Composition, kit and method for simultaneously detecting P4, P6 and P8 genotypes of group A rotaviruses

Abstract

The invention relates to the technical field of virus detection, in particular to a composition, a kit and a method for simultaneously detecting P4, P6 and P8 genotypes of group A rotaviruses. The composition comprises a component A, an upstream primer 1, a downstream primer 1 and a probe 1, wherein the upstream primer 1, the downstream primer 1 and the probe 1 are used for detecting the genotype of group A rotavirus P4; the component B comprises an upstream primer 2, a downstream primer 2 and a probe 2 which are used for detecting the genotype of the group A rotavirus P6; the component C comprises an upstream primer 3, a downstream primer 3 and a probe 3 which are used for detecting the genotype of the group A rotavirus P8; the component D is used for detecting an upstream primer 4, a downstream primer 4 and a probe 4 of a 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 P4 genotype (RV P4), P6 genotype (RV P6) and P8 genotype (RV P8). 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 is consistent with the genotype; the P serotype is inconsistent with the genotype, 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 P genotyping method mainly includes nested PCR and first-generation sequencing, but these methods all have the problems of long time consumption, high cost, complicated operation, insufficient sensitivity. There are few studies on the group A rotavirus P genotype in the existing patents. The patent with publication number CN116479184A simultaneously detects 3 P 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 the traditional electrophoresis-based typing method and sequencing-based typing method, this method only requires an ordinary PCR instrument, 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 much lower than 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, which is time-consuming and cannot adapt to general fluorescence quantitative instruments.

[0006] Therefore, it is necessary to establish a detection method that is simultaneously sensitive and rapid for human group A rotavirus P4, P6 and P8 genotypes. Summary of the invention

[0007] The present application provides a composition, a kit and a method for simultaneously detecting the P4, P6 and P8 genotypes of group A rotavirus. The purpose is to establish a detection method that is simultaneously targeted at three P genotypes (P4, P6 and P8) with high sensitivity and rapidity, and is initially applied to the detection of group A rotavirus P genotypes.

[0008] The present application performs a combined detection of the P4 genotype, P6 genotype and P8 genotype of the common group A rotavirus that infects humans, selects the conserved region of the virus to design primers, specifically detects the three genotypes of group A rotavirus, and can also achieve the purpose of distinguishing the three 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 P genotypes, and has high specificity and sensitivity, good repeatability, simple and rapid detection, and cost savings.

[0010] In a first aspect, the present application provides a composition for simultaneously detecting group A rotavirus P4, P6 and P8 genotypes, using the following technical solution:

[0011] A composition for simultaneously detecting group A rotavirus P4, P6 and P8 genotypes, the composition comprising the following components:

[0012] Component A: upstream primer 1, downstream primer 1 and probe 1 for detecting group A rotavirus P4 genotype, 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 P6 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 P8 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 human ribonucleoprotein complex (RNP), which are nucleotide sequences shown in SEQ ID NOs 10-12 respectively.

[0016] The present application selects the conserved regions of the RV P4, RV P6 and RV P8 genome sequences to design upstream and downstream primers and probes respectively, and the detection primer probe of RNP is used as an internal reference gene detection composition for monitoring the whole process of sample collection, extraction and detection. After analyzing the possible dimers, secondary structures, etc. of the four sets of primers and probes, a composition that can simultaneously detect three pathogens and internal reference genes is formed after selecting appropriate sequences. The composition or a kit including the composition can simultaneously identify the rotavirus P4 genotype, rotavirus P6 genotype and rotavirus P8 genotype with similar clinical symptoms caused by group A rotavirus infection, and has high specificity and sensitivity, good repeatability, and accurate and reliable detection results.

[0017] The principle of using the above composition to detect RV P4, RV P6 and RV P8 in this application is as follows:

[0018] Taking component A as an example, the upstream primer 1, downstream primer 1 and probe 1 in component A can all specifically bind to the genomic template of RVP4, and the binding site of probe 1 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 RVP4 exists in the sample to be tested, the upstream primer 1, downstream primer 1 and probe 1 are all bound to the genomic template of RVP4. As RT-PCR proceeds, the Taq enzyme encounters the probe 1 bound to the template during chain extension, and its 3'-5' exonuclease activity will cut off probe 1, 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 1, it indicates that RVP4 exists in the sample to be tested.

[0019] Since the probes of RV P4, RV P6 and RV P8 are all labeled with different fluorescence, the detection data of the three virus typing can be obtained through different fluorescence curves.

[0020] 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 of RV P4, RV P6 and RV P8 in the sample to be tested, greatly shortening the detection time and reducing the detection cost.

[0021] In a second aspect, the present application provides a kit for simultaneously detecting rotavirus P4 genotype (RV P4), rotavirus P6 genotype (RV P6) and rotavirus P8 genotype (RV P8), which comprises the above-mentioned composition.

[0022] Optionally, the kit further comprises an enzyme mixture, an amplification buffer, a positive quality control and a negative quality control.

[0023] Optionally, the enzyme mixture includes reverse transcriptase, RNase inhibitor, Taq DNA polymerase and enzyme buffer.

[0024] Optionally, the amplification buffer comprises a buffer, dNTPs, Mg 2+ .

[0025] Among them, positive quality control products and negative quality control products are used for quality control during the use of the kit.

[0026] Optionally, each component in the kit can be completely mixed into a fully premixed form.

[0027] 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.

[0028] In the present application, the three compositions of rotavirus P4 genotype (RV P4), rotavirus P6 genotype (RV P6) and rotavirus P8 genotype (RV P8) in the composition of the kit can be mixed and packaged, or they can be packaged separately. When packaged separately, the three compositions of rotavirus P4 genotype (RV P4), rotavirus P6 genotype (RV P6) and rotavirus P8 genotype (RV P8) can be used separately to detect RV P4, RV P6 and RV P8 in the sample to be tested separately.

[0029] In a third aspect, the present application provides a method for simultaneously detecting rotavirus P4 genotype, rotavirus P6 genotype and rotavirus P8 genotype in a sample to be tested using the above composition or the above kit, using the following technical scheme:

[0030] A method for simultaneously detecting rotavirus P4 genotype, rotavirus P6 genotype and rotavirus P8 genotype in a sample to be tested using the above composition or the above kit, the method specifically comprising the following steps:

[0031] (1) Using a nucleic acid extraction kit to extract nucleic acid from the sample to be tested to obtain viral RNA;

[0032] (2) Adding the extracted RNA to eight tubes pre-packed with reagents to form a reaction system;

[0033] (3) performing multiple real-time fluorescence quantitative RT-PCR amplification on the reaction system to obtain an amplification curve;

[0034] (4) Analyze the amplification curve, obtain the Ct value, and make a judgment.

[0035] In the present application, a multiplex real-time fluorescence quantitative RT-PCR detection method is designed, that is, in the same reaction system, specific primers and probes for three pathogens (RV P4, RV P6 and RV P8) and the conserved segment of the internal reference gene RNP are added, which solves the problem that the traditional real-time fluorescence quantitative RT-PCR detection method can only use a single pair of primers to amplify one pathogen RNA and can only detect one pathogen separately at a time, thereby reducing cross contamination caused during operation.

[0036] 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.

[0037] In the present application, the above-mentioned primers refer to upstream primers 1-4 and downstream primers 1-4 for RV P4, RV P6, RV P8 and internal reference gene RNP; the above-mentioned probes refer to probes 1-4 for RV P4, RV P6, RV P8 and internal reference gene RNP.

[0038] 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.

[0039] 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 RV P4, RV P6 and RV P8 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.

[0040] 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).

[0041] In some specific embodiments, in step (3), the amplification conditions include:

[0042] Reverse transcription at 50-55°C for 5-20 min;

[0043] Pre-denaturation at 90-95℃ for 1-5min;

[0044] 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.

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

[0046] Reverse transcription at 55°C for 15 min;

[0047] Pre-denaturation at 95°C for 1 min;

[0048] Denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, 40 cycles, and fluorescence was collected.

[0049] By adopting the above-mentioned amplification conditions, the present application can effectively amplify the RV P4, RV P6 and RV P8 genes in the sample to be tested.

[0050] In some specific embodiments, in step (4), the principle for analyzing and judging the amplification curve is:

[0051] 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 human rotavirus P4 genotype (RV P4) positive sample;

[0052] 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 rotavirus P6 genotype (RV P6) positive sample;

[0053] 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 judged to be a rotavirus P8 genotype (RV P8) positive sample.

[0054] 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.

[0055] It is worth noting that the method described in the present application is a method for the purpose of non-disease diagnosis.

[0056] In summary, the present application includes at least one of the following beneficial technical effects:

[0057] The group A rotavirus that infects humans can be divided into G genotype and P genotype, of which the most common P genotypes include P4, P6 and P8. Literature search found that the method used to detect P4, P6 and P8 alone or together is not a fluorescent quantitative method. The present application also detects the common group A rotavirus P genotype that infects 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 rows for easy operation. You only need to add the extracted nucleic acid to the eight rows to directly amplify it on the machine.

[0058] The composition provided in the present application for simultaneously detecting the human rotavirus P4 genotype, the rotavirus P6 genotype and the rotavirus P8 genotype is a primer and a probe with specificity for the conserved segments of the above three pathogens. When added to the same reaction system, the human rotavirus P4 genotype, the rotavirus P6 genotype and the rotavirus P8 genotype in the sample can be detected simultaneously 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 cross-contamination caused during operation.

[0059] 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.

[0060] 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

[0061] Figure 1 Amplification results of RV P4 - the first set of primers and probes (RV P4 (F1, R1, P1)).

[0062] Figure 2 Amplification results of RV P4 - the second set of primers and probes (RV P4 (F2, R2, P2)).

[0063] Figure 3 This is the amplification result of RV P4 - the third set of primers and probes (RV P4 (F3, R3, P3)).

[0064] Figure 4Amplification results of RV P6 - the first set of primers and probes (RV P6 (F1, R1, P1)).

[0065] Figure 5 Amplification results of RV P6 - the second set of primers and probes (RV P6 (F2, R2, P2)).

[0066] Figure 6 This is the amplification result of RV P6 - the third set of primers and probes (RV P6 (F3, R3, P3)).

[0067] Figure 7 Amplification results of RV P8 - the first set of primers and probes (RV P8 (F1, R1, P1)).

[0068] Figure 8 Amplification results of RV P8 - the second set of primers and probes (RV P8 (F2, R2, P2)).

[0069] Fig. 9 This is the amplification result of RV P8 - the third set of primers and probes (RV P8 (F3, R3, P3)).

[0070] Fig.10 is the amplification result of RNP (RNP(F,R,P)).

[0071] Fig.11 The concentration of RV P4-plasmid was 1×10 3 Amplification curves of the first multiplex detection system.

[0072] Fig.12 The concentration of RV P4-plasmid was 1×10 3 Amplification curves of the second multiplex detection system.

[0073] Fig.13 The concentration of RV P4-plasmid was 1×10 3 Amplification curves of the third multiplex detection system.

[0074] Fig.14 For RV P6-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0075] Fig.15 For RV P6-plasmid concentration 1×10 3 Amplification curves of the second multiplex detection system.

[0076] Fig.16 For RV P6-plasmid concentration 1×10 3 Amplification curves of the third multiplex detection system.

[0077] Fig.17 For RV P8-plasmid concentration 1×10 3 Amplification curves of the first multiplex detection system.

[0078] Fig.18 For RV P8-plasmid concentration 1×10 3 Amplification curves of the second multiplex detection system.

[0079] Fig.19 For RV P8-plasmid concentration 1×10 3 Amplification curves of the third multiplex detection system. DETAILED DESCRIPTION

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] The present application is further described in detail below in conjunction with the embodiments and test results.

[0087] Example 1

[0088] This example provides the design and synthesis of primers and probes.

[0089] Through literature search, some alternative primer and probe sequences were obtained. At the same time, the VP4 fragment sequences of the three P 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 three P genotypes (P4, P6, and P8) of group A rotavirus, 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 the structure was analyzed by primer premier 5 software. Finally, the Primer Blast 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 three P genotypes (P4, P6, and P8) of group A rotavirus and will not be degenerate and cause missed detection. Finally, three sets of primers and probes for the three P genotypes (P4, P6, and P8) of group A rotavirus were screened and determined, and the specific nucleotide sequences (5'-3') are shown in Table 1.

[0090] Table 1 Three sets of primers and probes for three P genotypes (P4, P6, P8) of group A rotavirus

[0091]

[0092]

[0093] Example 2

[0094] This example uses the primer probe of Example 1 to perform a single-plex test.

[0095] The AccurSTART U+One Step RT-qPCR Probe Kit (FOR FAST) sold by Novozymes was used as the amplification reaction system.

[0096] Three sets of primers and probes for group A rotavirus RV P4, RV P6, and RV P8 were tested in single-plex systems. Primer probe mix was prepared first, with the volumes of upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) being 2 μL respectively, and made up to 100 μL with nuclease-free water. 2 μL of primer probe mix, 4 μL of Novagen 5×One Step U+Mix, 1 μl of Novagen 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.

[0097] 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.

[0098] The amplification data of three sets of primers and probes for group A rotavirus RV P4, RV P6 and RV P8 and the internal reference gene RNP are shown in Tables 2 to 5 below.

[0099] The amplification results of RV P4 are shown in Table 2, and the results of each set of primers and probes are shown in Table 2. Figure 1-3 shown.

[0100] The amplification results of RV P6 are shown in Table 3, and the results of each set of primers and probes are shown in Table 3. Figure 4-6 shown.

[0101] The amplification results of RV P8 are shown in Table 4, and the results of each set of primers and probes are shown in Table 4. Figure 7-9 shown.

[0102] Among them, the amplification results of RNP are shown in Table 5. Fig.10 shown.

[0103] Table 2 Amplification results of RV P4

[0104] Plasmid concentration RV P4(F1,R1,P1) RV P4(F2,R2,P2) RV P4(F3,R3,P3) <![CDATA[1×10 3 (Average Ct)]]> 28.29 28.39 28.51 <![CDATA[1×10 2 (Average Ct)]]> 30.81 31.26 31.12 1×10(average Ct) 34.25 34.80 34.53 No template comparison Nt Nt Nt

[0105] Table 3 Amplification results of RV P6

[0106]

[0107]

[0108] Table 4 Amplification results of RV P8

[0109] Plasmid concentration RV P6(F1,R1,P1) RV P6(F2,R2,P2) RV P6(F3,R3,P3) <![CDATA[1×10 3 (Average Ct)]]> 28.73 28.74 28.58 <![CDATA[1×10 2 (Average Ct)]]> 31.96 31.60 31.56 1×10(average Ct) 35.60 35.41 35.38 No template comparison Nt Nt Nt

[0110] Table 5 RNP amplification results

[0111] Plasmid concentration RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 28.12 <![CDATA[1×10 2 (Average Ct)]]> 31.81 1×10(average Ct) 36.00 No template comparison Nt

[0112] From the amplification results in Tables 2 to 5, it can be seen that in the plasmid template test, the three sets of primer probes of RV P4, RV P6 and RV P8 and the single-plex system amplification effect of the internal reference gene are all good, the amplification curve is a typical S-shaped curve, and the Ct value is also close. It can be seen that all the above primer probes can be used as alternative primer probes for the establishment of the following multiplex system.

[0113] Example 3

[0114] This example uses the primers and probes of Example 1 to establish a multiplex detection system.

[0115] The AccurSTART U+One Step RT-qPCR Probe Kit (FOR FAST) sold by Novozymes was used as the amplification reaction system.

[0116] The three sets of primers and probes of group A rotavirus RV P4, RV P6, and RV P8 were used to establish a multiplex detection system with a quadruple system composed of the internal reference gene RNP. First, prepare the primer probe mix. The volume 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 is 2 μL, 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 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.

[0117] 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.

[0118] (1) First, three sets of alternative primer-probe combinations of RV P4 were selected, and the first set of primer-probe combinations of RV P6 and RV P8, as well as the primer-probe combination of the internal reference gene, to form the following combination to test the multiplex detection system. The amplification data are shown in Tables 6-8 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 11-13 shown.

[0119] Table 6 Multiplex detection system - the first set of candidate primers and probes for RV P4

[0120] Plasmid concentration RV P4(F1,R1,P1) RV P6(F1,R1,P1) RV P8(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 26.53 26.49 25.25 26.84 <![CDATA[1×10 2 (Average Ct)]]> 30.39 30.30 28.66 30.78 1×10(average Ct) 33.40 33.46 31.89 34.51 No template comparison Nt Nt Nt Nt

[0121] Table 7 Multiplex detection system - the second set of alternative primers and probes for RV P4

[0122] Plasmid concentration RV P4(F2,R2,P2) RV P6(F1,R1,P1) RV P8(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 26.58 26.50 25.29 26.76 <![CDATA[1×10 2 (Average Ct)]]> 30.14 30.27 28.67 30.50 1×10(average Ct) 33.50 33.53 32.14 34.33 No template comparison Nt Nt Nt Nt

[0123] Table 8 Multiplex detection system - the third set of alternative primers and probes for RV P4

[0124] Plasmid concentration RV P4(F3,R3,P3) RV P6(F1,R1,P1) RV P8(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 26.26 26.62 25.51 26.96 <![CDATA[1×10 2 (Average Ct)]]> 29.72 30.31 28.81 30.68 1×10(average Ct) 32.75 33.76 31.96 34.23 No template comparison Nt Nt Nt Nt

[0125] Combined with the test results of Tables 6 to 8 above and Figure 11-13 It can be seen that in the plasmid template test, the third set of the above three sets of multiplex detection systems has the best amplification effect, a smaller Ct value, the highest amplification sensitivity, and the three sets of primer-probe combinations are consistent in specificity. Therefore, the third set of primer-probe combination of RV P4 was selected.

[0126] Based on the above, the third primer-probe combination of RV P4 was fixed, the first primer-probe combination and the primer-probe combination of the internal reference RNP were used for RV P8, and the three primer-probe combinations of RV P6 were further screened.

[0127] (2) Next, the three alternative primer-probe combinations of RV P6 were selected, and the third primer-probe combination of RV P4, the first primer-probe combination of RV P8, and the primer-probe combination of the internal reference gene were respectively selected to form the following combination to test the multiplex detection system. The amplification data are shown in Tables 9-11 below. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 14-16 shown.

[0128] Table 9 Multiplex detection system - the first set of alternative primers and probes for RV P6

[0129]

[0130]

[0131] Table 10 Multiplex detection system - the second set of alternative primers and probes for RV P6

[0132] Plasmid concentration RV P4(F3,R3,P3) RV P6(F2,R2,P2) RV P8(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 26.52 26.37 25.18 26.67 <![CDATA[1×10 2 (Average Ct)]]> 29.94 29.98 28.41 30.58 1×10(average Ct) 33.12 33.48 31.60 34.63 No template comparison Nt Nt Nt Nt

[0133] Table 11 Multiplex detection system - the third set of alternative primers and probes for RV P6

[0134] Plasmid concentration RV P4(F3,R3,P3) RV P6(F3,R3,P3) RV P8(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 26.47 26.28 25.59 26.96 <![CDATA[1×10 2 (Average Ct)]]> 30.06 29.95 29.07 30.82 1×10(average Ct) 33.22 33.43 32.48 34.64 No template comparison Nt Nt Nt Nt

[0135] Combined with the test results of Tables 9 to 11 above and Figure 14-16 It can be seen that in the plasmid template test, the third set of the above three sets of multiplex detection systems has the best amplification effect, a smaller Ct value, the highest amplification sensitivity, and the three sets of primer-probe combinations are consistent in specificity. Therefore, the third set of primer-probe combination of RV P6 was selected.

[0136] On the basis of the above, the third primer-probe combination of RV P4 and the third primer-probe combination of RV P6 were fixed, and the three primer-probe combinations of RV P8 were further screened together with the primer-probe combination of the internal reference RNP.

[0137] (3) Finally, the three alternative primer-probe combinations of RV P8 were selected and combined with the third primer-probe combination of RV P4, the third primer-probe combination of RV P6, and the primer-probe combination of the internal reference gene to form the following combination to test the multiplex detection system. The amplification data are shown in Tables 12-14. Among them, the plasmid concentration was 1×10 3 The amplification curves of the multiplex detection system are Figure 17-19 shown.

[0138] Table 12 Multiplex detection system - the first set of alternative primers and probes for RV P8

[0139] Plasmid concentration RV P4(F3,R3,P3) RV P6(F3,R3,P3) RV P8(F1,R1,P1) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 27.01 26.93 24.94 27.19 <![CDATA[1×10 2 (Average Ct)]]> 30.81 30.74 28.75 30.99 1×10(average Ct) 33.94 34.08 32.06 34.23 No template comparison Nt Nt Nt Nt

[0140] Table 13 Multiplex detection system - the second set of alternative primers and probes for RV P8

[0141]

[0142]

[0143] Table 14 Multiplex detection system - the third set of alternative primers and probes for RV P8

[0144] Plasmid concentration RV P4(F3,R3,P3) RV P6(F3,R3,P3) RV P8(F3,R3,P3) RNP(F,R,P) <![CDATA[1×10 3 (Average Ct)]]> 26.25 25.80 24.05 26.05 <![CDATA[1×10 2 (Average Ct)]]> 29.87 29.54 27.68 29.80 1×10(average Ct) 33.23 32.79 31.02 33.52 No template comparison Nt Nt Nt Nt

[0145] Combined with the test results of Table 12-Table 14 and Figure 17-19 It can be seen that in the plasmid template test, the second and third sets of the above three sets of multiple detection systems have the best amplification effect, smaller Ct value, highest amplification sensitivity, and the three sets of primer-probe combinations are consistent in specificity. Therefore, the second and third sets of primer-probe combinations of RV P6 were selected.

[0146] Comprehensive analysis shows that two of the quadruple systems can be used as the optimal combination, namely: RV P4, RV P6 and RV P8 all choose the third primer combination; RV P4 and RV P6 choose the third primer combination and RV P8 chooses the second primer-probe combination. The obtained amplification curve presents a standard S-shaped curve with a smaller Ct value, optimal sensitivity and good specificity.

[0147] Therefore, two sets of multiplex detection systems were used for further studies in the following specific examples.

[0148] Example 4

[0149] This example uses the multiple detection system screened out in Example 3 to establish a multiple pathogen detection pre-packaging system.

[0150] 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.

[0151] The two sets of alternative multiplex amplification systems optimized in Example 3 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. Make 3 parallels for each gradient of mixed nucleic acid and 3 template-free controls.

[0152] 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.

[0153] 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.

[0154] (1) The amplification data of multiple pre-packaging detection system 1 [RV P4 (F3, R3, P3), RV P6 (F3, R3, P3), RV P8 (F3, R3, P3), RNP (F, R, P)] in October, April, August and December are shown in Tables 15-18 below.

[0155] Table 15 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RV P4)

[0156]

[0157] Table 16 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RV P6)

[0158]

[0159] Table 17 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RV P8)

[0160]

[0161] Table 18 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RNP)

[0162]

[0163] (2) The amplification data of multiple pre-packaging detection system 2 [RV P4 (F3, R3, P3), RV P6 (F3, R3, P3), RV P8 (F2, R2, P2), RNP (F, R, P)] in October, April, August and December are shown in Tables 19-22 below.

[0164] Table 19 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RV P4)

[0165]

[0166] Table 20 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RV P6)

[0167]

[0168] Table 21 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RV P8)

[0169]

[0170] Table 22 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RNP)

[0171]

[0172] Combined with the test results in Tables 15 to 22 above, it can be seen that in the plasmid template test, the shelf life of the pre-packaged detection system can be up to 12 months without changing the sensitivity and specificity of the kit.

[0173] Example 5

[0174] This example performs clinical sample testing.

[0175] Specific samples (4 cases of group B rotavirus, 4 cases of group C rotavirus, and 4 cases of group A rotavirus G9 genotype) were found from our company's sample library. Group A rotavirus RV P4, RV P6, and RV P8 samples quantified by digital PCR were diluted to 1×10 0 -1×10 2 copy / uL, 10 cases have been identified as group A rotavirus RV P4, RV P6 and RV P8. The pre-packaging multiplex system was evaluated in terms of sensitivity, specificity and sample detection rate.

[0176] Using the two sets of pre-packaging systems in Example 4, 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.

[0177] 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.

[0178] (1) Specificity experiment

[0179] After the two multiplex systems diluted the mixed nucleic acids of group A rotavirus RV P4, RV P6 and RV P8 samples quantified by digital PCR, the detected data are shown in the following Tables 23-24.

[0180] Table 23 Sample specificity test data of multiplex system 1

[0181] RV P4 RV P6 RV P8 Internal reference RNP Group B rotavirus sample 1 Nt Nt Nt 27.80 Group B rotavirus sample 2 Nt Nt Nt 29.20 Group B rotavirus sample 3 Nt Nt Nt 30.86 Group B rotavirus sample 4 Nt Nt Nt 32.72 Rotavirus group C sample 1 Nt Nt Nt 27.59 Rotavirus group C sample 2 Nt Nt Nt 29.14 Rotavirus group C sample 3 Nt Nt Nt 30.89 Rotavirus group C sample 4 Nt Nt Nt 32.95 Group A rotavirus G9 genotype-1 Nt Nt Nt 27.24 Group A rotavirus G9 genotype-2 Nt Nt Nt 28.83 Group A rotavirus G9 genotype-3 Nt Nt Nt 30.98 Group A rotavirus G9 genotype-4 Nt Nt Nt 33.14 No template comparison Nt Nt Nt Nt

[0182] Table 24 Multiplex system 2 sample specificity detection data

[0183]

[0184]

[0185] From the amplification results in Tables 23 and 24, it can be seen that among the 4 cases of group B rotavirus, 4 cases of group C rotavirus, and 4 cases of group A rotavirus G9 genotype, only the internal reference gene had obvious amplification curves, and both multiplex systems showed good specificity.

[0186] (2) Sensitivity test

[0187] After the two multiplex systems diluted the mixed nucleic acid of group A rotavirus RV P4, RV P6 and RV P8 samples quantified by digital PCR, the detected data are shown in the following Tables 25-26.

[0188] Table 25 Multiplex system 1 nucleic acid detection data

[0189] Nucleic acid copy number RV P4 RV P6 RV P8 Internal reference RNP <![CDATA[1×10 2 (Average Ct)]]> 30.08 28.63 28.61 30.13 <![CDATA[1×10 1 (Average Ct)]]> 33.65 32.39 32.23 33.86 <![CDATA[1×10 0 (Average Ct)]]> 34.58 33.69 33.16 34.81 No template comparison Nt Nt Nt Nt

[0190] Table 26 Multiplex system 2 nucleic acid detection data

[0191] Nucleic acid copy number RV P4 RV P6 RV P8 Internal reference RNP <![CDATA[1×10 2 (Average Ct)]]> 30.18 28.62 28.60 30.23 <![CDATA[1×10 1 (Average Ct)]]> 33.49 32.00 32.21 33.42 <![CDATA[1×10 0 (Average Ct)]]> 34.93 33.28 33.13 34.42 No template comparison Nt Nt Nt Nt

[0192] It can be seen from the amplification results in Tables 25 and 26 that in the nucleic acid template test, the sensitivity of the multiple detection pre-packaging detection system 1 is consistent with the sensitivity of system 2, and both systems perform the following sample tests.

[0193] (3) Sample testing

[0194] The two pre-packaging detection systems were used to test 10 samples that had been confirmed to be group A rotavirus RV P4, RV P6 and RV P8. The test results are shown in Tables 27-28.

[0195] Table 27 Multiple detection system 1 nucleic acid detection data

[0196]

[0197]

[0198] Table 28 Nucleic acid detection data of multiple detection system 2

[0199]

[0200]

[0201] From the amplification results in Tables 27 and 28, it can be seen that all samples of the multiplex detection system 1 were detected, while the detection rate of RVP4 samples of the multiplex detection system 2 was 80%, and the detection rates of RVP6 and RVP8 were both 70%.

[0202] Therefore, after the final sample testing and evaluation, it can be determined that the amplification effect of multiplex detection system 1 is better and the detection rate is higher. Therefore, the first multiplex detection system was finally selected.

[0203] 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.

[0204] 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 group A rotavirus P4, P6 and P8 genotypes, 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 P4 genotype, 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 P6 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 P8 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 human ribonucleoprotein complex, which are nucleotide sequences shown in SEQ ID NOs 10-12 respectively.

2. A kit for simultaneously detecting group A rotavirus P4, P6 and P8 genotypes, 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 rotavirus P4 genotype, rotavirus P6 genotype and rotavirus P8 genotype in a sample using the composition of claim 1 or the kit of any one of claims 2 to 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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