Composition, kit and method for simultaneously detecting NV GI, NV GII and NV GIV

Abstract

The invention relates to the technical field of virus detection, and particularly discloses a composition, a kit and a method for simultaneously detecting NV GI, NV GII and NV GIV. The composition provided by the invention comprises a component A, a component B and a component C, wherein the component A is used for detecting an upstream primer 1, a downstream primer 1 and a probe 1 of NV GI, and the nucleotide sequence is shown as SEQ ID NO.1-3; the component B 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 as SEQ ID NO.4-6; the component C is used for detecting an upstream primer 3, a downstream primer 3 and a probe 3 of the NV GIV, and the nucleotide sequences are shown as SEQ ID NO.7-9. The composition, the kit and the method can be used for simultaneously identifying NV GI, NV GII and NV GIV, and have the advantages of high specificity and sensitivity, good repeatability and accurate and reliable detection result.

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 is a virus of the genus Norovirus (NV) in the family Human Calicivirus (HuCV). It is a zoonotic pathogen that can cause acute gastroenteritis and severe diarrhea in humans and a variety of animals worldwide. It is also an important foodborne disease pathogen. The spread of Norovirus not only poses a serious threat to human health, but also causes huge social and economic 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 capable of spreading rapidly.

[0004] Norovirus-induced diarrhea is prevalent worldwide and can occur throughout the year. It mainly infects adults and school-age children, with a higher incidence in cold seasons. Norovirus-induced diarrhea is a self-limiting disease, for which there is no vaccine or specific medicine. The key to preventing this disease is to maintain good personal hygiene, food hygiene, and drinking water hygiene. It is important to develop healthy living habits such as washing hands frequently, not drinking raw water, keeping raw and cooked food separate, and avoiding cross contamination.

[0005] Norovirus has genetic diversity. The early classification was based on the differences in the nucleotide sequences of the RdRp and capsid protein coding regions of the NV genome. Norovirus is divided into five gene groups GⅠ~GⅤ, of which only GⅠ, GⅡ and GⅣ can infect humans, while GⅢ and GⅤ infect cattle and mice respectively. The gene groups can be further divided into genotypes. There are 8 genotypes in GI (GI.1~GI.8), 19 genotypes in GII (GII.1~GII.19), 2 genotypes in GIII (GIII.1~GIII.2), and only 1 genotype each in GⅣ and GⅤ.

[0006] Infectious diarrhea is diarrhea caused by a variety of 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, diarrhea-causing Escherichia coli, Vibrio parahaemolyticus, Salmonella, Campylobacter, Aeromonas and Shigella-like Plesiomonas, Bacillus cereus, Clostridium perfringens, Yersinia enterocolitica, etc. Parasites: Giardia, Entamoeba histolytica, Cryptosporidium, Cyclospora, etc. Antibiotic-associated diarrhea: Clostridium difficile, etc. The diarrhea symptoms caused by these pathogens are similar and difficult to diagnose through clinical manifestations. Therefore, it is necessary to clarify the diagnosis of norovirus or even the specific typing of norovirus, which requires laboratory testing to avoid misdiagnosis. Traditional laboratory diagnostic methods include virus culture methods, 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: 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 NO. 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, whose nucleotide sequences are shown in SEQ ID NO.7-9 respectively.

[0010] 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.

[0011] 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 infection of different types, and has high specificity and sensitivity, good repeatability, simple and rapid detection, and cost savings.

[0012] This application conducts a combined detection of the common norovirus subtypes GI, GII and GIV that infect humans, selects the conservative region of the virus for design, specifically detects the three subtypes of norovirus, and can distinguish the three subtypes at the same time. The current patents mainly focus on detecting norovirus GI or GII alone, and no patents for detecting the three subtypes of norovirus at the same time have been retrieved.

[0013] 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 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.

[0014] The principle of using the above composition to detect NV GI, NV GII and NV GIV in this application is as follows: 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 NV GI, 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 NV GI 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 NV GI. 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 NV GI exists in the sample to be tested.

[0015] Since the probes of NV GI, NV GII and NV GIV are all labeled with different fluorescence, the detection data of the three virus typing can be obtained through different fluorescence curves.

[0016] 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 NV GI, NV GII and NV GIV in the sample to be tested, greatly shortening the detection time and reducing the detection cost.

[0017] In a second aspect, the present application provides a kit for simultaneously detecting NV GI, NV GII and NV GIV for the purpose of non-disease diagnosis, wherein the kit comprises any of the compositions described above.

[0018] 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 RNase inhibitor, a Taq DNA polymerase and an enzyme buffer; the amplification buffer comprises a buffer, dNTPs, Mg 2+ ; The positive quality control product is a plasmid or pseudovirus containing amplified fragments of NV GI, NV GII and NV GIV; the negative quality control product is nuclease-free water.

[0019] Specifically, positive quality control products and negative quality control products play a role in quality control operation procedures and whether there is contamination during the use of the kit. 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-packed into 8-tube strips and covered with tube caps to form a pre-packed reagent; during use, the tube caps are opened, the extracted nucleic acid is added, the tube caps are covered and the reagent can be put into the machine.

[0020] The kit can achieve the effect of completely mixing the composition described in the first aspect, the enzyme mixture, the buffer solution, etc. together to form a fully premixed form.

[0021] The fully premixed reagents are pre-packed into 8-tube strips and capped to make them pre-packed. During 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; therefore, the number of operation steps and the probability of operation errors can be reduced.

[0022] 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 they 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.

[0023] 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 above compositions or any of the above kits for detection, specifically comprising 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 the pre-packed 8-tube strip containing 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.

[0024] 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 (NV GI, NV GII and NV GIV) 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.

[0025] 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.

[0026] In the present application, the above-mentioned 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-mentioned probes refer to probes 1-4 for NV GI, NV GII, NV GIV and internal reference gene RNP.

[0027] 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.

[0028] In some specific embodiments, the reaction system is 20 μL, specifically: The viral RNA solution extracted from the sample to be tested is 10uL, the enzyme mixture is 1μL, the upstream primers 1-4 with a concentration of 10μmol / L are 0.5μL each, the downstream primers 1-4 with a concentration of 10μmol / L are 0.5μL each, and the probes 1-4 with a concentration of 20μmol / L are 0.25μL each, including dNTPs and Mg 2+ 4 μL of buffer.

[0029] In some specific embodiments, the conditions for 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℃~95℃ for 5~15 seconds, annealing and extension at 50℃~60℃ for 30~45 seconds, repeat 35~45 cycles, and collect fluorescence.

[0030] In some preferred embodiments, the conditions for RT-PCR amplification include: Reverse transcription at 55°C for 15 min; Pre-denaturation at 95°C for 1 minute; Denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds, 40 cycles, and fluorescence was collected.

[0031] 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.

[0032] In some specific embodiments, the principle of analyzing and judging the amplification curve is: 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 norovirus type I positive sample; When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 2 of the sample to be tested is S-shaped, and the Ct value is within 37, the sample to be tested is judged to be a human 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 judged to be a human norovirus type IV positive sample.

[0033] In summary, the technical solution of this application has the following effects: The composition provided in the present application for simultaneously detecting human norovirus type I, norovirus type II and norovirus type IV is a composition with specific primers and probes 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, which 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.

[0034] The kit provided in the present application can achieve the purpose of completely mixing the composition for simultaneous detection of NV GI, NV GII and NV GIV, the enzyme mixture in the second aspect, the buffer solution, etc. together to form a fully premixed form. The fully premixed reagents are pre-packed into 8 strip tubes, covered with tube caps, and made into a pre-packed form. During use, it is only necessary to open the tube cap, add the extracted nucleic acid, cover the tube cap, and then put it on the machine, which greatly reduces the number of operating steps and the probability of operating errors.

[0035] The method for detection using the composition provided in the present application or a kit including the composition 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 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

[0036] Figure 1 This is the amplification curve of the first set of primers and probes NV GI (F1, R1, P1) of the single-plex test system of NV GI in Example 2.

[0037] Figure 2 This is the amplification curve of the second set of primers and probes NV GI (F2, R2, P2) of the single-plex test system of NV GI in Example 2.

[0038] Figure 3 This is the amplification curve of the third set of primers and probes NV GI (F3, R3, P3) of the single-plex test system of NV GI in Example 2.

[0039] Figure 4 This is the amplification curve of the first set of primers and probes NV GII (F1, R1, P1) of the single-plex test system of NV GII in Example 2.

[0040] Figure 5 This is the amplification curve of the second set of primers and probes NV GII (F2, R2, P2) of the single-plex test system of NV GII in Example 2.

[0041] Figure 6 This is the amplification curve of the third set of primers and probes NV GII (F3, R3, P3) of the single-plex test system of NV GII in Example 2.

[0042] Figure 7 This is the amplification curve of the first set of primers and probes NV GIV (F1, R1, P1) of the single-plex test system of NV GIV in Example 2.

[0043] Figure 8 This is the amplification curve of the second set of primers and probes NV GIV (F2, R2, P2) of the single-plex test system of NV GIV in Example 2.

[0044] Fig. 9 This is the amplification curve of the third set of primers and probes NV GIV (F3, R3, P3) of the NV GIV single-plex test system in Example 2.

[0045] Fig.10 This is a single-plex system amplification curve diagram of the internal reference genes of NV GI, NV GII and NV GIV in Example 2.

[0046] Fig.11 It 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.

[0047] Fig.12 It 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.

[0048] Fig.13It 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.

[0049] Fig.14 It 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.

[0050] Fig.15 It 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.

[0051] Fig.16 It 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.

[0052] Fig.17 It 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.

[0053] Fig.18 It 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.

[0054] Fig.19 It 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

[0055] 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 the present application. Example Example 1

[0056] Example 1 provides the design and synthesis of primers and probes for NV GI, NV GII and NV GIV.

[0057] Through literature search, some alternative primer probe sequences were obtained. The primer probe sequences designed in the literature are basically on the ORF1 or ORF2 gene. At the same time, 49 NV GI, 100 NV GII whole 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 multiple sequence alignment on the sequences obtained from Norovirus NV GI, NVGII and NV GIV to obtain multiple conserved fragments of each virus. 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 NV GI, NV GII and NV GIV and will not be degenerate and cause missed detection. 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.

[0058] Table 1 Nucleotide sequences of three sets of primers and probes for NV GI, NV GII and NV GIV

[0059] Example 2 provides a pathogen primer-probe singleplex test for NV GI, NV GII and NV GIV.

[0060] The AccurSTART U+ One Step RT-qPCR Probe Kit (FOR FAST) sold by Novozymes was used as the amplification reaction system.

[0061] Three sets of primers and probes for Norovirus NV GI, NV GII, and NV GIV were used to test the single-plex system. First, prepare the primer-probe mix. The volume of the upstream primer (100 μM), downstream primer (100 μM), and probe (100 μM) is 2 μL respectively, and fill it up to 100 μL with nuclease-free water. Add 2 μL of primer-probe mix, 4 μl of Novozyme 5×One Step U+ Mix, 1 μl of Novozyme One Step U+ Enzyme Mix, and 1×10 1 ~1×10 34 μL of plasmid with a copy / uL concentration and 9 μL of nuclease-free water. Make 3 parallels for each gradient of plasmid and 3 template-free controls.

[0062] 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.

[0063] The amplification data of three sets of primer probes for Norovirus NV GI, NV GII and NV GIV and the internal reference gene RNP are shown in Tables 2-5 below.

[0064] Table 2 Amplification data of the primer-probe single-plex test system of NV GI

[0065] Table 3 Amplification data of primer-probe single-plex test system of NV GII

[0066] Table 4 Amplification data of primer-probe single-plex test system of NV GIV

[0067] Table 5 Amplification data of the internal reference gene RNP of NV GI, NV GII and NV GIV

[0068] Figure 1 This is the amplification curve of the first set of primers and probes NV GI (F1, R1, P1) of 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) of the single-plex test system of NV GI in Example 2. Figure 3 This is the amplification curve of the third set of primers and probes NV GI (F3, R3, P3) of 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) of 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 NV GII (F2, R2, P2) of 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) of 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) of the single-plex test system of NV GIV in Example 2. Figure 8This is the amplification curve of the second set of primers and probes NV GIV (F2, R2, P2) of the single-plex test system of NV GIV in Example 2. Fig. 9 This is the amplification curve of the third set of primers and probes NV GIV (F3, R3, P3) of the NV GIV single-plex test system in Example 2. Fig.10 The single-plex system amplification curve of the internal reference genes of NV GI, NV GII and NV GIV in Example 2. It can be seen that in the plasmid template test, the three sets of primers of each pathogen and the single-plex system amplification of the internal reference gene have good effects, the amplification curve is a typical S-shaped curve, and the Ct value is also close. All primer probes can be used as alternative primer probes for the establishment of the following multiplex system. Example 3

[0069] Example 3 provides the establishment of a multiplex system for pathogen detection.

[0070] The AccurSTART U+ One Step RT-qPCR Probe Kit (FOR FAST) sold by Novozymes was used as the amplification reaction system.

[0071] The three sets of primer probes for Norovirus NV GI, NV GII, and NV GIV 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 (100μM), downstream primer (100μM), and probe (100μM) of the four sets of primer probes in the multiplex system is 2μL respectively, and fill it up to 100μL with nuclease-free water. Add 2μL of primer probe mix, 4μl of Novozyme 5×One Step U+ Mix, 1μl of Novozyme One Step U+Enzyme Mix, and 1×10 1 ~1×10 3 4 μL of plasmid with a copy / uL concentration and 9 μL of nuclease-free water. Make 3 parallels for each gradient of plasmid and 3 template-free controls.

[0072] 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.

[0073] First, three sets of alternative primer-probe combinations of Noro GI were selected, and the first set of primer-probe combinations of Noro GII and Noro GIV, as well as the primer-probe combination of the internal reference gene, to form the following three multiple combinations to test the multiple detection system. The amplification data are shown in Table 6 below.

[0074] Table 6 Amplification data of the multiplex detection system of the three alternative primer-probe combinations 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

[0075] Fig.11 It 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. Fig.12 It 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. Fig.13 It 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 set of the above three multiplex detection systems is the best, the Ct value is smaller, the amplification sensitivity is the highest, and the specificity of the three primer-probe combinations is consistent. Therefore, the third primer-probe combination of Noru GI is fixed, and the three primer-probe combinations of Noru GII continue to be screened.

[0076] Next, three sets of alternative primer-probe combinations of Norovirus GII were selected, respectively, and the third set of primer-probe combinations of Norovirus GI, the first set of primer-probe combinations of Norovirus GIV, and the primer-probe combinations of the internal reference gene were used to form the following three multiple combinations to test the multiple detection system. The amplification data are shown in Table 7 below.

[0077] Table 7 Amplification data of the multiplex detection system of the three alternative primer-probe combinations 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

[0078] Fig.14 It 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. Fig.15 It 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. Fig.16 It is an amplification curve diagram of the multiple 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 sets of multiple detection systems is the best, the Ct value is smaller, the amplification sensitivity is the highest, and the specificity of the three sets of primer-probe combinations is consistent. Therefore, the third set of primer-probe combinations of Norovirus NV GII is fixed, and the three sets of primer-probe combinations of Norovirus GIV are continuously screened.

[0079] Finally, three sets of alternative primer-probe combinations of Norovirus GIV were selected, and the third set of primer-probe combinations of Norovirus GI and GII, as well as the primer-probe combination of the internal reference gene, to form the following three multiple combinations to test the multiple detection system. The amplification data are shown in Table 8 below.

[0080] Table 8 Amplification data of the multiplex detection system of the three alternative primer-probe combinations 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

[0081] Fig.17 It 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. Fig.18 It 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. Fig.19 It is an amplification curve diagram of the multiple 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 sets of multiple detection systems is the best, the Ct value is smaller, the amplification sensitivity is the highest, and the specificity of the three sets of primer probe combinations is consistent. Therefore, the third set of primer probe combinations of Norovirus NV GII is fixed, and the three sets of primer probe combinations of Norovirus GIV are continuously screened.

[0082] Comprehensive analysis showed that Noru GI, GII, and GIV all selected the third set of primer combinations, and the obtained amplification curves showed a standard S-shaped curve, with a smaller Ct value, optimal sensitivity, and good specificity.

[0083] Example 4 Example 4 provides the establishment of a pre-packaging system for multiple pathogen detection.

[0084] 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.

[0085] 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 sets of primer-probes 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-packaging system. After the system was evenly mixed, 5μL / well was dispensed into eight-tube strips and capped. The eight-tube strips were stored at -20°C and the amplification results were tested in October, March, June, September, and December. 1×10 0 ~1×10 2 4 μL of mixed nucleic acid with a copy / uL concentration and 11 μL of nuclease-free water. Make 3 parallels for each gradient of mixed nucleic acid and 3 template-free controls.

[0086] 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, March, June, September, and December, respectively.

[0087] 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.

[0088] The amplification data of the multiple pre-packaging detection system in October, March, June, September, and December are shown in Tables 9-12 below. Table 9 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (NV GI)

[0089] Table 10 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (NV GII)

[0090] Table 11 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (NV GIV)

[0091] Table 12 Comparison of multiple pre-packaging (Pre) and multiple non-pre-packaging (NO-Pre) in different months (RNP)

[0092] In the mixed nucleic acid 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. Example 5

[0093] Example 5 provides clinical sample testing.

[0094] Specific samples (3 adenoviruses, 3 sazaviruses, 3 group A rotaviruses, and 3 astroviruses) were found from our company’s sample library. Norovirus GI, GII, and GIV samples quantified by digital PCR were diluted to 1×10 0 ~1×10 2 copy / uL, 21 samples of GI and GII typing confirmed as norovirus, 5 samples of GIV and 6 mixed positive samples of GI, GII and GIV. The pre-packaging multiplex system was evaluated in terms of sensitivity, specificity and sample detection rate.

[0095] Using the pre-packaging system 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.

[0096] 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.

[0097] 1. Specificity Experiment Among the 3 cases of adenovirus, 3 cases of sazavirus, 3 cases of group A rotavirus, and 3 cases of astrovirus, only the internal reference gene had an obvious amplification curve (as shown in Table 13), while no amplification was found for Norovirus GI, GII, and GIV, indicating that the detection specificity for common diarrhea viruses is good.

[0098] Table 13 Specific nucleic acid detection data

[0099] 2. Sensitivity experiment After the nucleic acid of Norovirus GI, GII and GIV samples quantified by digital PCR was diluted, the detected data are shown in Tables 14-16 below.

[0100] Table 14 Norovirus GI nucleic acid test data

[0101] Table 15 Norovirus GII nucleic acid test data

[0102] Table 16 Norovirus GIV nucleic acid detection data

[0103] In nucleic acid template testing, the pre-packaging detection system has high sensitivity and can distinguish different types.

[0104] 3. Single positive sample detection The pre-packaging detection system tested 21 samples of GI and GII type that had been confirmed as Norovirus and 5 samples of GIV type. The test results are shown in Tables 17-19.

[0105] Table 17 Norovirus GI sample nucleic acid test data

[0106] Table 18 Norovirus GII sample nucleic acid test data

[0107] Table 19 Norovirus GIV sample nucleic acid test data

[0108] All 21 samples of Norovirus GI and GII typing and 5 Norovirus GIV samples that had been confirmed to be Norovirus were detected, with a detection rate of 100%.

[0109] 4. Mixed positive sample detection The pre-packaging detection system was used to test 6 mixed positive samples of Norovirus GI, GII, and GIV. The test results are shown in Table 20.

[0110] Table 20 Nucleic acid test data of Norovirus mixed positive samples

[0111] All 6 mixed positive samples of Norovirus GI, GII, and GIV were detected, with a detection rate of 100%.

[0112] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A composition for simultaneously detecting NV GI, NV GII and NV GIV, characterized in that: The composition comprises the following components: 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 NO.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, whose nucleotide sequences are shown in SEQ ID NOs. 7-9 respectively.

2. The composition for simultaneously detecting NV GI, NV GII and NV GIV 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 its 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 simultaneously detecting NV GI, NV GII and NV GIV, characterized in that: The kit comprises the composition according to any one of claims 1-2.

4. The kit for simultaneously detecting NV GI, NV GII and NV GIV according to claim 3, characterized in that: The kit also includes: an enzyme mixture, an amplification buffer, a positive quality control product and a negative quality control product; the enzyme mixture includes a reverse transcriptase, an RNase inhibitor, a Taq DNA polymerase and an enzyme buffer; the amplification buffer includes a buffer, dNTPs, Mg 2+ ; The positive quality control product is a plasmid or pseudovirus containing amplified fragments of NV GI, NV GII and NV GIV; the negative quality control product is nuclease-free water.

5. The kit for simultaneously detecting NV GI, NV GII and NV GIV according to claim 3, characterized in that: 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-packed into 8-tube strips and covered with tube caps to form a pre-packed reagent; during use, the tube caps are opened, the extracted nucleic acid is added, the tube caps are covered and the reagent can be put into the machine.

6. 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 according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 5 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 the pre-packed 8-tube strip containing 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.

7. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 6, 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.

8. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 6, characterized in that: The reaction system is 20 μL, specifically: The viral RNA solution extracted from the sample to be tested is 10uL, the enzyme mixture is 1μL, the upstream primers 1-4 with a concentration of 10μmol / L are 0.5μL each, the downstream primers 1-4 with a concentration of 10μmol / L are 0.5μL each, and the probes 1-4 with a concentration of 20μmol / L are 0.25μL each, including dNTPs and Mg 2+ 4 μL of buffer.

9. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 6, 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℃~95℃ for 5~15 seconds, annealing and extension at 50℃~60℃ for 30~45 seconds, repeat 35~45 cycles, and collect fluorescence.

10. The method for simultaneously detecting NV GI, NV GII and NV GIV in a sample to be tested according to claim 6, 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 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 norovirus type I positive sample; When the amplification curve of the fluorescence channel of the fluorescent reporter group labeled on probe 2 of the sample to be tested is S-shaped, and the Ct value is within 37, the sample to be tested is judged to be a human 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 judged to be a human norovirus type IV positive sample.

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