Reagent card box for detecting mouse norovirus and mouse hepatitis virus

Abstract

The invention relates to a reagent card box for detecting murine norovirus and murine hepatitis virus, and relates to the technical field of molecular biology detection, the reagent card box comprises a primer probe composition for detecting murine norovirus and murine hepatitis virus, the primer probe composition for detecting murine norovirus comprises a forward primer SEQ ID NO: 1, a reverse primer SEQ ID NO: 2, a reverse primer SEQ ID NO: 3, a reverse primer SEQ ID NO: 4 and a reverse primer SEQ ID NO: 5, a probe: SEQ ID NO: 7; the primer and the probe for detecting the murine norovirus comprise a forward primer of SEQ ID NO: 3, a reverse primer of SEQ ID NO: 4 and a probe of SEQ ID NO: 8, the reagent card box disclosed by the invention adopts a POCT (Point-of-Care Testing) technology to carry out multiple real-time fluorescent PCR amplification detection on nucleic acids of the mouse norovirus and the mouse hepatitis virus, and has timeliness, closure, accuracy and convenience.

Description

Technical Field

[0001] This application relates to the field of molecular biology detection, and in particular to a reagent cartridge for the detection of mouse norovirus and mouse hepatitis virus. Background Technology

[0002] Murine norovirus (MNV), belonging to the Noroviridae family, is a single-stranded RNA virus whose primary hosts are rodents, especially mice. MNV infection in mice typically manifests as diarrhea, with loose or watery stools, accompanied by weight loss and decreased appetite. In some cases, respiratory symptoms may also occur. Furthermore, MNV infection can lead to intestinal flora imbalance and intestinal barrier dysfunction, causing multidimensional impacts on host health. Murine hepatitis virus (MHV), belonging to the Coronaviridae family, has an enveloped viral particle and also uses single-stranded RNA as its genetic material. When mice are infected with MHV, the symptoms are more complex and severe. Initially, fever, lethargy, and rough coat are observed, followed by significant weight loss and dehydration. Gastrointestinal symptoms are particularly prominent, including severe mucus or bloody diarrhea, vomiting, and interstitial pneumonia, leading to a decrease in lymphocytes and causing a double blow to the mouse's immune and respiratory systems.

[0003] Norovirus and mouse hepatitis virus can be transmitted via the fecal-oral route and respiratory tract, and there is a certain degree of cross-contamination in epidemic areas. In addition, the clinical symptoms caused by the two viruses are very similar, which brings difficulties to clinical detection and diagnosis.

[0004] Currently, routine detection methods for norovirus and murine hepatitis virus infection typically include virus isolation and culture, serological testing, and nucleic acid molecular detection. While these methods may offer good clinical value, they suffer from drawbacks such as being time-consuming, cumbersome, having low sensitivity, causing immune cross-reactivity, and requiring specific and expensive equipment, thus hindering their widespread clinical adoption. Using conventional PCR detection kits presents several limitations for grassroots testing laboratories: 1) they require specialized personnel and a biosafety level 2 laboratory; 2) specific nucleic acid extraction methods and conditions are prone to contamination and prolong the process; 3) single-sample detection kits for norovirus and murine hepatitis virus are time-consuming and labor-intensive for large-scale sample testing; 4) they cannot perform multiple tests per tube, requiring users to prepare reaction solutions according to specific formulas, demanding strict operational skills.

[0005] Therefore, there is an urgent need in this field to develop a time-saving, labor-saving, and highly sensitive POCT reagent cartridge for the simultaneous detection of mouse norovirus and mouse hepatitis virus. Summary of the Invention

[0006] The purpose of this application is to provide a time-saving, labor-saving, and highly sensitive reagent cartridge for the simultaneous detection of mouse norovirus and mouse hepatitis virus.

[0007] In a first aspect, this application provides a primer and probe composition for the detection of norovirus and mouse hepatitis virus, wherein the primers and probes for the detection of norovirus include: forward primer: SEQ ID NO:1, reverse primer: SEQ ID NO:2, probe: SEQ ID NO:7; and the primers and probes for the detection of norovirus include: forward primer: SEQ ID NO:3, reverse primer: SEQ ID NO:4, probe: SEQ ID NO:8.

[0008] Optionally, the primer-probe composition further includes primers and probes for IC, wherein the primers and probes for IC include: forward primer: SEQ ID NO:5, reverse primer: SEQ ID NO:6, and probe: SEQ ID NO:9.

[0009] Optionally, the 5' end of the probe includes a fluorescent reporter group, which is selected from FAM, HEX, ROX, JOE, VIC, TET, NED, FITC, CY3 or CY5.

[0010] Optionally, the 3' end of the probe includes a fluorescence quenching group selected from BHQ1, BHQ2, BHQ3, TAMRA, Eclipse, DABCYL, or MGB.

[0011] Secondly, this application provides a reagent cartridge for detecting mouse norovirus and mouse hepatitis virus, comprising the primer-probe composition described in the first aspect.

[0012] Optionally, the reagent cartridge is provided with a lysis buffer area, a washing buffer area, and an amplification area from top to bottom; the lysis buffer area contains lysis buffer, the washing buffer area contains washing buffer, and the amplification area contains PCR reaction solution and the primer and probe composition described in the first aspect.

[0013] Optionally, the PCR reaction solution includes a thermostable DNA polymerase with reverse transcription activity.

[0014] Optionally, the DNA polymerase is Tth DNA polymerase or rTth DNA polymerase.

[0015] Optionally, the PCR reaction solution is Hotstart Onestep RT-PCR Master Mix.

[0016] Optionally, the volume ratio of the PCR reaction solution to the primer-probe composition is 3-4:2-3.

[0017] Optionally, the amplification region further includes an encapsulation region, which encapsulates IC and Mn using a fusible material. 2+ The encapsulation fluid.

[0018] Optionally, the fusible material is selected from any one or more of paraffin, dodecane, tetradecane, and hexadecane.

[0019] This technical solution allows for the encapsulation of Mn at low temperatures. 2+ And IC, the Mn inside after high temperature melting 2+ IC can be mixed with PCR reaction solution. When used, it melts to form paraffin oil, which can prevent PCR products from forming aerosols and polluting the environment. Paraffin oil has low thermal conductivity, which can effectively ensure the temperature conditions for PCR. Its low-temperature solidification characteristics can fix the position of the magnetic beads after the temperature drops during the pullback.

[0020] Optionally, the encapsulation fluid consists of 2-400 mM Mn 2+ It is composed of IC and trehalose.

[0021] Optionally, the Mn 2+ It comes from any one or more of manganese sulfate, manganese acetate, and manganese chloride.

[0022] Optionally, the IC is synthetic DNA adapted to IC primers and probes.

[0023] Optionally, the IC sequence is as shown in SEQ ID NO:10, and the sequence is cloned from the PUC57 plasmid.

[0024] Optionally, the lysis buffer includes GTC, NaAc, NaCl, SDS, NP-40, Triton X-100, and PEG8000.

[0025] Optionally, the concentration of GTC is 0.8M-1.5M.

[0026] Optionally, the lysis buffer consists of 0.8M GTC, 0.5M NaAc, 1.0M NaCl, 1% SDS, 5% NP-40, 1% Triton X-100 and 2% PEG8000.

[0027] Optionally, the pH of the lysis solution is adjusted to 5.0–7.0 using acetic acid.

[0028] Optionally, the washing solution consists of Tris-HCl, KCl, PEG, and optionally BSA.

[0029] Optionally, the pH of the washing solution is adjusted to 5.0–8.0 using acetic acid.

[0030] Thirdly, this application provides a detection kit for murine norovirus and murine hepatitis virus, comprising the cartridge of the second aspect, as well as positive and negative control samples.

[0031] Optionally, the positive control is a diluted solution of engineered bacteria (inactivated) containing the target detection gene sequence of murine norovirus and murine hepatitis virus.

[0032] Optionally, the negative control product is pure water.

[0033] Fourthly, this application provides a detection method based on the reagent cartridge of the second aspect, the steps of which are as follows: 1. Collect samples and process them using reagent cartridges; 2. Real-time quantitative RT-PCR amplification; 3. Interpretation of test results.

[0034] Optionally, the sample may be derived from whole blood, plasma, or serum.

[0035] Optionally, the RT-PCR amplification procedure is as follows: In summary, this application includes at least one of the following beneficial technical effects: The fully automated nucleic acid extraction, amplification, and diagnostic integrated microfluidic cartridge involved in this invention employs POCT technology, enabling multiplex real-time fluorescent PCR amplification of norovirus and mouse hepatitis virus. Simultaneously, it identifies norovirus and mouse hepatitis virus based on the amplification results of labeled dual-color fluorescence and includes internal quality control functions to control false negatives and ensure the accuracy of sample collection and purification processes. The fully automated nucleic acid extraction, amplification, and diagnostic integrated microfluidic cartridge for norovirus and mouse hepatitis virus RT-PCR used in this invention is a powerful tool for next-generation nucleic acid detection. It surpasses other norovirus and mouse hepatitis virus molecular diagnostic kits in terms of detection timeliness, specificity, sensitivity, convenience, and technical parameters, as detailed below: 1. Fully Enclosed Modular Design: The microfluidic cartridge's automated control and multi-layered anti-contamination structure integrates nucleic acid extraction, amplification, and detection processes within a fully enclosed cartridge, structurally eliminating aerosol contamination, protecting the safety of testing personnel, and eliminating the need for a PCR laboratory. This significantly reduces the requirements for laboratory construction standards in nucleic acid testing and eliminates equipment contamination problems caused by nucleic acid extraction. 2. Timeliness: The integrated nucleic acid extraction and amplification technology and automatic judgment software allow the detection process to produce results in just 70-90 minutes, truly achieving full automation from sample processing to result reporting. It further enables the identification of mouse norovirus and mouse hepatitis virus using RT-PCR single-well and single-fluorescence detection channels, greatly reducing the professional skills required of testing personnel, improving detection efficiency, and making it suitable for rapid clinical diagnosis. 3. Accuracy: Dedicated primers and probes are designed specifically for the nucleic acid sequences of norovirus and hepatitis virus to ensure the specificity and sensitivity of the detection. At the same time, it can reflect the infection status of norovirus and hepatitis virus in the sample and confirm which of the two viruses is causing the infection. It can be used for the monitoring, prevention and control of norovirus and hepatitis virus and for clinical diagnosis, which helps in the early diagnosis and treatment of norovirus and hepatitis virus infection. 4. Convenience: Stored at 2-8℃, eliminating the need for conventional -20℃ freezing, which facilitates cold chain transportation and preservation of the product. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the integrated reagent cartridge of the present invention; Figure 2 This is a schematic diagram illustrating the use of the integrated reagent card holder provided in an embodiment of the present invention; Figure 3 Graphs showing the results of accelerated high-temperature aging (Ct values) for card cartridges with different designs; Figure 4 Figure showing the results (relative efficiency) of high-temperature accelerated aging of card cartridges with different designs; Figure 5 The graph shows the comparison results of different pyrolysis solutions; Figure 6 The results are from a fully automated nucleic acid detection analyzer that detects the three primer and probe combinations used in MNV. Figure 7 The results are from a fully automated nucleic acid detection analyzer that detects three primer-probe combinations for MHV. Detailed Implementation

[0037] To more clearly demonstrate the objectives, technical solutions, technical effects, and advantages of this invention, specific embodiments are further described below. The specific embodiments described herein are merely illustrative of this invention and are not intended to limit the invention. The invention is further illustrated below with reference to specific examples.

[0038] In this document, "reagent cartridge" refers to a fully automated nucleic acid extraction, amplification, and diagnostic integrated microfluidic cartridge. In this application, it is also referred to as "POCT reagent cartridge," "integrated reagent cartridge," "cartridge," or "microfluidic cartridge."

[0039] In this paper, "point-of-care testing (POCT)" is a new integrated gene analysis technology that combines nucleic acid extraction, real-time fluorescence gene detection and microfluidic reagent cartridges. It only requires manual sample addition, and the subsequent nucleic acid extraction, detection and analysis processes are all completed by the machine. It realizes a fully closed and automated output from sample to gene detection results. It has the characteristics of simple operation, high specificity, low cost, fast speed and accurate results.

[0040] Example 1 Primer and probe design and synthesis Upstream-specific primers, downstream-specific primers, and TaqMan fluorescent probes were designed based on known norovirus and mouse hepatitis virus sequences from NCBI. The internal control (IC) was a synthetic DNA sequence: ttgcagatttggacctg cgagcgggtt ctgacctgaa ggctctgcgc ggacttgtgg agacagccgc tcaccttggctattcagtt (SEQ ID NO:10). This IC sequence was cloned onto the PUC57 plasmid; the IC primers and probes were adapted to it for differentiation from the target gene signal.

[0041] The primers include: Norovirus upstream primer: 5'-ACTCCCCCAGGAGTTTGTCT-3' (SEQ ID NO:1); Downstream primer for norovirus: 5'-GGCGATGTAACCTTCGTTGT-3' (SEQ ID NO:2); upstream primer for mouse hepatitis virus: 5'-GCGGACAAGACCTATGAGGA-3' (SEQ ID NO:3); Downstream primer for mouse hepatitis virus: 5'-GGCACAGCATAGAATGCAGA-3' (SEQ ID NO:4); IC upstream primer: 5'-AGATTTGGACCTGCGAGCG-3' (SEQ ID NO:5); IC downstream primer: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 6); The TaqMan fluorescent probes are labeled with a three-color combination of fluorescent markers to achieve multiplex real-time fluorescent PCR amplification, including: Norovirus probe: 5'-GCGTTCACCGTGCAGTCCGA-3' (SEQ ID NO:7); Mouse hepatitis virus probe: 5'-TGGCATTCGATGCCATATACTCAGAGA-3' (SEQ ID NO:8); IC probe: 5'-TTCTGACCTGAAGGCTCTGCGCG-3' (SEQ ID NO: 9); The 5' end of the probe is labeled with fluorescent reporter groups FAM, ROX and HEX, respectively, and the 3' end of the probe contains fluorescent quencher groups MGB, BHQ2 and BHQ1, respectively.

[0042] Example 2: Establishment of the Reagent Cartridge 2.1 Assembly of reagent card boxes A POCT reagent cartridge for detecting norovirus and murine hepatitis virus, such as Figure 1 As shown, the assembly steps are as follows: (1) Dispensing PCR reaction solution and primer-probe composition of Example 1: Dispense 20 μL of PCR reaction solution and 15 μL of primer-probe composition into the bottom of the amplification region; (2) Embedding manganese ion solution and IC: 5 μL of encapsulation solution was embedded in the paraffin-encapsulated area (the upper layer of the PCR reaction solution) with paraffin; (3) Sealing: Seal the reaction solution with an appropriate amount of silicone oil; (4) Dispensing washing solution 2: Dispense 140μL of washing solution 2 into washing solution 2; (5) Sealing: Seal the washing solution 2 with an appropriate amount of silicone oil; (6) Dispensing washing solution 1: Dispense 140μL of washing solution 1 into washing solution 1; (7) Sealing: Seal the washing solution 1 with an appropriate amount of silicone oil; (8) Dispensing the lysis buffer: Dispense 800 μL of lysis buffer into the lysis buffer section.

[0043] 2.2 Reagents in the reagent cartridge 2.2.1 The specific components of the lysis buffer are as follows: GTC 0.8M NaAc 0.5M NaCl 1.0M SDS 1％ NP-40 5％ Triton X-100 (TX-100) 1％ PEG8000 2％ 2.2.2 The specific components of washing solution 1 are as follows: Tris-HCl (pH 8.3) 100mM KCl 100mM PEG 2％ 2.2.3 The specific components of washing solution 2 are as follows: Tris-HCl (pH 8.3) 100mM KCl 100mM PEG 2％ BSA 1.8％ 2.2.4 The PCR reaction solution was Hotstart Onestep RT-PCR Master Mix (from Hangzhou Suizeng Biotechnology Co., Ltd., product model B0027). HotStart Onestep RT-PCR Master Mix mainly contains HotStart rTth enzyme, which is active in Mn...2+ Under activated conditions, it exhibits highly efficient amplification activity.

[0044] The primer-probe composition is as follows: 2.2.5 The specific components of the encapsulation fluid are as follows: <![CDATA[Manganese(II) acetate tetrahydrate (CH3COO)2Mn·4H2O]]> 2.5mM Trehalose 1％ Internal standard template DNA (IC) 1-5 ng / μl Sterilized ultrapure water — Add to 5μl It should be understood that, depending on the different experimental needs, the specifications of each component in the embodiments of the present invention can be adjusted according to the above-mentioned content.

[0045] Example 3: Detection method based on reagent cartridges Rapid typing and identification were performed using a POCT reagent cartridge for detecting mouse norovirus and mouse hepatitis virus, as described in Example 2.

[0046] 3.1 Use of the test card box Whole blood from mice was collected using anticoagulant blood collection tubes as samples. For example... Figure 2 As shown, open the lid of the card box, then open the sealing film, add 10 μL of magnetic beads (shake to fully suspend them before use), then add 200 μL of sample, mix the reagents by pipetting, and then tighten the lid.

[0047] 3.2 Real-time fluorescent PCR instant detection The cartridge from Example 3.1 was subjected to fully automated RT-PCR nucleic acid extraction and amplification using the Life Ready K1000 fully automated nucleic acid detection instrument. The PCR amplification products were analyzed using a fully automated nucleic acid detection and analysis system, and the results were determined by combining the amplification curve and Ct value.

[0048] The fluorescence channel settings are as follows: The RT-PCR amplification program is set as follows: 3.3 Interpretation of test results After the detection procedure is completed, the instrument will automatically report the results. The results will show the detection results for norovirus (FAM), rat hepatitis virus (ROX), and internal quality control (HEX). If the norovirus (FAM) and / or rat hepatitis virus (ROX) results show a clear S-shaped amplification curve (including S-curves that have not reached the plateau phase), and Ct ≤ 38, then the corresponding result is considered positive. If both fluorescence (FAM, ROX) are negative and the internal quality control (HEX) Ct ≤ 38, then the corresponding result is considered negative. The internal standard HEX fluorescence channel detection result should show a clear S-shaped amplification curve (including S-curves that have not reached the plateau phase); otherwise, resampling and testing are required.

[0049] Example 4: Sensitivity and Specificity Testing Based on Reagent Cartridges 4.1 Specificity test Nucleic acid samples of the target pathogens (mice of norovirus MNV and mice of hepatitis virus MHV) were collected for positive sample verification; nucleic acid samples of non-target pathogens (nucleic acid samples of Sendai virus and Mycoplasma pneumoniae) were collected for specific detection. The detection of the target pathogens was positive in all cases, while the detection of the other non-target pathogens was negative. The results show that the method has good specificity, with a specificity of 100%.

[0050] 4.2 Sensitivity Test Different concentrations of positive control samples (inactivated diluted plasmid containing the target gene sequence of norovirus and mouse hepatitis virus) were used for detection. The results are shown in Tables 1 and 2.

[0051] Table 1 Table 2 According to the requirements of the guidelines for performance analysis of IVD products issued by the China Food and Drug Administration, the data in Tables 1 and 2 were statistically analyzed using the Probit regression method. The detection limit was set at the 95% detection rate sample concentration. The results are as follows: Detection limit for norovirus: 116.331 copies / ml, 95% confidence limit: 81.141 to 426.488 copies / ml; Detection limit for hepatitis virus: 145.221 copies / ml, 95% confidence limit: 102.153 to 408.751 copies / ml.

[0052] Example 5: Detection of Clinical Samples The detection method described in Example 3 was used to test multiple clinical samples that had been confirmed as norovirus and mouse hepatitis virus. The results are shown in Tables 3 and 4. The detection results of the reagent cartridge prepared in this invention showed high consistency with the results confirmed by conventional detection methods (mainly immunoassay), and the overall test concordance rate was the same. However, compared with conventional diagnostic methods, the detection time using the integrated reagent cartridge prepared in this invention is shorter and the sensitivity is higher.

[0053] Table 3 Table 4 Example 6: Optimization of the IC Built into the Card Box Three pre-installed IC methods were designed: 1.1 IC, MnAC and trehalose are encapsulated together in the paraffin-coated area. The elution and amplification area (hereinafter referred to as the amplification area) contains the PCR reaction solution and primers / probes. 1.2 The IC, primers, probes, and trehalose are encapsulated together in a paraffin-coated area, and the amplification area contains MnAC and PCR reaction solution; 1.3 MnAC and trehalose are encapsulated in the paraffin-coated area, and the IC, primers, probes, and PCR reaction solution are placed in the amplification area.

[0054] Three experimental cartridges were prepared according to the above three designs (with Mn as the activating ion). 2+ The PCR reaction solution was HotStartOnestep RT-PCR Master Mix. Each component was injected into the test cartridge (low / critical concentration of IC template was used to facilitate the detection of possible differences). Three test cartridges were designed according to methods 1.1, 1.2, and 1.3: IC cartridge 1 (method 1.1), IC cartridge 2 (method 1.2), and IC cartridge 3 (method 1.3).

[0055] High-temperature accelerated aging test results: Each cartridge was repeated twice (e.g., cartridge 1 is recorded as cartridge 1-1 and cartridge 1-2), and the results are as follows. Figure 3 and Figure 4 As shown, where, Figure 4 The efficiency standard of 100% was taken from the 0-day result of IC card box 1-1, and the relative efficiency was calculated based on the difference in Ct value (the larger the Ct, the lower the efficiency). The results show that IC card box 1 has the best efficiency, with almost no change in efficiency during the 56-day accelerated aging process; IC card box 2 has slightly worse preservation performance than IC card box 1; IC card box 3 is not effective, and its preservation performance cannot meet the established requirements.

[0056] The method for embedding the internal standard in the cartridge should be to combine the internal standard quality control (plasmid DNA) with Mn. 2+ They are encapsulated together in paraffin wax. When the detection program is running, the paraffin wax melts, and MnAC and IC are added to the amplification area. They are then mixed with the PCR reaction solution and primers / probes for fluorescent PCR amplification.

[0057] According to the conversion formula in the document "Basic Principles and Methods for Determining the Shelf Life of Medical Devices by Accelerated Aging Test" published in China Medical Device Information, Vol.14, No.5, 2008, it can be concluded that the cartridge designed in this application can be stably stored at 8℃ for at least 1 year, meeting the storage requirements of AIGS test cartridges.

[0058] Example 7: Optimization of the lysis buffer formulation When an excessive amount of whole blood is added, a large number of impurities such as hemoglobin are carried into the PCR reaction solution, inhibiting the amplification efficiency and potentially leading to false negatives (failure of internal standard IC gene detection).

[0059] Based on the goals of reducing protein adsorption and improving lysis efficiency, a new lysis buffer formulation was designed, as shown in Table 5: Table 5 Lysis buffers were prepared according to the designed formula and filled into cartridges. Genomic DNA from whole blood samples was detected using IC primers and probes. Anticoagulated whole blood samples from within one week were used as samples, with a loading volume of 200 μl per cartridge.

[0060] Test results: Formulas 4-7 were ineffective and failed to detect; Formula 8 showed amplification, but at a lower efficiency and was not as effective as Formula 1. The test results of the cartridges corresponding to formulations 1-3 (i.e., lysis buffer 1, lysis buffer 2, and lysis buffer 3) are as follows: Figure 5 As shown in Table 6, analysis of CT values ​​and amplification curves reveals that formulation 3 was ineffective, exhibiting low amplification efficiency and insufficient lysis capacity. Overall, formulation 2 demonstrated the best performance with stable amplification efficiency. Formulation 1 was less effective than 2, but still significantly superior to the original lysis buffer formulation (100mM acetate-sodium acetate, 1wt% polyethylene glycol 8000, 3.5M GTC, 5wt% Tween-20, and 0.5wt% sodium lauryl sulfate). Typically, the direct loading volume for (mammal) whole blood samples is ≤2μl, but after optimizing the lysis buffer formulation, the loading volume increased to ≥200μl, maintaining stable internal standard detection while preserving the POCT procedure for direct whole blood sample loading.

[0061] Table 6 Example 8: Screening of Primer-Probe Compositions Screening for the optimal primer-probe combination suitable for the detection of murine norovirus (MNV) and murine hepatitis virus (MHV) nucleic acids provides key parameters for the development of highly sensitive and specific joint detection kits.

[0062] 8.1 Primer and probe design MNV: Three sets of primers and probes were designed for the VP1 gene. The 5' end of the probe was labeled with the FAM fluorescent group and the 3' end was labeled with the MGB quencher group. MHV: Three sets of primers and probes were designed for the conserved region of ORF1ab. The 5' end of the probe was labeled with a ROX fluorescent probe, and the 3' end was labeled with a BHQ2 quencher group. IC: HEX-labeled primer probe, used to distinguish the target signal from the IC signal; the specific sequence is shown in Table 7.

[0063] Table 7 Primer and probe information 8.2 Experimental Methods The sensitivity of primer-probe combinations was evaluated by comparing the amplification performance of low-concentration samples (200 μL) in different cartridges: detection cartridges were prepared using 3 sets of primers and probes each for MNV and MHV → 3 sets of primers and probes for the same nucleic acid were added to the same low-concentration sample → detection was performed using a fully automated nucleic acid detection analyzer. Evaluation metrics: Ct value (cycle threshold; the lower the Ct value, the higher the amplification efficiency) and fluorescence growth curve (the greater the slope of the fluorescence signal rise, the stronger the amplification efficiency).

[0064] 8.3 Test Results (1) Amplification efficiency detection results The results of the fully automated nucleic acid detection analyzer using the MNV three-primer-probe combination are as follows: Figure 6 As shown, the detection results of the MHV three-primer-probe combination fully automated nucleic acid detection analyzer are as follows: Figure 7 As shown. According to Figure 6 and Figure 7 The results showed that all three primer and probe combinations of MNV / MHV could detect low-concentration samples. Among them, the optimal primer and probe combination showed a significant advantage in amplification efficiency: both MNV combination 3 and MHV combination 1 showed the highest target gene fluorescence growth value and the largest fluorescence growth slope. Therefore, the amplification efficiency of these two combinations in synergy with the internal standard was the best.

[0065] 1. Ct value results The Ct values ​​of the fully automated nucleic acid detection analyzer are summarized in Table 8. From the Ct value results, the target gene and internal standard Ct values ​​of MNV primer-probe combination 3 and MHV primer-probe combination 1 are the lowest, indicating that the amplification efficiency of both combined with the internal standard is the best.

[0066] Table 8 Through the above screening process, the optimal primer and probe combination can be determined for the norovirus / hepatitis virus combined detection kit, ensuring high sensitivity, specificity, and suitability for automated operation in clinical applications. Specifically, the optimal primer and probe combination for MNV is combination 3, and the optimal primer and probe combination for MHV is combination 1.

[0067] The embodiments described above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A primer probe composition for detection of murine norovirus and murine hepatitis virus, characterized in that, the primer and probe for detection of murine norovirus comprise: forward primer: SEQ ID NO: 1, reverse primer: SEQ ID NO: 2, probe: SEQ ID NO: 7; the primer and probe for detection of murine norovirus comprise: forward primer: SEQ ID NO: 3, reverse primer: SEQ ID NO: 4, probe: SEQ ID NO:

8.

2. The primer probe composition of claim 1, wherein the primer and probe of IC also comprise, the primer and probe of IC comprise: forward primer: SEQ ID NO: 5, reverse primer: SEQ ID NO: 6, probe: SEQ ID NO:

9.

3. The primer probe composition according to claim 1 or claim 2, characterized in that, the 5' end of the probe comprises a fluorescent reporter group selected from FAM, HEX, ROX, JOE, VIC, TET, NED, FITC, CY3 or CY5.

4. The primer probe composition according to claim 1 or claim 2, characterized in that, the 3' end of the probe comprises a fluorescent quencher group selected from BHQ1, BHQ2, BHQ3, TAMRA, Eclipse, DABCYL or MGB.

5. A reagent cartridge for detection of murine norovirus and murine hepatitis virus, characterized by, the reagent cartridge is provided with a lysis solution area, a washing solution area and an amplification area from top to bottom; the lysis solution area is separately provided with a lysis solution, the washing solution area is separately provided with a washing solution, and the amplification area is separately provided with a PCR reaction solution and the primer probe composition according to any one of claims 1-4.

6. The reagent cartridge of claim 5, wherein, The expansion region is also provided with a wrapping region that wraps the wrapping liquid including the IC and the Mn 2+ with a fusible material.

7. The reagent cartridge of claim 6, wherein, the fusible material is selected from any one or more of paraffin, dodecane, tetradecane and hexadecane.

8. The reagent cartridge of claim 5, wherein, the lysis solution comprises GTC, NaAc, NaCl, SDS, NP-40, Triton X-100 and PEG8000.

9. The reagent cartridge of claim 8, wherein, the lysis solution consists of 0.8M GTC, 0.5M NaAc, 1.0M NaCl, 1% SDS, 5% NP-40, 1% Triton X-100 and 2% PEG8000.

10. The reagent cartridge of claim 5, wherein, the washing solution consists of Tris-HCl, KCl, PEG and optionally BSA.

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