Composition, kit and method for simultaneously detecting seven G genotypes of group A rotavirus

Abstract

The invention relates to the technical field of virus detection, in particular to a composition, a kit and a method for simultaneously detecting seven G genotypes of group A rotaviruses. The composition comprises a component A-G and a component B-G, wherein the component A-G comprises upstream and downstream primers and probes for detecting genotypes of group A rotaviruses G1, G2, G3, G4, G8, G9 and G12 respectively; and the component H is used for detecting upstream and downstream primers and a probe of the human ribonucleoprotein complex. When being used for detection, the composition or the kit comprising the composition has the advantages of high sensitivity and specificity, simplicity and convenience in operation, short detection time, small required sample size, low pollution and the like, can be used for directly detecting nucleic acid extracted from a to-be-detected sample, and has relatively high application value in rapid detection of viruses.

Description

Technical Field

[0001] The present application relates to the technical field of virus detection, and in particular to a composition, a kit and a 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 Art

[0002] Rotavirus belongs to the Reoviridae family. Its particle diameter is 70-75nm, it has no envelope, a double-layer capsid, and 11 segments of double-stranded RNA. The total length of the rotavirus core RNA is about 18.5kb, 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), among which infections caused by rotavirus A account for more than 90% of rotavirus infection cases. Among the 6 structural proteins, glycoprotein VP7 and protease-sensitive protein VP4 are located in the outer capsid, which is the basis of the dual classification system of rotavirus.

[0003] In Group A rotavirus (RVA), different G serotypes can be divided according to the different VP7 antigenicity, among which G1-G4 are the most prevalent in the population; different P serotypes can be divided according to the different VP4 antigenicity, with P1A being the most common type. In clinical diagnosis and differentiation, virus genotyping is mainly used as the basis for diagnosis. The G serotype and genotype are expressed in the same way; the P serotype and genotype are expressed in different ways, and the genotype is represented 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 rotavirus infection rates of the seven G types (i.e., G1, G2, G3, G4, G8, G9, and G12) and the three P types (P[4], P[6], and P[8]) are higher.

[0004] Rotavirus is one of the main pathogens of childhood diarrhea worldwide. Group A rotavirus is one of the leading causes of death in infants under 5 years of age worldwide. All infants under 5 years of age have been infected with RVA at least once. Based on the differences in the 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 worldwide, 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 main G / P combinations G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8] account for more than 90% of the global RVA strains. At present, except for G12P[8], the other 5 G / P combinations of RVA are more common in my country. G9P[8] is the most prevalent strain of RVA in my country.

[0005] At present, the human group A rotavirus G genotyping methods mainly include nested PCR and first-generation sequencing, but these methods all have the problems of long time consumption, high cost, complex operation, insufficient sensitivity. There are few studies on the group A rotavirus G genotype in existing patents. The patent with publication number CN116479183A simultaneously detects 7 G genotypes, adopts PCR amplification coupled nucleic acid invasion reaction combined with nanogold color development (PCR-Invader-AuNP) method, which is a molecular detection method that can distinguish single base differences. Compared with traditional electrophoresis-based typing methods and sequencing-based typing methods, this method only requires ordinary PCR instruments, does not require special instruments and equipment, is simpler to operate, requires a shorter time, and the results can be distinguished by the naked eye. However, the technology used in this patent still has some problems. First of all, the sensitivity is not enough compared to the fluorescence quantitative method, and some low-concentration group A rotaviruses may not be typed. In addition, the entire operation process is more cumbersome than the fluorescence quantitative method, time-consuming, and cannot adapt to general fluorescence quantitative instruments.

[0006] Therefore, it is necessary to establish a detection method for the 7 G genotypes that is highly sensitive and rapid. Summary of the invention

[0007] The present application provides a composition, a kit and a method for simultaneously detecting seven G genotypes of group A rotavirus. The purpose is to establish a highly sensitive and rapid detection method for seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) at the same time, which is initially applied to the detection of group A rotavirus G genotypes.

[0008] The present application conducts a combined detection of the common group A rotavirus genotypes 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) that infect humans, selects the conservative region of the virus for design, and specifically detects the 7 genotypes of group A rotavirus, while being able to distinguish the 7 genotypes.

[0009] The composition provided in the present application or a kit including the composition can be used to 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 repeatability, simple and rapid detection, and cost savings.

[0010] In the first aspect, the present application provides a composition for simultaneously detecting seven G genotypes of group A rotavirus, using 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 the G1 genotype of group A rotavirus, which are nucleotide sequences shown in SEQ ID NOs 1-3 respectively; the 3' end of the probe 1 is modified with MGB;

[0013] Component B: upstream primer 2, downstream primer 2 and probe 2 for detecting group A rotavirus G2 genotype, which are nucleotide sequences shown in SEQ ID NOs 4-6 respectively; the 3' end of the 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 nucleotide sequences shown in SEQ ID NOs 7-9 respectively;

[0015] Component D: upstream primer 4, downstream primer 4 and probe 4 for detecting group A rotavirus G4 genotype, which are nucleotide sequences shown in SEQ ID NOs 10-12 respectively;

[0016] Component E: upstream primer 5, downstream primer 5 and probe 5 for detecting group A rotavirus G8 genotype, which are nucleotide sequences shown in SEQ ID NOs 13-15 respectively; 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 nucleotide sequences shown in SEQ ID NOs 16-18 respectively;

[0018] Component G: upstream primer 7, downstream primer 7 and probe 7 for detecting group A rotavirus G12 genotype, which are nucleotide sequences shown in SEQ ID NOs 19-21 respectively; the 3' end of the probe 7 is modified with MGB;

[0019] Component H: upstream primer 8, downstream primer 8 and probe 8 for detecting human ribonucleoprotein complex, which are nucleotide sequences shown in SEQ ID NOs 22-24 respectively.

[0020] The present application selects the conserved regions of the genome sequences of 7 G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus to design upstream and downstream primers and probes respectively, and the detection primer probe of human ribonucleoprotein complex (RNP) is used as an internal reference gene detection composition for monitoring sample collection, extraction and detection of the whole process. After analyzing the possible dimers, secondary structures, etc. of the primers and probes, a composition that can simultaneously detect 7 G genotypes of group A rotavirus and internal reference genes is formed after selecting a suitable sequence. The composition or a kit including the composition can simultaneously identify 7 G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus infection that cause similar clinical symptoms, and has high specificity and sensitivity, good repeatability, and accurate and reliable detection results.

[0021] The principle of using the above composition to detect 7 G genotypes (G1, G2, G3, G4, G8, G9 and G12) in this application is as follows:

[0022] Taking component B as an example, the upstream primer 2, downstream primer 2 and probe 2 in component B can all specifically bind to the genomic template of RV G2, and the binding site of probe 2 is 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 exists in the sample to be tested, the upstream primer 2, downstream primer 2 and probe 2 are all bound to the genomic template of RV G2. As RT-PCR proceeds, the Taq enzyme encounters the probe 2 bound to the template during chain extension, and its 3'-5' exonuclease activity cuts off the probe 2, and the fluorescent reporter group is away from the quencher group, and its energy cannot be absorbed, that is, a fluorescent signal is generated; and after each PCR cycle, the fluorescent signal and the target fragment have a synchronous exponential growth process. Therefore, if an S-shaped fluorescent signal curve is collected in the fluorescent channel corresponding to probe 2, it indicates that RV G2 exists in the sample to be tested.

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

[0024] The upstream and downstream primers and probes in the composition described in the present application have strong specificity and will not cause non-specific binding. Therefore, the above composition can be used to simultaneously perform specific detection on the seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) in the sample to be tested, greatly shortening the detection time and reducing the detection cost.

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

[0026] Optionally, the kit further comprises an enzyme mixture, an amplification buffer, a positive quality control and a negative quality control.

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

[0028] Optionally, the amplification buffer comprises a buffer, dNTPs, Mg 2+ .

[0029] Among them, positive quality control products and negative quality control products are used for quality control during the use of the kit.

[0030] Optionally, each component in the kit can be completely mixed into a fully premixed form.

[0031] The fully premixed reagents are pre-packed into 8-tube strips and capped to make them pre-packed. When in use, you only need to open the tube cap, add the extracted nucleic acid, and then cap the tube and put it on the machine. This reduces the number of operation steps and the probability of operation errors.

[0032] In the present application, the seven compositions of the seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus of the composition of the first aspect in the kit can be mixed and packaged, or they can be packaged separately. When packaged separately, 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 sample to be tested separately.

[0033] In a third aspect, the present application provides a method for simultaneously detecting seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) of rotavirus in a sample to be tested using the above composition or the above kit, using 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 sample to be tested using the above composition or the above kit, the method specifically comprising the following steps:

[0035] (1) Using a nucleic acid extraction kit to extract nucleic acid from the sample to be tested to obtain viral RNA;

[0036] (2) Adding the extracted RNA to eight tubes pre-packed with reagents to form a reaction system;

[0037] (3) performing multiple real-time fluorescence quantitative RT-PCR amplification on the reaction system to obtain an amplification curve;

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

[0039] In the present application, the designed multiple real-time fluorescence quantitative RT-PCR detection method is to add specific primers and probes for the seven G genotypes of group A rotavirus (G1, G2, G3, G4) and the conservative segment of the RNP of the internal reference gene in two reaction systems (G1, G2, G3, G4 constitute one reaction system, and G8, G9, G12 and the internal reference gene constitute another reaction system), thereby solving the problem that the traditional real-time fluorescence quantitative RT-PCR detection method can only use a single pair of primers to amplify the RNA of one pathogen and can only detect one pathogen separately at a time, thereby reducing the cross contamination caused during the operation.

[0040] In some specific embodiments, 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 the present application, the above-mentioned primers refer to upstream primers 1-8 and downstream primers 1-8 for 7 G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus and the internal reference gene RNP; the above-mentioned probes refer to probes 1-8 for 7 G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus and the internal reference gene RNP.

[0042] In some specific embodiments, the concentration of each primer in the reaction system is the same, which is 0.2 μmol / L, and the concentration of each probe is the same, which is 0.2 μmol / L.

[0043] In the present application, by controlling the concentrations of upstream and downstream primers and probes in the reaction system within the above range, the method has the best effect in detecting 7 G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus and the highest reaction efficiency; excessively high or low concentrations of upstream and downstream primers and probes in 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-packaged reagent (1 μL of enzyme mixture, 0.5 μL of upstream primers 1-4 and downstream primers 1-4 (both at a concentration of 10 μmol / L), 0.25 μL of probes 1-4 (both at a concentration of 20 μmol / L), dNTPs and Mg 2+ The buffer was 4 μL).

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

[0046] Reverse transcription at 50-55°C for 5-20 min;

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

[0048] Denaturation at 90-95°C for 5-15s, annealing and extension at 50-60°C for 30-45s, for 35-45 cycles, and fluorescence was collected.

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

[0050] Reverse transcription at 55°C for 15 min;

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

[0052] Denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, 40 cycles, and fluorescence was collected.

[0053] By adopting the above-mentioned amplification conditions, the present application can effectively amplify the specific genes of the seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus in the sample to be tested.

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

[0055] When the amplification curve of the fluorescence 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 fluorescence channel of the fluorescent reporter group 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 human group A rotavirus G2 genotype (RV G2) positive sample;

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

[0058] When the amplification curve of the fluorescence 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 human group A rotavirus G4 genotype (RV G4) positive sample;

[0059] When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 5 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 human group A rotavirus G8 genotype (RV G8) positive sample;

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

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

[0062] Compared with other detection methods, the detection method described in the present application has the advantages of high sensitivity and specificity, simple operation, short detection time, small sample amount required, and low pollution. It can directly detect the 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 the present application is a method for the purpose of non-disease diagnosis.

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

[0065] Literature search found few patents for the 7 G genotypes that infect humans, and only one patent used a method that was not a fluorescence quantitative method. This patent simultaneously detects the 7 common G genotypes that infect humans. The amplification system realizes the full premix form of enzyme, buffer and primer probe, and the premixed amplification system is pre-packed into eight-row sets for easy operation. You only need to add the extracted nucleic acid to the eight-row set to directly amplify it on the machine.

[0066] The composition provided in the present application for simultaneously detecting seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) of group A rotavirus is a primer and a probe with specificity for the conserved segments of the above seven pathogens. It is added into two reaction systems (G1, G2, G3, G4 constitute one reaction system, and G8, G9, G12 and an internal reference gene constitute another reaction system), and can simultaneously detect seven G genotypes (G1, G2, G3, G4, G8, G9 and G12) of human group A rotavirus in a sample without cross-reaction, thus solving the problem that the traditional fluorescent quantitative RT-PCR detection method can only use a single pair of primers to amplify the DNA of one pathogen and can only detect one pathogen separately at a time, thereby reducing the cross-contamination caused during the operation.

[0067] The kit can completely mix the above-mentioned composition, enzyme mixture, buffer, etc. together to make a fully premixed form. The fully premixed reagents are pre-packed into 8-tube strips and covered with tube caps to make a pre-packed form. During use, you only need to open the tube cap, add the extracted nucleic acid, cover the tube cap and put it on the machine. This reduces the number of operating steps and the probability of operating errors.

[0068] The method of detecting using the composition or a kit including the composition has the advantages of high sensitivity and specificity, simple operation, short detection time, small sample amount required, low pollution, etc. It can directly detect the nucleic acid extracted from the sample to be tested and has high application value in the rapid detection of viruses. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

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

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

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

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

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

[0081] Fig.13 This is the amplification result of RV G12 - the first set of primers and probes (RV G12 (F1, R1, P1)).

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

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

[0084] Fig.16 For RV G1-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0085] Fig.17 For RV G1-plasmid concentration 1×103 Amplification curves of the second multiplex detection system.

[0086] Fig.18 For RV G2-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0087] Fig.19 For RV G2-plasmid concentration 1×10 3 Amplification curves of the second multiplex detection system.

[0088] Fig. 20 The concentration of RV G3-plasmid was 1 × 10 3 Amplification curves of the first multiplex detection system.

[0089] Fig.21 The concentration of RV G3-plasmid was 1 × 10 3 Amplification curves of the second multiplex detection system.

[0090] Fig. 22 The concentration of RV G4-plasmid was 1 × 10 3 Amplification curves of the first multiplex detection system.

[0091] Fig.23 The concentration of RV G4-plasmid was 1 × 10 3 Amplification curves of the second multiplex detection system.

[0092] Fig.24 For RV G8-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0093] Fig.25 For RV G8-plasmid concentration 1×10 3 Amplification curves of the second multiplex detection system.

[0094] Fig.26 For RV G9-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0095] Fig. 27 For RV G9-plasmid concentration 1×10 3 Amplification curves of the second multiplex detection system.

[0096] Fig.28 For RV G12-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0097] Fig.29 For RV G12-plasmid concentration 1×103 Amplification curves of the second multiplex detection system. DETAILED DESCRIPTION

[0098] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0099] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is 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 these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0101] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0102] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0103] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0104] The present application is further described in detail below in conjunction with the embodiments and test results.

[0105] Example 1

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

[0107] Through literature search, some alternative primer and probe sequences were obtained. At the same time, the VP7 fragment sequences of 7 G genotypes were obtained from the NCBI nucleotide database. The bioinformatics software MEGA-X was used to perform multiple sequence alignment on the sequences obtained from the 7 G genotypes of group A rotavirus (G1, G2, G3, G4, G8, G9 and G12), and multiple conserved fragments of each virus were obtained. The conserved fragments need to meet the specific matching of specific typing and will not detect other typing. Upstream and downstream primers and probes were designed respectively, and structural analysis was performed using Primer Premier 5 software. Finally, the PrimerBlast function of NCBI was used to perform primer specificity analysis and matching analysis to ensure that the primers and probes are highly specific to the 7 G genotypes of group A rotavirus (G1, G2, G3, G4, G8, G9 and G12) and will not be degenerate and cause missed detection. Finally, 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 screened and determined, and their 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 example uses the primer probe of Example 1 to perform a single-plex test.

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

[0114] Two sets of primers and probes for each of the seven G genotypes of group A rotavirus (G1, G2, G3, G4, G8, G9, and G12) were tested in the single-plex system. First, prepare the primer probe mix. The volume of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) is 2 μL, respectively, and fill it up to 100 μL with nuclease-free water. Add 2 μL of primer probe mix, 4 μL of Novazon 5×One Step U+Mix, 1 μl of Novazon One Step U+Enzyme Mix, and 1×10 2 -1×10 34 μL of plasmid with a copy / μL concentration and 9 μL of nuclease-free water. Make 3 parallels for each gradient of plasmid and 3 template-free controls.

[0115] The amplification program was set as follows: 95°C pre-denaturation for 30 s; 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, for a total of 45 cycles. Fluorescence signal detection was performed at each annealing step.

[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 and the internal reference gene RNP are shown in Tables 2 to 9 below.

[0117] The amplification results of RV G1 are shown in Table 2, and the results of each set of primers and probes are shown in Table 2. Figure 1-2 shown.

[0118] The amplification results of RV G2 are shown in Table 3, and the results of each set of primers and probes are shown in Table 3. Figure 3-4 shown.

[0119] Among them, the amplification results of RV G3 are shown in Table 4, and the results of each set of primers and probes are shown in Table 4. Figure 5-6 shown.

[0120] Among them, the amplification results of RV G4 are shown in Table 5, and the results of each set of primers and probes are shown in Table 5. Figure 7-8 shown.

[0121] Among them, the amplification results of RV G8 are shown in Table 6, and the results of each set of primers and probes are shown in Table 6. Figure 9-10 shown.

[0122] Among them, the amplification results of RV G9 are shown in Table 7, and the results of each set of primers and probes are shown in Table 7. Figure 11-12 shown.

[0123] Among them, the amplification results of RV G12 are shown in Table 8, and the results of each set of primers and probes are shown in Table 8. Figure 13-14 shown.

[0124] Among them, the amplification results of RNP are shown in Table 9. Fig.15 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 comparison 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 comparison 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 comparison 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 comparison 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 comparison 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 comparison Nt Nt

[0140] Table 9 RNP amplification results

[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 comparison Nt

[0142] From the amplification results in Tables 2 to 9, it can be seen that in the plasmid template test, the two sets of primers for each pathogen and the single-plex system of the internal reference gene have good amplification effects, the amplification curve is a typical S-shaped curve, and the Ct value is also close. All primer probes can be used as alternative primer probes for the establishment of the following multiplex system.

[0143] Example 3

[0144] This example uses the primers and probes of 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 Novozymes was used as the amplification reaction system.

[0146] A multiplex detection system was established using a quadruple system 1 consisting of two sets of primers and probes for each of the four G genotypes (G1, G2, G3, and G4) of group A rotavirus. Primer probe mix was prepared first. The volumes of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) of the four sets of primer probes in the multiplex system were 2 μL, respectively, and the volume was made up to 100 μL with nuclease-free water. 2 μL of primer probe mix, 4 μl of Novazon 5×One Step U+Mix, 1 μL of Novazon One Step U+Enzyme Mix, and 1×10 1 -1×10 3 4 μL of plasmid with a copy / μL concentration and 9 μL of nuclease-free water. Make 3 parallels for each gradient of plasmid and 3 template-free controls.

[0147] The amplification program was set as follows: 95°C pre-denaturation for 30 s; 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, for a total of 45 cycles. Fluorescence signal detection was performed at each annealing step.

[0148] (1) First, two sets of alternative primer-probe combinations of RV G1 were selected and combined with the first set of primer-probe combinations of RV G2, RV G3, and RV G4 to form the following combinations for testing the multiplex detection system. The amplification data are shown in Tables 10-11 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 16-17 shown.

[0149] Table 10 Multiplex detection system - the first set of candidate 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 comparison Nt Nt Nt Nt

[0151] Table 11 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

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

[0154] On the basis of the above, the second set of primer-probe combination for RV G1 was fixed, the first set of primer-probe combination was used for RV G3 and RV G4, and the two sets of primer-probe combinations for RV G2 were further screened.

[0155] (2) Next, two alternative primer-probe combinations of RV G2 were selected and combined with the second primer-probe combination of RV G1 and the first primer-probe combination of RV G3 and RV G4 to form the following combination to test the multiplex detection system. The amplification data are shown in Tables 12-13. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 18-19 shown.

[0156] Table 12 Multiplex detection system - the first set of candidate 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 comparison Nt Nt Nt Nt

[0158] Table 13 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

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

[0161] On the basis of the above, the second set of primer-probe combinations of RV G1 and the second set of primer-probe combinations of RV G2 were fixed, the first set of primer-probe combinations of RV G4 were used, and the two sets of primer-probe combinations of RV G3 were further screened.

[0162] (3) The two sets of alternative primer probe combinations of RV G3 were selected and combined with the second set of primer probes of RV G1, the second set of primer probes of RV G2, and the first set of primer probes of RV G4 to form the following combination to test the multiple detection system. The amplification data are shown in Tables 14-15 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 20-21 shown.

[0163] Table 14 Multiplex detection system - the first set of candidate 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 comparison Nt Nt Nt Nt

[0165] Table 15 Multiplex detection system - the second set of alternative primers and probes for RV G3

[0166]

[0167]

[0168] Combined with the test results of Table 14-Table 15 and Figure 20-21 It can be seen that in the plasmid template test, the first set of the two sets of multiple detection systems has the best amplification effect, a smaller Ct value, the highest amplification sensitivity, and the specificity of the two sets of primer probe combinations is consistent. Therefore, the first set of probe combinations of RV G3 was selected.

[0169] (4) Finally, the two alternative primer-probe combinations of RV G4 were selected and combined with the second primer-probe combination of RV G1, the second primer-probe combination of RV G2, and the first primer-probe combination of RV G4 to form the following combination to test the multiplex detection system. The amplification data are shown in Tables 16-17 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 22-23 shown.

[0170] Table 16 Multiplex detection system - the first set of candidate 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 comparison Nt Nt Nt Nt

[0172] Table 17 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

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

[0175] Comprehensive analysis showed that group A rotavirus G1 and G2 selected the second set of primer-probe combinations, and G3 and G4 selected the first set of primer-probe combinations. The obtained amplification curves showed a standard S-shaped curve with a smaller Ct value, optimal sensitivity and good specificity.

[0176] Example 4

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

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

[0179] A multiplex detection system was established using a quadruple system 2 consisting of two sets of primer probes for each of the three G genotypes (G8, G9, and G12) of group A rotavirus and the internal reference gene RNP. Primer probe mix was prepared first. The volumes of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) of the four sets of primer probes in the multiplex system were 2 μL, respectively, and the volume was made up to 100 μL with nuclease-free water. 2 μL of primer probe mix, 4 μl of Novazon 5×One Step U+Mix, 1 μL of Novazon One Step U+Enzyme Mix, and 1×10 1 -1×10 3 4 μL of plasmid with a copy / μL concentration and 9 μL of nuclease-free water. Make 3 parallels for each gradient of plasmid and 3 template-free controls.

[0180] The amplification program was set as follows: 95°C pre-denaturation for 30 s; 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, for a total of 45 cycles. Fluorescence signal detection was performed at each annealing step.

[0181] (1) First, two sets of alternative primer probe combinations of RV G8 were selected and combined with the first set of primer probes of RV G9 and RV G12 and the primer probe of RNP to form the following combination to test the multiple detection system. The amplification data are shown in Tables 18-19 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 24-25 shown.

[0182] Table 18 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

[0184] Table 19 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

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

[0187] On the basis of the above, the second set of primer-probe combination for RV G8 was fixed, the first set of primer-probe and the primer-probe combination for RNP were used for RV G12, and the two sets of primer-probe combinations for RV G9 were further screened.

[0188] (2) Next, two alternative primer-probe combinations of RV G9 were selected and combined with the second primer-probe combination of RV G8, the first primer-probe combination of RV G12, and the primer-probe combination of RNP to form the following combination to test the multiplex detection system. The amplification data are shown in Tables 20-21 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 26-27 shown.

[0189] Table 20 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

[0191] Table 21 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

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

[0194] On the basis of the above, the second primer-probe combination of RV G8 and the second primer-probe combination of RV G9 were fixed, and the two primer-probe combinations of RV G12 were further screened together with the primer-probe combination of RNP.

[0195] (3) The two alternative primer probe combinations of RV G12 were selected and combined with the second primer probe of RV G8, the second primer probe of RV G9, and the primer probe of RNP to form the following combination to test the multiple detection system. The amplification data are shown in Tables 22-23 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 28-29 shown.

[0196] Table 22 Multiplex detection system - the 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 comparison Nt Nt Nt Nt

[0198] Table 23 Multiplex detection system - the second set of alternative primers and probes for RV G12

[0199]

[0200]

[0201] Combined with the test results of Table 22-Table 23 and Figure 28-29 It can be seen that in the plasmid template test, the first set of the two sets of multiplex detection systems has the best amplification effect, a smaller Ct value, the highest amplification sensitivity, and the specificity of the two sets of primer probe combinations is consistent. Therefore, the first set of probe combinations of RV G12 was selected.

[0202] Comprehensive analysis showed that group A rotavirus G8 and G9 selected the second set of primer-probe combinations, and G12 selected the first set of primer-probe combinations. The obtained amplification curves showed a standard S-shaped curve with a smaller Ct value, 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 Novozymes was used as the amplification reaction system. The amplification system combines an enzyme mixture (TaqDNA polymerase, reverse transcriptase, UNG enzyme, and reverse transcriptase inhibitor), dNTPs, Mg 2+ , buffer, etc. are all premixed into one tube.

[0206] The two sets of alternative multiplex amplification systems optimized in Example 3 and Example 4 were respectively formulated into primer-probe mixes. The volumes of the upstream primers (100 μM), downstream primers (100 μM) and probes (100 μM) of the four sets of primer probes were 4 μL respectively, and were made up to 100 μL with nuclease-free water. Add 1 μL of primer-probe mix and 4 μL of Novazon U+One Step RT-qPCR Probe 5×Master Mix to the reaction system to form a pre-packaging system. After mixing the system evenly, dispense 5 μL / well into eight-row tubes and cover them with tube caps. The pre-packed eight-row tubes were stored at -20°C, and the amplification conditions in October, April, August, and December were tested respectively. The quantified mixed nucleic acid was added to the eight-row pre-packaging system, and the nucleic acid was diluted to the following concentrations: 1×10 0 -1×10 2 4 μL of mixed nucleic acid with a copy / μL concentration and 11 μL of nuclease-free water. Three parallels were made for each gradient of mixed nucleic acid, and three template-free controls were made.

[0207] The non-pre-packaged system used as a control was consistent with the above operation process, but the primer probe mix and Novozymes' U+One Step RT-qPCR Probe 5×Master Mix were stored at -20°C, and the amplification conditions were tested in October, April, August, and December, respectively.

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

[0209] (1) The amplification data of multiple pre-packaging detection system 1 [RV G1 (F2, R2, P2), RV G2 (F2, R32, P2), RV G3 (F1, R1, P1), RV G4 (F1, R1, P1)] in October, April, August and December are shown in Tables 24-27 below.

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

[0211]

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

[0213]

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

[0215]

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

[0217]

[0218] (2) The amplification data of multiple pre-packaging detection system 2 [RV G8 (F2, R2, P2), RV G9 (F2, R2, P2), RV G12 (F1, R1, P1), RNP (F, R, P)] in October, April, August and December are shown in the following Tables 28-31.

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

[0220]

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

[0222]

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

[0224]

[0225] Table 31 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RNP)

[0226]

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

[0228] Example 6

[0229] This example performs 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 from our company's sample library. The mixed positive samples of group A rotavirus G1, G2, G3, and G4 quantified by digital PCR were diluted to 1×10 0- 1×10 2 copy / uL and the mixed positive samples of group A rotavirus G8, G9, G12 were diluted to 1×10 1 -1×10 3 copy / uL. Four samples of rotavirus genotype G1, G2, G3, G4, G8, G12 and eight samples of norovirus G9 were identified as group A. The pre-packaging multiplex system was evaluated in terms of sensitivity, specificity and sample detection rate.

[0231] Using the two sets of pre-packaging systems in Example 5, 10 μL of nucleic acid extracted from the above samples was directly added to the eight-tube series, mixed evenly, and tested on the machine.

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

[0233] (1) Specificity experiment

[0234] After the two multiplex systems diluted the mixed nucleic acid of 3 samples of group B rotavirus, 3 samples of group C rotavirus, and 6 samples of group A rotavirus P8 genotype quantified by digital PCR, the tested data are shown in the following Tables 32-33.

[0235] Table 32 Multiplex system 1 sample specificity detection data

[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 Rotavirus group C sample 1 Nt Nt Nt Nt Rotavirus group C sample 2 Nt Nt Nt Nt Rotavirus group C 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 comparison Nt Nt Nt Nt

[0237] Table 33 Multiplex system 2 sample specificity detection data

[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 Rotavirus group C sample 1 Nt Nt Nt 33.14 Rotavirus group C sample 2 Nt Nt Nt 27.27 Rotavirus group C 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 comparison Nt Nt Nt Nt

[0239] From the amplification results in Tables 32 and 33, it can be seen that among the 3 cases of group B rotavirus, 3 cases of group C rotavirus, and 6 cases of group A rotavirus P8 genotype, only the internal reference gene had obvious amplification curves, and both multiplex systems showed good specificity.

[0240] (2) Sensitivity test

[0241] After the two multiplex systems were used to quantify the mixed nucleic acid of group A rotavirus G1, G2, G3, G4 mixed positive samples and group A rotavirus G8, G9, G12 mixed positive samples by digital PCR, the detected data are shown in the following Tables 34-35.

[0242] Table 34 Multiplex system 1 nucleic acid detection data

[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 comparison Nt Nt Nt Nt

[0244] Table 35 Multiplex system 2 nucleic acid detection data

[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 comparison Nt Nt Nt Nt

[0246] It can be seen from the amplification results in Tables 34 and 35 that in nucleic acid template testing, the two sets of multiple pre-packaging detection systems have high sensitivity and can distinguish different types.

[0247] (3) Sample testing

[0248] Two sets of multiple pre-packaging detection systems were used to test 4 samples of G1, G2, G3, G4, G8, G12, which were confirmed to be group A rotavirus genotypes, and 8 samples of Norovirus G9. The test results are shown in Tables 35-36.

[0249] Table 35 Multiple detection system 1 nucleic acid detection data

[0250]

[0251]

[0252] Table 36 Multiple detection system 2 nucleic acid detection data

[0253]

[0254]

[0255] It can be seen from the amplification results in Tables 35 and 36 that all the above samples were detected with a detection rate of 100% and there was no cross reaction.

[0256] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0257] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A composition for simultaneously detecting seven 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 the G1 genotype of group A rotavirus, which are nucleotide sequences shown in SEQ ID NOs 1-3 respectively; the 3' end of the probe 1 is modified with MGB; Component B: upstream primer 2, downstream primer 2 and probe 2 for detecting group A rotavirus G2 genotype, which are nucleotide sequences shown in SEQ ID NOs 4-6 respectively; the 3' end of the probe 2 is modified with MGB; Component C: upstream primer 3, downstream primer 3 and probe 3 for detecting group A rotavirus G3 genotype, which are nucleotide sequences shown in SEQ ID NOs 7-9 respectively; Component D: upstream primer 4, downstream primer 4 and probe 4 for detecting group A rotavirus G4 genotype, which are nucleotide sequences shown in SEQ ID NOs 10-12 respectively; Component E: upstream primer 5, downstream primer 5 and probe 5 for detecting group A rotavirus G8 genotype, which are nucleotide sequences shown in SEQ ID NOs 13-15 respectively; the 3' end of the probe 5 is modified with MGB; Component F: upstream primer 6, downstream primer 6 and probe 6 for detecting group A rotavirus G9 genotype, which are nucleotide sequences shown in SEQ ID NOs 16-18 respectively; Component G: upstream primer 7, downstream primer 7 and probe 7 for detecting group A rotavirus G12 genotype, which are nucleotide sequences shown in SEQ ID NOs 19-21 respectively; the 3' end of the probe 7 is modified with MGB; Component H: upstream primer 8, downstream primer 8 and probe 8 for detecting human ribonucleoprotein complex, which are nucleotide sequences shown in SEQ ID NOs 22-24 respectively.

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

3. The kit according to claim 1, characterized in that The kit also includes an enzyme mixture, an amplification buffer, a positive quality control product, and a negative quality control product.

4. The kit according to claim 3, characterized in that The enzyme mixture includes reverse transcriptase, RNase inhibitor, Taq DNA polymerase and enzyme buffer.

5. The kit according to claim 3, characterized in that The amplification buffer comprises a buffer, dNTPs, Mg 2 + .

6. The kit according to claim 3, characterized in that Each component in the kit can be completely mixed into a fully premixed form.

7. A method for simultaneously detecting seven G genotypes of rotavirus in a sample using the composition of claim 1 or the kit of claims 2-6, characterized in that: The method specifically comprises the following steps: (1) Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested to obtain viral RNA; (2) Add the extracted RNA to the eight-tube strip containing pre-packed reagents to form a reaction system; (3) performing multiple real-time fluorescence quantitative RT-PCR amplification on the reaction system to obtain an amplification curve; (4) Analyze the amplification curve, obtain the Ct value, and make a judgment.

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