gene, and adenovirus Hexon gene).

[0066] III. RPA Amplification: Same as Example 4, amplification system and conditions are consistent; 50 μL of liquid paraffin oil is used to cover the surface of the reaction solution to prevent aerosol contamination.

[0067] IV. Preparation of sample loading buffer: Same as Example 4.

[0068] V. Preparation of lateral flow immunochromatographic strips: Same as Example 4.

[0069] VI. Hybridization and color development of the amplified products with the capture probes on the lateral flow immunochromatographic strip: Same as Example 4.

[0070] The principle of the RPA-lateral flow immunochromatographic strip technology of this invention is as follows: Figure 2As shown. When the target component is present, dsDNA amplicon labeled with FITC and NAT at both ends can be obtained through amplification. When analyzing the amplification products using LFS, the FITC at one end of the dsDNA amplicon can bind to the AuNP-Ab conjugate, and the NAT sequence at the other end can hybridize with the capture probe on the corresponding T line. The AuNP-Ab can be linked to the corresponding T line using the bridging effect of the dsDNA amplicon, making it appear red. Conversely, when the target component is not present, no double-labeled dsDNA amplicon will be generated, and none of the three T lines will be colored. Regardless of the presence or absence of the target component, excess AuNP-Ab conjugate can bind to the goat anti-mouse secondary antibody fixed on the C line, making the C line colored.

[0071] The final results of the lateral flow immunochromatographic strips are as follows: Figure 3 (In the figure, chromatographic strips 1-7 represent positive results for NoV GII, RVA, Ade, NoV GII+RVA, Ade+NoV GII, RVA+Ade, and Ade+RVA+NoV GII, respectively; strip 8 indicates that the analytical template was replaced with water.) See Table 1 and... Figure 3 As a result, it is possible to clearly determine which virus caused the diarrhea patient's infection.

[0072] Furthermore, using recombinant standard plasmids of three viruses as templates, the detection limits of the method of this invention for three diarrhea viruses were tested. Figure 4 It can be seen that the detection limit of the method of the present invention for norovirus type II and rotavirus group A is 10^1 copies / μL, and the detection limit of adenovirus is 10^1 copies / μL. 2 Copies / μL.

[0073] Meanwhile, to further verify the clinical applicability of the method of the present invention, stool samples were collected from 9 clinically diagnosed patients with diarrhea and tested using the above-mentioned detection method. The results are as follows. Figure 5 As shown: clear control lines (C lines) appeared on the chromatography strips of all samples, indicating that the detection system is stable and effective; among them, samples 1-3 showed color only on the T1 line (type II norovirus detection line), samples 4-6 showed color only on the T2 line (group A rotavirus detection line), and samples 7-9 showed color only on the T3 line (adenovirus detection line). The detection results were completely consistent with the clinical diagnosis results, confirming that the method of the present invention can be accurately applied to the virus typing detection of clinical patient samples with high clinical matching degree.

[0074] Furthermore, it was found that 11 common pathogens (Yersinia enterocolitica) were involved. Y. enterocolitica Bacillus cereus B.cereus Salmonella infantis S. Infantis Campylobacter jejuni C. jejuni Shigella flexneri S.flexneri Enterococcus faecalis E.faecium Escherichia coli E.coil Enterococcus faecalis E.fecalis Clostridium difficile C. difficile Positive samples of hepatitis B virus (HBV) and influenza A virus (FluA) were used as samples to test the detection results of non-type II norovirus, non-group A rotavirus, and non-adenovirus using the method of this invention. The results are as follows: Figure 6 As shown: the test lines for all 11 non-target pathogen samples did not develop color, only the control lines developed color normally, and all test results were negative; this indicates that the method of the present invention has extremely high specificity, specifically binding only to the target sequences of type II norovirus, group A rotavirus and adenovirus, and will not cross-react with other enteric pathogens, effectively avoiding false positive results caused by non-specific amplification or binding, and ensuring the accuracy of detection.

[0075] In summary, this invention enables rapid, accurate, and multiplex detection of three target viruses simultaneously, exhibiting high sensitivity and specificity. The detection limits for type II norovirus and group A rotavirus are 10-1. 1 Copies / μL, lower limit of detection for adenovirus is 10. 2 With a capacity of approximately 1000 μL, RPA is essentially identical to commercially available quantitative PCR kits, offering a low detection limit that greatly meets clinical needs. Furthermore, the entire detection process takes only 30-40 minutes. Compared to traditional PCR, RPA offers advantages such as ease of operation and the elimination of complex thermal cycling equipment, making it suitable for rapid on-site testing. In addition, this invention can simultaneously detect multiple diarrhea viruses, significantly improving detection efficiency and reducing potential missed diagnoses due to different pathogen types. Results are displayed through color changes or band patterns, providing a clear and intuitive understanding, greatly increasing its applicability and convenience in various medical environments. It can be widely applied in primary healthcare institutions, and even in mobile clinics and home healthcare settings, effectively improving the level of medical diagnosis in these areas.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus, characterized in that, The specific primer pairs designed for conserved genes of three pathogens—norovirus type II, rotavirus A, and adenovirus—have the following sequences: Type II norovirus ORF1 / ORF2 primer pair: ORF1 / ORF2-F: 5'-FITC-TTTACGTGCCCAGACAAGAGCCAATGTTCA-3'; ORF1 / ORF2-R:5'-TAGATAGATAGATAGA / iSpC12 / GTCACTCGACGCCATCTTCATTCACAAAAC-3'; Group A rotavirus VP6 gene primer pair: VP6-F: 5'-FITC-GAACGGAAATGACTTTCAAACTGGAGGAA-3'; VP6-R: 5'-CATTCTGCTTCCAAGT / iSpC12 / CTCAATCGTAGTTCTGGCGGTCTCAACAT-3'; Adenovirus Hexon gene primer pair: Hexon-F:5'-FITC-GAACTACATTGGCTTTAGGGATAACTTCATCG-3'; Hexon-R:5'-TCGACTGAGAATTGAC / iSpC12 / AACTGTCAACAGCCTGATTCCACATAGAA-3'.

2. The primer according to claim 1, characterized in that, In the downstream primer R of the three primer pairs, the 5' end is a labeled nucleic acid sequence. The nucleic acid sequence is designed with a C12 backbone structure modification between it and the adjacent unmodified sequence, namely the iSpC12 spacer, to prevent primer dimer formation.

3. The primer according to claim 1, characterized in that, The ORF1 / ORF2 primer pair, VP6 gene primer pair, and Hexon gene primer pair are used for the detection of type II norovirus, group A rotavirus, and adenovirus, respectively.

4. The use of a primer as described in any one of claims 1 to 3 in the preparation of a product for detecting infectious diarrhea-associated viruses, wherein the infectious diarrhea-associated viruses include type II norovirus, group A rotavirus, and adenovirus.

5. A probe for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus, characterized in that, The sequences of the ORF1 / ORF2 capture probe, VP6 capture probe, and Hexon capture probe are as follows: ORF1 / ORF2 capture probe: TCTATCTATCTATCTATTTTTT-Biotin; VP6 capture probe: ACTTGGAAGCAGAATGTTTTTT-Biotin; Hexon capture probe: GTCAATTCTCAGTCGATTTTTT-Biotin.

6. The probe according to claim 5, characterized in that, The ORF1 / ORF2 capture probe, VP6 capture probe, and Hexon capture probe are detection line probes designed for type II norovirus, group A rotavirus, and adenovirus, respectively, so that the amplification products can bind to the capture probes during the test strip chromatography process, thereby making the detection line colored.

7. The application of the probe as described in claim 5 or 6 in the preparation of a product for detecting infectious diarrhea-associated viruses, wherein the infectious diarrhea-associated viruses include norovirus type II, rotavirus group A, and adenovirus.

8. A kit for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus, characterized in that, Include: The primers as described in any one of claims 1 to 3; The probe as described in claim 5 or 6; Lateral flow immunochromatographic strips; RPA amplification reagents; Detection system for amplified product loading chromatography strips.

9. The reagent kit according to claim 8, characterized in that, The lateral flow immunochromatographic strip consists of a sample pad, a gold-labeled pad, an NC membrane, an absorbent pad, and a base plate. The sample pad is used to load the sample, the gold-labeled pad is used to store the AuNP-Ab conjugate, the NC membrane is used to fix the detection line and the control line, the absorbent pad is used to collect the aqueous phase and provide driving force for the flow of the reaction solution on the test strip, and the base plate serves as a backing to assemble the sample pad, gold-labeled pad, NC membrane, and absorbent pad into one unit.

10. A method for simultaneous detection of type II norovirus, group A rotavirus, and adenovirus for non-diagnostic purposes, characterized in that, Using the kit described in claim 8 or 9, the method is as follows: (1) Using the RNA of the sample to be tested as a template, the primers and RPA amplification reagents are used to form an RPA amplification system for RPA amplification; (2) Fix the probe to the NC membrane detection line position of the side-flow immunochromatographic strip; (3) The RPA amplification product obtained in step (1) is combined with the loading buffer, and the infection type of type II norovirus, group A rotavirus and adenovirus in the sample is determined by the detection results of the side-flow immunochromatographic strip detection system.

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