A qPCR primer and probe set for detecting multiple nova viruses, detection method and application

Abstract

This invention provides a primer-probe set, detection method, and application for qPCR detection of various nodaviruses, belonging to the field of molecular biology detection technology. This invention designs a primer-probe combination for various nodaviruses that can infect insect cell lines, including farm virus, Tn5 cell line virus, black beetle virus, Boralwa virus, Nodamura virus, and Macrobrachium rosenbergii nodavirus. In this combination, some viruses share upstream primers and probes, enabling simultaneous detection and identification of multiple target viruses in a single reaction system. This invention also provides a kit and detection method containing this primer-probe set. The detection method of this invention has advantages such as wide coverage, high detection sensitivity, strong specificity, and good resistance to matrix interference, and is suitable for the safety detection and quality control of nodavirus contamination in insect cell banks, cell cultures, and related biological products.

Description

Technical Field

[0001] This invention belongs to the field of molecular biological detection technology, specifically relating to a qPCR primer and probe set, detection method, and application for detecting various nova viruses. Background Technology

[0002] Insect cell expression systems have become an important platform for producing recombinant protein vaccines, virus-like particles, and certain gene therapy vectors due to their advantages such as low risk of cross-infection with human pathogens, high protein expression efficiency, and suitable post-translational modification capabilities. To ensure the safety of the final biological products, regulatory agencies such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), the U.S. Food and Drug Administration (FDA), and the Pharmacopoeia of the People's Republic of China have all explicitly required the detection and risk assessment of exogenous viral factors in the cell matrix used for production.

[0003] Viruses of the Nodaviridae family, particularly alpha-nodaviruses, are small, non-enveloped RNA viruses that primarily infect insects and other invertebrates. Common viruses that may contaminate insect cell lines (such as Sf9, Sf21, and HighFive™) include Flock House Virus (FHV), Tn5 Cell Line Virus (Tncl), Black Beetle Virus (BBV), Boolarra Virus (BoV), and Nodamura Virus (NoV). Furthermore, studies have shown that some nodaviruses that infect crustaceans, such as Macrobrachium rosenbergii nodavirus (MrNV), also possess the ability to infect insect cells. Latent contamination by these viruses can affect cell growth, product quality, and even pose unpredictable biosafety risks.

[0004] Currently, traditional methods for detecting novaviruses in insect cells include cell culture and observation of cytopathic effect (CPE), electron microscopy, immunological detection (such as ELISA), and conventional RT-PCR. However, these methods have significant limitations: cell culture is time-consuming (usually requiring several weeks), has limited sensitivity, and some viruses do not produce obvious CPE; electron microscopy is expensive and requires specialized operation; the specificity and sensitivity of immunological methods are limited by antibody quality; and conventional singleton RT-PCR has low throughput, making it difficult to meet the simultaneous screening needs of multiple viruses, lacking sensitivity when detecting low copy number viruses, and is susceptible to cross-contamination from cellular genomic DNA or during operation.

[0005] In recent years, real-time quantitative PCR (qPCR) technology has become the gold standard for pathogen detection due to its high sensitivity, high specificity, quantitative capability, and rapid efficiency. However, current technologies still lack a systematically validated qPCR detection protocol that can simultaneously cover multiple key nova viruses and is optimized for insect cells and their product matrices. Most existing primer-probe combinations target only a single or a few viruses, resulting in cumbersome detection procedures, increased costs, and the inability to identify and detect multiple target viruses in a single reaction. This fails to fully meet the urgent need of the biopharmaceutical industry for efficient and comprehensive viral safety assessment of cell matrices.

[0006] Therefore, there is an urgent need in this field to develop a qPCR detection primer-probe combination and supporting method that has broad coverage, high sensitivity, strong specificity, good resistance to matrix interference, and is suitable for the quality control of insect cells and their biological products. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a qPCR primer and probe set for detecting a variety of nova viruses, wherein the primer and probe set can detect a variety of nova viruses that can infect insect cells, and has high detection sensitivity, good specificity and strong durability.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a qPCR primer and probe set for detecting various nova viruses. The primer and probe set includes an upstream primer FHV-Tncl-F, a downstream primer FHV-R, a downstream primer Tncl-R, and a fluorescent probe FHV-Tncl-P for detecting farm viruses and Tn5 cell line viruses. The nucleotide sequence of the upstream primer FHV-Tncl-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer FHV-R is shown in SEQ ID NO.2, the nucleotide sequence of the downstream primer Tncl-R is shown in SEQ ID NO.3, and the nucleotide sequence of the fluorescent probe FHV-Tncl-P is shown in SEQ ID NO.4.

[0010] The upstream primer BBV-F, the downstream primer BBV-R, and the fluorescent probe BBV-P for detecting black beetle virus are shown in SEQ ID NO.5, the nucleotide sequence of the upstream primer BBV-F is shown in SEQ ID NO.6, and the nucleotide sequence of the fluorescent probe BBV-P is shown in SEQ ID NO.7.

[0011] The upstream primer BoV-F, the downstream primer BoV-R, and the fluorescent probe BoV-P for detecting Boralwa virus are shown in SEQ ID NO.8, the nucleotide sequence of the upstream primer BoV-F is shown in SEQ ID NO.9, and the nucleotide sequence of the fluorescent probe BoV-P is shown in SEQ ID NO.10.

[0012] The upstream primer NoV-F, the downstream primer NoV-R, and the fluorescent probe NoV-P used for detecting Nodamura virus have the following nucleotide sequences: upstream primer NoV-F as shown in SEQ ID NO.11, downstream primer NoV-R as shown in SEQ ID NO.12, and fluorescent probe NoV-P as shown in SEQ ID NO.13.

[0013] The upstream primer MrNV-F, the downstream primer MrNV-R, and the fluorescent probe MrNV-P are used to detect nodavirus in giant freshwater prawns. The nucleotide sequence of the upstream primer MrNV-F is shown in SEQ ID NO.14, the nucleotide sequence of the downstream primer MrNV-R is shown in SEQ ID NO.15, and the nucleotide sequence of the fluorescent probe MrNV-P is shown in SEQ ID NO.16.

[0014] Preferably, the fluorescent probe in the primer-probe set is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

[0015] More preferably, the fluorescent reporter group includes one of FAM, VIC or CY5; the fluorescent quencher group includes BHQ1 or BHQ2.

[0016] The present invention also provides a qPCR kit for detecting various nova viruses, the kit comprising the aforementioned qPCR primer and probe set.

[0017] Preferably, the detection system of the qPCR kit, in a total volume of 30 μL, comprises the following components: 2.3 μL of nucleic acid-free water, 16.2 μL of qPCR reaction solution, 1 μL of primer mixing working solution, 0.5 μL of probe mixing working solution, and 10 μL of template.

[0018] More preferably, for detecting scavenger virus, Tn5 cell line virus, and black beetle virus, the primer concentrations of the upstream primer FHV-Tncl-F, downstream primer FHV-R, and downstream primer Tncl-R in the primer mixture are all 10 μM, based on the effective final concentration of each primer sequence variant, and the primer concentrations of the upstream primer BBV-F and downstream primer BBV-R are 3 μM; for detecting Boralwa virus, the primer concentrations of the upstream primer BoV-F and downstream primer BoV-R in the primer mixture are 8 μM; for detecting Noda virus and Macrobrachium rosenbergii nodavirus, the primer concentrations of the upstream primer NoV-F, downstream primer NoV-R, upstream primer MrNV-F, and downstream primer MrNV-R in the primer mixture are 6 μM.

[0019] More preferably, for detecting farm virus, Tn5 cell line virus, and black beetle virus, the probe concentrations of fluorescent probe FHV-Tncl-P and fluorescent probe BBV-P in the probe mixture working solution are 10 μM and 3 μM, respectively; for detecting Boralwa virus, the probe concentration of fluorescent probe BoV-P in the probe mixture working solution is 8 μM; for detecting Nodamu virus and Macrobrachium rosenbergii Noda virus, the probe concentrations of fluorescent probe NoV-P and fluorescent probe MrNV-P in the probe mixture working solution are 6 μM and 6 μM, respectively.

[0020] This invention also provides a method for detecting various nodaviruses that can infect insect cells for non-diagnostic purposes, comprising the following steps: extracting RNA from the sample to be tested and reverse transcribing it into cDNA; using the cDNA as a template, employing the qPCR primer and probe set or the qPCR kit described above; collecting fluorescence signals, and determining whether the sample to be tested is positive for farm virus, Tn5 cell line virus, black beetle virus, Boralwa virus, Nodamura virus, and / or giant freshwater shrimp nodavirus based on the amplification curve and Cp value.

[0021] Preferably, the judgment criteria are as follows: if the Cp value of the sample to be tested is less than 40 and there is an obvious amplification curve, it is judged as positive; if the Cp value of the sample to be tested is greater than or equal to 40 or there is no obvious amplification curve, it is judged as negative.

[0022] Preferably, the qPCR reaction program is as follows: reverse transcription at 50℃ for 20 min; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s; annealing at 60℃ for 30 s; 45 cycles.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This invention provides a qPCR primer and probe set for detecting various nodaviruses. This primer and probe set has broad coverage and can detect multiple nodaviruses that can infect insect cells, namely Flock House Virus (FHV), Tn5 Cell Line Virus (Tncl), Black Beetle Virus (BBV), Boolarra Virus (BoV), Nodamura Virus (NoV), and Macrobrachium Rosenbergii Nodavirus (MrNV). It exhibits high detection sensitivity, with 100% detection of plasmid standards for each nodavirus at a reaction rate of 10 copies / reaction. The results show good specificity and robustness, and the nucleic acid extraction and detection processes are unaffected by cell matrix interference. Each nodavirus plasmid standard shows a positive S-shaped amplification curve in its corresponding qPCR reaction system, enabling precise molecular diagnosis of various nodaviruses. This method is suitable for quality control of insect cells and their biological products and has significant application value in pathogen detection and epidemiological investigation. Attached Figure Description

[0025] Figure 1 Standard curves for qPCR fluorescence quantitative detection of farm viruses and Tn5 cell line viruses;

[0026] Figure 2 A standard curve for qPCR fluorescence quantitative detection of black beetle virus;

[0027] Figure 3 A standard curve for qPCR quantitative detection of Boralwa virus;

[0028] Figure 4 The standard curve for qPCR fluorescence quantitative detection of Nodamu virus;

[0029] Figure 5 A standard curve for qPCR fluorescence quantitative detection of nodavirus in giant freshwater prawn;

[0030] Figure 6 The detection curves for qPCR fluorescence quantitative detection of farm viruses and Tn5 cell line viruses;

[0031] Figure 7 The detection curve for qPCR fluorescence quantitative detection of black beetle virus;

[0032] Figure 8 The detection curve for qPCR quantitative detection of Boralwa virus;

[0033] Figure 9The detection curve for qPCR fluorescence quantitative detection of Nodamu virus;

[0034] Figure 10 The detection curve for qPCR fluorescence quantitative detection of nodavirus in giant freshwater shrimp. Detailed Implementation

[0035] This invention provides a qPCR primer-probe set for detecting various nodaviruses. Using bioanalytical techniques, this invention classifies and compares all nucleic acid information of Flock House Virus (FHV), Tn5 Cell Line Virus (Tncl), Black Beetle Virus (BBV), Boolarra Virus (BoV), Nodamura Virus (NoV), and Macrobrachium Rosenbergii Nodavirus (MrNV) in Genebank. Conserved regions that maximally cover specific nodavirus species and their variants or subtypes are extracted for primer and probe design. Finally, primer-probe combinations for qPCR detection of each nodavirus are screened. The primer-probe set includes an upstream primer FHV-Tncl-F, a downstream primer FHV-R, a downstream primer Tncl-R, and a fluorescent probe FHV-Tncl-P for detecting farmhouse virus (FHV) and Tncl cell line virus (Tncl). The nucleotide sequence of the upstream primer FHV-Tncl-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer FHV-R is shown in SEQ ID NO.2, the nucleotide sequence of the downstream primer Tncl-R is shown in SEQ ID NO.3, and the nucleotide sequence of the fluorescent probe FHV-Tncl-P is shown in SEQ ID NO.4. The primer-probe set provided by this invention uses a shared upstream primer (FHV-Tncl-F) and detection probe (FHV-Tncl-P) for farmhouse virus (FHV) and Tncl cell line virus (Tncl), but uses specific downstream primers (FHV-R and Tncl-R) to distinguish them. When FHV-R and Tncl-R are added simultaneously to the same reaction system, this system can simultaneously screen for FHV and Tncl, with the presence of either virus generating a FAM fluorescence signal. For further identification of specific virus species, a detection system containing only one specific downstream primer can be used. This primer-probe set ensures high coverage and screening efficiency, and virus species identification can be achieved through simple experimental design.

[0036] The upstream primer BBV-F, the downstream primer BBV-R, and the fluorescent probe BBV-P for detecting black beetle virus are shown in SEQ ID NO.5, the nucleotide sequence of the upstream primer BBV-F is shown in SEQ ID NO.6, and the nucleotide sequence of the fluorescent probe BBV-P is shown in SEQ ID NO.7.

[0037] The upstream primer BoV-F, the downstream primer BoV-R, and the fluorescent probe BoV-P for detecting Boralwa virus are shown in SEQ ID NO.8, the nucleotide sequence of the upstream primer BoV-F is shown in SEQ ID NO.9, and the nucleotide sequence of the fluorescent probe BoV-P is shown in SEQ ID NO.10.

[0038] The upstream primer NoV-F, the downstream primer NoV-R, and the fluorescent probe NoV-P used for detecting Nodamura virus have the following nucleotide sequences: upstream primer NoV-F as shown in SEQ ID NO.11, downstream primer NoV-R as shown in SEQ ID NO.12, and fluorescent probe NoV-P as shown in SEQ ID NO.13.

[0039] The upstream primer MrNV-F, the downstream primer MrNV-R, and the fluorescent probe MrNV-P are used to detect nodavirus in giant freshwater prawns. The nucleotide sequence of the upstream primer MrNV-F is shown in SEQ ID NO.14, the nucleotide sequence of the downstream primer MrNV-R is shown in SEQ ID NO.15, and the nucleotide sequence of the fluorescent probe MrNV-P is shown in SEQ ID NO.16.

[0040] In this invention, the fluorescent probes in the primer-probe set are labeled with a fluorescent reporter group at their 5' end and a fluorescent quencher group at their 3' end. The presence or type of nova virus in the sample can be identified based on the fluorescence signal and amplification curve in the amplification product. This invention does not limit the specific fluorescent reporter and quencher groups contained in the probes; any group that can connect to the probe and play a corresponding characterizing role is acceptable. As a preferred embodiment, the fluorescent reporter group includes one of FAM, VIC, or CY5; the fluorescent quencher group includes BHQ1 or BHQ2. In some preferred embodiments, FHV-Tncl-P contains FAM, BBV-P contains VIC, BoV-P contains FAM, NoV-P contains VIC, and MrNV-P contains CY5; preferably, FHV-Tncl-P, BBV-P, BoV-P, and NoV-P contain BHQ1, and MrNV-P contains BHQ2.

[0041] This invention also provides a qPCR kit for detecting various nodaviruses, the kit comprising the aforementioned qPCR primer and probe set. The kit further includes at least one of a negative control, a positive control, and a qPCR reaction solution. In this invention, the positive control in the kit is a plasmid standard for nodavirus, and its detection sequences are shown in SEQ ID NO. 17-19. The qPCR reaction solution in the kit comprises commonly used components for qPCR reactions; specific examples of reaction reagents include reverse transcriptase, thermostable DNA polymerase, buffer, dNTPs, and Mg2+ for qPCR reactions. 2+ The present invention does not limit the use of reagents such as stabilizers. As a preferred embodiment, the qPCR reaction solution also includes the primer-probe combination provided by the present invention. The qPCR reaction solution containing the primer-probe combination provided by the present invention is pre-allocated; only the extracted genomic nucleic acid needs to be added during use, reducing potential contamination during preparation and providing convenience and speed. By setting up positive and negative controls, the present invention can effectively avoid false negative or false positive results, further ensuring the accuracy of the detection.

[0042] In this invention, the detection system of the qPCR kit preferably comprises the following components in a total volume of 30 μL: 2.3 μL of nucleic acid-free water, 16.2 μL of qPCR reaction solution, 1 μL of primer mixing working solution, 0.5 μL of probe mixing working solution, and 10 μL of template. The qPCR reaction solution comprises 15 μL of 2×Hifair® VC58P2 MP Buffer and 1.2 μL of Hifair® VC58P2 Enzyme Mix.

[0043] In this invention, for detecting veterinary virus, Tn5 cell line virus, and black beetle virus, the upstream primer FHV-Tncl-F in the primer mixture is a degenerate primer with the sequence TTGTCCGCAGGARACGAAA (SEQ ID NO: 1). In NO.1), "R" represents an A or G base. To ensure that the effective working concentration of each possible sequence variant in its degenerate sequence reaches 10 μM in the reaction, the primer concentration is set to 20 μM when preparing the primer mixture. The primer concentrations of the downstream primer FHV-R and downstream primer Tncl-R are 10 μM, and the primer concentrations of the upstream primer BBV-F and downstream primer BBV-R are preferably 3 μM. For detecting Boralwa virus, the primer concentrations of the upstream primer BoV-F and downstream primer BoV-R in the primer mixture are preferably 8 μM. For detecting Noda virus and Macrobrachium rosenbergii Nodavirus, the primer concentrations of the upstream primer NoV-F, downstream primer NoV-R, upstream primer MrNV-F, and downstream primer MrNV-R in the primer mixture are preferably 6 μM. In this invention, for detecting farm viruses, Tn5 cell line viruses, and black beetle viruses, the preferred probe concentrations of fluorescent probe FHV-Tncl-P and fluorescent probe BBV-P in the probe mixing working solution are 10 μM and 3 μM, respectively; for detecting Boralwa virus, the preferred probe concentration of fluorescent probe BoV-P in the probe mixing working solution is 8 μM; for detecting Nodamu virus and Macrobrachium rosenbergii nodavirus, the preferred probe concentrations of fluorescent probe NoV-P and fluorescent probe MrNV-P in the probe mixing working solution are 6 μM and 6 μM, respectively. This invention, by limiting the concentrations of primers and probes in the mixing working solution, reduces waste and effectively avoids the formation of primer dimers due to excessively high concentrations, which could affect the formation of amplification curves and the determination of Cp values.

[0044] This invention also provides a method for detecting various nodaviruses that can infect insect cells for non-diagnostic purposes, comprising the following steps: extracting RNA from the sample to be tested and reverse transcribing it into cDNA; using the cDNA as a template, employing the qPCR primer and probe set or the qPCR kit described above; collecting fluorescence signals, and determining whether the sample is positive for farm virus, Tn5 cell line virus, black beetle virus, Boralwa virus, Nodamura virus, and / or Macrobrachium rosenbergii nodavirus based on the amplification curve and Cp value. The method provided by this invention for detecting various nodaviruses that can infect insect cells is a method for non-disease diagnosis and treatment purposes, referring to the detection of nodavirus infection in non-insect animals, including but not limited to nodavirus contamination in environments / scenarios such as air, soil, biological products, and laboratory samples.

[0045] This invention does not impose specific limitations on the methods for extracting RNA from samples and for reverse transcription. Conventional methods well-known in the art can be used to extract RNA from the sample and perform reverse transcription; alternatively, commercially available kits can be used to extract RNA from the sample and perform reverse transcription. The detection method established by this invention can simultaneously detect and identify multiple target viruses in the same reaction system, and can also perform single detection of each virus. As a preferred embodiment, this invention utilizes qPCR detection to detect farm virus, Tn5 cell line virus, and black beetle virus; specifically, this invention utilizes dual qPCR detection to detect Nodamura virus and Macrobrachium rosenbergii nodavirus; it has advantages such as speed, sensitivity, and efficiency. In this invention, if multiple target viruses are detected simultaneously in the same reaction system, the template in the reaction system is a mixed template. Preferably, the templates of each target virus in the reaction system are mixed in an equal volume ratio. As a preferred embodiment, when detecting and identifying three viruses simultaneously—shed virus, Tn5 cell line virus, and black beetle virus—5-10 μL of each pathogen template is taken and thoroughly mixed, and the mixed template is used as the mixed template.

[0046] In this invention, the judgment criteria are preferably as follows: if the Cp value of the sample to be tested is less than 40 and there is an obvious amplification curve, it is judged as positive; if the Cp value of the sample to be tested is greater than or equal to 40 or there is no obvious amplification curve, it is judged as negative.

[0047] In this invention, the qPCR reaction program is as follows: reverse transcription at 50℃ for 20 min; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s; annealing at 60℃ for 30 s; 45 cycles. This invention optimizes the annealing temperature to ensure good stability of the amplification reaction.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0050] Example 1: Design of Primer-Probe Combinations

[0051] This invention summarizes a variety of novaviruses that can infect insect cells and designs primer-probe combinations for qPCR detection of these viruses. Information on these novaviruses that can infect insect cells is shown in Table 1 below.

[0052] Table 1. Types of Nodaviruses that can infect insect cells

[0053]

[0054] This invention employs bioanalytical techniques to classify and compare all nucleic acid information of the nodavirus species shown in Table 1 in Genebank, extracting conserved regions that can maximally cover specific nodavirus species and their variants or subtypes for primer and probe design. Finally, primer-probe combinations for qPCR detection targeting each nodavirus were screened, as shown in Table 2 below.

[0055] Table 2 Primer and probe combinations for nodaviruses that can infect insect cells.

[0056]

[0057]

[0058] Example 2: Detection of the linear range of nodavirus capable of infecting insect cells using primer-probe combinations

[0059] The corresponding plasmid standards for each virus were prepared from 1×10⁻⁶. 7 The concentration of copies / μL was sequentially diluted 10-fold in 7 gradients, namely 1×10⁻⁶ copies / μL. 7 1×10 6 1×10 5 1×10 4 1×10 3 1×10 2 and 1×10 1 Copies / μL were collected, and then 10μL was used for qPCR reaction to plot a standard curve.

[0060] The plasmid standards were pUC57-FHV-Tncl-BBV, pUC57-BoV, and pUC57-NoV-MrNV. The target gene sequences of the plasmid standards are shown in Table 3 below.

[0061] Table 3. Target gene sequences for plasmid standards.

[0062]

[0063] The premixed solutions for qPCR detection were prepared, and their composition is shown in Table 4 below. The total volume for each reaction was 30 μL, containing 20 μL of the premixed solution shown in the table below, and 10 μL of template (plasmid standard or sample cDNA). The primer and probe mixing working solutions for each system were prepared as shown in Table 5 below. The qPCR reaction program was as follows: reverse transcription at 50℃ for 20 min; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s; annealing at 60℃ for 30 s (fluorescence was collected at this step), for 45 cycles. The fluorescence detection channel was selected as FAM+VIC+CY5.

[0064] Table 4. Premixes for qPCR detection of insect cell-infectable nodavirus

[0065]

[0066] Table 5. Primer and probe mixtures for various systems capable of infecting insect cell nodavirus.

[0067]

[0068] Note: FHV-Tncl-F is a degenerate primer. In its sequence (SEQ ID NO.1), "R" represents an A or G base. To ensure that the effective working concentration of each possible sequence variant in its degenerate sequence reaches 10 μM in the reaction, the concentration of this primer is set to 20 μM when preparing the primer mixture working solution.

[0069] Experimental results for each virus are as follows Figure 1-5 As shown, the 1×10⁻⁶ standard plasmids for each virus can be observed. 7 -1×10 1 All seven gradients at copies / μL were detected, and the correlation coefficient R was [value missing]. 2 ≥0.99, with a slope between -3.1 and -3.58, indicating a good linear relationship.

[0070] Example 3: Detection Limit of Primer-Probe Combination for Infecting Insect Cell Nodavirus

[0071] This embodiment evaluates the detection limit of a method for detecting novaviruses that can infect insect cells. Using the qPCR system and method of Example 2, plasmid standards for each virus were tested at 10 copies / reaction. Two independent replicate tests were performed, 10 replicates per well, for a total of 20 replicates. At least 19 wells must be positive, i.e., a 95% detection rate. Simultaneously, the detection limit Cp value of each virus plasmid must meet a relative standard deviation (RSD) ≤ 5%, indicating repeatable and stable detection at the sensitivity points.

[0072] The test results are shown in Table 6-10. All plasmid standards for each Nodavirus 10 copies / reaction were detected in 100% of cases, and the RSD of the Cp values ​​were all ≤5%, indicating high detection sensitivity and good repeatability.

[0073] Table 6. Limit of Detection (LOD) and Cp Value of Nodavirus FHV-Tncl Corresponding Plasmid Standards

[0074]

[0075] Table 7. Limit of Detection (LOD) and Cp Value of Nodavirus BBV Corresponding Plasmid Standards

[0076]

[0077] Table 8. Limit of Detection (LOD) and Cp Value of Nodavirus BoV Corresponding Plasmid Standards

[0078]

[0079] Table 9. Limit of Detection (LOD) and Cp Values ​​of Nodavirus (NoV) Corresponding Plasmid Standards

[0080]

[0081] Table 10. Limit of Detection (LOD) and Cp Values ​​of Plasmid Standards Corresponding to Nodavirus (MrNV)

[0082]

[0083] Example 4: Verification of the robustness of primer-probe combination detection for nodavirus infecting insect cells.

[0084] This embodiment evaluates the robustness of the method for detecting nodaviruses that can infect insect cells.

[0085] This embodiment uses 10μL 1×10 2 Plasmid standards of copies / μL were added to 90μL of PBS, CHO cell lysis buffer, HEK 293 cell lysis buffer, and SF9 cell lysis buffer, respectively, as samples for testing. Sample extraction was performed using a commercially available viral DNA / RNA extraction kit (TransGen Biotech, catalog number ER201). Sample detection was conducted using a commercially available one-step RT-qPCR premix (Yisheng, catalog number 13650ES60) and the qPCR system and method described in Example 2. For matrix samples of the same virus, the average Cp values ​​of PBS matrix and other matrices were calculated and differencing to assess the interference of cell matrix on sample extraction and detection results. Robustness assessment required that the absolute value of the average Cp value difference (|ΔCp|) ≤ 1.5, and the relative standard deviation (RSD) of the Cp values ​​of the corresponding matrices ≤ 5%, indicating that the cell matrix does not interfere with detection, has good stability, and is suitable for the detection of such samples. The detection results are shown in Tables 11-25, demonstrating that the primer-probe combination provided by this invention is not affected by common cell matrices during detection and exhibits excellent robustness.

[0086] Table 11. Durability of Nodavirus FHV-Tncl (PBS matrix and CHO cell lysate)

[0087]

[0088] Table 12. Durability of Nodavirus FHV-Tncl (PBS matrix and HEK293 cell lysate)

[0089]

[0090] Table 13. Durability of Nodavirus FHV-Tncl (PBS matrix and SF9 cell lysate)

[0091]

[0092] Table 14. Durability of Nodavirus BBV (PBS matrix and CHO cell lysate)

[0093]

[0094] Table 15. Durability of Nodavirus BBV (PBS matrix and HEK293 cell lysate)

[0095]

[0096] Table 16. Durability of Nodavirus BBV (PBS matrix and SF9 cell lysate)

[0097]

[0098] Table 17. Durability of Nodavirus BoV (PBS matrix and CHO cell lysate)

[0099]

[0100] Table 18. Durability of Nodavirus BoV (PBS matrix and HEK293 cell lysate)

[0101]

[0102] Table 19. Durability of Nodavirus BoV (PBS matrix and SF9 cell lysate)

[0103]

[0104] Table 20. Durability of Nodavirus (NoV) (PBS matrix and CHO cell lysate)

[0105]

[0106] Table 21 Durability of Nodavirus (NoV) (PBS matrix and HEK293 cell lysate)

[0107]

[0108] Table 22 Durability of Nodavirus (NoV) (PBS matrix and SF9 cell lysate)

[0109]

[0110] Table 23 Durability of Nodavirus MrNV (PBS matrix and CHO cell lysate)

[0111]

[0112] Table 24 Durability of Nodavirus MrNV (PBS matrix and HEK293 cell lysate)

[0113]

[0114] Table 25 Durability of Nodavirus MrNV (PBS matrix and SF9 cell lysate)

[0115]

[0116] Example 5: Application of primer-probe combination in detecting Tncl virus in infected H5 cells of *Spodoptera litura*

[0117] Based on the aforementioned Tncl virus as an endogenous virus for *Spodoptera litura* H5 cells, this embodiment uses cultured *Spodoptera litura* H5 cell samples (approximately 1 × 10⁻⁶ cells). 4 100 cells were used as the sample to be tested; the sample was extracted using a commercial viral DNA / RNA extraction kit (TransGen Biotech, catalog number ER201); the sample was tested using a commercial one-step RT-qPCR premix (Yisheng, catalog number 13650ES60) and the qPCR system and method of Example 2 (i.e., the detection system for detecting farmhouse virus FHV, Tn5 cell line virus Tncl, and black beetle virus BBV); three independent replicate tests were performed, 9 replicates per test, for a total of 27 replicates. All sample results must be positive, i.e., 100% detection rate; at the same time, the Cp value of all replicates must meet the relative standard deviation (RSD) ≤ 5%, that is, the cell samples can be reproducibly and stably detected. The test results are shown in Table 26. It can be seen that the primer and probe combination provided by this invention can detect 100% of all cell samples during the test, the Cp value RSD ≤ 5%, and no value was detected in the BBV detection channel, which has very good stability and specificity.

[0118] Table 26 Examples of the application of Tncl virus infecting H5 cells of *Spodoptera litura*.

[0119]

[0120] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A qPCR primer probe set for detecting multiple Nodaviruses, characterized in that, the nucleotide sequence of the upstream primer FHV-Tncl-F is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer FHV-R is shown as SEQ ID NO. 2, the nucleotide sequence of the downstream primer Tncl-R is shown as SEQ ID NO. 3, and the nucleotide sequence of the fluorescent probe FHV-Tncl-P is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer BBV-F is shown as SEQ ID NO. 5, the nucleotide sequence of the downstream primer BBV-R is shown as SEQ ID NO. 6, and the nucleotide sequence of the fluorescent probe BBV-P is shown as SEQ ID NO. 7; the nucleotide sequence of the upstream primer BoV-F is shown as SEQ ID NO. 8, the nucleotide sequence of the downstream primer BoV-R is shown as SEQ ID NO. 9, and the nucleotide sequence of the fluorescent probe BoV-P is shown as SEQ ID NO. 10; the nucleotide sequence of the upstream primer NoV-F is shown as SEQ ID NO. 11, the nucleotide sequence of the downstream primer NoV-R is shown as SEQ ID NO. 12, and the nucleotide sequence of the fluorescent probe NoV-P is shown as SEQ ID NO. 13; the nucleotide sequence of the upstream primer MrNV-F is shown as SEQ ID NO. 14, the nucleotide sequence of the downstream primer MrNV-R is shown as SEQ ID NO. 15, and the nucleotide sequence of the fluorescent probe MrNV-P is shown as SEQ ID NO.

16.

2. The qPCR primer probe set of claim 1, wherein, The 5' end of the fluorescent probe in the primer probe set is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

3. The qPCR primer probe set of claim 2, wherein, The fluorescent reporter group comprises one of FAM, VIC or CY5; and the fluorescent quencher group comprises BHQ1 or BHQ2.

4. A qPCR kit for detecting multiple Nodaviruses, characterized in that, The kit comprises the qPCR primer probe set according to any one of claims 1-3.

5. The qPCR kit according to claim 4, characterized in that, The detection system of the kit for qPCR comprises the following components in a total volume of 30 μL: nucleic acid-free water 2.3 μL, qPCR reaction solution 16.2 μL, primer mixed working solution 1 μL, probe mixed working solution 0.5 μL, and template 10 μL.

6. The qPCR kit according to claim 5, characterized in that, For detecting FHV, Tn5 cell line virus, black beetle virus, the primer concentration of upstream primer FHV-Tncl-F, downstream primer FHV-R and downstream primer Tncl-R in the primer mixed working solution is 10 μM in terms of effective final concentration of each primer sequence variant, and the primer concentration of upstream primer BBV-F and downstream primer BBV-R is 3 μM; for detecting Boval virus, the primer concentration of upstream primer BoV-F and downstream primer BoV-R in the primer mixed working solution is 8 μM; for detecting Nomura virus, Macrobrachium rosenbergii nodavirus, the primer concentration of upstream primer NoV-F, downstream primer NoV-R, upstream primer MrNV-F and downstream primer MrNV-R in the primer mixed working solution is 6 μM.

7. The qPCR kit according to claim 5, characterized in that, For detecting FHV, Tn5 cell line virus, black beetle virus, the probe concentration of fluorescent probe FHV-Tncl-P and fluorescent probe BBV-P in the probe mixed working solution is 10 μM and 3 μM respectively; for detecting Boval virus, the probe concentration of fluorescent probe BoV-P in the probe mixed working solution is 8 μM; for detecting Nomura virus, Macrobrachium rosenbergii nodavirus, the probe concentration of fluorescent probe NoV-P and fluorescent probe MrNV-P in the probe mixed working solution is 6 μM and 6 μM respectively.

8. A method of detecting a Nodavirus that can infect a variety of insect cells for non-diagnostic purposes, characterized in that, The method comprises the following steps: extracting RNA of a sample to be detected and reverse transcribing into cDNA; using the qPCR primer probe group according to any one of claims 1-3 or the qPCR kit according to any one of claims 4-7 as a template; collecting fluorescence signals, and determining whether FHV, Tn5 cell line virus, black beetle virus, Boval virus, Nomura virus and / or Macrobrachium rosenbergii nodavirus in the sample to be detected is positive according to the amplification curve and Cp value.

9. The method of claim 8, wherein, The determination criteria are as follows: if the Cp value of the sample to be detected is less than 40 and there is an obvious amplification curve, it is determined to be positive; if the Cp value of the sample to be detected is greater than or equal to 40 or there is no obvious amplification curve, it is determined to be negative.

10. The method of claim 8, wherein, The reaction program of the qPCR is as follows: 50℃ reverse transcription for 20 min; 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 45 cycles. The reaction program of the qPCR is as follows: 50℃ reverse transcription for 20 min; 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 45 cycles.

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