A method for detecting GI and GII norovirus on-site typing

Abstract

The application discloses a method for GI and GII type norovirus on-site typing detection, comprising the following steps: in the presence of primers, carrying out double RT-RPA reaction on a template, then adding the amplification product after reaction into a CRISPR / Cas12a detection system of GI and GII type norovirus respectively, carrying out fluorescence analysis, and completing GI and GII type norovirus on-site typing detection. The application covers each subtype of norovirus GI and GII, has no cross reaction with common gastroenteritis viruses such as rotavirus and enterovirus, can effectively distinguish the two genotypes of GI and GII, and has no off-target phenomenon, and the minimum detection limit of the application for the GI and GII genotype norovirus is 10 2 copies / µL and 1 copies / µL respectively. The whole detection process can be completed within 35 min under the condition of 37 DEG C constant temperature, can be directly used for on-site detection by means of an ultraviolet flashlight, and does not need laboratory instruments and professional operation.

Description

Technical Field

[0001] This invention pertains to biotechnology and relates to a method for on-site typing and detection of norovirus types GI and GII. Specifically, it relates to a method using dual RT-RPA combined with CRISPR / Cas12a technology for on-site typing and detection of norovirus types GI and GII. Background Technology

[0002] Norovirus (NoV) belongs to the genus Norovirus in the family Caliciviridae and is one of the major pathogens causing nonbacterial gastroenteritis in humans [Randazzo W, D'souza DH, Sanchez G. Norovirus: The Burden of the Unknown[J]. Adv Food Nutr Res, 2018, 86: 13-53]. Norovirus has diverse transmission routes, low infectious doses, long viral shedding time, strong environmental resistance, rapid viral mutation, and short duration of immune protection, making it highly infectious and capable of rapid transmission. Norovirus (Norovirus) contains three open reading frames (ORFs), encoding non-structural proteins, the capsid protein (Virus particle 1, VP1), and microstructural proteins, respectively. Based on the nucleic acid sequences of the norovirus capsid protein region and RNA polymerase (RdRp) region, Norovirus can be classified into 10 genotypes. Genotypes GI and GII are the most common infecting humans, with GII being the most prevalent worldwide. Compared to GI norovirus, GII is considered more likely to cause severe diarrhea. Currently, there are no effective drugs or vaccines against norovirus; early detection and diagnosis are the most effective ways to control norovirus outbreaks.

[0003] Currently, the main detection methods for norovirus include electron microscopy, immunological methods, and molecular biological detection techniques. Among them, electron microscopy and immunological methods have poor specificity and low sensitivity, and are prone to false positives and false negatives [Morillo S G, Luchs A, Cilli A, et al. Norovirus 3rd Generation kit: an improvement for rapid diagnosis of sporadic gastroenteritis cases and valuable for outbreak detection[J]. J Virol Methods, 2011, 173(1): 13-6]. With the rapid development of molecular biology, a variety of sensitive and specific detection methods have been established. Among them, qPCR is regarded as the gold standard for virus detection, with high specificity and sensitivity, and is suitable for laboratory testing. POCT technology is of great value in the handling of sudden outbreaks of infectious diseases. It can overcome the limitations of existing detection technologies on personnel and locations, shorten the testing time, improve convenience, promote the forward and downward movement of diagnosis, and realize the rapid diagnosis of suspected patients and on-site screening of close contacts. Some isothermal amplification methods, such as RT-LAMP and RT-RPA, have reaction conditions suitable for point-of-care testing (POCT), but due to the low reaction temperature, they are prone to non-specific amplification, leading to false positives. CRISPR (clustered regularly interspaced short palindromic repeats), initially known for its use in gene editing, has recently been applied to nucleic acid detection. CRISPR / Cas nucleic acid detection systems have low sensitivity when used alone and often require pre-amplification steps to improve sensitivity. Currently, biosensor systems combining pre-amplification with CRISPR / Cas technology can be broadly categorized into the SHERLOCK platform for detecting RNA targets and the HOLMES and DETECTR platforms for detecting DNA targets. Existing technology has established a detection method for norovirus GII.4 genotype based on CRISPR / Cas12a, but the detection limit needs improvement, and because it can only detect one subtype of the GII genotype, this method is difficult to use for assessing norovirus outbreaks. Summary of the Invention

[0004] This invention combines RT-RPA isothermal amplification technology with a CRISPR / Cas nucleic acid detection system to develop a POCT technology platform suitable for RNA typing detection.

[0005] The present invention adopts the following technical solution:

[0006] A method for on-site typing detection of norovirus types GI and GII includes the following steps: in the presence of primers, the template is subjected to a dual RT-RPA reaction, and then the amplification products after the reaction are added to the CRISPR / Cas12a detection system for norovirus types GI and GII, respectively, followed by fluorescence analysis to complete the on-site typing detection of norovirus types GI and GII.

[0007] In this invention, the primer sequences are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0008] In this invention, the template is a plasmid template or a nucleic acid template.

[0009] In this invention, the dual RT-RPA reaction uses a 50 μL system, with primer concentrations ranging from 100 nM to 700 nM. Preferably, the concentrations of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:3, and SEQ ID NO:6 are 100 nM to 300 nM; these concentrations are final concentrations. The template solution is 3–8 μL, with a concentration of 1–10 μL. 10 Copy / µL, preferably, the template solution is 4–6 µL, and the concentration of the template solution is 1–10. 7 Copy / µL. The dual RT-RPA reaction also includes standard reaction components such as buffer, water, magnesium acetate, and reverse transcriptase.

[0010] In this invention, the temperature of the dual RT-RPA reaction is 37°C, and the time is 15 to 30 minutes, preferably 20 to 25 minutes.

[0011] In this invention, the CRISPR / Cas12a system is a 10 µL system, comprising crRNA, ssDNA probe, amplification product, and other conventional reagents. In the CRISPR / Cas12a detection system for G1 type norovirus, the crRNA sequence is SEQ ID NO:7; in the CRISPR / Cas12a detection system for G2 type norovirus, the crRNA sequence is SEQ ID NO:8. The ssDNA probe sequence is SEQ ID NO:9, consistent for both G1 and G2 types. Preferably, in the CRISPR / Cas12a system, the concentration of Cas12a is 200–300 nM, the concentration of crRNA is 400–600 nM, the concentration of the ssDNA probe is 3–5 µM, and the amplification product is 1–2 µL; all concentrations represent the final concentration of each component in the 10 µL CRISPR / Cas12a system.

[0012] In this invention, fluorescence analysis is performed using a fluorescence PCR instrument. Specifically, the reaction is carried out at 37°C for 15 minutes, and the fluorescence intensity is measured every 10 seconds. Based on the fluorescence intensity curve, the on-site typing detection of type GI and type GII norovirus is completed.

[0013] In this invention, fluorescence analysis is performed using a conventional PCR instrument. After the reaction is completed, images are taken using an imaging instrument or under ultraviolet light to complete the on-site typing detection of type GI and type GII norovirus.

[0014] This invention combines RT-RPA isothermal amplification technology with a CRISPR / Cas nucleic acid detection system to develop a point-of-care testing (POCT) platform suitable for RNA genotyping. This invention successfully and sensitively detects norovirus GI and GII genotypes within 35 minutes at a constant temperature of 37°C, with clinical sample results consistent with the RT-qPCR method. The detection targets of this invention cover all subtypes of norovirus GI and GII, which is of great significance for the timely confirmation and control of norovirus outbreaks. Furthermore, direct diagnosis of norovirus GI and GII genotypes can provide more accurate diagnostic and treatment information for clinicians. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the dual RT-RPA / CRISPR / Cas detection method of the present invention.

[0016] Figure 2 To assess the susceptibility of single and double RPA assays for GI and GII noroviruses, plasmids serially diluted 10-fold were used as templates for single or double RPA reactions. The RPA products were purified and analyzed by agarose gel electrophoresis. (a) GI.1 plasmid as template (10-fold dilution) 5 10 4 10 3 10 2 (a) Single RPA was performed using 10,1 copies / µL; (b) GII.3 type plasmid was used as template (10,1 copies / µL) 5 10 4 10 3 10 2 (c) Single RPA was performed using GI.1 (left) and GII.3 (right) plasmids as templates (10, 1 copies / µL); 4 10 3 10 2 Single-weight RPA was performed using 10, 1 copies / µL. Specific RPA products are marked with a red dashed box; NC, template-free negative control; M, DNA marker.

[0017] Figure 3To assess the sensitivity of dual RPA-CRISPR / Cas12a for the detection of GI and GII noroviruses, dual RPA reactions were performed using serially diluted plasmids as templates at 37°C for 20 min using a standard PCR instrument. The amplified products were then added to GI and GII type-specific CRISPR / Cas12a detection systems and reacted at 37°C for 15 min using a Q160 fluorescence PCR instrument. Immediately after the reaction, the reaction tubes were photographed under UV light. The experiment was repeated three times. The figure shows the endpoint fluorescence detection results of the CRISPR / Cas12a system after 15 min of reaction under UV light (top) and fluorescence PCR instrument detection (bottom). A two-tailed t-test was used to analyze the endpoint fluorescence detection results measured by the fluorescence PCR instrument. Asterisks indicate the significance level compared to the NC control group (***). P <0.001, **** P <0.0001). ns, no statistical significance. NC, no template negative control.

[0018] Figure 4 To ensure the specificity of the dual RT-RPA-CRISPR / Cas12a assay, extracted viral sample nucleic acid was used as a template for dual RT-RPA reactions. These reactions were performed using a standard PCR instrument at 37°C for 20 min. The amplified products were then added to GI and GII type-specific CRISPR / Cas12a detection systems, respectively, and reacted using a Q160 fluorescence PCR instrument at 37°C for 15 min. Immediately after the reaction, the reaction tubes were placed under UV light for imaging. The experiment was repeated three times. The figure shows the endpoint fluorescence detection results of the CRISPR / Cas12a system after 15 min of reaction under UV light (top) and fluorescence PCR instrument detection (bottom). A two-tailed t-test was used to analyze the endpoint fluorescence detection results measured by the fluorescence PCR instrument. Asterisks indicate the level of significance compared to the NC control group (****). P <0.0001). RV, rotavirus; EAdv, enteric adenovirus; NC, template-free negative control.

[0019] Figure 5 Results of single-phase RPA primer experiments for norovirus types GI and GII. All primers used in the single-phase RPA system were at a final concentration of 200 nM. A standard PCR instrument was used, and the reaction was carried out at 37°C for 20 min. After purification of the RPA products, the amplification effect of the primers was analyzed by agarose gel electrophoresis. The red dashed boxes indicate specific RPA products; M stands for DNA marker. A4: II-F1 / II-R1, C3: II-F3 / II-R3, C4: II-F4 / II-R3, D2: I-F5 / I-R1, D6: II-F4 / I-R3.

[0020] Figure 6 Results of dual RPA primer experiments for norovirus types GI and GII. All primers used in the dual RPA system were at a final concentration of 200 nM. A standard PCR instrument was used, and the reaction was carried out at 37°C for 20 min. The RPA products were purified and analyzed by agarose gel electrophoresis to determine the amplification efficiency. The red dashed boxes indicate specific RPA products; M stands for DNA marker. C3: II-F3 / II-R3, C4: II-F4 / II-R3, D2: I-F5 / I-R1.

[0021] Figure 7 To optimize the RT-RPA system, the CRISPR / Cas12a system was used to analyze the RT-RPA products. The CRISPR / Cas12a reaction was performed in a Q160 fluorescence PCR instrument at 37°C for 15 min, and the endpoint fluorescence signal measured by the fluorescence PCR instrument at 15 min was counted. (a) Screening of GI RT primers. Using the extracted GI norovirus sample (Ct value 30) nucleic acid as a template, GI single RT-RPA system containing different RT primers (I-RT-1, I-RT-2 and I-RT-3) was added, and the reaction was carried out at 37°C for 20 min. The amplification products were then added to a GI-type specific CRISPR / Cas12a system for detection. (b) Screening of GII RT primers. Using extracted GII norovirus sample (Ct value 35) nucleic acid as a template, a GII single RT-RPA system containing different RT primers (II-RT-1, II-RT-2, and II-RT-3) was added. The reaction was carried out at 37°C for 20 min, and the amplification product was added to a GII-type specific CRISPR / Cas12a system for detection. (c) Optimization of primer concentrations in the dual RT-RPA reaction system. Using extracted GI (Ct value 30) and GII norovirus sample (Ct value 35) nucleic acids as templates, dual RT-RPA systems with different primer concentration combinations (combinations A, B, C, and D) were added. The reaction was carried out at 37°C for 20 min, and the amplification product of the GI (GII) sample was added to a GI (GII)-type specific CRISPR / Cas12a system for detection. A, B, C, and D represent four different primer combinations. The above experiments were repeated three times, and the results are expressed as mean ± standard deviation.

[0022] Figure 8 Optimization for the CRISPR / Cas12a system. Using the GII.4 subtype (10 6The CRISPR / Cas12a system was optimized using a dual RPA product (copies / µL) as an activator. The initial CRISPR / Cas12a reaction system consisted of: 250 nM cas12a, 500 nM crRNA, 2 µM ssDNA probe (6-FAM-TTTTTTTT-BHQ1), 1 µL 10×NEB Buffer 2.1, 1.5 µL amplification product, and nuclease-free water to bring the total to 10 µL. A Q160 fluorescence PCR instrument was used, and the reaction was carried out at 37℃ for 15 min. The endpoint fluorescence signal measured by the fluorescence PCR instrument at 15 min was recorded. (a) Optimization of Cas12a concentration. With other initial conditions unchanged, three different concentrations of cas12a (125, 250, and 500 nM) were set. (b) Optimization of ssDNA probe concentration. Five groups of ssDNA probes with different concentrations (1, 2, 3, 4, and 5 µM) were set up with Cas12a concentration of 250 nM and other initial conditions unchanged. The above experiments were repeated three times, and the results are expressed as mean ± standard deviation.

[0023] Figure 9 To detect GI and GII noroviruses in human fecal samples using a dual RT-RPA-CRISPR / Cas12a and RT-qPCR method. (a) Using extracted viral nucleic acid as a template, a dual RT-RPA system was added and reacted at 37°C for 20 min. The figure shows the agarose gel electrophoresis results of the purified amplified products. (b) The unpurified RT-RPA products from (a) were added to the GI and GII type CRISPR / Cas12a systems, respectively, and reacted at 37°C for 15 min using a Q160 fluorescence PCR instrument. The fluorescence intensity was measured every 10 s for real-time fluorescence detection. (c) The endpoint fluorescence signal measured by the fluorescence PCR instrument at 15 min of reaction was counted. (d) The extracted viral nucleic acid was detected by RT-qPCR using a norovirus nucleic acid assay kit (fluorescent PCR method). S1-S7, clinical samples 1-7. NC, negative control without template. Detailed Implementation

[0024]

[0025] This invention combines RT-RPA isothermal amplification technology with a CRISPR / Cas nucleic acid detection system to develop a POCT technology platform suitable for RNA genotyping detection. See also Figure 1This invention successfully and sensitively detects norovirus GI and GII genotypes within 35 minutes at a constant temperature of 37°C. The specific experimental methods used are conventional techniques, including nucleic acid extraction, plasmid construction, RPA reaction, and fluorescence analysis. Except for the primer sequences disclosed in this invention, all components involved in the reaction are existing products.

[0026] Nucleic acid extraction. Human anal swab samples were provided by Suzhou Center for Disease Control and Prevention, including norovirus types GI and GII (GI.1, GII.P12-GII.3, GII.P16-GII.4, GII.P16-GII.2, GII.13, etc.) and other viruses (rotavirus, enteric adenovirus, as negative controls). Viral nucleic acid was extracted according to the instructions of the viral genomic DNA / RNA extraction kit (Tiangen), and the nucleic acid was stored at -80°C.

[0027] RT-qPCR. Clinical samples were tested using a norovirus nucleic acid assay kit (fluorescent PCR method). The RT-qPCR assay was performed on a Q160 fluorescence PCR instrument, with the system configuration and program settings following the instruction manual. Cyclic parameters were set as follows: 50°C for 30 min (reverse transcription), 95°C for 10 min, 45 cycles: 95°C for 10 sec, 55°C for 40 sec. Single-point fluorescence detection was performed at 55°C.

[0028] Plasmid template construction. The junction of the norovirus RdRp gene and the VP1 gene was selected as the detection target. Plasmid templates were constructed based on the norovirus nucleic acid sequences of subtypes GII.4 (GenBank accession number GU991353.1, 4968-5228), GII.3 (GenBank accession number KF306213.1, 4906-5241), and GII.1 (GenBank accession number MH638229.1, 5169-5515), using standard techniques.

[0029] Example 1

[0030] 1. Single RPA reaction, using a 50 μL system.

[0031] Mix 1 μL primer II-F4 (10 µM), 1 μL primer II-R3 (10 µM), 29.5 μL Primer Free Rehydration buffer, and 11 μL nuclease-free water, then add the mixture to the main reaction reagent (TwistAmp® Basic kit - TwistAmp® basic reaction) and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL plasmid template (GII.4 or GII.3 subtype), and react at 37°C for 20 min.

[0032] Mix 1 μL primer I-F5 (10 µM), 1 μL primer I-R1 (10 µM), 29.5 μL Primer Free Rehydration buffer, and 11 μL nuclease-free water, add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL plasmid template (GI.1 subtype), and react at 37 °C for 20 min.

[0033] 2. Double RPA reaction, using a 50μL system.

[0034] Mix 1 μL primer II-F4 (10 µM), 1 μL primer II-R3 (10 µM), 1 μL primer I-F5 (10 µM), 1 μL primer I-R1 (10 µM), 29.5 μL Primer Free Rehydration buffer, and 9 μL nuclease-free Water, then add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL plasmid template (GII.4 or GII.3 subtype), and react at 37 °C for 20 min.

[0035] Mix 1 μL primer II-F4 (10 µM), 1 μL primer II-R3 (10 µM), 1 μL primer I-F5 (10 µM), 1 μL primer I-R1 (10 µM), 29.5 μL Primer Free Rehydration buffer, and 9 μL nuclease-free Water, then add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL plasmid template (GI.1 subtype), and react at 37 °C for 20 min.

[0036] 3. CRISPR / Cas12a reaction: At the final concentration, use 250 nM cas12a, 500 nM crRNA, 4 µM ssDNA probe (6-FAM-TTTTTTTT-BHQ1), 1 µL 10×NEB Buffer 2.1, and 1.5 µL of amplification product. Bring the total volume to 10 µL with nuclease-free water. Use a Q160 fluorescence PCR instrument at 37℃ for 15 min, measuring fluorescence intensity every 10 s. In the CRISPR / Cas12a detection system for type GI norovirus, the crRNA sequence is SEQ ID NO:7; in the CRISPR / Cas12a detection system for type GII norovirus, the crRNA sequence is SEQ ID NO:8.

[0037] 4. Sensitivity

[0038] Dilute the plasmid template to 10 using DEPC-concentrated water. 5 10 4 10 3 10 2 10 1 A gradient concentration of 1 copy / µL was used. Norovirus plasmids of GI.1 or GII.3 subtypes at the above gradient concentrations were used as templates for single-component and double-component RPA reactions, respectively. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis. This invention tested the amplification effect of the primer system applied to the double-component RPA system, performed sensitivity experiments, and compared the results with those of the single-component RPA reaction. The results showed that the single-component RPA reaction achieved a detection sensitivity of 1 copy / µL for both GI and GII type plasmid templates. Figure 2 (a, b), while the detection sensitivities of the dual RPA reaction for GI and GII are 10, respectively. 2 copies / µL and 1 copy / µL ( Figure 2 c).

[0039] Dilute the plasmid template to 10 using DEPC-concentrated water. 4 10 3 10 2 10 1Gradient concentrations of 1 copy / µL were used. The GI.1 or GII.4 subtype norovirus plasmids at these gradient concentrations were used as templates for duplex RT-RPA. The amplified products were added to GI and GII norovirus CRISPR / Cas12a systems, respectively. A Q160 fluorescence PCR instrument was used, and the reaction was carried out at 37℃ for 15 min, with fluorescence intensity measured every 10 s. The results showed that the detection limits for GI and GII norovirus using this invention were 10 copies / µL. 2 copies / µL and 1 copy / µL ( Figure 3 ).

[0040] 5. Specificity

[0041] Nucleic acids from norovirus (G1), norovirus (G2), rotavirus, and adenovirus samples were used in a dual RT-RPA-CRISPR / Cas12a system (with the nucleic acid template replacing the plasmid template) to analyze specificity. Figure 4 As shown, the specific CRISPR / Cas12a system for GI and GII noroviruses only produces effective fluorescent detection signals for the dual RT-RPA amplification products of the target sample. It does not cross-react with common gastroenteritis viruses such as rotavirus and enteric adenovirus, and can effectively distinguish between the GI and GII genotypes without off-target effects. This indicates that the present invention has the specificity for genotyping detection of GI and GII noroviruses.

[0042] Comparative Example 1

[0043] With subtypes GII.4 and GII.3 (10 6 Using copies / µL) as templates and II-F1 / II-R1 (A4) as primers, a single RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0044] With subtypes GII.4 and GII.3 (10 6 Using copies / µL as templates and II-F3 / II-R3 (C3) as primers, a single RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0045] With subtypes GII.4 and GII.3 (10 6 Using copies / µL as templates and II-F4 / II-R3 (C4) as primers, a single RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0046] With GI.1 subtype (10 6Using copies / µL as templates and I-F5 / I-R1 (D2) as primers, a single RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0047] With GI.1 subtype (10 6 Using copies / µL) as templates and II-F4 / I-R3 (D6) as primers, a single RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0048] The gelation results for the above single RPA reaction (all primers were concentrated at a final concentration of 200 nM, and the rest were consistent with the above) can be found in [link to documentation]. Figure 5 .

[0049] Comparative Example 2

[0050] With subtype GII.4 (10 3 Using copies / µL as templates and II-F4 / II-R3 (C4) and I-F5 / I-R1 (D2) as primers, a double RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0051] With GI.1 subtype (10 3 Using copies / µL as templates and II-F4 / II-R3 (C4) and I-F5 / I-R1 (D2) as primers, a double RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0052] With subtype GII.4 (10 3 Using copies / µL) as templates and II-F3 / II-R3 (C3) and I-F5 / I-R1 (D2) as primers, a double RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0053] With GI.1 subtype (10 3 Using copies / µL) as templates and II-F3 / II-R3 (C3) and I-F5 / I-R1 (D2) as primers, a double RPA reaction was performed. The amplified products were purified (phenol:chloroform = 1:1) and analyzed by gel electrophoresis.

[0054] The gelation results for the above double RPA reaction (all primers were concentrated at a final concentration of 200 nM, and the rest were consistent with the above) can be found in [link to documentation]. Figure 6 As can be seen, the primers specified in this invention achieve significantly better technical results.

[0055] Primer design follows certain experimental principles, such as RPA primer length of 30-35 bp and amplicon length of 80-150 bp. However, conventional methods yield many primers that meet these principles based on the site, but the technical effects of these primers are unpredictable. This invention limits the primers to II-F4 / II-R3 (C4) and I-F5 / I-R1 (D2). The results of the dual RPA reaction show that the product bands are relatively clear and without tailing, achieving unexpected technical progress compared to other primers.

[0056] Using routinely extracted clinical sample nucleic acid (RT-qPCR detection CT value) as a template, double (single) RT-RPA reactions were performed, and the amplification products were added to the CRISPR / Cas12a detection system for G1 and G2 noroviruses, respectively. The reaction was carried out at 37℃ for 15 min using a Q160 fluorescence PCR instrument, and the fluorescence intensity was measured every 10 s.

[0057] Example 2: Detection of clinical samples (single RT-RPA-CRISPR / Cas12a)

[0058] Mix 2.5 μL primer II-F4 (10 µM), 1.5 μL primer II-R3 (10 µM), 3 μL primer II-RT-2 (10 µM), 29.5 μL Primer Free Rehydration buffer, 5.5 μL nuclease-free water, and 0.5 μL M-MuLV Reverse Transcriptase (200 U / µL, NEB), add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL nucleic acid template (GII subtype), and react at 37°C for 20 min.

[0059] Mix 2.5 μL primer I-F5 (10 µM), 1.5 μL primer I-R1 (10 µM), 3 μL primer I-RT-2 (10 µM), 29.5 μL Primer Free Rehydration buffer, 5.5 μL nuclease-free water, and 0.5 μL L-MuLV Reverse Transcriptase (200 U / µL, NEB), add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL nucleic acid template (GI subtype), and react at 37°C for 20 min.

[0060] Comparative Example 3: Testing of clinical samples (single-weighted RT-RPA-CRISPR / Cas12a)

[0061] Mix 2.5 μL primer II-F4 (10 µM), 1.5 μL primer II-R3 (10 µM), 3 μL primer II-RT-1 or II-RT-3 (10 µM), 29.5 μL Primer Free Rehydration buffer, 5.5 μL nuclease-free Water, and 0.5 μL M-MuLV Reverse Transcriptase (200 U / µL, NEB), add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL nucleic acid template (GII subtype), and react at 37°C for 20 min.

[0062] Mix 2.5 μL primer I-F5 (10 µM), 1.5 μL primer I-R1 (10 µM), 3 μL primer I-RT-1 or I-RT-3 (10 µM), 29.5 μL Primer Free Rehydration buffer, 5.5 μL nuclease-free Water, and 0.5 μL M-MuLV Reverse Transcriptase (200 U / µL, NEB), add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL nucleic acid template (GI subtype), and react at 37°C for 20 min.

[0063] The above single RT-RPA products were subjected to a CRISPR / Cas12a reaction: 250 nM cas12a, 500 nM crRNA, 4 µM ssDNA probe (6-FAM-TTTTTTTT-BHQ1), 1 µL 10×NEB Buffer 2.1, 1.5 µL of amplified product, and nuclease-free water was added to bring the total to 10 µL. A Q160 fluorescence PCR instrument was used, and the reaction was carried out at 37℃ for 15 min, with fluorescence intensity measured every 10 s. In the CRISPR / Cas12a detection system for G1 norovirus, the crRNA sequence is SEQ ID NO:7; in the CRISPR / Cas12a detection system for G2 norovirus, the crRNA sequence is SEQ ID NO:8. Figure 7 a, Figure 7b represents the test results for GI and GII subtype templates, and the fluorescence intensity at 15 min.

[0064] Example 3: Detection of clinical samples (dual RT-RPA-CRISPR / Cas12a)

[0065] A mixture of 1 μL primer II-F4 (10 µM), 1 μL primer II-R3 (10 µM), 1 μL primer I-F5 (10 µM), 1 μL primer I-R1 (10 µM), 1 μL primer II-RT-2 (10 µM), 1 μL primer I-RT-2 (10 µM), 29.5 μL primer-free rehydration buffer, 6.5 μL nuclease-free water, and 0.5 μL M-MuLVReverse Transcriptase (200 U / µL, NEB) was added to the main reaction reagent and allowed to dissolve completely. Finally, 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL nucleic acid template were added, and the reaction was carried out at 37 °C for 20 min. The nucleic acid template was of subtype GII or subtype GI.

[0066] By changing the concentration of the above primers, other dual RT-RPA products were obtained.

[0067] The above dual RT-RPA products were subjected to a CRISPR / Cas12a reaction: 250 nM cas12a, 500 nM crRNA, 4 µM ssDNA probe (6-FAM-TTTTTTTT-BHQ1), 1 µL 10×NEB Buffer 2.1, 1.5 µL of amplified product, and nuclease-free water was added to bring the total to 10 µL. A Q160 fluorescence PCR instrument was used, and the reaction was carried out at 37℃ for 15 min, with fluorescence intensity measured every 10 s. In the CRISPR / Cas12a detection system for G1 norovirus, the crRNA sequence was SEQ ID NO:7; in the CRISPR / Cas12a detection system for G2 norovirus, the crRNA sequence was SEQ ID NO:8. Figure 7 c represents the test results of GI and GII subtype templates at different primer concentrations, and the fluorescence intensity at 15 min.

[0068] Example 4: Detection of plasmid template

[0069] Mix 1 μL primer II-F4 (10 µM), 1 μL primer II-R3 (10 µM), 1 μL primer I-F5 (10 µM), 1 μL primer I-R1 (10 µM), 29.5 μL Primer-Free Rehydration buffer, 8.5 μL nuclease-free Water, and 0.5 μL M-MuLV Reverse Transcriptase (200 U / µL, NEB), add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL GII.4 isotype (10 µM). 6 Using copies / µL as templates, the reaction was carried out at 37℃ for 20 min.

[0070] The above-mentioned dual RPA products were subjected to a CRISPR / Cas12a reaction: 250 nM cas12a, 500 nM crRNA, 2 µM ssDNA probe (6-FAM-TTTTTTTT-BHQ1), 1 µL 10×NEB Buffer 2.1, 1.5 µL of amplified product, and nuclease-free water was added to bring the total to 10 µL. A Q160 fluorescence PCR instrument was used, and the reaction was carried out at 37℃ for 15 min, with fluorescence intensity measured every 10 s. In the CRISPR / Cas12a detection system for G1 norovirus, the crRNA sequence is SEQ ID NO:7; in the CRISPR / Cas12a detection system for G2 norovirus, the crRNA sequence is SEQ ID NO:8.

[0071] By varying the concentration of cas12a in the above CRISPR / Cas12a system, detection results were obtained for other CRISPR / Cas12a systems. The results showed that the fluorescence signal was strongest at a cas12a concentration of 250 nM. Figure 8 a); By changing the ssDNA probe concentration in the above CRISPR / Cas12a system, detection results were obtained for other CRISPR / Cas12a systems. The results showed that the fluorescence signal reached saturation at an ssDNA probe concentration of 4 µM. Figure 8 b).

[0072] This invention adds specific RT primers and optimizes the concentration of each primer in the dual RT-RPA reaction, thereby improving the sensitivity of the dual RT-RPA-CRISPR / Cas12a system in detecting samples. In particular, the concentration of each primer at 200 nM can simultaneously produce obvious detection fluorescence for two clinical samples, GI (ct value 30) and GII (ct value 35).

[0073] Example 5: Dual RT-RPA-CRISPR / Cas12a detection of clinical samples

[0074] Mix 1 μL primer II-F4 (10 µM), 1 μL primer II-R3 (10 µM), 1 μL primer I-F5 (10 µM), 1 μL primer I-R1 (10 µM), 1 μL primer II-RT-2 (10 µM), 1 μL primer I-RT-2 (10 µM), 29.5 μL primer-free rehydration buffer, 6.5 μL nuclease-free water, and 0.5 μL M-MuLVReverse Transcriptase (200 U / µL, NEB), add the mixture to the main reaction reagent and allow it to dissolve completely. Finally, add 2.5 μL MgOAc aqueous solution (280 mM) and 5 μL template, and react at 37 °C for 20 min.

[0075] The CRISPR / Cas12a system consisted of the following components at final concentrations: 250 nM Cas12a, 500 nM crRNA, 4 µM ssDNA probe (6-FAM-TTTTTTTT-BHQ1), 1 µL 10×NEB Buffer 2.1, 1.5 µL of amplification product, and nuclease-free water to a final volume of 10 µL. The reaction was performed using a Q160 fluorescence PCR instrument at 37°C for 15 min, with fluorescence intensity measured every 10 s. After the reaction, images were taken using an imaging system or under UV light.

[0076] like Figure 9 As shown, the detection efficacy of the invented dual RT-RPA-CRISPR / Cas12a method on seven clinical samples of norovirus types GI and GII (ct values ​​ranging from 23 to 35) was evaluated and compared with the RT-qPCR method (Norovirus Nucleic Acid Assay Kit, Shanghai ZJ Bio-Tech Co., Ltd.). It can be seen that the sample detection results of the present invention and the RT-qPCR method are completely consistent, with two samples showing GI type and five showing GII type. Notably, sample number 7, a GII type sample with a ct value of 35, was also effectively detected by the present invention.

[0077] The sequences involved in the embodiments and comparative examples of this invention are shown in Table 1.

[0078]

[0079] Norovirus is one of the leading causes of acute gastroenteritis outbreaks worldwide. Currently, there are no effective antiviral drugs or commercial vaccines for NoVs. This means that point-of-care testing (POCT) for NoVs, enabling rapid diagnosis of suspected patients and on-site screening of close contacts, is crucial for controlling its spread.

[0080] This invention combines RT-RPA isothermal amplification technology with a CRISPR / Cas12a nucleic acid detection system to develop a POCT technology platform suitable for RNA detection. This improves the sensitivity of the entire detection reaction, enabling specific recognition and cleavage of target nucleic acids, providing specificity assurance for the entire reaction. After activating its auxiliary cleavage activity, it indiscriminately cleaves ssDNA in the system. By introducing FAM-modified single-stranded reporter DNA molecules into the reaction system, FAM fluorescence and UV flashlight can be used for on-site detection, eliminating the need for laboratory instruments and professional personnel for operation and result interpretation.

[0081] Due to the significant sequence differences among the subtypes of norovirus GI and GII, primer selection is a major challenge. This invention uses a dual RT-RPA preamplification system. The single RPA reaction achieves a detection sensitivity of 1 copies / µL for both GI and GII plasmid templates, while the dual RPA reaction achieves a detection sensitivity of 10 copies / µL for both GI and GII plasmid templates. 2 The 1 copy / µL and 1 copy / µL concentrations mitigate the problem of decreased amplification efficiency caused by the interaction between the two primer pairs in dual RPA. In existing technologies, both single and dual RT-RPA methods exhibit poor amplification efficiency for clinical samples, failing to effectively amplify samples with a ct value exceeding 30. This is related to low reverse transcription efficiency, as, similar to RT-PCR, reverse primers are used as reverse transcription primers in RT-RPA reactions. However, RPA primers are generally over 30 nt, and direct use for reverse transcription may lead to decreased efficiency. This invention adds specific primers and limits primer concentration, improving the sensitivity for detecting clinical samples, enabling the detection of clinical samples with a ct value as high as 35.

[0082] This invention fully utilizes the cis and trans-cleavage activities of the CRISPR / Cas12a system, effectively distinguishing between genotypes GI and GII without off-target effects. While the design positions of GI and GII crRNA and PAM sequences are not highly conserved, they can still be effectively detected. Common pathogens causing viral gastroenteritis, besides norovirus, include rotavirus and enteric adenovirus. This invention generates effective fluorescent detection signals only for GI and GII norovirus samples, showing no cross-reactivity with common gastroenteritis viruses such as rotavirus and enteric adenovirus. It effectively distinguishes between the GI and GII genotypes without off-target effects, with detection limits of 10 for GI and GII genotype norovirus, respectively. 2 The results were obtained using two methods: 1 copy / µL and 1 copy / µL. Seven clinically positive norovirus samples were tested using a dual RT-RPA-CRISPR / Cas12a method. The results were consistent with RT-qPCR. Notably, GII samples with a ct value of 35 were also effectively detected. Furthermore, fluorescence detection results showed that this method has a very low background fluorescence signal, allowing for visual differentiation between negative and positive results under UV conditions.

[0083] This invention is more suitable for POCT applications. First, M-MuLV Reverse Transcriptase is directly added to the RPA isothermal amplification system for a one-step RT-RPA reaction, eliminating the need for a separate step to reverse transcribe RNA into cDNA, thus simplifying the pre-amplification system. Second, the dual RT-RPA amplification system allows for dual amplification of GI and GII noroviruses in a single tube, saving both cost and time.

[0084] In summary, this invention successfully and sensitively detects norovirus genotypes GI and GII within 35 minutes at a constant temperature of 37°C, with clinical sample detection results consistent with the RT-qPCR method. This invention's dual RT-RPA-CRISPR / Cas12a detection method is a POCT technology platform suitable for RNA genotyping detection, applicable to high-traffic areas such as kindergartens, schools, nursing homes, and hotels, as well as emergency scenarios such as mobile vehicles, medical ships, makeshift hospitals, and disaster sites. Compared to other rapid norovirus detection methods, this method has a more comprehensive detection range, covering all subtypes of norovirus GI and GII, which is of great significance for norovirus epidemic prevention and control. Furthermore, this method can directly diagnose norovirus GI and GII genotypes, providing more accurate diagnostic information for clinical practice.

Claims

1. Primers for on-site typing detection of norovirus types GI and GII, characterized in that, The primer sequences are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

2. The application of the primers described in claim 1 in the preparation of a reagent for on-site typing detection of type GI and GII norovirus using dual RT-RPA combined with CRISPR / Cas12a.

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