A composition, kit and method for simultaneous detection of NV GI, NV GII and NV GIV

Abstract

The application relates to the technical field of virus detection, and specifically discloses a composition, a kit and a method for simultaneously detecting NV GI, NV GII and NV GIV. The composition provided by the application comprises: component A, which is used for detecting an upstream primer 1, a downstream primer 1 and a probe 1 of NV GI, and the nucleotide sequences are shown in SEQ ID NO. 1-3; component B, which is used for detecting an upstream primer 2, a downstream primer 2 and a probe 2 of NV GII, and the nucleotide sequences are shown in SEQ ID NO. 4-6; and component C, which is used for detecting an upstream primer 3, a downstream primer 3 and a probe 3 of NV GIV, and the nucleotide sequences are shown in SEQ ID NO. 7-9. The composition, the kit and the method provided by the application can simultaneously identify NV GI, NV GII and NV GIV, have high specificity and sensitivity, are good in repeatability, and are accurate and reliable in detection results.

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 NV GI, NV GII and NVGIV. Background Art

[0002] Norovirus, a member of the genus Norovirus (NV) in the family Human Caliciviridae (HuCV), is a zoonotic pathogen that can cause acute gastroenteritis and severe diarrhea in humans and a variety of animals worldwide. It is also a major cause of foodborne illness. Norovirus epidemics pose a serious threat to human health and result in significant socioeconomic losses.

[0003] Norovirus has a variety of transmission routes, a low infection dose, a long detoxification time, strong environmental resistance, rapid viral mutation, and a short immune protection time. It is highly contagious and has the ability to spread rapidly.

[0004] Norovirus-induced diarrhea is prevalent worldwide and can occur year-round, primarily affecting adults and school-age children, with a higher incidence in colder months. Norovirus-induced diarrhea is a self-limiting disease with no vaccine or specific medication. Maintaining good personal, food, and water hygiene is key to preventing the disease. Healthy habits include frequent handwashing, avoiding raw water, keeping raw and cooked food separate, and avoiding cross-contamination.

[0005] Noroviruses are genetically diverse. Early classifications were based on nucleotide sequence differences in the RdRp and capsid protein coding regions of the NV genome. Noroviruses are divided into five genogroups, GⅠ to GV. Of these, only GⅠ, GⅡ, and GⅣ can infect humans, while GⅢ and GV infect cattle and mice, respectively. Genogroups can be further divided into genotypes. There are eight genotypes in the GI group (GI.1 to GI.8), 19 genotypes in the GII group (GII.1 to GII.19), two genotypes in the GIII group (GIII.1 to GIII.2), and only one genotype each in GIV and GV.

[0006] Infectious diarrhea is diarrhea caused by various pathogens such as viruses, bacteria, fungi, and protozoa. Common viruses: Norovirus, group A rotavirus and adenovirus, astrovirus, certain respiratory viruses, etc. Common bacteria: Vibrio cholerae, Shigella dysenteriae, diarrheagenic Escherichia coli, Vibrio parahaemolyticus, Salmonella, Campylobacter, Aeromonas and Shigella-like bacteria, Bacillus cereus, Clostridium perfringens, Yersinia enterocolitica, etc. Parasites: Giardia lamblia, Entamoeba histolytica, Cryptosporidium, Cyclospora, etc. Antibiotic-associated diarrhea: Clostridium difficile, etc. The diarrhea symptoms caused by these pathogens are similar, and it is difficult to diagnose through clinical manifestations. Therefore, it is necessary to clarify the diagnosis of norovirus or even the specific type of norovirus, which requires laboratory testing to avoid misdiagnosis. Traditional laboratory diagnostic methods include virus culture, etc., which are relatively complicated and time-consuming. With the development of molecular diagnostic technology, most laboratories have begun to use fluorescent quantitative RT-PCR detection, but most of them are single-plex fluorescent detection, which can only detect a single pathogen, increasing the experimental operation time and the required sample volume, or can only detect norovirus GI and GII at a time, and there is no method to detect GIV at the same time.

[0007] Therefore, it is necessary to establish a highly sensitive and rapid detection method for norovirus type I (NV GI), norovirus type II (NV GII) and norovirus type IV (NV GIV) for the detection of norovirus. Summary of the Invention

[0008] In order to solve the above technical problems, the present application provides a composition, a kit and a method for simultaneously detecting NV GI, NV GII and NV GIV.

[0009] In a first aspect, the present application provides a composition for simultaneously detecting NV GI, NV GII and NV GIV, the composition comprising the following components:

[0010] Component A: upstream primer 1, downstream primer 1 and probe 1 for detecting NV GI, the nucleotide sequences of which are shown in SEQ ID NOs. 1-3 respectively;

[0011] Component B: upstream primer 2, downstream primer 2 and probe 2 for detecting NV GII, the nucleotide sequences of which are shown in SEQ ID NOs. 4-6 respectively;

[0012] Component C: upstream primer 3, downstream primer 3 and probe 3 for detecting NV GIV, whose nucleotide sequences are shown in SEQ ID NOs. 7-9 respectively.

[0013] Preferably, the composition for simultaneously detecting NV GI, NV GII and NV GIV also includes component D, which is an upstream primer 4, a downstream primer 4 and a probe 4 for detecting human ribonucleoprotein complex RNP, and their nucleotide sequences are shown in SEQ ID NO.10-12 respectively; the component D is used as an internal reference gene detection composition.

[0014] In order to overcome the defects existing in the prior art, the present application provides a composition for simultaneously detecting norovirus type I (NV GI), norovirus type II (NV GII) and norovirus type IV (NV GIV) using multiple real-time fluorescence quantitative RT-PCR. The composition or a kit including the composition can simultaneously identify viral pathogens with similar clinical symptoms caused by norovirus infections of different types, and has high specificity and sensitivity, good repeatability, simple and rapid detection, and cost-saving.

[0015] This application combines detection of common human norovirus subtypes GI, GII, and GIV. By selecting conserved regions of the virus for design, the system specifically detects and distinguishes between the three subtypes. Current patents primarily focus on detecting either GI or GII alone; no patents have been found for simultaneous detection of all three subtypes.

[0016] This application selects the conserved regions of the NV GI, NV GII and NV GIV genome sequences to design upstream and downstream primers and probes respectively, and the detection primer probe of the human ribonucleoprotein complex (RNP) is used as an internal reference gene detection composition to monitor the entire process of sample collection, extraction and detection. After analyzing the possible dimers, secondary structures, etc. of the four sets of primers and probes, a suitable sequence is selected to form a composition that can simultaneously detect three pathogens and internal reference genes. The composition or a kit including the composition can be used to simultaneously identify norovirus type I, norovirus type II and norovirus type IV with similar clinical symptoms caused by norovirus infection, and has high specificity and sensitivity, good repeatability, and accurate and reliable test results.

[0017] The principle of using the above composition to detect NV GI, NV GII and NV GIV in this application is as follows:

[0018] For example, the upstream primer 1, the downstream primer 1 and the probe 1 in the component A can all specifically bind to the genomic template of NV GI, and the binding site of the probe 1 is between the two primers. When the probe is complete, the fluorescence energy emitted by the fluorescent reporter group is absorbed by the quencher, and no signal can be detected by the instrument. During the RT-PCR, if the sample to be tested contains NV GI, the upstream primer 1, the downstream primer 1 and the probe 1 all bind to the genomic template of NV GI. With the progress of the RT-PCR, the 3'-5' exonuclease activity of the Taq enzyme will cut the probe 1 when it encounters the probe 1 combined with the template during the strand extension, and the fluorescent reporter group is away from the quencher, and its energy cannot be absorbed, that is, a fluorescence signal is generated. And every time a PCR cycle is performed, the fluorescence signal and the target fragment have a synchronous exponential growth process. Therefore, if an S-shaped fluorescence signal curve is collected in the fluorescence channel corresponding to the probe 1, it indicates that the sample to be tested contains NV GI.

[0019] Since the probes of NV GI, NV GII and NV GIV are labeled with different fluorescence, the detection data of the three virus types can be obtained through different fluorescence curves.

[0020] The upstream primer and the downstream primer and the probe in the composition described in the present application have high specificity and will not cause non-specific binding. Therefore, the composition described above can be used to simultaneously and specifically detect NV GI, NV GII and NV GIV 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 NV GI, NV GII and NV GIV for non-disease diagnosis purposes, the kit comprising the composition described in any one of the above.

[0022] Preferably, the kit further comprises an enzyme mixture, an amplification buffer, a positive quality control and a negative quality control; the enzyme mixture comprises a reverse transcriptase, an RNAase inhibitor, a Taq DNA polymerase and an enzyme buffer; the amplification buffer comprises a buffer, dNTPs, Mg 2+ ; the positive quality control is a plasmid containing NV GI, NV GII and NV GIV amplification fragments, a pseudo virus; and the negative quality control is nuclease-free water.

[0023] Specifically, the positive quality control and the negative quality control play a role in the quality control operation process and whether there is pollution during the use of the kit

[0024] Preferably, the kit completely mixes the composition, the enzyme mixture, and the amplification buffer together to form a fully premixed reagent; the fully premixed reagent is pre-packaged into 8 strip tubes, and the tubes are capped to make pre-packaged reagents; during use, the tube caps are opened, the extracted nucleic acid is added, the tube caps are closed, and the reagents can be loaded onto the instrument.

[0025] The kit can achieve the goal of completely mixing the composition described in the first aspect, the enzyme mixture, the buffer solution, etc. together to form a fully premixed form.

[0026] Aliquot the premixed reagents into 8-tube strips and cap them. This pre-packaging method allows for easy insertion of the reagents into the tubes, reducing the number of steps and the potential for error.

[0027] In the present application, the three compositions of norovirus type I (NV GI), norovirus type II (NV GII), and norovirus type IV (NV GIV) of the composition of the first aspect in the kit can be mixed and packaged or can be packaged separately. When packaged separately, the three compositions of components norovirus type I (NV GI), norovirus type II (NV GII), and norovirus type IV (NV GIV) can be used separately to detect NV GI, NV GII, and NV GIV in the sample to be tested separately.

[0028] In a third aspect, the present application provides a method for simultaneously detecting NV GI, NV GII, and NV GIV in a sample to be tested, using any of the compositions described above or any of the kits described above, specifically comprising the following steps:

[0029] Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested to obtain viral RNA;

[0030] The extracted viral RNA is added to 8 pre-packed tube strips containing the composition and other raw materials to form a reaction system;

[0031] Performing multiple real-time fluorescence quantitative RT-PCR amplification on the reaction system to obtain an amplification curve;

[0032] The amplification curve is analyzed to obtain the Ct value and make a judgment.

[0033] In this application, a multiplex real-time fluorescence quantitative RT-PCR detection method was designed, that is, in the same reaction system, specific primers and probes for three pathogens (NV GI, NV GII and NV GIV) and the conserved segment of the internal reference gene RNP were added. This solves 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 cross-contamination caused during operation.

[0034] 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.

[0035] In this application, the above primers refer to upstream primers 1-4 and downstream primers 1-4 for NV GI, NV GII, NV GIV and internal reference gene RNP; the above probes refer to probes 1-4 for NV GI, NV GII, NV GIV and internal reference gene RNP.

[0036] In some specific embodiments, the concentration of each primer in the reaction system is the same, both are 0.2 μmol / L, and the concentration of each probe is the same, both are 0.2 μmol / L.

[0037] In some specific embodiments, the reaction system is 20 μL, specifically:

[0038] The viral RNA solution extracted from the sample to be tested is 10uL, 1μL of enzyme mixture, 0.5μL of upstream primers 1-4 with a concentration of 10μmol / L, 0.5μL of downstream primers 1-4 with a concentration of 10μmol / L, 0.5μL of probes 1-4 with a concentration of 20μmol / L, 0.25μL of probes 1-4 with a concentration of 20μmol / L, containing dNTPs and Mg 2+ 4 μL of buffer.

[0039] In some embodiments, the conditions for RT-PCR amplification include:

[0040] Reverse transcription at 50-55°C for 5-20 minutes;

[0041] Pre-denaturation at 90℃~95℃ for 1~5 minutes;

[0042] Denaturation at 90°C-95°C for 5-15 seconds, annealing and extension at 50°C-60°C for 30-45 seconds, for 35-45 cycles, and fluorescence was collected.

[0043] In some preferred embodiments, the conditions for RT-PCR amplification include:

[0044] Reverse transcription at 55°C for 15 minutes;

[0045] Pre-denaturation at 95°C for 1 minute;

[0046] Denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds, 40 cycles, and fluorescence was collected.

[0047] The present application can effectively amplify the NV GI, NV GII and NV GIV genes in the sample to be tested by adopting the above-mentioned amplification conditions.

[0048] In some specific embodiments, the principles for analyzing and judging the amplification curve are:

[0049] When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 1 of the test sample is S-shaped and the Ct value is within 37, the test sample is judged to be a human norovirus type 1 positive sample;

[0050] When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 2 of the test sample is S-shaped and the Ct value is within 37, the test sample is judged to be a human norovirus type II positive sample;

[0051] When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on 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 norovirus type IV positive sample.

[0052] In summary, the technical solution of this application has the following effects:

[0053] The composition provided in this application for the simultaneous detection of human norovirus type I, norovirus type II and norovirus type IV is a primer and probe with specificity for the conserved segments of the above three pathogens. When added to the same reaction system, human norovirus type I, norovirus type II and norovirus type IV in the sample can be detected simultaneously without cross-reaction. This solves 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 problem of cross-contamination caused during operation.

[0054] The kit provided herein can be used to completely mix the composition for simultaneous detection of NV GI, NV GII, and NV GIV, the enzyme mixture described in the second aspect, the buffer, and the like, to create a fully premixed form. The fully premixed reagents are pre-packed into eight strip tubes and capped to create a pre-packed form. During use, the tester only needs to open the tube cap, add the extracted nucleic acid, and cap the tube to be ready for use, significantly reducing the number of steps and the probability of operational errors.

[0055] The method for detecting by using the composition or the kit comprising the composition has the advantages of high sensitivity and specificity, simple operation, short detection time, small required sample amount, low pollution, etc., can directly detect the extracted nucleic acid in the to-be-detected sample, and has high application value in rapid detection of viruses. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 The amplification curve diagram of the single test system of the first set of primer probes NV GI (F1, R1, P1) of NV GI in Example 2.

[0057] Figure 2 The amplification curve diagram of the single test system of the second set of primer probes NV GI (F2, R2, P2) of NV GI in Example 2.

[0058] Figure 3 The amplification curve diagram of the single test system of the third set of primer probes NV GI (F3, R3, P3) of NV GI in Example 2.

[0059] Figure 4 The amplification curve diagram of the single test system of the first set of primer probes NV GII (F1, R1, P1) of NV GII in Example 2.

[0060] Figure 5 The amplification curve diagram of the single test system of the second set of primer probes NV GII (F2, R2, P2) of NV GII in Example 2.

[0061] Figure 6 The amplification curve diagram of the single test system of the third set of primer probes NV GII (F3, R3, P3) of NV GII in Example 2.

[0062] Figure 7 The amplification curve diagram of the single test system of the first set of primer probes NV GIV (F1, R1, P1) of NV GIV in Example 2.

[0063] Figure 8 The amplification curve diagram of the single test system of the second set of primer probes NV GIV (F2, R2, P2) of NV GIV in Example 2.

[0064] Figure 9 The amplification curve diagram of the single test system of the third set of primer probes NV GIV (F3, R3, P3) of NV GIV in Example 2.

[0065] Figure 10 The amplification curve diagram of the single system of the internal reference gene of NV GI, NV GII and NV GIV in Example 2.

[0066] Figure 11 This is an amplification curve diagram of the multiplex detection system of the first primer-probe combination of NV GI, the first primer-probe combination of NV GII, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3.

[0067] Figure 12 This is an amplification curve diagram of the multiplex detection system of the second primer-probe combination of NV GI, the first primer-probe combination of NV GII, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3.

[0068] Figure 13 This is an amplification curve diagram of the multiplex detection system of the third primer-probe combination of NV GI, the first primer-probe combination of NV GII, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3.

[0069] Figure 14 This is an amplification curve diagram of the multiplex detection system of the first primer-probe combination of NV GII, the third primer-probe combination of NV GI, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3.

[0070] Figure 15 This is an amplification curve diagram of the multiplex detection system of the second primer-probe combination of NV GII, the third primer-probe combination of NV GI, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3.

[0071] Figure 16 This is an amplification curve diagram of the multiplex detection system of the third primer-probe combination of NV GII, the third primer-probe combination of NV GI, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3.

[0072] Figure 17 This is an amplification curve diagram of the multiplex detection system of the first primer-probe combination of NV GIV, the third primer-probe combination of NV GI, the third primer-probe combination of NV GII, and the primer-probe combination of the internal reference gene in Example 3.

[0073] Figure 18 This is an amplification curve diagram of the multiplex detection system of the second primer-probe combination of NV GIV, the third primer-probe combination of NV GI, the third primer-probe combination of NV GII, and the primer-probe combination of the internal reference gene in Example 3.

[0074] Figure 19This is an amplification curve diagram of the multiplex detection system of the third primer-probe combination of NV GIV, the third primer-probe combination of NV GI, the third primer-probe combination of NV GII, and the primer-probe combination of the internal reference gene in Example 3. DETAILED DESCRIPTION

[0075] The present application is further described in detail below in conjunction with examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in this application. Example Example 1

[0076] Example 1 provides the design and synthesis of primers and probes for NV GI, NV GII and NV GIV.

[0077] A literature search identified several candidate primer and probe sequences. The primer and probe sequences designed in the literature were generally located on the ORF1 or ORF2 genes. 49 NV GI, 100 NV GII full-genome sequences, and 50 NV GIV nucleic acid sequences were obtained from the NCBI nucleotide database. The bioinformatics software MEGA-X was used to perform a multiple sequence alignment of the sequences obtained for norovirus NV GI, NVGII, and NV GIV, obtaining multiple conserved segments for each virus. These conserved segments needed to specifically match a specific subtype and not detect other subtypes. Separate upstream and downstream primers and probes were designed, and structural analysis was performed using Primer Premier 5 software. Finally, primer specificity and compatibility analysis was performed using the NCBI Primer Blast function to ensure that the primers and probes were highly specific for NV GI, NV GII, and NV GIV and that degeneracy could prevent missed detections. The specific nucleotide sequences (5'-3') of the three sets of primers and probes for NV GI, NV GII and NV GIV that were finally screened and determined are shown in Table 1.

[0078] Table 1 Nucleotide sequences of three sets of primers and probes for NV GI, NV GII, and NV GIV

[0079]

[0080] Example 2 provides a pathogen primer-probe singleplex assay for NV GI, NV GII, and NV GIV.

[0081] The AccurSTART U+ One Step RT-qPCR Probe Kit (FOR FAST) sold by Novozymes was used as the amplification reaction system.

[0082] Three sets of primers and probes for norovirus NV GI, NV GII, and NV GIV were tested in single-plex systems. First, prepare the primer-probe mix. The volumes of upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) were 2 μL each, and the volume was made up to 100 μL with nuclease-free water. Add 2 μL of primer-probe mix, 4 μL of Novozymes 5× One Step U+ Mix, 1 μL of Novozymes One Step U+ Enzyme Mix, and 1×10 1 ~1×10 3 4 μL of plasmid at a copy / μL concentration and 9 μL of nuclease-free water. Three replicates are performed for each plasmid gradient, and three no-template controls are also included.

[0083] The amplification program was set as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, and 60°C annealing and extension for 30 s, for a total of 45 cycles. Fluorescence signal detection was performed during each annealing step.

[0084] The amplification data of three sets of primers and probes for norovirus NV GI, NV GII and NV GIV and the internal reference gene RNP are shown in Tables 2-5 below.

[0085] Table 2 Amplification data of the primer-probe single-plex test system of NV GI

[0086]

[0087] Table 3 Amplification data of the primer-probe single-plex test system of NV GII

[0088]

[0089] Table 4 Amplification data of the primer-probe single-plex test system of NV GIV

[0090]

[0091] Table 5 Amplification data of the internal reference gene RNP of NV GI, NV GII and NV GIV

[0092]

[0093] Figure 1 This is the amplification curve of the first set of primers and probes NV GI (F1, R1, P1) in the single-plex test system of NV GI in Example 2. Figure 2 This is the amplification curve of the second set of primers and probes NV GI (F2, R2, P2) in the single-plex test system of NV GI in Example 2. Figure 3This is the amplification curve of the third set of primers and probes NV GI (F3, R3, P3) in the single-plex test system of NV GI in Example 2. Figure 4 This is the amplification curve of the first set of primers and probes NV GII (F1, R1, P1) in the single-plex test system of NV GII in Example 2. Figure 5 This is the amplification curve of the second set of primers and probes for NV GII (F2, R2, P2) in the single-plex test system of NV GII in Example 2. Figure 6 This is the amplification curve of the third set of primers and probes NV GII (F3, R3, P3) in the single-plex test system of NV GII in Example 2. Figure 7 This is the amplification curve of the first set of primers and probes NV GIV (F1, R1, P1) in the single-plex test system of NV GIV in Example 2. Figure 8 This is the amplification curve of the second set of primers and probes for NV GIV (F2, R2, P2) in the single-plex test system of NV GIV in Example 2. Figure 9 This is the amplification curve of the third set of primers and probes NV GIV (F3, R3, P3) in the single-plex test system of NV GIV in Example 2. Figure 10 Figure 2 shows the amplification curves for the singleplex system of the internal reference genes NV GI, NV GII, and NV GIV in Example 2. As can be seen, the three primer sets for each pathogen and the internal reference genes all performed well in the singleplex system for plasmid template testing, with typical S-shaped amplification curves and similar Ct values. All primer probes can be used as alternative primer probes for establishing the following multiplex system. Example 3

[0094] Example 3 provides the establishment of a multiplex system for pathogen detection.

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

[0096] A multiplex detection system was established by using three sets of primer probes for each of Norovirus NV GI, NV GII, and NV GIV and a quadruple system consisting 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 respectively, and fill it up to 100 μL with nuclease-free water. Add 2 μL of primer probe mix, 4 μl of Novozymes 5×One Step U+ Mix, 1 μl of Novozymes One Step U+Enzyme Mix, and 1×10 1 ~1×10 34 μL of plasmid at a copy / μL concentration and 9 μL of nuclease-free water. Three replicates are performed for each plasmid gradient, and three no-template controls are also included.

[0097] The amplification program was set as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, and 60°C annealing and extension for 30 s, for a total of 45 cycles. Fluorescence signal detection was performed during each annealing step.

[0098] First, we selected three alternative primer-probe combinations for Norovirus GI, combined them with the first primer-probe combinations for Norovirus GII and Norovirus GIV, and the primer-probe combination for the internal reference gene, to form the following three multiplex combinations for testing the multiplex detection system. The amplification data are shown in Table 6 below.

[0099] Table 6 Amplification data of the multiplex detection system using three alternative primer-probe combinations for NV GI, the first primer-probe combination for NV GII, the first primer-probe combination for NV GIV, and the primer-probe combination for the internal reference gene

[0100]

[0101] Figure 11 This is an amplification curve diagram of the multiplex detection system of the first primer-probe combination of NV GI, the first primer-probe combination of NV GII, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3. Figure 12 This is an amplification curve diagram of the multiplex detection system of the second primer-probe combination of NV GI, the first primer-probe combination of NV GII, the first primer-probe combination of NVGIV, and the primer-probe combination of the internal reference gene in Example 3. Figure 13 This is an amplification curve diagram of the multiplex detection system of the third primer-probe combination of NV GI, the first primer-probe combination of NV GII, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3. It can be seen that in the plasmid template test, the amplification effect of the third of the three multiplex detection systems is the best, with a smaller Ct value, the highest amplification sensitivity, and the same specificity among the three primer-probe combinations. Therefore, the third primer-probe combination of Norovirus GI is fixed, and the three primer-probe combinations of Norovirus GII are further screened.

[0102] Next, we selected three alternative primer-probe combinations for Norovirus GII, combined them with the third primer-probe combination for Norovirus GI, the first primer-probe combination for Norovirus GIV, and the primer-probe combination for the internal reference gene, to form the following three multiplex combinations for testing the multiplex detection system. The amplification data are shown in Table 7 below.

[0103] Table 7 Amplification data of the multiplex detection system using three alternative primer-probe combinations for NV GII, the third primer-probe combination for NV GI, the first primer-probe combination for NV GIV, and the primer-probe combination for the internal reference gene

[0104]

[0105] Figure 14 This is an amplification curve diagram of the multiplex detection system of the first primer-probe combination of NV GII, the third primer-probe combination of NV GI, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3. Figure 15 This is an amplification curve diagram of the multiplex detection system of the second primer-probe combination of NV GII, the third primer-probe combination of NV GI, the first primer-probe combination of NVGIV, and the primer-probe combination of the internal reference gene in Example 3. Figure 16 This is an amplification curve diagram of the multiplex detection system of the third primer-probe combination of NV GII, the third primer-probe combination of NV GI, the first primer-probe combination of NV GIV, and the primer-probe combination of the internal reference gene in Example 3. It can be seen that in the plasmid template test, the amplification effect of the third set of the above three multiplex detection systems is the best, with a smaller Ct value, the highest amplification sensitivity, and the same specificity among the three sets of primer-probe combinations. Therefore, the third set of primer-probe combination of Norovirus NV GII is fixed, and the three sets of primer-probe combinations of Norovirus GIV are further screened.

[0106] Finally, we selected three alternative primer-probe combinations for Norovirus GIV, along with the third primer-probe combination for Norovirus GI and GII, and the primer-probe combination for the internal reference gene, to form the following three multiplex combinations for testing the multiplex detection system. The amplification data are shown in Table 8 below.

[0107] Table 8 Amplification data of the multiplex detection system using three alternative primer-probe combinations for NV GIV, the third primer-probe combination for NV GI, the third primer-probe combination for NV GII, and the primer-probe combination for the internal reference gene

[0108]

[0109] Figure 17 This is an amplification curve diagram of the multiplex detection system of the first primer-probe combination of NV GIV, the third primer-probe combination of NV GI, the third primer-probe combination of NV GII, and the primer-probe combination of the internal reference gene in Example 3. Figure 18This is an amplification curve diagram of the multiplex detection system of the second primer-probe combination of NV GIV, the third primer-probe combination of NV GI, the third primer-probe combination of NV GII, and the primer-probe combination of the internal reference gene in Example 3. Figure 19 This is an amplification curve diagram of the multiplex detection system of the third primer probe combination of NV GIV, the third primer probe combination of NV GI, the third primer probe combination of NV GII, and the primer probe combination of the internal reference gene in Example 3. It can be seen that in the plasmid template test, the amplification effect of the third set of the above three multiplex detection systems is the best, the Ct value is smaller, the amplification sensitivity is highest, and the specificity of the three sets of primer probe combinations is consistent. Therefore, the third set of primer probe combination of Norovirus NV GII is fixed, and the three sets of primer probe combinations of Norovirus GIV are further screened.

[0110] Comprehensive analysis showed that the third primer combination was selected for Noru GI, GII, and GIV, and the obtained amplification curve showed a standard S-shaped curve with a smaller Ct value, optimal sensitivity, and good specificity.

[0111] Example 4

[0112] Example 4 provides the establishment of a pre-packaging system for multiple pathogen detection.

[0113] The AccurSTART U+ One Step RT-qPCR Super PreMix (PreMix) kit sold by Novozymes was used as the amplification reaction system. This 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.

[0114] The optimized multiplex amplification system 3 in Example 3 was prepared into primer-probe mix. The volume of the upstream primer (100 μM), downstream primer (100 μM) and probe (100 μM) of the four primer-probe sets was 4 μL respectively, and the volume was made up to 100 μL with nuclease-free water. 1 μL of primer-probe mix was added to the reaction system, and Novozyme U + One Step RT-qPCR Probe 5×MasterMix 4μl was used to form a pre-aliquot system. After mixing the system evenly, 5μL / well was dispensed into eight strip tubes and capped. The eight strips were stored at -20°C and amplification was tested in October, March, June, September, and December. 1×10 0 ~1×10 24 μL of nucleic acid mixture at a copy / μL concentration and 11 μL of nuclease-free water. Three replicates of each nucleic acid mixture were performed, along with three no-template controls.

[0115] 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 results were tested in 0, 3, 6, 9, and 12 months, respectively.

[0116] The amplification program was set as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, and 60°C annealing and extension for 30 s, for a total of 45 cycles. Fluorescence signal detection was performed during each annealing step.

[0117] The amplification data of the multiple pre-packaging detection system in October, March, June, September, and December are shown in Tables 9-12 below.

[0118] Table 9 Comparison of multiple pre-packed (Pre) and multiple non-pre-packed (NO-Pre) in different months (NV GI)

[0119]

[0120] Table 10 Comparison of multiple pre-packed (Pre) and multiple non-pre-packed (NO-Pre) products in different months (NV GII)

[0121]

[0122] Table 11 Comparison of multiple pre-packed (Pre) and multiple non-pre-packed (NO-Pre) products in different months (NV GIV)

[0123]

[0124] Table 12 Comparison of multiple pre-packed (Pre) and multiple non-pre-packed (NO-Pre) in different months (RNP)

[0125]

[0126] In mixed 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. Example 5

[0127] Example 5 provides clinical sample testing.

[0128] Specificity samples (adenovirus 3 cases, sapovirus 3 cases, group A rotavirus 3 cases, astrovirus 3 cases) were found from the sample library of the company, and Norovirus GI, GII and GIV samples quantified by digital PCR were diluted to 1 x 10 0 1 x 10 2 copies / uL, 21 GI, GII and GIV samples determined by Norovirus typing and 5 Norovirus GIV samples and 6 mixed positive samples of Norovirus GI, GII and GIV were determined. The pre-packaged multiplex system was evaluated from sensitivity, specificity, and sample detection rate.

[0129] Using the pre-packaged system in Example 4, 10 uL of nucleic acid extracted from the above samples was directly added to the eight-row tube, mixed uniformly, and detected by machine.

[0130] The amplification program was set as follows: reverse transcription reaction at 55℃ for 15 min; pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, a total of 45 cycles. The annealing step of each cycle was detected by fluorescence signal.

[0131] 1. Specificity experiment

[0132] Adenovirus 3 cases, sapovirus 3 cases, group A rotavirus 3 cases, astrovirus 3 cases were detected only with obvious amplification curve of the internal reference gene (shown in Table 13), and Norovirus GI, GII and GIV had no amplification, indicating good specificity for detection of common viruses causing diarrhea.

[0133] Table 13 Specific nucleic acid detection data

[0134]

[0135] 2. Sensitivity experiment

[0136] After dilution of Norovirus GI, GII and GIV sample nucleic acids quantified by digital PCR, the detection data are shown in Tables 14-16.

[0137] Table 14 Norovirus GI nucleic acid detection data

[0138]

[0139] Table 15 Norovirus GII nucleic acid detection data

[0140]

[0141] Table 16 Norovirus GIV nucleic acid detection data

[0142]

[0143] The pre-packaged detection system has high sensitivity in nucleic acid template testing and can distinguish different types.

[0144] 3. Single positive sample detection

[0145] The pre-packaged detection system was used to detect 21 samples of GI and GII types and 5 samples of GIV type. The detection results are shown in Tables 17-19.

[0146] Table 17. Nucleic acid detection data of norovirus GI samples

[0147]

[0148] Table 18. Nucleic acid detection data of norovirus GII samples

[0149]

[0150] Table 19. Nucleic acid detection data of norovirus GIV samples

[0151]

[0152] All of the 21 samples of GI and GII types and 5 samples of GIV type were detected, with a detection rate of 100%.

[0153] 4. Mixed positive sample detection

[0154] The pre-packaged detection system was used to detect 6 samples of mixed norovirus GI, GII and GIV types. The detection results are shown in Table 20.

[0155] Table 20. Nucleic acid detection data of norovirus mixed positive samples

[0156]

[0157] All of the 6 samples of mixed norovirus GI, GII and GIV types were detected, with a detection rate of 100%.

[0158] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of the present application.

Claims

1. A composition for simultaneous detection of NV GI, NV GII and NV GIV by multiplex real-time fluorescence quantitative RT-PCR, characterized in that: The composition comprises the following components: Component A: upstream primer 1, downstream primer 1 and probe 1 for detecting NV GI, whose nucleotide sequences are shown in SEQ ID NOs. 1-3 respectively; Component B: upstream primer 2, downstream primer 2 and probe 2 for detecting NV GII, the nucleotide sequences of which are shown in SEQ ID NOs. 4-6 respectively; Component C: upstream primer 3, downstream primer 3 and probe 3 for detecting NV GIV, the nucleotide sequences of which are shown in SEQ ID NOs. 7-9 respectively; Among them, the probes of NV GI, NV GII and NV GIV are all labeled with different fluorescent markers.

2. The composition for simultaneous detection of NV GI, NV GII and NV GIV by multiplex real-time fluorescence quantitative RT-PCR according to claim 1, characterized in that: It also includes component D, which is an upstream primer 4, a downstream primer 4 and a probe 4 for detecting human ribonucleoprotein complex RNP, and their nucleotide sequences are shown in SEQ ID NO.10-12 respectively; the component D is used as an internal reference gene detection composition.

3. A kit for simultaneous detection of NV GI, NV GII and NV GIV by multiplex real-time fluorescence quantitative RT-PCR, characterized in that: The kit comprises the composition according to any one of claims 1 to 2.

4. A method for simultaneously detecting NV GI, NV GII and NV GIV in a sample for non-disease diagnosis purposes, characterized in that: Detection using the composition as claimed in claim 1 specifically comprises the following steps: Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested to obtain viral RNA; adding the extracted viral RNA to a mixture of the composition and other raw materials to form a reaction system; Performing multiple real-time fluorescence quantitative RT-PCR amplification on the reaction system to obtain an amplification curve; The amplification curve is analyzed to obtain the Ct value and make a judgment.

5. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 4, characterized in that: 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.

6. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 4, characterized in that: The conditions for the RT-PCR amplification include: Reverse transcription at 50-55°C for 5-20 minutes; Pre-denaturation at 90℃~95℃ for 1~5 minutes; Denaturation at 90°C-95°C for 5-15 seconds, annealing and extension at 50°C-60°C for 30-45 seconds, for 35-45 cycles, and fluorescence was collected.

7. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 4, characterized in that: The principles for analyzing and judging the amplification curve are: When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 1 of the test sample is S-shaped and the Ct value is within 37, the test sample is judged to be a human norovirus type 1 positive sample; When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 2 of the test sample is S-shaped and the Ct value is within 37, the test sample is judged to be a human norovirus type II positive sample; When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on 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 norovirus type IV positive sample.

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