A composition, kit, and method for simultaneous detection of seven G genotypes of group A rotavirus.

Abstract

This application relates to the technical field of virus detection, specifically to a composition, kit, and method for simultaneously detecting seven G genotypes of group A rotavirus. The composition comprises: components A-G for detecting upstream and downstream primers and probes for group A rotavirus G1, G2, G3, G4, G8, G9, and G12 genotypes; and component H for detecting the upstream and downstream primers and probes for human ribonucleoprotein complex. Detection using this composition or a kit containing it offers advantages such as high sensitivity and specificity, ease of operation, short detection time, small sample volume required, and low contamination. It can directly detect nucleic acids extracted from the sample to be tested, and has high application value in rapid virus detection.

Description

Technical Field

[0001] This application relates to the technical field of virus detection, and more particularly to a composition, kit, and method for simultaneously detecting group A rotavirus G1 genotype (RV G1), G2 genotype (RV G2), G3 genotype (RV G3), G4 genotype (RV G4), G8 genotype (RV G8), G9 genotype (RV G9), and G12 genotype (RV G12). Background Technology

[0002] Rotavirus belongs to the Reoviridae family. Its particle diameter is 70-75 nm, it is non-enveloped, has a double capsid, and contains 11 segments of double-stranded RNA. The rotavirus core RNA is approximately 18.5 kb in length, encoding 11-12 proteins, including 6 structural proteins (VP1-VP4, VP6, VP7) and 5 non-structural proteins (NSP1-NSP4, NSP5 / NSP6). There are 10 subtypes of rotavirus (rotavirus AJ), with rotavirus A accounting for over 90% of rotavirus infections. Among the 6 structural proteins, the glycoprotein VP7 and the protease-sensitive protein VP4 are located in the outer capsid, forming the basis of the rotavirus dual taxonomy.

[0003] In Group A Rotavirus (RVA), different G serotypes can be distinguished based on the antigenicity of VP7, with G1-G4 being the most prevalent in the population; different P serotypes can be distinguished based on the antigenicity of VP4, with P1A being the most common. In clinical diagnosis and differentiation, viral genotyping is mainly used as the diagnostic basis. The description of G serotype is consistent with that of genotype; the description of P serotype is inconsistent with that of genotype, and genotype is indicated by square brackets "[]" (e.g., serotype P1A is genotype P[8], P1B is genotype P[4], and P2A is genotype P[6]). Among them, the infection rate of rotaviruses of the 7 G types (i.e., G1, G2, G3, G4, G8, G9 and G12) and the 3 P types (P[4], P[6] and P[8]) is higher.

[0004] Rotavirus is one of the major pathogens causing childhood diarrhea worldwide. Group A rotavirus is one of the leading causes of death in children under 5 years old worldwide, and all children under 5 years old have been infected with RVA at least once. Based on the differences in VP7 and VP4 sequences of RVA, RVA is further divided into different genotypes. To date, 41 G genotypes and 57 P genotypes of RVA have been identified globally, of which 7 G types (G1, G2, G3, G4, G8, G9 and G12) and 3 P types (P[4], P[6] and P[8]) are dominant. The 6 major G / P combinations G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8] account for more than 90% of global RVA strains. Currently, except for G12P[8], the other 5 G / P combinations of RVA are relatively common in my country. G9P[8] is the most prevalent RVA strain in my country.

[0005] Currently, the main methods for typing the G genotype of human group A rotavirus include nested PCR and first-generation sequencing. However, these methods suffer from drawbacks such as long processing time, high cost, complex operation, and insufficient sensitivity. Existing patents rarely study the G genotype of group A rotavirus. Patent CN116479183A simultaneously detects seven G genotypes using a PCR amplification coupled with nucleic acid invasion reaction combined with gold nanoparticle colorimetry (PCR-Invader-AuNP), a molecular detection method capable of distinguishing single-base differences. Compared to traditional electrophoresis-based and sequencing-based typing methods, this method requires only a standard PCR instrument, no special equipment, is simpler to operate, requires less time, and the results are visually distinguishable. However, the technology used in this patent still has some limitations. First, its sensitivity is insufficient compared to quantitative fluorescence methods, and it may not be able to type some low concentrations of group A rotavirus. Second, the entire operation process is more cumbersome and time-consuming than quantitative fluorescence methods, making it unsuitable for general-purpose quantitative fluorescence instruments.

[0006] Therefore, there is a need to develop a detection method that is both highly sensitive and rapid for the seven G genotypes. Summary of the Invention

[0007] This application provides a composition, kit, and method for the simultaneous detection of seven G genotypes of group A rotavirus. The aim is to establish a highly sensitive and rapid detection method targeting all seven G genotypes (G1, G2, G3, G4, G8, G9, and G12), initially applied to the detection of group A rotavirus G genotypes.

[0008] This application performs combined detection on common group A rotavirus genotypes that infect humans: G1 genotype (RV G1), G2 genotype (RV G2), G3 genotype (RV G3), G4 genotype (RV G4), G8 genotype (RV G8), G9 genotype (RV G9), and G12 genotype (RVG12). The design selects conserved regions of the virus and specifically detects seven genotypes of group A rotavirus, while also being able to distinguish between the seven genotypes.

[0009] The composition provided in this application or the kit containing the composition can simultaneously identify viral pathogens with similar clinical symptoms caused by group A rotavirus infection of different G genotypes, and has high specificity and sensitivity, good reproducibility, and is simple, fast and cost-effective.

[0010] In a first aspect, this application provides a composition for simultaneously detecting seven G genotypes of group A rotavirus, employing the following technical solution:

[0011] A composition for simultaneously detecting seven G genotypes of group A rotavirus, the composition comprising the following components:

[0012] Component A: Upstream primer 1, downstream primer 1, and probe 1 for detecting group A rotavirus G1 genotype, which are respectively the nucleotide sequences shown in SEQ ID NO 1-3; MGB modification is performed on the 3' end of probe 1;

[0013] Component B: Upstream primer 2, downstream primer 2, and probe 2 for detecting group A rotavirus G2 genotype, respectively, are the nucleotide sequences shown in SEQ ID NO 4-6; the 3' end of probe 2 is modified with MGB;

[0014] Component C: Upstream primer 3, downstream primer 3, and probe 3 for detecting group A rotavirus G3 genotype, which are respectively the nucleotide sequences shown in SEQ ID NO 7-9;

[0015] Component D: Upstream primer 4, downstream primer 4, and probe 4 for detecting group A rotavirus G4 genotype, which are respectively the nucleotide sequences shown in SEQ ID NO 10-12;

[0016] Component E: Upstream primer 5, downstream primer 5, and probe 5 for detecting group A rotavirus G8 genotype, which are respectively the nucleotide sequences shown in SEQ ID NO 13-15; the 3' end of the probe 5 is modified with MGB;

[0017] Component F: Upstream primer 6, downstream primer 6, and probe 6 for detecting group A rotavirus G9 genotype, which are respectively the nucleotide sequences shown in SEQ ID NO 16-18;

[0018] Component G: Upstream primer 7, downstream primer 7, and probe 7 for detecting group A rotavirus G12 genotype, which are respectively the nucleotide sequences shown in SEQ ID NO 19-21; the 3' end of the probe 7 is modified with MGB;

[0019] Component H: upstream primer 8, downstream primer 8 and probe 8 used for detecting human ribonucleoprotein complex, which are respectively the nucleotide sequences shown in SEQ ID NO 22-24.

[0020] This application selects conserved regions of the genomic sequences of seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus to design upstream and downstream primers and probes, respectively. The detection primers and probes for the human ribonucleoprotein complex (RNP) are used as internal reference genes in the detection composition to monitor the entire process of sample collection, extraction, and detection. After analyzing the possible dimers and secondary structures of the primers and probes, suitable sequences were selected to form a composition capable of simultaneously detecting the seven G genotypes of group A rotavirus and the internal reference gene. Using this composition or a kit containing this composition, seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) causing similar clinical symptoms due to group A rotavirus infection can be simultaneously identified, exhibiting high specificity and sensitivity, good reproducibility, and accurate and reliable detection results.

[0021] The principle of using the above composition to detect seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) in this application is as follows:

[0022] Taking component B as an example, upstream primer 2, downstream primer 2, and probe 2 in component B can all specifically bind to the RV G2 genomic template, and the binding site of probe 2 is located between the two primers. When the probe is intact, the fluorescence energy emitted by the fluorescent reporter group is absorbed by the quencher group, and the instrument cannot detect the signal. During RT-PCR, if RV G2 is present in the sample, upstream primer 2, downstream primer 2, and probe 2 all bind to the RV G2 genomic template. As RT-PCR proceeds, Taq polymerase encounters probe 2 bound to the template during chain extension, and its 3'-5' exonuclease activity cleaves probe 2. The fluorescent reporter group is far from the quencher group, and its energy cannot be absorbed, thus generating a fluorescence signal. Moreover, after each PCR cycle, the fluorescence signal, like the target fragment, undergoes a synchronous exponential growth process. Therefore, if an S-shaped fluorescence signal curve is collected in the fluorescence channel corresponding to probe 2, it indicates the presence of RV G2 in the sample.

[0023] Since the G1, G2, G3, G4 probes of the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) are all labeled with different fluorescence, and the G8, G9, and G12 probes and the internal reference gene RNP are also labeled with different fluorescence, the detection data of the seven viral genotypes can be obtained by using different fluorescence curves.

[0024] The upstream and downstream primers and probes in the composition described in this application have high specificity, and non-specific binding will not occur. Therefore, the above composition can be used to simultaneously and specifically detect seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) in the test sample, which greatly shortens the detection time and reduces the detection cost.

[0025] Secondly, this application provides a kit for simultaneously detecting seven G genotypes of group A rotavirus, comprising the above-mentioned composition.

[0026] Optionally, the kit may also include an enzyme mixture, amplification buffer, positive control, and negative control.

[0027] Optionally, the enzyme mixture includes reverse transcriptase, RNase inhibitor, Taq DNA polymerase, and enzyme buffer.

[0028] Optionally, the amplification buffer includes buffer, dNTPs, and Mg. 2+ .

[0029] Positive and negative control samples are used for quality control during the use of the reagent kit.

[0030] Optionally, all components in the kit can be fully mixed to form a premixed form.

[0031] The fully premixed reagents are pre-aliped into 8-tube bundles and capped. During use, simply open the cap, add the extracted nucleic acid, and close the cap again before instrumentation. This reduces the number of steps and the probability of errors.

[0032] In this application, the seven compositions of the first aspect of the kit containing the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus can be packaged together or individually. When individually packaged, the seven compositions of components RV G1, RV G2, RV G3, RV G4, RV G8, RV G9, and RV G12 can be used separately to detect RV G1, RV G2, RV G3, RV G4, RV G8, RV G9, and RV G12 in the test sample.

[0033] Thirdly, this application provides a method for simultaneously detecting seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of rotavirus in a test sample using the above-described composition or kit, employing the following technical solution:

[0034] A method for simultaneously detecting seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of rotavirus in a test sample using the above-described composition or kit, the method specifically comprising the following steps:

[0035] (1) Nucleic acid was extracted from the sample to be tested using a nucleic acid extraction kit to obtain viral RNA;

[0036] (2) Add the extracted RNA to the pre-allocated reagents in an eight-tube bundle to form a reaction system;

[0037] (3) Perform multiplex real-time quantitative RT-PCR amplification on the reaction system to obtain the amplification curve;

[0038] (4) Analyze the amplification curve, obtain the Ct value, and make a judgment.

[0039] In this application, a multiplex real-time quantitative RT-PCR detection method is designed, which involves adding primers and probes specific to the seven G genotypes (G1, G2, G3, G4) of group A rotavirus and the conserved RNP region of the internal reference gene to two reaction systems (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus and the internal reference gene to two reaction systems (G8, G9, G12 and the internal reference gene to two reaction systems). This solves the problem that traditional real-time quantitative RT-PCR detection methods can only use a single pair of primers to amplify the RNA of one pathogen and can only detect one pathogen at a time, thus reducing cross-contamination during operation.

[0040] In some specific implementations, the concentration of each primer in the reaction system is 0.2-0.25 μmol / L, and the concentration of each probe is 0.2-0.25 μmol / L.

[0041] In this application, the primers mentioned above refer to upstream primers 1-8 and downstream primers 1-8 targeting the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus and the internal reference gene RNP; the probes mentioned above refer to probes 1-8 targeting the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus and the internal reference gene RNP.

[0042] In some specific implementations, the concentrations of all primers in the reaction system are the same, at 0.2 μmol / L, and the concentrations of all probes are the same, at 0.2 μmol / L.

[0043] In this application, by controlling the concentrations of upstream and downstream primers and probes within the above-mentioned range, the method achieves the best detection effect and the highest reaction efficiency for the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus; excessively high or low concentrations of upstream and downstream primers and probes within the reaction system will reduce the detection effect.

[0044] In some specific embodiments, in step (2), the reaction system is 20 μL, specifically: 10 μL of RNA solution extracted from the sample to be tested, 10 μL of pre-aliquoted reagent (1 μL of enzyme mixture, 0.5 μL each of upstream primers 1-4 and downstream primers 1-4 (concentration of 10 μmol / L), and 0.25 μL each of probes 1-4 (concentration of 20 μmol / L), containing dNTPs and Mg 2+ The buffer solution is 4 μL.

[0045] In some specific implementations, the amplification conditions in step (3) include:

[0046] Reverse transcription at 50-55℃ for 5-20 minutes;

[0047] Pre-denaturation at 90-95℃ for 1-5 minutes;

[0048] Denaturation at 90-95℃ for 5-15 seconds, annealing and extension at 50-60℃ for 30-45 seconds, 35-45 cycles, then fluorescence collection.

[0049] In some preferred embodiments, the amplification conditions in step (3) include:

[0050] Reverse transcription at 55℃ for 15 minutes;

[0051] Pre-denaturation at 95℃ for 1 min;

[0052] Denaturation at 95℃ for 5 seconds, annealing and extension at 60℃ for 30 seconds, 40 cycles, fluorescence collected.

[0053] By employing the aforementioned amplification conditions, this application enables the effective amplification of the specific genes of the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus in the test sample.

[0054] In some specific implementations, the principle for analyzing and judging the amplification curve in step (4) is as follows:

[0055] When the amplification curve of the fluorescent channel of the fluorescent reporter group labeled on probe 1 of the sample to be tested is S-shaped and the Ct value is within 37, the sample to be tested is judged to be a positive sample of human group A rotavirus G1 genotype (RV G1).

[0056] When the amplification curve of the fluorescent reporter channel labeled on probe 2 of the sample to be tested is S-shaped and the Ct value is within 37, the sample to be tested is judged to be a positive sample of human group A rotavirus G2 genotype (RV G2).

[0057] When the amplification curve of the fluorescent reporter channel labeled on probe 3 of the sample is S-shaped and the Ct value is within 37, the sample is judged to be a positive sample of human group A rotavirus G3 genotype (RV G3).

[0058] When the amplification curve of the fluorescent channel of the fluorescent reporter group labeled on probe 4 of the sample to be tested is S-shaped and the Ct value is within 37, the sample to be tested is judged to be a positive sample of human group A rotavirus G4 genotype (RV G4).

[0059] When the amplification curve of the fluorescent channel of the fluorescent reporter group labeled on probe 5 of the test sample is S-shaped and the Ct value is within 37, the test sample is judged to be a positive sample of human group A rotavirus G8 genotype (RV G8).

[0060] When the amplification curve of the fluorescent reporter group labeled on probe 6 of the sample is S-shaped and the Ct value is within 37, the sample is judged to be a positive sample of human group A rotavirus G9 genotype (RV G9).

[0061] When the amplification curve of the fluorescent reporter channel labeled on probe 7 of the sample is S-shaped and the Ct value is within 37, the sample is judged to be a positive sample of human group A rotavirus G12 genotype (RV G12).

[0062] Compared with other detection methods, the detection method described in this application has the advantages of high sensitivity and specificity, simple operation, short detection time, small sample volume required, and low pollution. It can directly detect RNA extracted from the sample to be tested and has high application value in the rapid detection of viruses.

[0063] It is worth noting that the method described in this application is for purposes other than disease diagnosis.

[0064] In summary, this application includes at least one of the following beneficial technical effects:

[0065] A literature search revealed very few patents related to the seven G genotypes that infect humans, with only one patent using a method that did not employ quantitative fluorescence. This patent simultaneously detects the seven common G genotypes that infect humans. The amplification system is a fully premixed form of enzymes, buffers, and primers / probes, pre-alimentsed into eight-linked arrays for easy operation. Simply add the extracted nucleic acid to the eight-linked arrays for direct amplification.

[0066] The composition provided in this application for the simultaneous detection of seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus consists of primers and probes specific to conserved regions of the above seven pathogens. Adding these to two reaction systems (G1, G2, G3, and G4 form one reaction system, while G8, G9, G12 and an internal reference form another) allows for the simultaneous detection of all seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of human group A rotavirus in a sample without cross-reaction. This solves the problem of traditional quantitative RT-PCR methods, which can only amplify the DNA of one pathogen using a single primer pair and can only detect one pathogen at a time, thus reducing cross-contamination during operation.

[0067] The kit allows for complete premixing of the above-mentioned composition, enzyme mixture, buffer, etc. The premixed reagents are pre-aliped into 8-tube arrays and capped. During use, simply open the tube cap, add the extracted nucleic acid, and cap the tube again before instrumentation. This reduces the number of steps and the probability of errors.

[0068] The detection method using this composition or a kit including this composition has the advantages of high sensitivity and specificity, simple operation, short detection time, small sample volume required, and low contamination. It can directly detect nucleic acids extracted from the sample to be tested and has high application value in the rapid detection of viruses. Attached Figure Description

[0069] Figure 1 The amplification results for RV G1 - the first set of primers and probes (RV G1(F1,R1,P1)).

[0070] Figure 2 The amplification results for RV G1 - the second set of primers and probes (RV G1(F2,R2,P2)).

[0071] Figure 3 The amplification results for RV G2 - the first set of primers and probes (RV G2(F1,R1,P1)).

[0072] Figure 4The amplification results for RV G2 - the second set of primers and probes (RV G2(F2,R2,P2)).

[0073] Figure 5 The amplification results for RV G3 - the first set of primers and probes (RV G3(F1,R1,P1)).

[0074] Figure 6 The amplification results for RV G3 - the second set of primers and probes (RV G3(F2,R2,P2)).

[0075] Figure 7 The amplification results for RV G4 - the first set of primers and probes (RV G4(F1,R1,P1)).

[0076] Figure 8 The amplification results for RV G4 - the second set of primers and probes (RV G4(F2,R2,P2)).

[0077] Figure 9 The amplification results for RV G8 - the first set of primers and probes (RV G8(F1,R1,P1)).

[0078] Figure 10 The amplification results for RV G8 - the second set of primers and probes (RV G8(F2,R2,P2)).

[0079] Figure 11 The amplification results for RV G9 - the first set of primers and probes (RV G9(F1,R1,P1)).

[0080] Figure 12 The amplification results for RV G9 - the second set of primers and probes (RV G9(F2,R2,P2)).

[0081] Figure 13 The amplification results for RV G12 - the first set of primers and probes (RV G12(F1,R1,P1)).

[0082] Figure 14 The amplification results for RV G12 - the second set of primers and probes (RV G12(F2,R2,P2)).

[0083] Figure 15 The result is the amplification of RNP (RNP(F,R,P)).

[0084] Figure 16 The concentration of RV G1- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0085] Figure 17 The concentration of RV G1- plasmid is 1×10⁻⁶.3 Amplification curves of the second set of multiplex detection systems.

[0086] Figure 18 The concentration of RV G2- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0087] Figure 19 The concentration of RV G2- plasmid is 1×10⁻⁶. 3 Amplification curves of the second set of multiplex detection systems.

[0088] Figure 20 The concentration of RV G3- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0089] Figure 21 The concentration of RV G3- plasmid is 1×10⁻⁶. 3 Amplification curves of the second set of multiplex detection systems.

[0090] Figure 22 The concentration of RV G4- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0091] Figure 23 The concentration of RV G4- plasmid is 1×10⁻⁶. 3 Amplification curves of the second set of multiplex detection systems.

[0092] Figure 24 The concentration of RV G8- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0093] Figure 25 The concentration of RV G8- plasmid is 1×10⁻⁶. 3 Amplification curves of the second set of multiplex detection systems.

[0094] Figure 26 The concentration of RV G9- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0095] Figure 27 The concentration of RV G9- plasmid is 1×10⁻⁶. 3 Amplification curves of the second set of multiplex detection systems.

[0096] Figure 28 The concentration of RV G12- plasmid is 1×10⁻⁶. 3 The amplification curve of the first set of multiplex detection systems.

[0097] Figure 29 The concentration of RV G12- plasmid is 1×10⁻⁶.3 Amplification curves of the second set of multiplex detection systems. Detailed Implementation

[0098] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0099] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0100] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0101] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0102] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0103] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0104] The present application will be further described in detail below with reference to the embodiments and test results.

[0105] Example 1

[0106] This embodiment provides the design and synthesis of primers and probes.

[0107] Literature review yielded a pool of candidate primer and probe sequences. Simultaneously, VP7 fragment sequences for seven G genotypes were obtained from the NCBI nucleotide database. Multiple sequence alignment was performed on the sequences obtained for the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus using the bioinformatics software MEGA-X. Multiple conserved fragments for each virus were identified. These conserved fragments needed to specifically match the chosen genotype without detecting other genotypes. Upstream and downstream primers and probes were designed and their structures were analyzed using Primer Premier 5 software. Finally, NCBI's PrimerBlast function was used for primer specificity and matching analysis to ensure that the primers and probes were highly specific for the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus and that degeneracy would not lead to missed detections. The final two sets of primers and probes for each of the seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus were selected through screening. The specific nucleotide sequences (5'-3') are shown in Table 1.

[0108] Table 1. Two sets of primers and probes for each of the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus.

[0109]

[0110]

[0111] Example 2

[0112] This embodiment uses the primers and probes from Example 1 for single-weight testing.

[0113] The AccurSTART U+One Step RT-qPCR Probe Kit (FOR FAST) sold by Novizan was used as the amplification reaction system.

[0114] Two sets of primers and probes for each of the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus were tested in single-component systems. First, a primer-probe mix was prepared, with 2 μL each of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM), and then brought to a final volume of 100 μL with nuclease-free water. To the reaction system, 2 μL of the primer-probe mix, 4 μL of Novizumab 5× One Step U+Mix, 1 μL of Novizumab One Step U+Enzyme Mix, and 1×10⁻⁶ ppm of the primer-probe mix were added. 2 -1×10 34 μL of plasmid with a plasmid concentration of 1 copy / μL and 9 μL of nuclease-free water were prepared. Each plasmid gradient was prepared in triplicate, and 3 template-free controls were also prepared.

[0115] The amplification program was set as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s; 60℃ annealing extension for 30s, for a total of 45 cycles. Fluorescence signal was detected during the annealing step of each cycle.

[0116] The amplification data of two sets of primers and probes for each of the seven G genotypes (G1, G2, G3, G4, G8, G9, and G12) of group A rotavirus, as well as the internal reference gene RNP, are shown in Tables 2-9 below.

[0117] The amplification results of RV G1 are shown in Table 2, and the results of each primer and probe group are as follows: Figure 1-2 As shown.

[0118] The amplification results of RV G2 are shown in Table 3, and the results of each primer and probe group are as follows: Figure 3-4 As shown.

[0119] The amplification results of RV G3 are shown in Table 4, and the results of each primer and probe group are as follows: Figure 5-6 As shown.

[0120] The amplification results of RV G4 are shown in Table 5, and the results of each primer and probe group are as follows: Figure 7-8 As shown.

[0121] The amplification results of RV G8 are shown in Table 6, and the results of each primer and probe group are as follows: Figure 9-10 As shown.

[0122] The amplification results of RV G9 are shown in Table 7, and the results of each primer and probe group are as follows: Figure 11-12 As shown.

[0123] The amplification results of RV G12 are shown in Table 8, and the results of each primer and probe group are as follows: Figure 13-14 As shown.

[0124] The amplification results of RNP are shown in Table 9. Figure 15 As shown.

[0125] Table 2 Amplification results of RV G1

[0126] plasmid concentration RV G1(F1,R1,P1) RV G1(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 27.70 27.84 <![CDATA[1×10 2 (Average Ct) 31.30 31.37 1×10 (average Ct) 34.28 34.35 No template provided Nt Nt

[0127] Table 3 Amplification results of RV G2

[0128] plasmid concentration RV G2(F1,R1,P1) RV G2(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 29.59 29.33 <![CDATA[1×10 2 (Average Ct) 32.56 32.34 1×10 (average Ct) 37.66 37.52 No template provided Nt Nt

[0129] Table 4 Amplification results of RV G3

[0130]

[0131]

[0132] Table 5 Amplification results of RV G4

[0133] plasmid concentration RV G4(F1,R1,P1) RV G4(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 27.18 27.26 <![CDATA[1×10 2 (Average Ct) 31.46 31.44 1×10 (average Ct) 36.00 35.42 No template provided Nt Nt

[0134] Table 6 Amplification results of RV G8

[0135] plasmid concentration RV G8(F1,R1,P1) RV G8(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 27.14 27.22 <![CDATA[1×10 2 (Average Ct) 31.04 31.26 1×10 (average Ct) 34.12 35.45 No template provided Nt Nt

[0136] Table 7 Amplification results of RV G9

[0137] plasmid concentration RV G9(F1,R1,P1) RV G9(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 27.05 26.92 <![CDATA[1×10 2 (Average Ct) 31.82 31.85 1×10 (average Ct) 36.61 36.51 No template provided Nt Nt

[0138] Table 8 Amplification results of RV G12

[0139] plasmid concentration RV G12(F1,R1,P1) RV G12(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 25.71 25.66 <![CDATA[1×10 2 (Average Ct) 30.21 30.06 1×10 (average Ct) 33.15 34.06 No template provided Nt Nt

[0140] Table 9. Amplification results of RNP

[0141] plasmid concentration RNP(F,R,P) <![CDATA[1×10 3 (Average Ct) 26.21 <![CDATA[1×10 2 (Average Ct) 30.17 1×10 (average Ct) 35.72 No template provided Nt

[0142] As shown in Tables 2-9, the amplification results of the singlet systems using two sets of primers and the internal reference gene for each pathogen were good in the plasmid template test, with typical S-shaped curves and similar Ct values. All primers and probes can be used as candidate primers and probes for establishing the following multiplex systems.

[0143] Example 3

[0144] This embodiment utilizes the primers and probes from Example 1 to establish a multiplex detection system (G1, G2, G3, G4).

[0145] The AccurSTART U+One Step RT-qPCR Probe Kit (FOR FAST) sold by Novizan was used as the amplification reaction system.

[0146] A quadruple detection system was established using two sets of primers and probes for each of the four G genotypes (G1, G2, G3, G4) of group A rotavirus. First, a primer-probe mix was prepared. The volumes of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) for each of the four primer-probe mixes in the multiplex system were 2 μL each, and the volume was brought to 100 μL with nuclease-free water. Then, 2 μL of the primer-probe mix, 4 μL of Novizumab 5×One Step U+Mix, 1 μL of Novizumab One Step U+Enzyme Mix, and 1×10⁻⁶ ppm of the primer-probe mix were added to the reaction system. 1 -1×10 3 4 μL of plasmid with a plasmid concentration of 1 copy / μL and 9 μL of nuclease-free water were prepared. Each plasmid gradient was prepared in triplicate, and 3 template-free controls were also prepared.

[0147] The amplification program was set as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s; 60℃ annealing extension for 30s, for a total of 45 cycles. Fluorescence signal was detected during the annealing step of each cycle.

[0148] (1) First, two sets of candidate primer-probe combinations for RV G1 were selected and combined with the first sets of primer-probe combinations for RV G2, RV G3, and RV G4, respectively, to form the following combinations for testing of the multiplex detection system. The amplification data are shown in Table 10-11 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figure 16-17 As shown.

[0149] Table 10. Multiple Detection System – The First Set of Alternative Primers and Probes for RV G1

[0150] plasmid concentration RV G1(F1,R1,P1) RV G2(F1,R1,P1) RV G3(F1,R1,P1) RV G4(F1,R1,P1) <![CDATA[1×10 3 (Average Ct) 22.00 24.02 22.78 23.30 <![CDATA[1×10 2 (Average Ct) 25.86 27.61 26.68 27.03 1×10 (average Ct) 29.59 31.08 30.39 30.60 No template provided Nt Nt Nt Nt

[0151] Table 11 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G1

[0152] plasmid concentration RV G1(F2,R2,P2) RV G2(F1,R1,P1) RV G3(F1,R1,P1) RV G4(F1,R1,P1) <![CDATA[1×10 3 (Average Ct) 21.86 23.10 21.83 22.79 <![CDATA[1×10 2 (Average Ct) 25.24 27.18 25.56 26.15 1×10 (average Ct) 28.81 30.95 29.11 29.61 No template provided Nt Nt Nt Nt

[0153] Combining the test results in Tables 10 and 11 above, and Figure 16-17 It can be seen that, in plasmid template testing, the second set of multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the second primer-probe combination for RV G1 was selected.

[0154] Based on the above, the second primer-probe combination for RV G1 is fixed, while the first primer-probe combination is used for RV G3 and RV G4. The two primer-probe combinations for RV G2 are then screened.

[0155] (2) Next, two sets of candidate primer-probe combinations for RV G2 were selected and combined with the second set of primer-probe combinations for RV G1 and the first set of primer-probe combinations for RV G3 and RV G4, respectively, to form the following combinations for testing of the multiplex detection system. The amplification data are shown in Tables 12-13 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figure 18-19 As shown.

[0156] Table 12 Multiplex Detection System – First Set of Alternative Primers and Probes for RV G2

[0157] plasmid concentration RV G1(F2,R2,P2) RV G2(F1,R1,P1) RV G3(F1,R1,P1) RV G4(F1,R1,P1) <![CDATA[1×10 3 (Average Ct) 21.72 22.98 22.38 22.38 <![CDATA[1×10 2 (Average Ct) 25.68 27.26 26.34 26.34 1×10 (average Ct) 29.35 30.73 29.87 29.87 No template provided Nt Nt Nt Nt

[0158] Table 13 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G2

[0159] plasmid concentration RV G1(F2,R2,P2) RV G2(F2,R2,P2) RV G3(F1,R1,P1) RV G4(F1,R1,P1) <![CDATA[1×10 3 (Average Ct) 21.76 22.51 21.86 21.86 <![CDATA[1×10 2 (Average Ct) 25.75 27.17 25.67 25.67 1×10 (average Ct) 28.90 30.59 29.19 29.19 No template provided Nt Nt Nt Nt

[0160] Combining the test results in Tables 12-13 above and Figure 18-19 It can be seen that, in plasmid template testing, the second set of multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the second primer-probe combination for RV G2 was selected.

[0161] Based on the above, the second primer-probe combination of RV G1 and the second primer-probe combination of RV G2 were fixed, and the first primer-probe combination of RV G4 was used to continue screening the two primer-probe combinations of RV G3.

[0162] (3) Two sets of candidate primer-probe combinations for RV G3 were selected and combined with the second set of primers-probes for RV G1, the second set of primers-probes for RV G2, and the first set of primers-probes for RV G4 to form the following combinations for testing of a multiplex detection system. The amplification data are shown in Tables 14-15 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figure 20-21 As shown.

[0163] Table 14 Multiplex Detection System – First Set of Alternative Primers and Probes for RV G3

[0164] plasmid concentration RV G1(F2,R2,P2) RV G2(F2,R2,P2) RV G3(F1,R1,P1) RV G4(F1,R1,P1) <![CDATA[1×10 3 (Average Ct) 21.78 22.95 21.75 21.75 <![CDATA[1×10 2 (Average Ct) 25.72 27.01 25.43 25.43 1×10 (average Ct) 27.83 30.38 29.04 29.04 No template provided Nt Nt Nt Nt

[0165] Table 15 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G3

[0166]

[0167]

[0168] Combining the test results in Tables 14-15 above and Figure 20-21 It can be seen that, in plasmid template testing, the first set of the two multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the first probe combination of RV G3 is selected.

[0169] (4) Finally, two sets of candidate primer-probe combinations for RV G4 were selected and combined with the second set of primer-probe combinations for RV G1, the second set of primer-probe combinations for RV G2, and the first set of primer-probe combinations for RV G4, respectively, to form the following combinations for testing of the multiplex detection system. The amplification data are shown in Tables 16-17 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figure 22-23 As shown.

[0170] Table 16 Multiplex Detection System – First Set of Alternative Primers and Probes for RV G4

[0171] plasmid concentration RV G1(F2,R2,P2) RV G2(F2,R2,P2) RV G3(F1,R1,P1) RV G4(F1,R1,P1) <![CDATA[1×10 3 (Average Ct) 21.86 23.15 21.49 22.89 <![CDATA[1×10 2 (Average Ct) 25.81 27.12 25.15 26.81 1×10 (average Ct) 29.08 30.73 28.76 30.36 No template provided Nt Nt Nt Nt

[0172] Table 17 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G4

[0173] plasmid concentration RV G1(F2,R2,P2) RV G2(F2,R2,P2) RV G3(F1,R1,P1) RV G4(F2,R2,P2) <![CDATA[1×10 3 (Average Ct) 21.61 22.67 21.90 22.34 <![CDATA[1×10 2 (Average Ct) 25.39 26.58 25.66 26.18 1×10 (average Ct) 28.93 30.15 29.14 29.76 No template provided Nt Nt Nt Nt

[0174] Combining the test results in Tables 16 and 17 above, and Figure 22-23 It can be seen that, in plasmid template testing, the first set of the two multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the first probe combination of RV G4 is selected.

[0175] Comprehensive analysis shows that for group A rotavirus G1 and G2, the second primer-probe combination was selected, while for G3 and G4, the first primer-probe combination was used. The resulting amplification curves were standard S-shaped curves with smaller Ct values, optimal sensitivity, and good specificity.

[0176] Example 4

[0177] This embodiment utilizes the primers and probes from Example 1 to establish a multiplex detection system (G8, G9, G12, and internal reference RNP).

[0178] The AccurSTART U+One Step RT-qPCR Probe Kit (FOR FAST) sold by Novizan was used as the amplification reaction system.

[0179] A multiplex detection system was established using a quadruple system consisting of two sets of primers and probes for each of the three G genotypes (G8, G9, G12) of group A rotavirus and an internal reference gene RNP. First, a primer-probe mix was prepared. The volumes of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) for each of the four primer-probe mixes in the multiplex system were 2 μL each, and the volume was brought to 100 μL with nuclease-free water. Then, 2 μL of the primer-probe mix, 4 μL of Novizumab 5×One Step U+Mix, 1 μL of Novizumab One Step U+Enzyme Mix, and 1×10⁻⁶ ppm of the reagent were added to the reaction system. 1 -1×10 3 4 μL of plasmid with a plasmid concentration of 1 copy / μL and 9 μL of nuclease-free water were prepared. Each plasmid gradient was prepared in triplicate, and 3 template-free controls were also prepared.

[0180] The amplification program was set as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s; 60℃ annealing extension for 30s, for a total of 45 cycles. Fluorescence signal was detected during the annealing step of each cycle.

[0181] (1) First, two sets of candidate primer-probe combinations for RV G8 were selected and combined with the first sets of primer-probe combinations for RV G9 and RV G12, and the primer-probe combination for RNP, respectively, to form the following combination for testing of the multiplex detection system. The amplification data are shown in Tables 18-19 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figure 24-25 As shown.

[0182] Table 18 Multiplex Detection System – First Set of Alternative Primers and Probes for RV G8

[0183] plasmid concentration RV G8(F1,R1,P1) RV G9(F1,R1,P1) RV G12(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct) 21.95 21.32 22.79 23.38 <![CDATA[1×10 2 (Average Ct) 27.36 24.24 26.67 27.41 1×10 (average Ct) 30.80 28.88 30.13 31.04 No template provided Nt Nt Nt Nt

[0184] Table 19 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G8

[0185] plasmid concentration RV G8(F2,R2,P2) RV G9(F1,R1,P1) RV G12(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct) 20.89 22.22 22.64 23.31 <![CDATA[1×10 2 (Average Ct) 26.78 26.08 26.50 27.38 1×10 (average Ct) 30.02 29.60 29.94 30.92 No template provided Nt Nt Nt Nt

[0186] Combining the test results in Tables 18-19 above and Figure 24-25 It can be seen that, in plasmid template testing, the second set of multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the second primer-probe combination for RV G8 was selected.

[0187] Based on the above, the second set of primer and probe combinations for RV G8 is fixed, and the first set of primer and probe combinations and the primer and probe combination of RNP are used for RV G12 to continue screening the two sets of primer and probe combinations for RV G9.

[0188] (2) Next, two sets of candidate primer-probe combinations for RV G9 were selected and combined with the second set of primer-probe combinations for RV G8, the first set of primer-probe combinations for RV G12, and the primer-probe combination for RNP, respectively, to form the following combinations for testing of the multiplex detection system. The amplification data are shown in Tables 20-21 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figure 26-27 As shown.

[0189] Table 20 Multiple Detection System – First Set of Alternative Primers and Probes for RV G9

[0190] plasmid concentration RV G8(F2,R2,P2) RV G9(F1,R1,P1) RV G12(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct) 23.60 22.23 22.68 23.26 <![CDATA[1×10 2 (Average Ct) 29.15 26.09 26.52 27.23 1×10 (average Ct) 30.60 29.93 29.92 31.17 No template provided Nt Nt Nt Nt

[0191] Table 21 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G9

[0192] plasmid concentration RV G8(F2,R2,P2) RV G9(F2,R2,P2) RV G12(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct) 21.74 21.25 21.83 23.25 <![CDATA[1×10 2 (Average Ct) 26.86 24.32 24.72 27.32 1×10 (average Ct) 30.57 29.42 26.33 30.90 No template provided Nt Nt Nt Nt

[0193] Combining the test results in Tables 20-21 above and Figure 26-27 It can be seen that, in plasmid template testing, the second set of multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the second primer-probe combination for RV G9 was selected.

[0194] Based on the above, the second primer-probe combination of RV G8 and the second primer-probe combination of RV G9 were fixed, and the primer-probe combination of RNP was used to continue screening the two primer-probe combinations of RV G12.

[0195] (3) Two sets of candidate primer-probe combinations for RV G12 were selected and combined with the second set of primers and probes for RV G8, the second set of primers and probes for RV G9, and the primer-probe combination for RNP, respectively, to form the following combinations for testing of a multiplex detection system. The amplification data are shown in Tables 22-23 below. The plasmid concentration was 1×10⁻⁶. 3 The amplification curves of the multiplex detection system are as follows: Figures 28-29 As shown.

[0196] Table 22 Multiplex Detection System – First Set of Alternative Primers and Probes for RV G12

[0197] plasmid concentration RV G8(F2,R2,P2) RV G9(F2,R2,P2) RV G12(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct) 21.08 20.09 22.02 23.12 <![CDATA[1×10 2 (Average Ct) 24.22 23.94 24.34 27.13 1×10 (average Ct) 30.14 28.54 28.90 30.76 No template provided Nt Nt Nt Nt

[0198] Table 23 Multiplex Detection System – Second Set of Alternative Primers and Probes for RV G12

[0199]

[0200]

[0201] Combining the test results in Tables 22 and 23 above, and Figures 28-29 It can be seen that, in plasmid template testing, the first set of the two multiplex detection systems exhibits the best amplification effect, with a smaller Ct value, the highest amplification sensitivity, and consistent specificity between the two primer-probe combinations. Therefore, the first probe combination of RV G12 was selected.

[0202] Comprehensive analysis shows that for group A rotavirus G8 and G9, the second primer-probe combination was selected, while for G12, the first primer-probe combination was used. The resulting amplification curves were standard S-shaped curves with smaller Ct values, optimal sensitivity, and good specificity.

[0203] Example 5

[0204] This embodiment establishes a pre-packaging system for multiple pathogen detection.

[0205] The AccurSTART U+One Step RT-qPCR Super PreMix (PreMix) kit, sold by Novizan, was used as the amplification reaction system. This amplification system contains an enzyme mixture (Taq DNA polymerase, reverse transcriptase, UNG enzyme, and a reverse transcriptase inhibitor), dNTPs, and Mg... 2+ All components, including buffers, were premixed into one tube.

[0206] The two optimized multiplex amplification systems from Examples 3 and 4 were prepared into primer-probe mixes. The volumes of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) for each of the four primer-probe mixes were 4 μL, and the volume was brought to 100 μL with nuclease-free water. 1 μL of primer-probe mix and 4 μL of Novizan U+ One Step RT-qPCR Probe 5×Master Mix were added to the reaction system to form a pre-allotted system. After thorough mixing, 5 μL / well was aliquoted into eight-tube strips and capped. The pre-allotted eight-tube strips were stored at -20°C, and amplification was tested at 0, 4, 8, and 12 months. Quantified mixed nucleic acid was added to the pre-allotted eight-tube strip system, and the nucleic acid was diluted to the following concentration: 1×10⁻⁶. 0 -1×10 2 4 μL of mixed nucleic acid at a concentration of copy / μL and 11 μL of nuclease-free water were prepared. Each gradient of mixed nucleic acid was prepared in triplicate, and three template-free controls were also prepared.

[0207] The non-pre-mixed system used as a control was operated in the same way as described above, but the primer probe mix and Novizan's U+One Step RT-qPCR Probe 5×Master Mix were stored at -20℃ and the amplification was tested at 0 months, 4 months, 8 months and 12 months respectively.

[0208] The amplification program was set as follows: reverse transcription reaction at 55℃ for 15 min; pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 10 s; annealing and extension at 60℃ for 30 s, for a total of 45 cycles. Fluorescence signal was detected during the annealing step of each cycle.

[0209] (1) The amplification data for the multiple pre-packed detection system 1 [RV G1(F2,R2,P2), RV G2(F2,R32,P2), RV G3(F1,R1,P1), RV G4(F1,R1,P1)] in 0 months, 4 months, 8 months and 12 months are shown in Table 24-27 below.

[0210] Table 24 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G1)

[0211]

[0212] Table 25 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G2)

[0213]

[0214] Table 26 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G3)

[0215]

[0216] Table 27 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G4)

[0217]

[0218] (2) The amplification data for the multiple pre-packaged detection system 2 [RV G8(F2,R2,P2), RV G9(F2,R2,P2), RV G12(F1,R1,P1), RNP(F,R,P)] in 0 months, 4 months, 8 months and 12 months are shown in Table 28-31 below.

[0219] Table 28 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G8)

[0220]

[0221] Table 29 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G9)

[0222]

[0223] Table 30 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) for different months (RV G12)

[0224]

[0225] Table 31 Comparison of Multiple Pre-packaging (Pre) and Multiple Non-Pre-packaging (NO-Pre) by Month (RNP)

[0226]

[0227] Based on the test results in Tables 24-31 above, it can be seen that the shelf life of the pre-packaged detection system for nucleic acid template testing can reach 12 months without changing the sensitivity and specificity of the kit.

[0228] Example 6

[0229] This embodiment involved clinical sample testing.

[0230] Specific samples (3 cases of group B rotavirus, 3 cases of group C rotavirus, and 6 cases of group A rotavirus P8 genotype) were found in our sample bank. Mixed positive samples of group A rotavirus G1, G2, G3, and G4, quantified by digital PCR, were diluted to 1×10⁻⁶. 0- 1×10 2 The copy / uL and mixed positive samples of group A rotavirus G8, G9, and G12 were diluted to 1×10⁻⁶. 1 -1×10 3 The pre-alcoholized multiplex system consisted of 4 samples each of group A rotavirus genotypes G1, G2, G3, G4, G8, and G12, and 8 norovirus G9 samples. Sensitivity, specificity, and sample detection rate were evaluated.

[0231] Using the two pre-dispensing systems described in Example 5, 10 μL of the extracted nucleic acid from the above samples was directly added to an eight-tube strip, mixed thoroughly, and then tested.

[0232] The amplification program was set as follows: reverse transcription reaction at 55℃ for 15 min; pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 10 s; annealing and extension at 60℃ for 30 s, for a total of 45 cycles. Fluorescence signal was detected during the annealing step of each cycle.

[0233] (1) Specificity test

[0234] The data obtained from the mixed nucleic acid dilution of three group B rotavirus samples, three group C rotavirus samples, and six group A rotavirus P8 genotype samples quantified by digital PCR using two multiplex systems are shown in Tables 32-33 below.

[0235] Table 32 Sample Specificity Detection Data for Multiple Systems

[0236] RV G1 RV G2 RV G3 RV G4 Group B rotavirus sample 1 Nt Nt Nt Nt Group B rotavirus sample 2 Nt Nt Nt Nt Group B rotavirus sample 3 Nt Nt Nt Nt Group C rotavirus sample 1 Nt Nt Nt Nt Group C rotavirus sample 2 Nt Nt Nt Nt Group C rotavirus sample 3 Nt Nt Nt Nt Group A rotavirus P8 genotype-1 Nt Nt Nt Nt Group A rotavirus P8 genotype-2 Nt Nt Nt Nt Group A rotavirus P8 genotype-3 Nt Nt Nt Nt Group A rotavirus P8 genotype-4 Nt Nt Nt Nt Group A rotavirus P8 genotype-5 Nt Nt Nt Nt Group A rotavirus P8 genotype-6 Nt Nt Nt Nt No template provided Nt Nt Nt Nt

[0237] Table 33. Sample Specificity Detection Data for Multiple Systems 2

[0238] RV G8 RV G9 RV G12 Internal reference RNP Group B rotavirus sample 1 Nt Nt Nt 27.17 Group B rotavirus sample 2 Nt Nt Nt 28.63 Group B rotavirus sample 3 Nt Nt Nt 30.27 Group C rotavirus sample 1 Nt Nt Nt 33.14 Group C rotavirus sample 2 Nt Nt Nt 27.27 Group C rotavirus sample 3 Nt Nt Nt 28.88 Group A rotavirus P8 genotype-1 Nt Nt Nt 30.76 Group A rotavirus P8 genotype-2 Nt Nt Nt 33.88 Group A rotavirus P8 genotype-3 Nt Nt Nt 28.13 Group A rotavirus P8 genotype-4 Nt Nt Nt 30.22 Group A rotavirus P8 genotype-5 Nt Nt Nt 32.50 Group A rotavirus P8 genotype-6 Nt Nt Nt 33.77 No template provided Nt Nt Nt Nt

[0239] As shown in Tables 32-33, only the internal reference gene showed a clear amplification curve in the 3 cases of group B rotavirus, 3 cases of group C rotavirus, and 6 cases of group A rotavirus P8 genotype. Both multiplex systems showed good specificity.

[0240] (2) Sensitivity Experiment

[0241] The data obtained from the mixed dilution of nucleic acid in two multiplex systems for quantifying group A rotavirus G1, G2, G3, G4 mixed positive samples and group A rotavirus G8, G9, G12 mixed positive samples by digital PCR are shown in Tables 34-35 below.

[0242] Table 34 Nucleic Acid Detection Data from Multiple Systems 1

[0243] Nucleic acid copy number RV G1 RV G2 RV G3 RV G4 <![CDATA[1×10 2 (Average Ct) 19.15 23.08 20.38 22.69 <![CDATA[1×10 1 (Average Ct) 22.76 27.00 24.19 26.49 <![CDATA[1×10 0 (Average Ct) 27.40 30.55 27.92 30.18 No template provided Nt Nt Nt Nt

[0244] Table 35 Nucleic Acid Detection Data from Multiple Systems 2

[0245] Nucleic acid copy number RV G8 RV G9 RV G12 Internal reference RNP <![CDATA[1×10 2 (Average Ct) 24.09 22.07 21.43 23.67 <![CDATA[1×10 1 (Average Ct) 28.69 25.80 25.15 27.63 <![CDATA[1×10 0 (Average Ct) 31.90 29.17 28.68 31.34 No template provided Nt Nt Nt Nt

[0246] As shown in Tables 34-35, the two multiplex pre-dispensed detection systems have high sensitivity in nucleic acid template testing and can distinguish different subtypes.

[0247] (3) Sample testing

[0248] Two multiplex pre-dispensed detection systems were used to test four samples each of G1, G2, G3, G4, G8, and G12 (all identified as group A rotavirus genotypes) and eight norovirus G9 samples. The results are shown in Tables 35-36.

[0249] Table 35 Nucleic Acid Detection Data for Multiplex Detection System

[0250]

[0251]

[0252] Table 36 Nucleic Acid Detection Data for Multiplex Detection System 2

[0253]

[0254]

[0255] As shown in Tables 35 and 36, all of the above samples were detected, with a detection rate of 100%, and there was no cross-reactivity.

[0256] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0257] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A composition for simultaneous detection of 7 G genotypes of group A rotavirus, characterized in that, The composition comprises the following components: Component A: upstream primer 1, downstream primer 1 and probe 1 for detecting Group A rotavirus G1 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 1-3 in order; MGB modification is performed at the 3' end of the probe 1; Component B: upstream primer 2, downstream primer 2 and probe 2 for detecting Group A rotavirus G2 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 4-6 in order; MGB modification is performed at the 3' end of the probe 2; Component C: upstream primer 3, downstream primer 3 and probe 3 for detecting Group A rotavirus G3 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 7-9 in order; Component D: upstream primer 4, downstream primer 4 and probe 4 for detecting Group A rotavirus G4 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 10-12 in order; Component E: upstream primer 5, downstream primer 5 and probe 5 for detecting Group A rotavirus G8 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 13-15 in order; MGB modification is performed at the 3' end of the probe 5; Component F: upstream primer 6, downstream primer 6 and probe 6 for detecting Group A rotavirus G9 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 16-18 in order; Component G: upstream primer 7, downstream primer 7 and probe 7 for detecting Group A rotavirus G12 genotype, which are respectively represented by the nucleotide sequences of SEQ ID NOs 19-21 in order; MGB modification is performed at the 3' end of the probe 7; Component H: upstream primer 8, downstream primer 8 and probe 8 for detecting human ribonucleoprotein complex, which are respectively represented by the nucleotide sequences of SEQ ID NOs 22-24 in order; The above components are divided into two reaction systems, one reaction system comprising Component A, Component B, Component C and Component D, and the other reaction system comprising Component E, Component F, Component G and Component H.

2. A kit for simultaneous detection of seven G genotypes of group A rotavirus, characterized in that, The kit comprises the composition of claim 1.

3. The kit of claim 2, wherein The kit further comprises an enzyme mixture, an amplification buffer, a positive quality control and a negative quality control.

4. The kit of claim 3, wherein The enzyme mixture comprises a reverse transcriptase, an RNase inhibitor, a Taq DNA polymerase and an enzyme buffer.

5. The kit of claim 3, wherein The amplification buffer includes a buffer, dNTPs, Mg 2 + .

6. The kit of claim 3, wherein Each component in the kit is fully mixed into a full premix form.

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